Note: Descriptions are shown in the official language in which they were submitted.
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Tissue carbohydrate compositions and analysis thereof
FIELD OF INVENTION
The present invention reveals novel methods for producing novel carbohydrate
compositions,
glycomes, from animal tissues. The tissue substrate materials can be total
tissue samples and
fractionated tissue parts, or artificial models of tissues such as cultivated
cell lines. The
invention is further directed to the compositions and compositions produced by
the methods
according to the invention. The invention further represent methods for
analysis of the
glycomes, especially mass spectrometric methods.
BACKGROUND
Multiple methods to produce and analyze oligosaccharides from isolated
glycoproteins are
known. The present invention is directed to specific methods to release and
purify total
oligosaccharide pools quantitatively from tissues. The invention is
specifically directed to
methods using very low amounts of tissues. It is realized that purification of
an
oligosaccharide mixture from complex tissue samples to level of purity useful
for analysis is
more complex task than isolation of the oligosaccharides from purified
proteins. It is further
realized that the purification methods are novel and useful for the effective
analysis of protein
derived glycans.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Example of glycan signal analysis of MALDI-TOF mass spectrometric
data. A.
Mass spectrometric raw data showing a window of neutral N-glycan mass spectrum
in
positive ion mode, B. Glycan profile generated from the data in A.
Figure 2. Example of glycan signal analysis of MALDI-TOF mass spectrometric
data. A.
Mass spectrometric raw data showing a window of sialylated N-glycan mass
spectrum in
negative ion mode, B. Glycan profile generated from the data in A.
Figure 3. SW 480 (human colon adenocarcinoma cell line) neutral N-glycans
(passage n + 4).
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Figure 4. Glycan profiles of neutral (light columns) and combined neutral and
desialylated
(sialylated) glycans (dark columns) of SW 480 cells (passage n + 39).
Figure 5. Neutral protein-linked glycans of SW 480 cells. Differently shaded
columns from
left to right; Light columns: young cell line in growth phase (passage n + 4);
Dark columns:
starvated (passage n + 8); Blank columns: confluent (passage n + 8); Light
columns: old cell
line in growth phase (passage n + 39).
Figure 6. Sialylated N-glycan fraction of SW 480 cells (passage n + 4).
Figure 7. Neutral protein-linked glycans of human lung tissue.
Figure 8. Neutral protein-linked glycans of human ovary tissue.
Figure 9. Neutral protein-linked glycans of human ovary tissue with abnormal
growth.
Figure 10. Neutral protein-linked glycans of human liver tissue.
Figure 11. Neutral protein-linked glycans of human stomach tissue from blood
group specific
donors.
Figure 12. Neutral protein-linked glycans of human stomach tissue from blood
group specific
donors.
Figure 13. Neutral protein-linked glycans of human stomach tissue from blood
group specific
donors.
Figure 14. Neutral protein-linked glycans of healthy lung tissue (light
columns) and lung
cancer tumor (dark columns).
Figure 15. Neutral protein-linked glycans of human tissues, A. stomach, and B.
colon.
Figure 16. Neutral protein-linked glycans of bovine milk glycoproteins from A.
total milk,
and B. lactoferrin isolated from total milk.
Figure 17. Reference neutral N-glycan structures for NMR analysis (A-D).
Figure 18. Reference acidic N-glycan structures for NMR analysis (A-E).
Figure 19. Neutral and acidic N-glycan profiles of lysosomal protein sample.
Figure 20. Neutral glycosphingolipid glycan profile from human leukocytes.
Figure 21. Acidic glycosphingolipid glycan profile from human leukocytes.
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SUMMARY OF THE INVENTION
The present invention reveals novel methods for producing novel carbohydrate
compositions,
glycomes from animal tissues, preferably from vertebrates, more preferably
human and
mammalian tissues. The tissue substrate materials can be total tissue samples
and fractionated
tissue parts, such as serums, secretions and isolated differentiated cells
from the tissues, or
artificial models of tissues such as cultivated cell lines. In a preferred
embodiment the
invention is directed to special methods for the analysis of the surfaces of
tissues. The
invention is further directed to the compositions and compositions produced by
the methods
according to the invention. The invention further represent preferred methods
for analysis of
the glycomes, especially mass spectrometric methods.
The invention represents effective methods for purification of oligosaccharide
fractions from
tissues, especially in very low scale. The prior art has shown analysis of
separate glycome
components from tissues, but not total glycomes. It is further realized that
the methods
according to the invention are useful for analysis of glycans from isolated
proteins or
peptides. The invention represents effective methods for the practical
analysis of glycans from
isolated proteins especially from very small amounts of samples.
The invention is further directed to novel quantitative analysis methods for
glycomes. The
glycome analysis produces large amounts of data. The invention reveals methods
for the
analysis of such data quantitatively and comparision of the data between
different samples.
The invention is especially directed to quantitative two-dimensional
representation of the
data.
The present invention is specifically directed to glycomes of tissues
according to the
invention comprising glycan material with monosaccharide composition for each
of glycan
mass components according to the Formula Mn:
[Ma2]i1[Ma3]õ2
{[Ma2]i3[M(x6)]õ4}[Ma6]õ5{[Ma2]i6[Ma2]õ7[M(x3]õ8)M(34GN(34([{Fuca6}]mGNyR2)Z
wherein p, nl, n2, n3, n4, n5, n6, n7, n8, and m, and z are either
independently 0 or 1; with
the proviso that when n2 is 0, also nl is 0; when n4 is 0, also n3 is 0; when
n5 is 0, also nl,
n2, n3, and n4 are 0; when n7 is 0, also n6 is 0; when n8 is 0, also n6 and n7
are 0;
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y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacid and/or peptides derived from protein;
[] and ( indicates determinant either being present or absent depending on the
value of nl,
n2, n3, n4, n5, n6, n7, n8, and m; and
{} indicates a branch in the structure,
with the provisio that is 0 indicatin soluble mannose-G1cNAc1-glycome or
there is 5, more preferably 4 or less mannose residues or m is 1 and there is
6 or less mannose
units.
Typical glycomes comprise of subgroups of glycans, including N-glycans, 0-
glycans,
glycolipid glycans, and neutral and acidic subglycomes.
The preferred analysis method includes:
1) Preparing a tissue sample containing glycans for the analysis
2) Releasing total glycans or total glycan groups from a tissue sample, or
extracting free
glycans from a tissue sample
3) Optionally modifying glycans
4) Purification of the glycan fraction/fractions from biological material of
the sample
5) Optionally modifying glycans
6) Analysis of the composition of the released glycans preferably by mass
spectrometry
7a) Optionally presenting the data about released glycans quantitatively and
7b) Comparing the quantitative data set with another data set from another
tissue sample
or
8) Comparing data about the released glycans quantitatively or qualitatively
with data
produced from another tissue sample
The invention is directed to diagnosis of clinical state of tissue samples,
based on analysis of
glycans present in the samples. The invention is especially directed to
diagnosing cancer and
the clinical state of cancer.
The invention is further directed to structural analysis of glycan mixtures
present in tissue
samples.
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DESCRIPTION OF THE INVENTION
Tissue derived glycomes
5
Glycomes - novel glycan mixtures from tissue samples
The present invention reveals novel methods for producing novel carbohydrate
compositions,
glycomes from animal tissues, preferably from vertebrates, more preferably
human and
mammalian tissues. The tissue substrate materials can be
total tissue samples and
fractionated tissue parts, such as serums, secretions and isolated
differentiated cells from the
tissues, or
artificial models of tissues such as cultivated cell lines.
The invention revealed that the glycan structures on cell surfaces vary
between the various
tissues and same tissues under changing conditions.
The glycan structures on cell surfaces in general have been known to have
numerous
biological roles. Thus the knowledge about exact glycan mixtures from cell or
tissue surfaces
is important for knowledge about the status of cells. The invention revealed
that multiple
conditions affect the cells and cause changes in their glycomes.
Molecular weight distribution and structure groups of the glycomes
Preferred monosaccharide compositions of the glycomes
General compositions
The inventors were able to release or isolate various glycan fractions from
tissue materials,
which are useful for the characterization of the cellular material. The
glycans or major part
thereof are released preferably from glycoproteins or glycolipids of tissue
samples. The
invention is specifically directed to such glycan fractions.
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The glycan fractions of tissue samples comprise typically multiple, at least
about 10 "glycan
mass components" typically corresponding at least ten glycans and in most
cases clearly more
than 10 glycan structures.
Glycan mass components and corresponding monosaccharide compositions
The glycan mass components correspond to certain molecular weights observable
by mass
spectrometry and further correspond to specific monosaccharide composition or
monosaccharide compositions. Each monosaccharide component is normally present
in a
glycan as glycosidically linked monosaccharide residue in the nonreducing end
part of glycan
and the reducing end monosaccharide may be in free alditol form or modified
for example by
reduction or conjugated to an reducing end modifying reagent well known in the
art or to one,
two or several amino acids in case of glycopeptides. Monosaccharide
composition can be
obtained from molecular mass in a mass spectrum (glycan mass component) after
correcting
potential effect of the ion forms observable by the specific mass spectrometry
technologue
such as protonation/deprotonation, Na+, K+, Li+, or other adduct combinations,
or isotope
pattern derived effects. The monosaccharide compositions are calculated by
fitting mixtures
of individual monosaccharide (residue) masses and modification groups to
corrected
molecular mass of glycan mass component. Typically the molecular mass of
fitting
composition and the experimental mass correspond to each other very closely
with similar
first and even second decimals with optimal calibration.
The fitting may be further checked by measuring the experimental mass
difference from the
smaller and/or larger glycan mass component next in the putative biosynthetic
serie of a
glycan type and comparing the difference with the exact molecular mass of
corresponding
monosaccharide unit (residue), typically the mass differences of fitting
components in a good
quality mass spectrum and with correct marking of peaks in decimals, preferaby
in second or
third decimal of the mass number depending on the resolution of the specific
mass
spectrometric method. For optimal mass accuracy, an internal calibration may
be used, where
two or more known component's mass peaks are used to re-calculate masses for
each
components in the spectrum. Such calibration components are preferably
selected among the
most abundant glycan signals present in the glycan profiles, in the case of
human or other
animal cell derived glycan profiles most preferably selected among the most
abundant glycan
signals present in Figures described in the present invention.
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The monosaccharide composition includes monosaccharide component names and
number,
typically as subscript, indicating how many of the individual mass components
is present in
the monosaccharide composition; and names of assigned modifying groups and
numbers
indicating their abundance.
It is further realized that the masses of glycan mass component may be
obtained as exact
monoisotopic mass of usually smallest isotope of the glycan mass component or
as an average
mass of the isotope distribution of the glycan mass component. Exact mass is
calculated form
exact masses of individual mass components and average from masses average
masses of
individual mass components. Person skilled in art can recognize from the peak
shapes (i.e. by
the resolution obtained) in the mass spectrum whether to use monoisotopic or
average masses
to interpret the spectra. It is further realized that average and exact masses
can be converted to
each other when isotope abundances of molecules are known, typically natural
abundance
without enrichment of isotopes can be assumed, unless the material is
deliberately labelled
with radioactive or stable isotopes.
It is further realized that specific rounded mass numbers can be used as names
for glycan
mass components. The present invention uses preferably mass numbers rounded
down from
the exact mass of the monosaccharide composition (and usually observable or
observed mass)
to closest integer as names of glycan mass components.
The masses of gylcan mass components are obtained by calculating molecular
mass of
individual monosaccharide components (Hex, HexNAc, dHex, sialic acids) from
the known
atom compositions (for example hexose (Hex) corresponds to C6H1206) and
subtracting for
water in case of monosaccharide residue, followed by calculating the sum of
the
monosaccharide components (and possible modifications such as SO3 or PO3H) .It
is further
realized that molecular masses of glycans may be calculated from atomic
compositions or any
other suitable mass units corresponding molecular masses of these. The
molecular masses and
calculation thereof are known in the art and masses of monosaccharide
components/residues
are available in tables with multiple decimals from various sources.
It is further realized that many of the individual monosaccharide compositions
described in
the present invention further correspond to several isomeric individual
glycans. In addition,
there exist also monosaccharide compositions that have nearly equal masses,
for example
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dHex2 and NeuAc monosaccharide residues that have nearly equal masses, and
other
examples can be presented by a person skilled in the art. It is realized that
the ability to
differentiate compositions with nearly equal masses depends on
instrumentation, and the
present method is especially directed to a possibility to select also such
compositions in place
of proposed compositions.
The preferred glycans in glycomes comprise at least two of following
monosaccharide
component residues selected from group: Hexoses (Hex) which are Gal, Glc and
Man; N-
acetylhexosamines (HexNAc) which are G1cNAc and Ga1NAc; pentose, which is Xyl;
Hexuronic acids which are G1cA and IdoA; deoxyhexoses (dHex), which is fucose
and sialic
acids which are NeuAc and/NeuGc; and further modification groups such as
acetate (Ac),
sulphate and phosphate forming esters with the glycans. The monosaccharide
residues are
further grouped as major backbone monosaccharides including G1cNAc, HaxA, Man
and Gal;
and specific terminal modifying monosaccharide units Glc, Ga1NAc, Xyl and
sialic acids.
Detection of glycan modifications
The present invention is directed to analyzing glycan components from
biological samples,
preferably as mass spectrometric signals. Specific glycan modifications can be
detected among the detected signals by determined indicative signals as
exemplified below.
Modifications can also be detected by more specific methods such as chemical
or physical
methods, for example mass spectrometric fragmentation or glycosidase detection
as disclosed
in the present invention. In a preferred form of the present method, glycan
signals are
assigned to monosaccharide compositions based on the detected m/z ratios of
the glycan
signals, and the specific glycan modifications can be detected among the
detected
monosaccharide compositions.
In a further aspect of the present invention, relative molar abundances of
glycan components
are assigned based on their relative signal intensities detected in mass
spectrometry as
described in the Examples, which allows for quantification of glycan
components with
specific modifications in relation to other glycan components. The present
method is also
directed to detecting changes in relative amounts of specific modifications in
cells at different
time points to detect changes in cell glycan compositions.
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Glycome glycan fraction further comprising monosaccharides
The invention is specifically directed to glycan compositions, which further
comprise at least
one monosaccharide component in free form, preferably a preferred
monosaccharide
component described above. The monosaccharide comprising compositions are in a
preferred
embodiment derived from a cell material or released glycomes, which has been
in contact
with monosaccharide releasing chemicals or enzymes, preferably with
exoglycosidase
enzymes or chemicals such as oxidating reagents and/or acid, more preferably
with a
glycosidase enzyme. The invention is further directed to compositions
comprising a specific
preferred monosaccharide according to the invention, an exoglycosidase enzyme
capable
releasing all or part of the specific monosaccharide and an glycan composition
according to
the invention from which at least part of the terminal specific monosaccharide
has been
released.
Limit of detection for glycome components
It is further realized that by increasing the sensitivity of detection the
number of glycan mass
components can be increased. The analysis according to the invention can be in
most cases
performed from major or significant components in the glycome mixture. The
present
invention is preferably directed to detection of glycan mass components from a
high quality
glycan preparation with optimised experimental condition, when the glycan mass
components
have abundance at least higher than 0.01 % of total amount of glycan mass
components, more
preferably of glycan mass components of abundance at least higher than 0.05%,
and most
preferably at least higher than 0.10% are detected. The invention is further
directed practical
quality glycome compositions and analytic process directed to it, when glycan
mass
components of at least about 0.5 %, of total amount of glycan mass components,
more
preferably of glycan mass components of abundance at least higher than 1.0 %,
even more
preferably at least higher than 2.0%, most preferably at least higher than
4.0% (presenting
lower range practical quality glycome), are detected. The invention is further
directed to
glycomes comprising preferred number of glycan mass components of at least the
abundance
of observable in high quality glycomes, and in another embodiment glycomes
comprising
preferred number of glycan mass components of at least the abundance of
observable in
practical quality glycomes.
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Subglycomes obtainable by purification or specific release method
It further realized that fractionation or differential specific release
methods of glycans from
glycoconjugates can be applied to produce subglycomes containing part of
glycome.
5 The subglycomes produced by fractionation of glycomes are called
"fractionated
subglycomes".
The glycomes produced by specific release methods are "linkage-subglycomes".
The
invention is further directed to combinations of linkage-subglycomes and
fractionated
subglycomes to produce "fractionated linkage-subglycomes", for example
preferred
10 fractionated linkage-subglycomes includes neutral 0-glycans, neutral N-
glycans, acidic 0-
glycans, and acidic N-glycans, which were found very practical in
characterising target
material according to the invention.
The fractionation can be used to enrich components of low abundance. It is
realized that
enrichment would enhance the detection of rare components. The fractionation
methods may
be used for larger amounts of cell material. In a preferred embodiment the
glycome is
fractionated based on the molecular weight, charge or binding to carbohydrate
binding agents.
These methods have been found useful for specific analysis of specific
subglycomes and
enrichment more rare components. The present invention is in a preferred
embodiment
directed to charge based separation of neutral and acidic glycans. This method
gives for
analysis method, preferably mass spectroscopy material of reduced complexity
and it is useful
for analysis as neutral molecules in positive mode mass spectrometry and
negative mode mass
spectrometry for acidic glycans.
Differential release methods may be applied to get separately linkage specific
subglycomes
such as 0-glycan, N-glycan, glycolipid or proteoglycan comprising fractions or
combinations
thereof. Chemical and enzymatic methods are known for release of specific
fractions,
furthermore there are methods for simultaneous release of 0-glycans and N-
glycans.
Novel complete compositions
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It is realized that at least part of the glycomes have novelty as novel
compositions of very
large amount of components. The glycomes comprising very broad range
substances are
referred as complete glycomes.
Preferably the composition is a complete composition comprising essentially
all degrees of
polymerisation in general from at least about about disaccharides, more
preferably from
trisaccharides to at least about 25-mers in a high resolution case and at
least to about 20-mers
or at least about 15-mer in case of medium and practical quality preparations.
It is realized that especially the lower limit, but also upper limit of a
subglycome depend on
the type of subglycome and /or method used for its production. Different
complete ranges
may be produced in scope of general glycomes by fractionation, especially
based on size of
the molecules.
Novel compositions with new combinations of subglycomes and preferred glycan
groups
It is realized that several glycan types are present as novel glycome
compositions produced
from the tissue samples. The invention is specifically directed to novel
mixture composition
comprising different subglycomes and preferred glycan groups
Novel quantitative glycome compositions
It is realised that the glycome compossitiona as described in examples
represent quantitatively
new data about glycomes from the preferred tissue sample types. The
proportions of various
components cannot be derived from background data and are very useful for the
analysis
methods according to the invention. The invention is specifically directed to
glycome
compositions according to the examples when the glycan mass components are
present in
essentially similar relative amounts.
Preferred composition formulas
The present invention is specifically directed to glycomes of tissue samples
according to the
invention comprising glycan material with monosaccharide composition for each
of glycan
mass components according to the Formula I:
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NeuAc,nNeuGcnHexoHexNAcpdHexqHexArPensActModXX, (I)
where m, n, o, p, q, r, s, t, , and x are independent integers with values > 0
and less than about
100,
with the proviso that
for each glycan mass components at least two of the backbone monosaccharide
variables o, p,
or r is greater than 0, and
ModX represents a modification (or N different modifications Mod1, Mod2, ...,
ModN),
present in the composition in an amount of x (or in independent amounts of xl,
x2, ..., xN),
Preferably examples of such modifications (Mod) including for example SO3 or
PO3H
indicating esters of sulfate and phosphate, respectively
and the glycan composition is preferably derived from isolated human tissue
samples or
preferred subpopulations thereof according to the invention.
It is realized that usually glycomes contain glycan material for which the the
variables are less
much less than 100, but large figures may be obtained for polymeric material
comprising
glycomes with repeating polymer structures, for example ones comprising
glycosaminoglycan
type materials. It is further realized that abundance of the glycan mass
components with
variables more than 10 or 15 is in general very low and observation of the
glycome
components may require purification and enrichment of larger glycome
components from
large amounts of samples.
Broad mass range glycomes
In a preferred embodiment the invention is directed to broad mass range
glycomes comprising
polymeric materials and rare individual components as indicated above.
Observation of large
molecular weight components may require enrichment of large molecular weight
molecules
comprising fraction. The broad general compositions according to the Formula I
are as
described above,
with the proviso that
m, n, o, p, q, r, s, t, and x are independent integers with preferable values
between 0 and 50,
with the proviso that for each glycan mass components at least two of o, p, or
r is at least 1,
and the sum of the monosaccharide variables; m, n, o, p, q, r, and s,
indicating the degree of
polymerization or oligomerization, for each glycan mass component is less than
about 100
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and the glycome comprises at least about 20 different glycans of at least
disaccharides.
Practical mass range glycomes
In a preferred embodiment the invention is directed to practical mass range
and high quality
glycomes comprising lower molecular weight ranges of polymeric material. The
lower
molecular weight materials at least in part and for preferred uses are
observable by mass
spectrometry without enrichment.
In a more preferred general composition according to the Formula I as
described above,
m, n, o, p, q, r, s, t, and x are independent integers with preferable values
between 0 and about
20, more preferably between 0 and about 15, even more preferably between 0 and
about 10,
with the proviso that at least two of o, p, or r is at least 1,
and the sum of the monosaccharide variables; m, n, o, p, q, r, and s,
indicating the degree of
polymerization or oligomerization, for each glycan mass component is less than
about 50 and
more preferably less than about 30,
and the glycome comprises at least about 50 different glycans of at least
trisaccharides.
In a preferred embodiment the invention is directed to practical mass range
high quality
glycomes which may comprise some lower molecular weight ranges of polymeric
material.
The lower molecular weight materials at least in part and for preferred uses
are observable by
mass spectrometry without enrichment.
In a more preferred general composition according to the Formula I as
described above,
m, n, o, p, q, r, s, t, and x are independent integers with preferable values
between 0 and about
10, more preferably between 0 and about 9, even more preferably, between 0 and
about 8,
with the proviso that at least two of o, p, or r is at least 1,
and the sum of the monosaccharide variables; m, n, o, p, q, r, and s,
indicating the degree of
polymerization or oligomerization, for each glycan mass component is less than
about 30 and
more preferably less than about 25,
and the glycome comprises at least about 50 different glycans of at least
trisaccharides.
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The practical mass range glycomes may typically comprise tens of components,
for example
in positive ion mode MALDI-TOF mass spectrometry for neutral subglycomes it is
usually
possible to observe even more than 50 molecular mass components, even more
than 100 mass
component corresponding to much larger number of potentially isomeric glycans.
The number
of components detected depends on sample size and detection method.
Preferred Subglycomes
The present invention is specifically directed to subglycomes of tissue sample
glycomes
according to the invention comprising glycan material with monosaccharide
compositions for
each of glycan mass components according to the Formula I and as defined for
broad and
practical mass range glycomes. Each subglycome has additional characteristics
based on
glycan core structures of linkage-glycomes or fractionation method used for
the fractionated
glycomes. The preferred linkage glycomes includes:
N-glycans, 0-glycans, glycolipid glycans, neutral and acidic subglycomes,
N-glycan subglycome
Protein N-glycosidase releases N-glycans comprising typically two N-
acetylglycosamine
units in the core, optionally a core linked fucose unit and typically then 2-3
hexoses (core
mannoses), after which the structures may further comprise hexoses being
mannose or in
complex -type N-glycans further N-acetylglycosamines and optionally hexoses
and sialic
acids.
N-glycan subglycomes relased by protein N-glycosidase comprise N-glycans
containing N-
glycan core structure and are releasable by protein N-glycosidase from cells.
The N-glycan core structure is Man(34GIcNAc(3(Fuc(X6)n4GIcNAc, wherein n is 0
orl and the
N-glycan structures can be elongated from the Man(34 with additional
mannosylresidues. The
protein N-glycosidase cleaves the reducing end GIcNAc from Asn in proteins. N-
glycan
subglycomes released by endo-type N-glycosidases cleaving between GIcNAc units
contain
Man(34GIcNAc(3-core, and the N-glycan structures can be elongated from the
Man(34 with
additional mannosylresidues.
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In case the Subglycome and analysis representing it as Glycan profile is
formed from N-
glycans liberated by N-glycosidase enzyme, the preferred additional
constraints for Formula
I are:
p > 0, more preferably 1< p< 100, typically p is between 2 and about 20, but
polymeric
5 structures containing glycomes may comprise larger amounts of HexNAc and
it is realized that in typical core of N-glycans indicating presence of at
least partially complex
type structure
when p> 3 it follows that o> 1.
10 Glycolipid subglycome
In case the Subglycome and analysis representing it as Glycan profile is
formed from lipid-
linked glycans liberated by endoglycoceramidase enzyme, the preferred
additional constraints
for Formula I are:
15 o> 0, more preferably 1< o< 100, and
when p> 1 it follows that o> 2.
Typically glycolipids comprise two hexoses (a Iactosylresidue) at the core.
The degree of
oligomerization in a usual practical glycome from glycolipds is under about 20
and more
preferably under 10. Very large structures comprising glycolipids,
polyglycosylceramides,
may need enrichment for effective detection.
Neutral and acidic subglycomes
Most preferred fractionated Subglycomes includes 1) subglycome of neutral
glycans and 2)
subglycome of acidic glycans. The major acidic monosaccharide unit is in most
cases a sialic
acid, the acidic fraction may further comprise natural negatively charged
structure/structures
such as sulphate(s) and/phosphate(s).
In case the Subglycome and analysis representing it as Glycan profile is
formed from
sialylated glycans, the preferred additional constraints for Formula I are:
(m + n) > 0, more preferably 1<(m + n) < 100.
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Large amounts of sialic acid in a glycan mass component would indicate
presence of
polysailic acid type structures. Practical and high resolutions acidic
glycomes usually have
m+n values for individual major glycan mass components with preferred
abundance between
1 and 10, more preferably and of the between 1-5 and most preferably between 1-
4 for a
usual glycomes according to the invention. For neutral glycans, (m + n) = 0,
and they do not
contain negatively charged groups as above.
Preferred structure groups observable in glycome profiles
The present invention is specifically directed to the glycomes of tissue
samples according to
the invention comprising as major components at least one of structure groups
selected
from the groups described below.
Glycan groups
According to the present invention, the Glycan signals are optionally
organized into Glycan
groups and Glycan group profiles based on analysis and classification of the
assigned
monosaccharide and modification compositions and the relative amounts of
monosaccharide
and modification units in the compositions, according to the following
classification rules:
10 The glycan structures are described by the formulae:
HexmHexNAcndHex NeuAcpNeuGcqPenrMod l sM a1 Mod2sM d2 ... ModXSM ax,
wherein m, n, o, p, q, individual sMod, and X. are each independent variables,
and Mod is a
functional group covalently linked to the glycan structure.
2 Glycan structures in general are classified as follows:
a. Structures (p,q = 0) are classified as "non-sialylated",
b. Structures (p,q > 0) are classified as "sialylated",
c. Structures (q > 0) are classified as "NeuGc-containing",
d. Relation [2 (p + q) : (m + n)] describes the general sialylation degree of
a
glycan structure,
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C. In the case of mammalian glycans, structures (o = 0) are classified as "non-
fucosylated",
f. In the case of mammalian glycans, structures (o > 0) are classified as
"fucosylated",
g. Structures (Mod = Ac and sAc > 0) are classified as `acetylated',
h. Structures (Mod = SO3 and sSO3 > 0) are classified as `sulfated', and
i. Structures (Mod = PO3H and sPO3H > 0) are classified as `phosphorylated'.
30 N-glycan glycan structures, generated e.g. by the action of peptide-N-
glycosidases, are classified as follows:
a. Structures (n = 2 and m> 0 and p,q = 0) are classified as "mannose-
terminated
N-glycans",
b. Structures (n = 2 and m> 5 and o,p,q = 0) are classified as "high-mannose N-
glycans",
c. Structures (n = 2 and m> 5 and o > 0 and p,q = 0) are classified as
"fucosylated high-mannose N-glycans",
d. Structures (n = 2 and 4> m> 1 and p,q = 0) are classified as "low-mannose N-
glycans",
C. Structures (n = 2 and 4> m> 1 and o> 0 and p,q = 0) are classified as
"fucosylated low-mannose N-glycans",
f. Structures (n = 3 and m> 2) are classified as "hybrid-type or monoantennary
N-glycans",
g. Structures (n > 4 and m> 3) are classified as "complex-type N-glycans",
h. Structures (n > m> 2) are classified as "N-glycans containing non-reducing
terminal N-acetylhexosamine",
i. Structures (n = m> 5) are classified as "N-glycans potentially containing
bisecting N-acetylglucosamine",
j. In the case of mammalian N-glycans, structures (o > 2) are classified as "N-
glycans containing a2-, a3-, or a4-linked fucose",
k. Relation [2 (p + q) : (m + n - 5)] describes the "overall sialylation
degree" of a
sialylated N-glycan structure, and
1. Specifically, sum (p + q) describes the "sialylation degree" of a
sialylated
hybrid-type or monoantennary N-glycan structure.
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40 Mucin-type 0-glycan structures, generated e.g. by alkaline 0-elimination,
are
classified as follows:
a. Structures (n = m), with (N = n = m), are classified as "Type N 0-glycans",
b. More specifically, structures (n = m = 1) are classified as "Type 1 0-
glycans",
c. More specifically, structures (n = m = 2) are classified as "Type 2 0-
glycans",
d. More specifically, structures (n = m = 3) are classified as "Type 3 0-
glycans",
C. Relation [2 (p + q) : (m + n)] describes the overall sialylation degree of
a
sialylated N-glycan structure, and
f. Specifically, relation [(p + q) : N] describes the sialylation degree of a
sialylated Type N 0-glycan structure.
Lipid-linked can also be classified into structural groups based on their
monosaccharide
compositions, as adopted from the classifications above according to the
invention.
For example, glycan signal corresponding to a tissue sample N-glycan
structure:
Hex5HexNAc4dHex2NeuAc1Ac1,
is classified as belonging to the following Glycan Groups:
- sialylated (general sialylation degree: 2/9),
- fucosylated,
- acetylated,
- complex-type N-glycans (overall sialylation degree: 0.5),
- N-glycans containing a2-, a3-, or a4-linked fucose.
Glycomes comprising novel glycan types
The present invention revealed novel unexpected components among in the
glycomes studied.
The present invention is especially directed to glycomes comprising such
unusual materials
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Preferred glycome types
Derivatized glycomes
It is further realized that the glycans may be derivatized chemically during
the process of
release and isolation. Preferred modifications include modifications of the
reducing end and
or modifications directed especially to the hydroxyls- and/or N-atoms of the
molecules. The
reducing end modifications include modifications of reducing end of glycans
involving
known derivatization reactions, preferably reduction, glycosylamine,
glycosylamide, oxime
(aminooxy-) and reductive amination modifications. Most preferred
modifications include
modification of the reducing end. The derivatization of hydroxyl- and/or amine
groups, such
as produced by methylation or acetylation methods including permethylation and
peracetylation has been found especially detrimental to the quantitative
relation between
natural glycome and the released glycome.
Non-derivatized released glycomes
In a preferred embodiment the invention is directed to non-derivatized
released glycomes. The
benefit of the non-derivatized glycomes is that less processing needed for the
production. The
non-derivatized released glycomes correspond more exactly to the natural
glycomes from
which these are released. The present invention is further directed to
quantitative purification
according to the invention for the non-derivatized releases glycomes and
analysis thereof.
The present invention is especially directed to released glycomes when the
released glycome
is not a permodified glycome such as permethylated glycome or peracetyated
glycome. The
released glycome is more preferably reducing end derivatized glycome or a non
derivatized
glycome, most preferably non-derivatized glycome.
Novel cell surface glycomes and released glycomes of the target material
The present invention is further directed to novel total compositions of
glycans or
oligosaccharides referred as glycomes and in a more specific embodiment as
released
glycomes observed from or produced from the target material according to the
invention. The
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released glycome indicates the total released glycans or total specific glycan
subfractions
released from the target material according to the invention. The present
invention is
specifically directed to released glycomes meaning glycans released from the
target material
according to the invention and to the methods according to the invention
directed to the
5 glycomes.
The present invention preferably directed to the glycomes released as
truncated and/or non-
truncated glycans and/or derivatized according to the invention.
10 The invention is especially directed to N-linked and/or 0-linked and/or
Lipid linked released
glycomes from the target material according to the invention. The invention is
more
preferably directed to released glycomes comprising glycan structures
according to the
invention, preferably glycan structures as defined in formula I, . The
invention is more
preferably directed to N-linked released glycomes comprising glycan structures
according to
15 the invention, preferably glycan structures as defined in formula I.
Non-derivatized released cell surface glycomes and production
In a preferred embodiment the invention is directed to non-derivatized
released cell surface
glycomes. The non-derivatized released cell surface glycomes correspond more
exactly to the
20 fractions of glycomes that are localized on the cell surfaces, and thus
available for biological
interactions. These cell surface localized glycans are of especial importance
due to their
availability for biological interactions as well as targets for reagents (e.g.
antibodies, lectins
etc...) targeting the cells or tissues of interest. The invention is further
directed to release of
the cell surface glycomes, preferably from intact cells by hydrolytic enzymes
such as
proteolytic enzymes, including proteinases and proteases, and/or glycan
releasing enzymes,
including endo-glycosidases or protein N-glycosidases. Preferably the surface
glycoproteins
are cleaved by proteinase such as trypsin and then glycans are analysed as
glycopeptides or
preferably relased further by glycan relasing enzyme.
Analysis of the glycomes
Analysis of the glycan mixtures by physical means, preferably by mass
spectrometry
The present invention is directed to analysis of glycan mixtures present in
tissue samples.
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Quantitative and qualitative analysis of glycan profile data
The invention is directed to novel methods for qualitative analysis of glycome
data. The
inventors noticed that there are specific components in glycomes according to
the invention,
the presence or absence of which are connected or associated with specific
cell type or cell
status. It is realized that qualitative comparison about the presence of
absence of such signals
are useful for glycome analysis. It is further realized that signals either
present or absent that
are derived from a general glycome analysis may be selected to more directed
assay
measuring only the qualitatively changing component or components optionally
with a more
common component or components useful for verification of data about the
presence or
absence of the qualitative signal.
The present invention is further specifically directed to quantitative
analysis of glycan data
from tissue samples. The inventors noted that quantitative comparisons of the
relative
abundances of the glycome components reveal substantial differences about the
glycomes
useful for the analysis according to the invention.
Essential steps of the glycome analysis
The process contains essential key steps which should be included in every
process according
to the present invention.
The essential key steps of the analysis are:
1. Release of total glycans or total glycan groups from a tissue sample
2. Purification of the glycan fraction/fractions from biological material of
the sample,
preferably by a small scale column array or an array of solid-phase extraction
steps
3. Analysis of the composition of the released glycans, preferably by mass
spectrometry
In most cases it is useful to compare the data with control sample data. The
control sample
may be for example from a healthy tissue or cell type and the sample from same
tissue altered
by cancer or another disease. It is preferable to compare samples from same
individual
organism, preferably from the same human individual.
Specific types of the glycome analysis
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Comparative analysis
The steps of a comparative analysis are:
1. Release of total glycans or total glycan groups from tissue sample
2. Purification of the glycan fraction/fractions from biological material of
the sample,
preferably by a small scale column array or an array of solid-phase extraction
steps
3. Analysis of the composition of the released glycans, preferably by mass
spectrometry
4. Comparing data about the released glycans quantitatively or qualitatively
with data
produced from another tissue sample
It may be useful to analyse the glycan structural motifs present in the
sample, as well as their
relative abundances. The ability to elucidate structural motifs results from
the quantitative
nature of the present analysis procedure, comparison of the data to data from
previously
analyzed samples, and knowledge of glycan biosynthesis.
Analysis including characterization of structural motives
The glycome analysis may include characterization of structural motives of
released glycans.
The structural motif analysis may be performed in combination with structural
analysis.
Preferred methods to reveal specific structural motifs include
a) direct analysis of specific structural modifications of the treatment of
glycans
preferably by exo- or endoglycosidases and/or chemical modification or
b) indirect analysis by analysis of correlating factors for the structural
motives for such as
mRNA-expression levels of glycosyltransferases or enzymes producing sugar
donor
molecules for glycosyltransferases.
The direct analyses are preferred as they are in general more effective and
usually more
quantitative methods, which can be combined to glycome analysis.
In a preferred embodiment the invention is directed to combination of analysis
of structural
motifs and glycome analysis.
The steps of a structural motif analysis are:
1. Release of total glycans or total glycan groups from a tissue sample
2. Purification of the glycan fraction/fractions from biological material of
the sample,
preferably by a small scale column array or an array of solid-phase extraction
steps
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3. Analysis of the composition of the released glycans, preferably by mass
spectrometry
4. Analysis of structural motifs present in of the glycan mixture, and
optionally their
relative abundancies
5. Optionally, comparing data about the glycan structural motifs with data
produced from
another tissue sample
The steps 3 and 4 may be combined or performed in order first 4 and then 3.
Preferred detailed glycome analysis including quantative data analysis
Detailed preferred glycome analysis according to the invention
More detailed preferred analysis method include following analysis steps:
1. Preparing a tissue sample containing glycans for the analysis
2. Release total glycans or total glycan groups from a tissue sample
3. Optionally modifying glycans or part of the glycans.
4. Purification of the glycan fraction/fractions
from biological material and reagents of the sample by a small scale column
array
5. Optionally modifying glycans and optionally purifying modified glycans
6. Analysis of the composition of the released glycans preferably by mass
spectrometry
using at least one mass spectrometric analysis method
7. a) Optionally presenting the data about released glycans quantitatively and
7. b) Comparing the quantitative data set with another data set from another
tissue sample
and/or alternatively to 7a) and 7b)
8. Comparing data about the released glycans quantitatively or qualitatively
with data
produced from another tissue sample
The present methods further allow the possibility to use part of the non-
modified material or
material modified in step 3 or 5 for additional modification step or step and
optionally
purified after modification step or steps, optionally combining modified
samples, and analysis
of additionally modified samples, and comparing results from differentially
modified samples.
As mentioned above, It is realized that many of the individual monosaccharide
compositions
in a given glycome further corresponds to several isomeric individual glycans.
The present
methods allow for generation of modified glycomes. This is of particular use
when
modifications are used to reveal such information about glycomes of interest
that is not
directly available from a glycan profile alone (or glycome profiles to
compare). Modifications
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can include selective removal of particular monosaccharides bound to the
glycome by a
defined glycosidic bond, by degradation by specific exoglycosidases or
selective chemical
degradation steps such as e.g. periodic acid oxidation. Modifications can also
be introduced
by using selective glycosyltransferase reactions to label the free acceptor
structures in
glycomes and thereby introduction of a specific mass label to such structures
that can act as
acceptors for the given enzyme. In preferred embodiment several of such
modifications steps
are combined and used to glycomes to be compared to gain further insights of
glycomes and
to facilitate their comparison.
Quantitative presentation of glycome analysis
The present invention is specifically directed to quantitative presentation of
glycome data.
Two-dimensional presentation by quantitation and component indicators
The quantitative presentation means presenting quantitative signals of
components of the
glycome, preferably all major components of the glycome, as a two -dimensional
presentation
including preferably a single quantitative indicator presented together with
component
identifier.
The preferred two dimensional presentations includes tables and graphs
presenting the two
dimensional data. The preferred tables list quantitative indicators in
connection with,
preferably beside or under or above the component identifiers, most preferably
beside the
identifier because in this format the data comprising usually large number of
component
identifier - quantitation indicator pairs.
Quantitation indicator
The quantitation indicator is a value indicating the relative abundance of the
single glycome
component with regard to other components of total glycome or subglycome. The
quantitation
indicator can be directly derived from quatitative experimental data, or
experimental data
corrected to be quantitative.
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Normalized quantitation indicator
The quantitation indicator is preferably a normalized quantitation indicator.
The normalized
quantitation indicator is defined as the experimental value of a single
experimental
quantitation indicator divided by total sum of quantitation indicators
multiplied by a constant
5 quantitation factor.
Preferred quantitation factors includes integer numbers from 1- 1000 0000 000,
more
preferably integer numbers 1, 10 or 100, and more preferably 1 or 100, most
preferably 100.
The quantitation number one is preferred as commonly understandable portion
from 1 concept
10 and the most preferred quantitation factor 100 corresponds to common
concept of per cent
values.
The quantitation indicators in tables are preferably rounded to correspond to
practical
accuracy of the measurements from which the values are derived from. Preferred
rounding
15 includes 2-5 meaningful accuracy numbers, more preferably 2-4 numbers and
most preferably
2-3 numbers.
Component indicators
The preferred component indicators may be experimentally derived component
indicators.
20 Preferred components indicators in the context of mass spectrometric
analysis includes mass
numbers of the glycome components, monosaccharide or other chemical
compositions of the
components and abbreviation corresponding to thereof, names of the molecules
preferably
selected from the group: desriptive names and abbreviations; chemical names,
abbreviations
and codes; and molecular formulas including gaphic representations of the
formulas.
25 It is further realized that molecular mass based component indicators may
include multiple
isomeric structures. The invention is in a preferred embodiment directed to
practical analysis
using molecular mass based component indicators. In more specific embodiment
the
invention is further directed to chemical or enzymatic modification methods or
indirect
methods according to the invention in order to resolve all or part of the
isomeric components
corresponding to a molecular mass based component indicators.
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Glycan signals
The present invention is directed to a method of accurately defining the
molecular masses of
glycans present in a sample, and assigning monosaccharide compositions to the
detected
glycan signals.
The Glycan signals according to the present invention are glycan components
characterized
by:
1 mass-to-charge ratio (m/z) of the detected glycan ion,
2 molecular mass of the detected glycan component, and/or
3 monosaccharide composition proposed for the glycan component.
Glycan profiles
The present invention is further directed to a method of describing mass
spectrometric raw
data of Glycan signals as two-dimensional tables of:
1 monosaccharide composition, and
2 relative abundance,
which form the Glycan profiles according to the invention. Monosaccharide
compositions are
as described above. For obtaining relative abundance values for each Glycan
signal, the raw
data is recorded in such manner that the relative signal intensities of the
glycan signals
represent their relative molar proportions in the sample. Methods for relative
quantitation in
MALDI-TOF mass spectrometry of glycans are known in the art (Naven & Harvey,
19xx;
Papac et al., 1996) and are described in the present invention. However, the
relative signal
intensities of each Glycan signal are preferably corrected by taking into
account the potential
artefacts caused by e.g. isotopic overlapping, alkali metal adduct
overlapping, and other
disturbances in the raw data, as described below.
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By forming these Glycan profiles and using them instead of the raw data,
analysis of the
biological data carried by the Glycan profiles is improved, including for
example the
following operations:
1 identification of glycan signals present in the glycan profile,
2 comparison of glycan profiles obtained from different samples,
3 comparison of relative intensities of glycan signals within the glycan
profile, and
4 organizing the glycan signals present in the glycan profile into subgroups
or subprofiles.
Analysis of associated signals to produce single quantitative signal
(quantitation
indicator)
Analysis of associated signals: isotope correction
Glycan signals and their associated signals may have overlapping isotope
patterns.
Overlapping of isotope patterns is corrected by calculating the experimental
isotope patterns
and subtracting overlapping isotope signals from the processed data.
Analysis of associated signals: adduct ion correction in positive ion mode
Glycan signals may be associated with signals arising from multiple adduct
ions in positive
ion mode, e.g. different alkali metal adduct ions. Different Glycan signals
may give rise to
adduct ions with similar m/z ratios: as an example, the adduct ions [Hex+Na]+
and [dl4ex+K]+
have m/z ratios of 203.05 and 203.03, respectively. Overlapping of adduct ions
is corrected by
calculating the experimental alkali metal adduct ion ratios in the sample and
using them to
correct the relative intensities of those Glycan signals that have overlapping
adduct ions in the
experimental data. Preferably, the major adduct ion type is used for
comparison of relative
signal intensities of the Glycan signals, and the minor adduct ion types are
removed from the
processed data. The calculated proportions of minor adduct ion types are
subtracted from the
processed data.
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Analysis of associated signals: adduct ion correction in negative ion mode
Also in negative ion mode mass spectrometry, Glycan signals may be associated
with signals
arising from multiple adduct ions. Typically, this occurs with Glycan signals
that correspond
to multiple acidic group containing glycan structures. As an example, the
adduct ions
[NeuAc2-H+Na]- at m/z 621.2 and [NeuAc2-H+K]- at m/z 637.1, are associated
with the
Glycan signal [NeuAc2-H]- at m/z 599.2. These adduct ion signals are added to
the Glycan
signal and thereafter removed from the processed data. In cases where
different Glycan
signals and adduct ion signals overlap, this is corrected by calculating the
experimental alkali
metal adduct ion ratios in the sample and using them to correct the relative
intensities of those
Glycan signals that have overlapping adduct ions in the experimental data.
Analysis of associated signals: removal of elimination products
Glycan signals may be associated with signals, e.g. elimination of water (loss
of H20), or
lack of methyl ether or ester groups (effective loss of CH2), resulting in
experimental m/z
values 18 or 14 mass units smaller than the Glycan signal, respectively. These
signals are not
treated as individual Glycan signals, but are instead treated as associated
signals and removed
from the processed data.
Classification of Glycan signals into Glycan groups
According to the present invention, the Glycan signals are optionally
organized into Glycan
groups and Glycan group profiles based on analysis and classification of the
assigned
monosaccharide and modification compositions and the relative amounts of
monosaccharide
and modification units in the compositions, according to the classification
rules described
above:
Generation of Glycan group profiles.
To generate Glycan group profiles, the proportions of individual Glycan
signals belonging to
each Glycan group are summed. The proportion of each Glycan group of the total
Glycan
signals equals its prevalence in the Glycan profile. The Glycan group profiles
of two or more
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samples can be compared. The Glycan group profiles can be further analyzed by
arranging
Glycan groups into subprofiles, and analyzing the relative proportions of
different Glycan
groups in the subprofiles. Similarly formed subprofiles of two or more samples
can be
compared.
Specific technical aspects of tissue glycome analysis
Preferred sample sizes
The present invention is especially useful when low sample amounts are
available. Practical
cellular or tissue material may be available for example for diagnostic only
in very small
amounts.
Sample sizes for preferred pico-scale preparation methods
The inventors found surprisingly that glycan fraction could be produced and
analysed
effectively from samples containing low amount of material, for example 100
000-1 000 000
cells or a cubic millimetre (microliter) of the cells.
The combination of very challenging biological samples and very low amounts of
samples
forms another challenge for the present analytic method. The yield of the
purification process
must be very high. The estimated yields of the glycan fractions of the
analytical processes
according to the present invention varies between about 50% and 99 %. Combined
with
effective removal of the contaminating various biological materials even more
effectively
over the wide preferred mass ranges according to the present invention show
the ultimate
performance of the method according to the present invention.
Isolation of glycans and glycan fractions
The present invention is directed to a method of preparing an essentially
unmodified glycan
sample for analysis from the glycans present in a given sample.
A preferred glycan preparation process consists of the following steps:
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1 isolating a glycan-containing fraction from the sample,
2 ...Optionally purification the fraction to useful purity for glycome
analysis
5 The preferred isolation method is chosen according to the desired glycan
fraction to be
analyzed. The isolation method may be either one or a combination of the
following methods,
or other fractionation methods that yield fractions of the original sample:
1 extraction with water or other hydrophilic solvent, yielding water-soluble
glycans or
10 glycoconjugates such as free oligosaccharides or glycopeptides,
2 extraction with hydrophobic solvent, yielding hydrophilic glycoconjugates
such as
glycolipids,
3 N-glycosidase treatment, especially Flavobacterium meningosepticum N-
glycosidase F
treatment, yielding N-glycans,
15 4 alkaline treatment, such as mild (e.g. 0.1 M) sodium hydroxide or
concentrated ammonia
treatment, either with or without a reductive agent such as borohydride, in
the former case in
the presence of a protecting agent such as carbonate, yielding 0-elimination
products such as
0-glycans and/or other elimination products such as N-glycans,
5 endoglycosidase treatment, such as endo-(3-galactosidase treatment,
especially Escherichia
20 freundii endo-(3-galactosidase treatment, yielding fragments from poly-N-
acetyllactosamine
glycan chains, or similar products according to the enzyme specificity, and/or
6 protease treatment, such as broad-range or specific protease treatment,
especially trypsin
treatment, yielding proteolytic fragments such as glycopeptides.
25 The released glycans are optionally divided into sialylated and non-
sialylated subfractions and
analyzed separately. According to the present invention, this is preferred for
improved
detection of neutral glycan components, especially when they are rare in the
sample to be
analyzed, and/or the amount or quality of the sample is low. Preferably, this
glycan
fractionation is accomplished by graphite chromatography.
According to the present invention, sialylated glycans are optionally modified
in such manner
that they are isolated together with the non-sialylated glycan fraction in the
non-sialylated
glycan specific isolation procedure described above, resulting in improved
detection
simultaneously to both non-sialylated and sialylated glycan components.
Preferably, the
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modification is done before the non-sialylated glycan specific isolation
procedure. Preferred
modification processes include neuraminidase treatment and derivatization of
the sialic acid
carboxyl group, while preferred derivatization processes include amidation and
esterification
of the carboxyl group.
Glycan release methods
The preferred glycan release methods include, but are not limited to, the
following methods:
Free glycans - extraction of free glycans with for example water or suitable
water-solvent
mixtures.
Protein-linked glycans including 0- and N-linked glycans - alkaline
elimination of protein-
linked glycans, optionally with subsequent reduction of the liberated glycans.
Mucin-type and other Ser/Thr 0-linked glycans - alkaline 0-elimination of
glycans, optionally
with subsequent reduction of the liberated glycans.
N-glycans - enzymatic liberation, optionally with N-glycosidase enzymes
including for
example N-glycosidase F from C. meningosepticum, Endoglycosidase H from
Streptomyces,
or N-glycosidase A from almonds.
Lipid-linked glycans including glycosphingolipids - enzymatic liberation with
endoglycoceramidase enzyme; chemical liberation; ozonolytic liberation.
Glycosaminoglycans - treatment with endo-glycosidase cleaving
glycosaminoglycans such as
chondroinases, chondroitin lyases, hyalurondases, heparanases, heparatinases,
or
keratanases/endo-beta-galactosidases ;or use of 0-glycan release methods for 0-
glycosidic
Glycosaminoglycans; or N-glycan release methods for N-glycosidic
glycosaminoglycans or
use of enzymes cleaving specific glycosaminoglycan core structures; or
specific chemical
nitrous acid cleavage methods especially for amine/N-sulphate comprising
glycosaminoglycans
Glycan fragments - specific exo- or endoglycosidase enzymes including for
example
keratanase, endo-(3-galactosidase, hyaluronidase, sialidase, or other exo- and
endoglycosidase
enzyme; chemical cleavage methods; physical methods
Effective purification process
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The invention describes special purification methods for glycan mixtures from
tissue samples.
Previous glycan sample purification methods have required large amounts of
material and
involved often numerous chromatographic steps and even purification of
specific proteins. It
is known that protein glycosylation varies protein specifically and single
protein specific data
can thus not indicate the total tissue level glycosylation. Purification of
single protein is a
totally different task than purifying the glycan fraction according to the
present invention.
When the purification starts from a tissue or cells, the old processes of
prior art involve often
laborious homogenisation steps affecting the quality of the material produced.
The present
purification directly from a biological sample such as cell or tissue
material, involves only a
few steps and allows quick purification directly from the biological material
to analysis
preferably by mass spectrometry.
Purification from cellular materials of cells and/or tissues
The cellular material contains various membranes, small metabolites, various
ionic materials,
lipids, peptides, proteins etc. All of the materials can prevent glycan
analysis by mass
spectrometry if these cannot be separated from the glycan fraction. Moreover,
for example
peptide or lipid materials may give rise to mass spectrometric signals within
the preferred
mass range within which glycans are analysed. Many mass spectrometric methods,
including
preferred MALDI-mass spectrometry for free glycan fractions, are more
sensitive for peptides
than glycans. With the MALDI method peptides in the sample may be analysed
with
approximately 1000-fold higher sensitivity in comparision to methods for
glycans. Therefore
the method according to the present invention should be able to remove for
example potential
peptide contaminations from free glycan fractions most effectively. The method
should
remove essential peptide contaminations from the whole preferred mass range to
be analysed.
Purification suitable for mass spectrometry, especially MALDI-TOF mass
spectrometry
The inventors discovered that the simple purification methods would separate
released
glycans from all possible cell materials so that
1) The sample is technically suitable for mass spectrometric analysis.
This includes two major properties,
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a) the samples is soluble for preparation of mass spectrometry sample and
b) does not have negative interactions with chemicals involved in the mass
spectrometric method, preferably the sample dries or crystallizes properly
with matrix
chemical used in MALDI-TOF mass spectrometry
When using MALDI-technologies, the sample does not dry or crystallize properly
if the
sample contains harmful impurity material in a significant amount.
2) The purity allows production of mass spectrum of suitable quality.
a)The sample has so low level impurities that it gives mass spectrometric
signals. Especially
when using MALDI-TOF mass spectrometry, signals can be suppressed by
background so
that multiple components/peaks cannot be obtained.
b) the sample is purified so that there is no major impurity signals in the
preferred mass
ranges to be measured.
Preferably the present invention is directed to analysis of unusually small
sample amounts.
This provides a clear benefit over prior art,when there is small amount amount
of sample
available from a small region of diseased tissue or diagnostic sample such as
tissue slice
produced for microscopy or biopsy sample. Methods to achieve such purity
(purity being a
requirement for the sensitivity needed for such small sample amounts) from
tissue or cell
samples (or any other complex biological matices e.g. serum, saliva) has not
been described in
the prior art.
In a preferred embodiment the method includes use of non-derived glycans and
avoiding
general derived glycans. There are methods of producing glycan profiles
including
modification of all hydroxyl groups in the sample such as permethylation. Such
processes
require large sample amounts and produces chemical artefacts such as
undermethylated
molecules lowering the effectivity of the method. These artefact peaks cover
all minor signals
in the spectra, and they can be misinterpreted as glycan structures. It is of
importance to note
that in glycome analyses the important profile-to profile differences often
reside in the minor
signals.. In a specific embodiment the present invention is directed to site
specific
modification of the glycans with effective chemical or enzyme reaction,
preferably a
quantitative reaction.
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Preferred analytical technologies for glycome analysis
Mass spectrometric analysis of glycomes
The present invention is specifically directed to quantitative mass
spectrometric methods for
the analysis of glycomes. Most preferred mass spectrometric methods are MALDI-
TOF mass
spectrometry methods.
MALDI-TOF analysis
The inventors were able to optimise MALDI-TOF mass spectrometry for glycome
analysis.
The preferred mass spectrometric analysis process is MALDI-TOF mass
spectrometry, where
the relative signal intensities of the unmodified glycan signals represent
their relative molar
proportions in the sample, allowing relative quantification of both neutral
(Naven & Harvey,
19xx) and sialylated (Papac et al., 1996) glycan signals. Preferred
experimental conditions
according to the present invention are described under Experimental procedures
of Examples
listed below.
Preferred mass ranges for MALDI-TOF analysis and released non-modified
glycomes
For MALDI-TOF mass spectrometry of unmodified glycans in positive ion mode,
optimal
mass spectrometric data recording range according to the present invention is
over m/z 200,
more preferentially between m/z 200 - 10000, or even more preferably between
m/z 200 -
4000 for improved data quality. In the most preferred form according to the
present invention,
the data is recorded between m/z 700 - 4000 for accurate relative
quantification of glycan
signals.
For MALDI-TOF mass spectrometry of unmodified glycans in negative ion mode,
optimal
mass spectrometric data recording range according to the present invention is
over m/z 300,
more preferentially between m/z 300 - 10000, or even more preferably between
m/z 300 -
4000 for improved data quality. In the most preferred forms according to the
present
invention, the data is recorded between m/z 700 - 4000 or most preferably
between m/z 800 -
4000 for accurate relative quantification of glycan signals.
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Practical mz-ranges
The practical ranges comprising most of the important signals, as observed by
the present
invention may be more limited than these. Preferred practical ranges includes
lower limit of
about m/z 400, more preferably about m/z 500, and even more preferably about
m/z 600, and
5 most preferably m/z about 700 and upper limits of about m/z 4000, more
preferably m/z about
3500 (especially for negative ion mode),, even more preferably m/z about 3000
(especially for
negative ion mode), and in particular at least about 2500 (negative or
positive ion mode) and
for positive ion mode to about m/z 2000 (for positive ion mode analysis). The
preferred range
depends on the sizes of the sample glycans, samples with high branching or
polysaccharide
10 content or high sialylation levels are preferably analysed in ranges
containing higher upper
limits as described for negative ion mode. The limits are preferably combined
to form ranges
of maximum and minimum sizes or lowest lower limit with lowest higher limit,
and the other
limits analogously in order of increasing size
Preferred analysis modes for MALDI-TOF for effective glycome analysis
15 The inventors were able to show effective quantitative analysis in both
negative and positive
mode mass spectrometry.
Sample handling
The inventors developed optimised sample handling process for preparation of
the samples
20 for MALDI-TOF mass spectrometry.
Glycan purification
The glycan purification method according to the present invention consists of
at least one of
25 purification options, preferably in specific combinations described below,
including the
following purification options:
1) Precipitation-extraction;
2) Ion-exchange;
3) Hydrophobic interaction;
30 4) Hydrophilic interaction; and
5) Affinity to graphitized carbon.
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1) Precipitation-extraction may include precipitation of glycans or
precipitation of
contaminants away from the glycans. Preferred precipitation methods include:
1. Glycan material precipitation, for example acetone precipitation of
glycoproteins,
oligosaccharides, glycopeptides, and glycans in aqueous acetone,
preferentially ice-cold over
80 % (v/v) aqueous acetone; optionally combined with extraction of glycans
from the
precipitate, and/or extraction of contaminating materials from the
precipitate;
2. Protein precipitation, for example by organic solvents or trichloroacetic
acid, optionally
combined with extraction of glycans from the precipitate, and/or extraction of
contaminating
materials from the precipitate;
3. Precipitation of contaminating materials, for example precipitation with
trichloroacetic
acid or organic solvents such as aqueous methanol, preferentially about 2/3
aqueous methanol
for selective precipitation of proteins and other non-soluble materials while
leaving glycans in
solution;
2) Ion-exchange may include ion-exchange purification or enrichment of glycans
or removal
of contaminants away from the glycans. Preferred ion-exchange methods include:
1. Cation exchange, preferably for removal of contaminants such as salts,
polypeptides, or
other cationizable molecules from the glycans; and
2. Anion exchange, preferably either for enrichment of acidic glycans such as
sialylated
glycans or removal of charged contaminants from neutral glycans, and also
preferably for
separation of acidic and neutral glycans into different fractions.
3) Hydrophilic interaction may include purification or enrichment of glycans
due to their
hydrophilicity or specific adsorption to hydrophilic materials, or removal of
contaminants
such as salts away from the glycans. Preferred hydrophilic interaction methods
include:
1. Hydrophilic interaction chromatography, preferably for purification or
enrichment of
glycans and/or glycopeptides;
2. Adsorption of glycans to cellulose in hydrophobic solvents for their
purification or
enrichment, preferably to microcrystalline cellulose, and even more preferably
using an n-
butanol:methanol:water or similar solvent system for adsorption and washing
the adsorbed
glycans, in most preferred system n-butanol:methanol:water in relative volumes
of 10:1:2,
and water or water:ethanol or similar solvent system for elution of purified
glycans from
cellulose.
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4) Affinity to graphitized carbon may include purification or enrichment of
glycans due to
their affinity or specific adsorption to graphitized carbon, or removal of
contaminants away
from the glycans. Preferred graphitized carbon affinity methods include porous
graphitized
carbon chromatography.
Preferred purification methods according to the invention include combinations
of one or
more purification options. Examples of the most preferred combinations include
the following
combinations:
1) For neutral underivatized glycan purification: 1. cation exchange of
contaminants, 2.
hydrophobic adsorption of contaminants, and 3. graphitized carbon affinity
purification of
glycans.
1) For sialylated underivatized glycan purification: 1. cation exchange of
contaminants, 2.
hydrophobic adsorption of contaminants, 3. adsorption of glycans to cellulose,
and 4.
graphitized carbon affinity purification of glycans.
NMR-analysis of glycomes
The present invention is directed to analysis of released glycomes by
spectrometric method
useful for characterization of the glycomes. The invention is directed to NMR
spectroscopic
analysis of the mixtures of released glycans. The inventors showed that it is
possible to
produce a released glycome from tissue samples in large scale enough and
useful purity for
NMR-analysis of the glycome.
In a preferred embodiment the NMR-analysis of the tissue glycome is one
dimensional proton
NMR-analysis showing structural reporter groups of the major components in the
glycome.
The present invention is further directed to combination of the mass
spectrometric and NMR
analysis of small scale tissue samples.
Analysis of changes related to animal individuals, animal species and animal
status
The inventors further realized major glycome differences between samples from
the same
species. The invention is specifically directed to analysis of individual
differences between
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animals. The invention is further directed to the use of the information in
breeding of animals,
especially production animals.
The inventors further realized major glycome differences between samples from
animals
related to the status of the animal. The invention is especially directed to
the analysis of
biological status related changes of animal.
The inventors further noticed major species specific differences in the total
released glycomes
analysed. It is realized that species specific glycome differences are useful
for analysis of
effects of glycosylations when animal materials from different species are in
contact with
each other.
In a preferred embodiment the glycosylation is analysed when one animal is
consumed as
food or feed of another and the analysis is directed to potential allergic or
immunogenic
effects in the animal consuming the other animal. Preferably the invention is
directed to the
use of analysis of animal derived human feeds and feeds derived from other
animals.
In another embodiment the invention is directed to analysis the invention is
directed to
situations when one animal species is exposed to material from another animal
in air,
especially in context of allergy inducing air mediated animal contacts.
Preferred target species, especially animals for tissue analysis
The invention revealed that glycome oligosaccharide mixtures can be produced
effectively
from eukaryotic species especially animal tissues.
Plant and insect differentiated cells are separately preferred eukaryotic
materials.
Preferred animals include vertebrate animals, more preferably mammals, more
preferably
domestic or farm animals or human, analysis of human samples is most
preferred. The
preference of animals is based on similarity of sample compositions and
availability animal
materials and presence of individual, species and status related changes.
Most preferred domestic or farm animals includes pets such as cat, dog, pet
rodents (such as
mouse, hamster, rat) and production/farm animals such as animals selected from
the group:
pig, ruminats (especially ones producing milk such as cow, buffalo); avian
production animals
(such as hen(chicken), turkey, duck), and horse.
The invention is especially directed to analysis of specific tissues of animal
in context of
breeding of animals especially production animals, horse and cats or dogs. The
invention is
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especially directed to analysis of specific tissues of animal in context of
breeding of especially
production animals, and major pets under extensive breeding preferably cats or
dogs.
The invention is further directed to analysis of species specific differences
between the
preferred domestic or farm animals in two preferred contexts either in context
when the
animal is context of food contact with human or in context of air contact with
human. The
preferred animal in food contact are major production/farm animals, which are
also preferred
in air contact with animals as well as major pets cats and dogs.
The invention is further directed to analysis of domestic and farm animals in
context of the
status of the animal. It is realized that it is useful to analyze of status of
the cells, when the
health or physiological status of the animal is needed to revealed.
The invention is in a preferred embodiment directed to analysis of human type
primates such
as monkeys especially apes (examples include chimpanzee, pygmy chimpanzee,
gorilla,
orangutan) and human, the preference is based on close similarity of primates
and human on
genetic and cell biological level, providing similarity for samples to be
analysed and
scientifically important evolution based glycosylation changes between similar
species.
The invention is further directed to analysis of animals useful for
development of
pharmaceutical and therapeutic materials. The preferred animals include
rodents (such as
mouse, hamster, rat) and human type primates. It is further preferred to
analyze these animals
in context of air mediated contact with human or other animals.
The invention is further directed to animals involved in sports, especially
horses and dogs. It
is realized that development of animals in sports involves especially analysis
of individual
and animal status related changes. It is further preferred to analyze these
animals in context of
air mediated contact with human or other animals.
Targets of analysis- Tissue materials
The present invention refers as "tissue materials" all preferred target tissue
related material
including for example tissues, secretions and cultivated differentiated cells
Preferred tissue type
The present invention is preferably directed to specific tissue types for the
analysis according
to the invention. The tissue type are found to be very suitable and feasible
for the analysis
according to the invention. The analysis is especially directed to analysis of
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1) tissues of gastrointestinal track, preferably mouth, larynx, stomach, large
and small
intestine
2) internal organs such as ovarian tissue, liver, lungs, or kidney
3) tissues of circulatory system, especially blood
5 4) cultivated cell line models of the differentiated tissues
Preferred tissue parts
The present invention is preferably directed to specific parts of tissue for
the analysis
according to the invention. The inventors realized that it is possible perform
glycomics
10 analysis of specific parts of tissues and reveal differences useful for
studies of diseases and
disease induced changes and other changes or presence of receptor structures
on specific
subtissues. Preferred subtissues includes
1) tissues surfaces, especially epithelia of gastrointestinal tract and cell
surfaces and
2) components of circulatory system, preferably serum/plasma, and blood cells,
especially red
15 cells and white blood cells
Preferred tissue derivatives to be analysed including liquid secretions
The invention is further directed to material produced by tissues.
20 Preferably the invention is directed to the analysis of secretions of
tissues, preferably liquid
secretions of tissues, preferably milk, saliva or urine. It is realized that
liquid secretions form
a specific group of tissue derived materials found especially useful for the
glycome analysis
methods according to the invention.
Milk is especially preferred as a food material consumed by animals and human
and analysis
25 with regard to each of individual specific, animal status specific and
species specific
differences.
The invention is under separate preferred embodiment directed to the analysis
of specific
conjugated glycomes such as protein or lipid derived glycomes, from the
secretions and in
30 another preferred embodiment free soluble glycomes of the secretions.
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Soluble glycome materials: tissue and/or secretion materials, especially with
high
protein content
The invention is in a preferred embodiment directed to specific methods
developed for the
analysis of soluble glycome material from tissues and secretions. This group
includes
background for purification different from solid tissue and cell derived
materials. The group
includes tissue solutions such as blood serum/plasma and liquid secretions
such as milk,
saliva and urine.
The invention is further directed to the soluble glycome materials with high
protein content
including preferably milk and serum/plasma. The materials are especially
directed as specific
target of technologies directed to analysis of high protein content solutions,
in separate
preferred embodiments the technologies are directed to analysis human serum or
bovine milk.
Milk glycomes
The present invention is specifically directed to glycome analysis of milks
form human and
other animals, preferably from ruminant animals such as bovine, buffalo,
sheep, goat, and
camel, the most common milk production animals bovine and buffalo are
preferred. Most
preferably the common bovine milk is analysed.
Preferred ruminant milk glycomes
The invention is specifically directed to analysis of colostrums and regular
milks of ruminant
milks. The invention represent novel total glycomics analysis methods for both
secreted and
conjugated glycomes. The invention is further directed to glycome analysis
according to the
invention to food production fractions and specific milk products of ruminant
milks.
Bovine milk glycomes
The invention is especially directed to novel total glycomics analysis methods
for both
secreted and conjugated glycomes from bovine milks. The invention specifically
represents
novel glycomes released from proteins of bovine milks.
The invention is further directed to glycomes released from proteins from
regular milk and in
a separate embodiment to glycomes released from proteins of bovine colostrums.
The
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invention is further directed to glycome analysis according to the invention
to food production
fractions and specific milk products of bovine milk such whey, low fat milk,
or buttermilk.
Subcomponents of glycomes, especially from secreted proteins such as milks
The invention is further directed to methods for selecting specific components
of glycomes
and searching enriched fractions such as specific protein fraction comprising
the specific
glycome components.
As a specific example and embodiment of a purified glycome component the
invention is
directed to protein, referred as mannose protein, containing enriched with
mannose glycans
such as high-mannose or low mannose glycans isolated from bovine milk. The
invention is
especially directed to bovine Iactoferrin carrying almost exclusively mannose
glycans. It was
further revealed that the Iactoferrin is expressed only in certain milk
batches.
The present invention is further directed to analysis of milks to reveal
specific animals and
conditions for effective production of mannose proteins, especially mannose
Iactoferrin. The
invention is further directed to single step chromatographic purification of
the mannose
Iactoferrin.
Tissue surface glycomes
In a preferred embodiment the invention is directed to special methods for the
analysis of the
surfaces of tissues.
The preferred tissue surfaces includes
1)epithelia of the preferred gastrointestinal tract tissues
and
2) surfaces of cells according to cells on surface of tissues or separable
homogeneously from
tissue, such as blood cells and
3) surfaces of cultivated cells which may be used as models for differentiated
tissues.
Non-derivatized released target material surface glycomes and production
In a preferred embodiment the invention is directed to non-derivatized
released cell surface
glycomes. The non-derivatized released cell surface glycomes correspond more
exactly to the
fractions of glycomes that are localized on the cell surfaces, and thus
available for biological
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interactions. These cell surface localized glycans are of especial importance
due to their
availability for biological interactions as well as targets for reagents (e.g.
antibodies, lectins
etc...) targeting the cells or tissues of interest. The invention is further
directed to release of
the cell surface glycomes, preferably from intact cells by hydrolytic enzymes
such as
proteolytic enzymes, including proteinases and proteases, and/or glycan
releasing enzymes,
including endo-glycosidases or protein N-glycosidases. Preferably the surface
glycoproteins
are cleaveed by proteinase such as trypsin and then glycans are analysed as
glycopeptides or
preferably relased further by glycan relasing enzyme.
Cell models of differentiated tissues
The invention is further directed to cultured cells corresponding to
differentiated cells. Such
cells may be used as models for differentiated cells. The differentiated cells
include
differentiated cell models of cancer.
Stably growing differentiated cultured cell lines are also used in production
mammalian
proteins and for other biotechnical production for example in cell therapies.
It is realized that the differentiated cells would need to be controlled with
regard to cells status
and individual cell line specific differences. It is realized that cell status
would need to be
checked with regard to numerous factors related to cell status. It is further
realized that there
is differences between individual cell lines when these are derived from
different animal
individuals.
In case the differentiated cells would be used in context of contact with
other animals from
different species than from which the cell line is derived there is need for
controlling species
specific differences.
It is especially realized that it would be useful to check status of
differentiated cells used in
production of biotechnical products, such as recombinant therapeutic proteins
such as
antibodies, growth factors and recombinant receptors including recombinant TNF-
alpha
receptors. The invention is especially directed to the analysis of
differentiated cell lines
producing recombinant proteins.
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The glycome compositions
The invention is further directed to the compositions and compositions
produced by the
methods according to the invention. The invention further represent preferred
methods for
analysis of the glycomes, especially mass spectrometric methods.
The invention is specifically directed to released glycomes derived conjugated
glycans from
preferred tissue materials and cell models of differentiated tissues.
Purification method
The invention represents effective methods for purification of oligosaccharide
fractions from
tissues, especially in very low scale. The prior art has shown analysis of
separate glycome
components from tissues, but not total glycomes. It is further realized that
the methods
according to the invention are useful for analysis of glycans from isolated
proteins or
peptides.
Analysis of glycomes
The invention is further directed to novel quantitative analysis methods for
glycomes. The
glycome analysis produces large amounts of data. The invention reveals methods
for the
analysis of such data quantitatively and comparision of the data between
different samples.
The invention is especially directed to quantitative two-dimensional
representation of the
data.
Integrated glycome analysis
The invention is further directed to integrated glycomics or glycome analysis
process
including
1) Optional release of glycans from tissues
2) isolation/purification of glycans from sample,
3) analysis of the glycome
4) quantitative presentation of the data
The first step is optional as the method is further directed to analysis of
known and novel
secretion derivable soluble glycomes.
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Application of the methods for analysis of proteins
The invention represents effective methods for the practical analysis of
glycans from isolate
proteins especially from very small amounts of samples. The invention is
especially directed
to the application of the methods for the analysis of proteins using the
purification method,
5 analysis methods and/or integrated glycome analysis.
Product by process
The present invention is specifically directed to the glycan fraction produced
according to the
10 present invention from the pico scale tissue material sample according to
the present
invention. The preferred glycan fraction is essentially devoid of signals of
contaminating
molecules within the preferred mass range when analysed by MALDI mass
spectrometry
according to the present invention.
15 Preferred uses of glycomes and analysis thereof with regard to status of
cells
In the present invention the word cell refer to cells of tissue material
according to the
invention, especially cultivated differentiated cells.
20 Product by process
The present invention is specifically directed to the glycan fraction produced
according to the
present invention from the pico scale tissue material sample according to the
present
invention. The preferred glycan fraction is essentially devoid of signals of
contaminating
molecules within the preferred mass range when analysed by MALDI mass
spectrometry
25 according to the present invention.
The glycome products from tissue samples according to present invention are
produced
preferably directely from complete tissue material cells or membrane fractions
thereof, more
preferably directly from intact cells as effectively shown in examples. In
another preferred
embodiment the glycome fractions are cell surface glycomes and produced
directly from
30 surfaces of complete tissue material cells, preferably intact or
essentially intact cells of tissue
materials or surfaces of intact tissues according to the invention. In another
embodiment the
glycome products according to the invention are produced directly from
membrane fraction
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Preferred uses of glycomes and analysis thereof with regard to status of cells
Search of novel of novel carbohydrate marker structures
It is further realized that the analysis of glycome is useful for search of
most effectively
altering glycan structures in the tissue materials for analysis by other
methods.
The glycome component identified by glycome analysis according to the
invention can be
further analysed/verified by known methods such as chemical and/or glycosidase
enzymatic
degradation(s) and further mass spectrometric analysis and by fragmentation
mass
spectrometry, the glycan component can be produced in larger scale by know
chromatographic methods and structure can be verified by NMR- spectroscopy.
The other methods would preferably include binding assay using specific
labelled
carbohydrate binding agents including especially carbohydrate binding proteins
(lectins,
antibodies, enzymes and engineered proteins with carbohydrate binding
activity) and other
chemicals such as peptides or aptamers aimed for carbohydrate binding. It is
realized that the
novel marker structure can be used for analysis of cells, cell status and
possible effects of
contaminats to cell with similar indicative value as specific signals of the
glycan mass
components in glycome analysis by mass spectrometry according to the
invention.
The invention is especially directed to search of novel carbohydrate marker
structures from
cell/tissue surfaces, preferably by using cell surface profiling methods. The
cell surface
carbohydrate marker structures would be further preferred for the analysis
and/or sorting of
cells.
Control of cell status and potential contaminations by glycosylation analysis
Control of cell status
Contamination/harmful effect due to nature of raw material for producing a
cell population
Species specific, tissue specific, and individual specific differences in
glycan structures are
known. The difference between the origin of the cell material and the
potential recipient of
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transplanted material may cause for example immunologic or allergic problems
due to
glycosylation differences. It is further noticed that culture of cells may
cause changes in
glycosylation. When considering human derived cell materials according to the
present
invention, individual specific differences in glycosylation are a potent
source of harmful
effects.
Control of raw material cell population
The present invention is directed to control of glycosylation of cell
populations to be used in
therapy.
The present invention is specifically directed to control of glycosylation of
cell materials,
preferably when
1) there is difference between the origin of the cell material and the
potential recipient of
transplanted material. In a preferred embodiment there are potential inter-
individual specific
differences between the donor of cell material and the recipient of the cell
material. In a
preferred embodiment the invention is directed to animal or human, more
preferably human
specific, individual person specific glycosylation differences. The individual
specific
differences are preferably present in tissue materials. The invention is
preferably not directed
to observation of known individual specific differences such as blood group
antigens changes
on erythrocytes.
2) There is possibility in variation due to disease specific variation in the
materials. The
present invention is specifically directed to search of glycosylation
differences in the tissue
materials according to the present invention associated with infectious
disease, inflammatory
disease, or malignant disease. Part of the inventors have analysed numerous
types of early
human cells and observed similar glycosylation types in certain cancers and
tumors.
3) There is a possibility of specific inter-individual biological differences
in the animals,
preferably humans, from which the cell are derived for example in relation to
species, strain,
population, isolated population, or race specific differences in the cell
materials.
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Time dependent changes during cultivation of cells
Furthermore during long term cultivation of cells spontaneous mutations may be
caused in
cultivated cell materials. It is noted that mutations in cultivated cell lines
often cause harmful
defects on glycosylation level.
It is further noticed that cultivation of cells may cause changes in
glycosylation. It is realized
that minor changes in any parameter of cell cultivation including quality and
concentrations of
various biological, organic and inorganic molecules, any physical condition
such as
temperature, cell density, or level of mixing may cause difference in cell
materials and
glycosylation. The present invention is directed to monitoring glycosylation
changes
according to the present invention in order to observe change of cell status
caused by any cell
culture parameter affecting the cells.
The present invention is in a preferred embodiment directed to analysis of
glycosylation
changes when the density of cells is altered. The inventors noticed that this
has a major impact
of the glycosylation during cell culture.
In case there is heterogeneity in cell material this may cause observable
changes or harmful
effects in glycosylation.
Furthermore, the changes in carbohydrate structures, even non-harmful or
functionally
unknown, can be used to obtain information about the exact genetic status of
the cells.
The present invention is specifically directed to the analysis of changes of
glycosylation,
preferably changes of sialylation according to the present invention in order
to observe
changes of cell status during cell cultivation.
Contaminations or alterations in cells due to process conditions
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Conditions and reagents inducingharmful glycosylation or harmful glycosylation
related
effects to cells duringcell handling
The inventors further revealed conditions and reagents inducing harmful
glycans to be
expressed by cells with same associated problems as the contaminating glycans.
The inventors
found out that several reagents used in a regular cell purification process
caused changes cells
being purified.
It is realized, that the materials during cell handling may affect the
glycosylation of cell
materials. This may be based on the adhesion, adsorption, or metabolic
accumulation of the
structure in cells under processing.
In a preferred embodiment the cell handling reagents are tested with regard to
the presence
glycan component being antigenic or harmfull structure such as cell surface
NeuGc, Neu-O-
Ac or mannose structure.
The inventors note effects of various effector molecules in cell culture on
the glycans
expressed by the cells if absorption or metabolic transfer of the carbohydrate
structures have
not been performed. The effectors typically mediate a signal to cell for
example through
binding a cell surface receptor.
The effector molecules include various cytokines, growth factors, and their
signalling
molecules and co-receptors. The effector molecules may be also carbohydrates
or
carbohydrate binding proteins such as lectins.
Controlled cell isolation/purification and culture conditions to avoid
contaminations with
harmful glycans or other alteration in glycome level
Stress caused by cell handling
It is realized that cell handling including isolation/purification, and
handling in context of cell
storage and cell culture processes are not natural conditions for cells and
cause physical and
chemical stress for cells. The present invention allows control of potential
changes caused by
the stress. The control may be combined by regular methods may be combined
with regular
checking of cell viability or the intactness of cell structures by other
means.
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Examples of physical and/or chemical stress in cell handling step
Washing and centrifuging cells cause physical stress which may break or harm
cell membrane
structures. Cell purifications and separations or analysis under non-
physiological flow
conditions also expose cells to certain non-physiological stress. Cell storage
processes and
5 cell preservation and handling at lower temperatures affects the membrane
structure. All
handling steps involving change of composition of media or other solution,
especially
washing solutions around the cells affect the cells for example by altered
water and salt
balance or by altering concentrations of other molecules effecting biochemical
and
physiological control of cells.
Observation and control of glycome changes by stress in cell handling
processes
The inventors revealed that the method according to the invention is useful
for observing
changes in cell membranes which usually effectively alter at least part of the
glycome
observed according to the invention. It is realized that this related to exact
organization and
intact structures cell membranes and specific glycan structures being part of
the organization.
The present invention is specifically directed to observation of total glycome
and/or cell
surface glycomes, these methods are further aimed for the use in the analysis
of intactness of
cells especially in context of stressfull condition for the cells, especially
when the cells are
exposed to physical and/or chemical stress. It is realized that each new cell
handling step
and/or new condition for a cell handling step is useful to be controlled by
the methods
according to the invention. It is further realized that the analysis of
glycome is useful for
search of most effectively altering glycan structures for analysis by other
methods such as
binding by specific carbohydrate binding agents including especially
carbohydrate binding
proteins (lectins, antibodies, enzymes and engineered proteins with
carbohydrate binding
activity).
Controlled cell preparation (isolation or purification) with regard to
reagents
The inventors analysed process steps of common cell preparation methods.
Multiple sources
of potential contamination by animal materials were discovered.
The present invention is specifically directed to carbohydrate analysis
methods to control of
cell preparation processes. The present invention is specifically directed to
the process of
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51
controlling the potential contaminations with animal type glycans, preferably
N-
glycolylneuraminic acid at various steps of the process.
The invention is further directed to specific glycan controlled reagents to be
used in cell
isolation
The glycan-controlled reagents may be controlled on three levels:
1. Reagents controlled not to contain observable levels of harmful glycan
structure,
preferably N-glycolylneuraminic acid or structures related to it
2. Reagents controlled not to contain observable levels of glycan structures
similar to the
ones in the cell preparation
3. Reagent controlled not to contain observable levels of any glycan
structures.
The control levels 2 and 3 are useful especially when cell status is
controlled by glycan
analysis and/or profiling methods. In case reagents in cell preparation would
contain the
indicated glycan structures this would make the control more difficult or
prevent it. It is
further noticed that glycan structures may represent biological activity
modifying the cell
status.
Cell preparation methods including glycan-controlled reagents
The present invention is further directed to specific cell purification
methods including
glycan-controlled reagents.
Storage induced changes causing harmful glycosylations or change in the status
of cells
It was realized that storage of the cell materials may cause harmful changes
in glycosylation
or changes in cell status observable by glycosylation analysis according to
the present
invention.
Changes observable in context of low temperature storage or handling of cells
The inventors discovered that keeping the cells in lower temperatures alters
the status of cells
and this is observable by analysing the chemical structures of cells,
preferably the
glycosylation of the cells. The lower temperatures usually vary between 0 -36
degrees of
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Celsius including for example incubator temperature below about 36 degrees of
Celsius more
preferably below 35 degrees of Celsius, various room temperatures, cold room
and fridge
temperatures typically between 2-10 degrees of Celsius, and temperatures from
incubation on
ice close to 0 degrees of Celsius typically between 0-4 degrees of Celsius.
The lowered
temperatures are typically needed for processing of cells or temporary storage
of the preferred
cells.
The present invention is specifically directed to analysis of the status of
cells kept in low
temperatures in comparison to natural body temperatures. In a preferred
embodiment the
control is performed after certain time has passed from process in lower
temperature in order
to confirm the recovery of the cells from the lower temperature. In another
preferred
embodiment the present invention is directed to development of lower
temperature methods
by controlling the chemical structures of cells, preferably by controlling
glycosylation
according to the present invention.
ChanQes observable in context of cryopreservation
The inventors discovered that cryopreservation alters the status of cells and
this observable
analysing the chemical structures of cells, preferably the glycosylation of
the cells. The
present invention is specifically directed to analysis of the status of
cryopreserved cells. In a
preferred embodiment the control is performed after certain time has passed
from preservation
in order to confirm the recovery of the cells from the cryopreservation. In
another preferred
embodiment the present invention is directed to development of
cryopreservetion methods by
controlling the chemical structures of cells, preferably by controlling
glycosylation according
to the present invention.
Contaminations with harmful glycans such as antigenic animal type glycans
Several glycans structures contaminating cell products may weaken the
biological activity of
the product.
The harmful glycans can affect the viability during handling of cells, or
viability and/or
desired bioactivity and/or safety in therapeutic use of cells.
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The harmful glycan structures may reduce the in vitro or in vivo viability of
the cells by
causing or increasing binding of destructive lectins or antibodies to the
cells. Such protein
material may be included e.g. in protein preparations used in cell handling
materials.
Carbohydrate targeting lectins are also present on human tissues and cells,
especially in blood
and endothelial surfaces. Carbohydrate binding antibodies in human blood can
activate
complement and cause other immune responses in vivo. Furthermore immune
defence lectins
in blood or leukocytes may direct immune defence against unusual glycan
structures.
Additionally harmful glycans may cause harmful aggregation of cells in vivo or
in vitro. The
glycans may cause unwanted changes in developmental status of cells by
aggregation and/or
changes in cell surface lectin mediated biological regulation.
Additional problems include allergenic nature of harmful glycans and
misdirected targeting of
cells by endothelial/cellular carbohydrate receptors in vivo.
Contaminations from reagents
The present invention is specifically directed to control of the reagents used
to prevent
contamination by harmful glycan structures. The harmful glycan structures may
originate
from reagents used during cell handling processes such as cell preservation,
cell preparation,
and cell culture.
Preferred reagents to be controlled according to the present invention include
cell blocking
reagents, such as antibody receptor blocking reagents, washing solutions
during cell
processing, material blocking reagents, such as blocking reagents for
materials like for
example magnetic beads. Preferably the materials are controlled:
1. so that these would not contain a contaminating structure, or more
specifically
preferred glycan structure according to the invention
2. so that the materials contain very low amounts or do not contain any
potentially
harmful structures according to the invention.
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Structural features derived from the glycome compositions
Novel Glycomes from tissues
The present invention revealed that it is possible to profile tissues by
released glycomes from
tissues. The invention revealed novel glycan groups which haven't been
observed in any
similar composition derived from biological materials.
The novel glycan groups include
1. Degraded mannose glycans including
a. novel low mannose glycan group,
b. novel Soluble mannose oligomer comprising glycome comprising single
reducing terminal G1cNAc- unit, soluble mannose-G1cNAc1-glycome
2. non-sialylated acidic (sulphated and/or fosforylated) glycans and
3. N-glycans containing terminal glucose structures
The invention invention is directed to glycome compositions, derived from
tissue material
according to the invention, wherein the glycome composition contain at least
one of the
preferred novel glycan groups in combination with other glycan groups; such as
neutral
glycans including high mannose glycan, G1cNAc(32Man-glycans, complex type-
glycans,
hybrid type glycans acidic glycans; according to the invention obtainable from
tissue
materials according to the invention.
Most preferred novel glycan group degraded mannose glycan. The most preferred
mannose
glycans includes Mannose type glycans containing less than 6 mannose units
including
low mannose glycans, fucosylated low mannose (up to 4-5 mannose residues) or
fucosylated
high mannose structures (4-5 mannose residues)
and soluble mannose-G1cNAc 1-glycome
Most preferred Mannose type glycan including, high- and low mannose type
structures are
according to the Formula Mn:
[Ma2]i1[Ma3]õ2 {[Ma2]i3[M(x6)]õ4 }[Ma6]õ5 {[Ma2]i6[Ma2]õ7[M(x3]õ8 )M(34GN(34([
{Fuca6 }]mGNyR2).
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wherein p, nl, n2, n3, n4, n5, n6, n7, n8, and m, and z are either
independently 0 or 1; with
the proviso that when n2 is 0, also nl is 0; when n4 is 0, also n3 is 0; when
n5 is 0, also nl,
n2, n3, and n4 are 0; when n7 is 0, also n6 is 0; when n8 is 0, also n6 and n7
are 0;
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
5 R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacid and/or peptides derived from protein;
[] and ( indicates determinant either being present or absent depending on the
value of nl,
n2, n3, n4, n5, n6, n7, n8, and m; and
10 {} indicates a branch in the structure,
with the provisio that is 0 indicating soluble mannose-G1cNAc1-glycome or
there is 5, more preferably 4 or less mannose residues or m is 1 and there is
6 or less mannose
units.
15 Marker structures and glycomes
The invention revealed individual glycan structures and structure groups,
which are novel
markers for the cell materials according to the invention. The present
invention is directed to
the use of the marker structures and their combinations for analysis, for
labelling and for cell
separation, as modification targets and for other methods according to
invention.
The present invention revealed large groups of glycans, which can be derived
from cells
according to the invention. The present invention is especially directed to
release of various
protein or lipid linked oligosaccharide and/or polysaccharide chains as free
glycan, glycan
reducing end derivative or glycopeptide fractions referred as glycomes from
the cell material
according to the invention. The glycans can be released separately from
differently linked
glycan groups on proteins and or glycolipids or in combined process producing
several
isolated glycome fractions and/or combined glycome fractions, which comprise
glycans
released at least from two different glycomes. The relative amounts of various
components/component groups observable in glycan profiling as peaks in mass
spectra and in
quantitative presentations of glycan based profiling information, especially
in analysis of
mass spectrometric and/or NMR-data were revealed to be characteristic for
individual cell
types. The glycomes was further revealed to contain glycan subgroups or
subglycomes which
are very useful for characterization of the cell materials according to the
invention.
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GI, c~types based on linkage structures
The invention revealed four major glycome types based on the linkage
structures. Two protein
linked glycomes are N-linked glycomes and 0-linked glycomes. The majority of
the
glycosaminoglycan (gag) glycomes (gagomes) are also linked to certain proteins
by specific
core and linkage structures. The glycolipid glycome is linked to lipids,
usually sphingolipids.
Core structures of glycomes and terminal glycome specific and common
structures
The invention has revealed specific glycan core structures for the specific
subglycomes
studied. The various structures in specific glycomes were observed to contain
common
reducing end core structures such as N-glycan and 0-glycan, Glycosaminoglycan
and
glycolipid cores. The cores are elongated with varying glycan chains usually
comprising
groups of glycans with different chain length. The presence of a core
structures is often
observably as a characteristic monosaccharide composition as monosaccharide
composition of
the core structure causing different relation of monosaccharide residues in
speficic glycan
signals of glycomes when profiled by mass spectrometry according to the
invention. The
present invention further revealed specific non-reducing end terminal
structures of specific
marker glycans. Part of the non-reducing end terminal structures are
characteristic for several
glycomes, for example N-acetylactosamine type terminal structures, including
fucosylated
and sialylated variants were revealed from complex N-glycans, 0-glycan and
Glycolipid
glycomes. Part of the structures are specific for glycomes such terminal Man-
structures in
Low-mannose and High-mannose N-glycans.
Combined analysis of different glycomes
The invention revealed similar structures on protein and lipid linked glycomes
in the cell
materials according to the invention. It was revealed that combined analysis
of the different
glycomes is useful characterization of specific cell materials according to
the invention. The
invention specifically revealed similar lactosamine type structures in
glycolipid and
glycoprotein linked glycomes.
The invention further revealed glycosaminoglycan glycome and glycome profile
useful for the
analysis of the cell status and certain synergistic characteristics
glycosaminoglycan glycomes
and other protein linked glycomes such as non-sialic acid containing acidic
structures in N-
Iiked glycomes. The biological roles of glycosaminoglycans and glycolipids in
regulation of
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57
cell biology and their biosynthetic difference and distance revealed by
glycome analysis
make these a useful combination for analysis of cell status. It is further
realized that
combination of all all glycomes inclding 0-glycan and N-glycan glycomes,
glycolipid
glycome and glycosaminoglycan glycome are useful for analysis of cells
according to the
invention. The invention further revealed common chemical structural features
in the all
glycomes according invention supporting the effective combined production,
purification and
analysis of glycomes according to the invention.
In a preferred embodiment the invention is directed to combined analysis of
following
glycome combinations, more preferably the glycomes are analysed from same
sample to
obtain exact information about the status of the cell material:
1. Two protein linked glycomes: N-glycan and 0-glycan glycomes
2. Glycolipid glycomes with protein linked glycomes, especially preferred
glycolipid
glycomes and N-glycan glycomes
3. Protein linked glycome or glycomes with glycosaminoglycan glycome, in
preferred
embodiment a glycosaminoglycan glycome and N-glycan glycome.
4. Lipid linked glycome or glycomes with glycosaminoglycan glycome
5. Protein linked 0-glycan and N-glycan glycomes, glycolipid glycome and
glycosaminoglycan glycome.
The invention further revealed effective methods for the analysis of different
glycomes. It was
revealed that several methods developed for sample preparation are useful for
both lipid and
protein linked glycomes, in a preferred embodiment proteolytic treatment is
used for both
production of protein linked glycome and a lipid linked glycome, especially
for production of
cell surface glycomes. For production of Total cell glycomes according to the
invention the
extraction of glycolipids is preferably used for degradation of cells and
protein fraction
obtained from the lipid extraction is used for protein linked glycome
analysis. The invention
is further directed to the chemical release of glycans, preferably for
simultaneous release of
both 0-linked and N-linked glycans. Glycolipid and other glycomes, especially
N-linked
glycome, can be effectively released enzymatically, the invention is directed
to sequential
release of glycans by enzymes, preferably including step of inactivating
enzymes between the
treatments and using glycan contolled enzymes to avoid contamination or
controlling
contamination of glycans originationg from enzymes.
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Common structural features of all glycomes and preferred common subfeatures
The present invention reveals useful glycan markers for tissue materials and
combinations
thereof and glycome compositions comprising specific amounts of key glycan
structures. The
invention is furthermore directed to specific terminal and core structures and
to the
combinations thereof.
The preferred glycome glycan structure(s) and/or glycomes from cells according
to the
invention comprise structure(s) according to
the formula CO:
R1Hex(3z{R3 }n1Hex(NAc)õ2XyR2,
Wherein X is glycosidically linked disaccharide epitope (34(Fuc(x6)nGN,
wherein n is 0 or 1,
or X is nothing and
Hex is Gal or Man or GIcA,
HexNAc is GIcNAc or Ga1NAc,
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon,
z is linkage position 3 or 4, with the provision that when z is 4 then HexNAc
is GIcNAc and
then Hex is Man or Hex is Gal or Hex is GIcA, and
when z is 3 then Hex is G1cA or Gal and HexNAc is G1cNAc or Ga1NAc;
nl is 0 or 1 indicating presence or absence of R3;
n2 is 0 or 1, indicating the presence or absence of NAc, with the proviso that
n2 can be 0 only
when Hex(3z is Ga1(34, and n2 is preferably 0, n2 structures are preferably
derived from
glycolipids;
Rl indicates 1-4, preferably 1-3, natural type carbohydrate substituents
linked to the core
structures or nothing;
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagine N-
glycoside
aminoacids and/or peptides derived from protein, or natural serine or
threonine linked 0-
glycoside derivative such as serine or threonine linked 0-glycosides including
asparagine N-
glycoside aminoacids and/or peptides derived from protein, or when n2 is 1 R2
is nothing or a
ceramide structure or a derivetive of a ceramide structure, such as lysolipid
and amide
derivatives thereof;
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R3 is nothing or a branching structure respesenting a G1cNAc(36 or an
oligosaccharide with
G1cNAc(36 at its reducing end linked to Ga1NAc (when HexNAc is Ga1NAc); or
when Hex is
Gal and HexNAc is G1cNAc, and when z is 3 then R3 is Fuca4 or nothing, and
when z is 4
R3 is Fuca3 or nothing.
The preferred disaccharide epitopes in the glycan structures and glycomes
according to the
invention include structures Gal(34G1cNAc, Man(34G1cNAc, G1cA(34G1cNAc,
Gal(33G1cNAc,
Gal(33Ga1NAc, G1cA(33G1cNAc, G1cA(33Ga1NAc, and Gal(34G1c, which may be
further
derivatized from reducing end carbon atom and non-reducing monosaccharide
residues and is
in a separate embodiment branched from the reducing end residue. Preferred
branched
epitopes include Ga1(34(Fuc(x3)G1cNAc, Ga1(33(Fuc(x4)G1cNAc, and
Ga1(33(G1cNAc(36)Ga1NAc, which may be further derivatized from reducing end
carbon atom
and non-reducing monosaccharide residues.
Preferred epitopes for methods according to the invention
N-acetyllactosamine Gal,83/4G1cNAc terminal epitopes
The two N-acetyllactosamine epitopes Gal(34G1cNAc and/or Gal(33G1cNAc
represent
preferred terminal epitopes present on tissue materials or backbone structures
of the preferred
terminal epitopes for example further comprising sialic acid or fucose
derivatisations
according to the invention. In a preferred embodiment the invention is direted
to fucosylated
and/or non-substituted glycan non-reducing end forms of the terminal epitopes,
more
preferably to fucosylated and non-substutituted forms. The invention is
especially directed to
non-reducing end terminal (non-susbtituted) natural Gal(34G1cNAc and/or
Gal(33G1cNAc-
structures from human tissue material glycomes. The invention is in a specific
embodiment
directed to non-reducing end terminal fucosylated natural Gal(34G1cNAc and/or
Gal(33G1cNAc-structures from human tissue material glycomes.
Preferred fucosylated N-acetyllactosamines
The preferred fucosylated epitopes are according to the Formula TF:
(Fuc(x2)nl Gal(33/4(Fuc(x4/3 )õ2G1cNAc(3-R
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Wherein
nl is 0 or 1 indicating presence or absence of Fuca2;
n2 is 0 or 1, indicating the presence or absence of Fuca4/3 (branch), and
R is the reducing end core structure of N-glycan, 0-glycan and/or glycolipid.
5
The preferred structures thus include type 1 lactosamines (Gal(33G1cNAc
based):
Ga1(33(Fuc(x4)G1cNAc (Lewis a), Fuca2Ga1(33G1cNAc H-type 1, structure and,
Fuca2Ga1(33(Fuc(x4)G1cNAc (Lewis b) and
type 2 lactosamines (Gal(34G1cNAc based):
10 Ga1(34(Fuc(x3)G1cNAc (Lewis x), Fuca2Ga1(34G1cNAc H-type 2, structure and,
Fuca2Ga1(34(Fuc(x3)G1cNAc (Lewis y).
The type 2 lactosamines (fucosylated and/or terminal non-substituted) form an
especially
preferred group in context of tissue materials. Type 1 lactosamines
(Gal(33G1cNAc -
15 structures) are especially preferred in context of tissue materials.
Lactosamines Gal,83/4GlcNAc and glycolipid structures comprising lactose
structures
(Gal,(#Glc)
The lactosamines form a preferred structure group with lactose-based
glycolipids. The
20 structures share similar features as products of (33/4Ga1-transferases. The
(33/4 galactose based
structures were observed to produce characteristic features of protein linked
and glycolipid
glycomes.
The invention revealed that furthermore Ga1(33/4G1cNAc-structures are a key
feature of
25 glycolipids of human cells. Such glycolipids comprise two preferred
structural epitopes
according to the invention. The most preferred glycolipid types include thus
lactosylceramide
based glycosphingolipids and especially lacto- (Gal(33G1cNAc), such as
lactotetraosylceramide Gal(33G1cNAc(33Ga1(34Glc(3Cer, prefered structures
further including
its non-reducing terminal structures selected from the group:
Ga1(33(Fuc(x4)G1cNAc (Lewis
30 a), Fuca2Ga1(33G1cNAc (I4-type 1), structure and, Fuca2Ga1(33(Fuc(x4)G1cNAc
(Lewis b) or
sialylated structure SAa3Ga1(33G1cNAc or SAa3Ga1(33(Fuc(x4)G1cNAc, wherein SA
is a
sialic acid, preferably Neu5Ac preferably replacing Gal(33G1cNAc of
lactotetraosylceramide
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61
and its fucosylated and/or elogated variants such as preferably
according to the Formula:
(Sac(x3)õ5(Fuca2)i1Ga1(33 (Fuca4)i3G1cNAc(33 [Ga1(33/4(Fuca4/3 )õZG1cNAc (33
]õ4Ga1(34G1c(3Cer
wherein
nl is 0 or 1, indicating presence or absence of Fuca2;
n2 is 0 or 1, indicating the presence or absence of Fuca4/3 (branch),
n3 is 0 or 1, indicating the presence or absence of Fuca4 (branch)
n4 is 0 or 1, indicating the presence or absence of (fucosylated) N-
acetyllactosamine
elongation;
n5 is 0 or 1, indicating the presence or absence of Saca3 elongation;
Sac is terminal structure, preferably sialic acid, with 0- linkage, with the
proviso that when
Sac is present, n5 is 1, then nl is 0
and
neolacto (Gal(34GIcNAc)-comprising glycolipids such as
neolactotetraosylceramide Gal(34G1cNAc(33Ga1(34Glc(3Cer, preferred structures
further
including its non-reducing terminal Gal(34(Fuc(x3)G1cNAc (Lewis x),
Fuca2Ga1(34GIcNAc
H-type 2, structure and, Fuca2Ga1(34(Fuc(x3)G1cNAc (Lewis y)
and
its fucosylated and/or elogated variants such as preferably
(Sac(x3/6)õ5(Fuc(x2)n1Ga1(34(Fuc(x3)n3G1cNAc(33[Gat(34(Fuc(x3)õ2GIcNAc(33]n4Ga1
(34G1c(3Cer
nl is 0 or 1 indicating presence or absence of Fuca2;
n2 is 0 or 1, indicating the presence or absence of Fuca3 (branch),
n3 is 0 or 1, indicating the presence or absence of Fuca3 (branch)
n4 is 0 or 1, indicating the presence or absence of (fucosylated) N-
acetyllactosamine
elongation,
n5 is 0 or 1, indicating the presence or absence of Saca3/6 elongation;
Sac is terminal structure, preferably sialic acid (SA) with 0- linkage, or
sialic acid with a6-
linkage, with the proviso that when Sac is present, n5 is 1, then nl is 0, and
when sialic acid is
bound by a6- linkage preferably also n3 is 0.
Preferred human cell glycosphingolipid glycan profiles, compositions, and
marker structures
The inventors were able to describe human cell glycolipid glycomes by mass
spectrometric
profiling of liberated free glycans, revealing about 80 glycan signals from
different cell types.
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The proposed monosaccharide compositions of the neutral glycans were composed
of 2-7
Hex, 0-5 HexNAc, and 0-4 dHex. The proposed monosaccharide compositions of the
acidic
glycan signals were composed of 0-2 NeuAc, 2-9 Hex, 0-6 HexNAc, 0-3 dHex,
and/or 0-1
sulphate or phosphate esters. The present invention is especially directed to
analysis and
targeting of such human cell glycan profiles and/or structures for the uses
described in the
present invention with respect to human cells. The present invention is
further specifically
directed to glycosphingolipid glycan signals specific to human cell types.
Terminal glycan epitopes that were demonstrated in the present experiments in
human cell
glycosphingolipid glycans are useful in recognizing cells or specifically
binding to the cells
via glycans, and other uses according to the present invention, including
terminal epitopes:
Gal, Gal04G1c (Lac), Gal(34G1cNAc (LacNAc type 2), Ga1(33, Non-reducing
terminal
HexNAc, Fuc, al,2-Fuc, al,3-Fuc, Fuca2Gal, Fuca2Ga1p4G1cNAc (H type 2),
Fuca2Ga1(34G1c (2'-fucosyllactose), Fuca3GlcNAc, Ga1(34(Fuca3)G1cNAc (Lex),
Fuca3Glc,
Ga1(34(Fuca3)Glc (3-fucosyllactose), Neu5Ac, Neu5Aca2,3, and Neu5Aca2,6. The
present
invention is further directed to the total terminal epitope profiles within
the total human cell
glycosphingolipid glycomes and/or glycomes.
The present invention revealed characteristic variations (increased or
decreased expression in
comparision to similar control cell or a contaminatiog cell or like) of both
structure types in
various tissue and cell materials according to the invention. The structures
were revealed with
characteristic and varying expression in three different glycome types: N-
glycans, 0-glycans,
and glycolipids. The invention revealed that the glycan structures are a
charateristic feature of
tissue materials and are useful for various analysis methods according to the
invention.
Amounts of these and relative amounts of the epitopes and/or derivatives
varies between
tissue materials or between cells exposed to different conditions during
growing, storage, or
induction with effector molecules such as cytokines and/or hormones.
The preferred glycome glycan structure(s) and/or glycomes from cells according
to the
invention comprise structure(s) according to
the formula C 1:
R1Hex(3z{R3 }n1HexNAcXyR2,
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Wherein X is glycosidically linked disaccharide epitope (34(Fuc(x6)nGN,
wherein n is 0 or 1,
or X is nothing and
Hex is Gal or Man or G1cA,
HexNAc is G1cNAc or Ga1NAc,
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon,
z is linkage position 3 or 4, with the provision that when z is 4 then HexNAc
is G1cNAc and
then Hex is Man or Hex is Gal or Hex is G1cA, and
when z is 3 then Hex is G1cA or Gal and HexNAc is G1cNAc or Ga1NAc,
Rl indicates 1-4, preferably 1-3, natural type carbohydrate substituents
linked to the core
structures,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacids and/or peptides derived from protein, or natural serine or
threonine linked O-
glycoside derivative such as serine or threonine linked 0-glycosides including
asparagines N-
glycoside aminoacids and/or peptides derived from protein.
R3 is nothing or a branching structure respesenting a G1cNAc(36 or an
oligosaccharide with
G1cNAc(36 at its reducing end linked to Ga1NAc (when HexNAc is Ga1NAc) or when
Hex is
Gal and HexNAc is G1cNAc the then when z is 3 R3 is Fuca4 or nothing and when
z is 4 R3
is Fuca3 or nothing.
The preferred disaccharide epitopes in the glycan structures and glycomes
according to the
invention include structures Gal(34G1cNAc, Man(34G1cNAc, G1cA(34G1cNAc,
Gal(33G1cNAc,
Gal(33Ga1NAc, G1cA(33G1cNAc and G1cA(33Ga1NAc, which may be further
derivatized from
reducing end carbon atom and non-reducing monosaccharide residues and is
separate
embodinment branched from the reducing end residue. Preferred branched
epitopes include
Ga1(34(Fuc(x3)G1cNAc, Ga1(33(Fuc(x4)G1cNAc, Ga1(33(G1cNAc(36)Ga1NAc, which may
be
further derivatized from reducing end carbon atom and non-reducing
monosaccharide
residues.
The preferred disaccharide epitopes of glycoprotein or glycolipid structures
present on
glycans of human cells according to the invention comprise structures based on
the formula C2:
R1Hex(34G1cNAcXyR2,
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Wherein Hex is Gal OR Man and when Hex is Man then X is glycosidically linked
disaccharide epitope (34(Fuc(x6)nGN, wherein n is 0 or 1, or X is nothing and
when Hex is Gal then X is (33Ga1NAc of 0-glycan core or (32/4/6Mana3/6
terminal of N-
glycan core (as in formula NC3)
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon,
Rl indicates 1-4, preferably 1-3, natural type carbohydrate substituents
linked to the core
structures,
when Hex is Gal preferred R1 groups include structures SAa3/6,
SAa3/6Ga1(34G1cNAc(33/6,
when Hex is Man preferred Rl groups include Mana3, Mana6, branched structure
Mana3 {Mana6 } and elongated variants thereof as described for low mannose,
high-mannose
and complex type N-glycans below,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacids and/or peptides derived from protein, or natural serine or
threonine linked O-
glycoside derivative such as serine or threonine linked 0-glycosides including
asparagines N-
glycoside aminoacids and/or peptides derived from protein.
Structures of N-linked glycomes
Common core structure of N-linked glycomes
The inventors revealed that the N-glycans released by specific N-glycan
release methods from
the cells according to the invention, and preferred cells according to the
invention, comprise
mostly a specific type of N-glycan core structure.
The preferred N-glycan structure of each cell type is characterised and
recognized by treating
cells with a N-glycan releasing enzyme releasing practically all N-glycans
with core type
according to the invention. The N-glycan relasing enzyme is preferably protein
N-glycosidase
enzyme, preferably by protein N-glycosidase releasing effectively the N-
glycomes according
to the invention, more preferably protein N-glycosidase with similar
specificity as protein N-
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glycosidase F, and in a specifically preferred embodiment the enzyme is
protein N-
glycosidase F from F. meningosepticum. Alternative chemical N-glycan release
method was
used for controlling the effective release of the N-glycomes by the N-glycan
relasing enzyme.
5 The inventors used the NMR glycome analysis according to the invention for
further
characterization of released N-glycomes from small cell samples available. NMR
spectroscopy revealed the N-glycan core signals of the preferred N-glycan core
type of the
cells according to the invention.
10 The minimum formula
The present invention is directed to glycomes derived from cells and
comprising a common
N-glycosidic core structures. The invention is specifically directed to
minimum formulas
covering both GNl-glycomes and GN2-glycomes with difference in reducing end
structures.
15 The minimum core structure includes glycans from which reducing end G1cNAc
or
Fuca6GlcNAc has been released.These are referred as GNl-glycomes and the
components
thereof as GNl-glycans. The present invention is specifically directed to
natural N-glycomes
from cells comprising GNl-glycans. In a preferred embodiment the invention is
directed to
purified or isolated practically pure natural GNl-glycome from human cells.
The release of
20 the reducing end G1cNAc-unit completely or partially may be included in the
production of
the N-glycome or N-glycans from cells for analysis. The invention is
specifically directed to
soluble high/low mannose glycome of GNl-type.
The glycomes including the reducing end G1cNAc or Fuca6GlcNAc are referred as
GN2-
25 glycomes and the components thereof as GN2-glycans. The present invention
is also
specifically directed to natural N-glycomes from cells and tissues comprising
GN2-glycans. In
a preferred embodiment the invention is directed to purified or isolated
practically pure
natural GN2-glycome from cells.
30 The preferred N-glycan core structure(s) and/or N-glycomes from cells
according to the
invention comprise structure(s) according to
the formula NC 1:
R1M(34GNXyR2,
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Wherein X is glycosidically linked disaccharide epitope (34(Fuc(x6)nGN,
wherein n is 0 or 1,
or X is nothing and
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
Rl indicates 1-4, preferably 1-3, natural type carbohydrate substituents
linked to the core
structures,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacids and/or peptides derived from protein.
It is realized that when the invention is directed to a glycome, the formula
indicates mixture of
several or typically more than ten or even higher number of different
structures according to
the Formulas describing the glycomes according to the invention.
The possible carbohydrate substituents Rl comprise at least one mannose (Man)
residue, and
optionally one or several GIcNAc, Gal, Fuc, SA and/GaINAc residues, with
possible sulphate
and or phosphate modifications.
When the glycome is released by N-glycosidase the free N-glycome saccharides
comprise in a
preferred embodiment reducing end hydroxyl with anomeric linkage A having
structure a
and/or (3, preferably both a and P. In another embodiment the glycome is
derivatized by a
molecular structure which can be reacted with the free reducing end of a
released glycome,
such as amine, aminooxy or hydrazine or thiol structures. The derivatizing
groups comprise
typically 3 to 30 atoms in aliphatic or aromatic structures or can form
terminal group spacers
and link the glycomes to carriers such as solid phases or microparticels,
polymeric carries
such as oligosaccharides and/or polysaccharide, peptides, dendrimer, proteins,
organic
polymers such as plastics, polyethyleneglycol and derivatives, polyamines such
as
polylysines.
When the glycome comprises asparagine N-glycosides, A is preferably beta and R
is linked
asparagine or asparagine peptide. The peptide part may comprise multiple
different aminoacid
residues and typically multiple forms of peptide with different sequences
derived from natural
proteins carrying the N-glycans in cell materials according to the invention.
It is realized that
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for example proteolytic release of glycans may produce mixture of
glycopeptides. Preferably
the peptide parts of the glycopeptides comprises mainly a low number of amino
acid residues,
preferably two to ten residues, more preferably two to seven amino acid
residues and even
more preferably two to five aminoacid residues and most preferably two to four
amino acid
residues when "mainly" indicates preferably at least 60 % of the peptide part,
more preferably
at least 75 % and most preferably at least 90 % of the peptide part comprising
the peptide of
desired low number of aminoacid residues.
The preferred GN2- N-glycan core structure(s)
The preferred GN2- N-glycan core structure(s) and/or N-glycomes from cells
according to the
invention comprise structure(s) according to
the formula NC2:
R1M(34GN(34(Fuc(x6)nGNyR2,
wherein n is 0 or 1 and
wherein y is anomeric linkage structure a and/or (3 or linkage from
derivatized anomeric
carbon and
Rl indicates 1-4, preferably 1-3, natural type carbohydrate substituents
linked to the core
structures,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacid and/or peptides derived from protein.
The preferred compositions thus include one or several of the following
structures
NC2a: Ma3 {Ma6 }M(34GN(34 {Fuca6 }n1GNyR2
NC2b: Ma6M(34GN(34{Fuca6}n1GNyR2
NC2c: Ma3M(34GN(34{Fuca6}n1GNyR2
More preferably compositions comprise at least 3 of the structures or most
preferably both
structures according to the formula NC2a and at least both fucosylated and non-
fucosylated
with core structure(s) NC2b and/or NC2c.
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The preferred GNl- N-glycan core structure(s)
The preferred GNl- N-glycan core structure(s) and/or N-glycomes from cells
according to the
invention comprise structure(s) according to
the formula NC3:
R1M(34GNyR2,
wherein y is anomeric linkage structure a and/or (3 or linkage from
derivatized anomeric
carbon and
Rl indicates 1-4, preferably 1-3, natural type carbohydrate substituents
linked to the core
structures,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagine N-
glycoside
aminoacids and/or peptides derived from protein.
Multi-mannose GNI- N-glycan core structure(s)
The invention is specifically directed glycans and/or glycomes derived from
preferred cells
according to the present invention when the natural glycome or glycan
comprises Multi-
mannose GNl- N-glycan core structure(s) structure(s) according to
the formula NC4:
[R1Ma3]n3 {R3Ma6 }õ2M(34GNXyR2,
Rl and R3 indicate nothing or one or two, natural type carbohydrate
substituents linked to the
core structures, when the substituents are a-linked mannose monosaccharide
and/or
oligosaccharides and the other variables are as described above.
Furthermore common elongated GN2- N-glycan core structures are preferred types
of
glycomes according to the invention
The preferred N-glycan core structures further include differently elongated
GN2- N-glycan
core structures according to the
formula NC5:
[R1Ma3]n3 {R3Ma6 }õ2M(34GN(34 {Fuca6 }n1GNyR2,
wherein nl, n2 and n3 are either 0 or 1 and
wherein y is anomeric linkage structure a and/or (3 or linkage from
derivatized anomeric
carbon and
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Rl and R3 indicate nothing or 1-4, preferably 1-3, most preferably one or two,
natural type
carbohydrate substituents linked to the core structures,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagine N-
glycoside
aminoacids and/or peptides derived from protein,
GN is G1cNAc, M is mannosyl-, [] indicate groups either present or absent in a
linear
sequence.
{}indicates branching which may be also present or absent.
with the provision that at least n2 or n3 is 1. Preferably the invention is
directed to
compositions comprising with all possible values of n2 and n3 and all
saccharide types when
Rl and/or are R3 are oligosaccharide sequences or nothing.
Preferred N-glycan types in glycomes comprising N-glycans
The present invention is preferably directed to N-glycan glycomes comprising
one or several
of the preferred N-glycan core types according to the invention. The present
invention is
specifically directed to specific N-glycan core types when the compositions
comprise N-
glycan or N-glycans from one or several of the groups Low mannose glycans,
High mannose
glycans, Hybrid glycans, and Complex glycans, in a preferred embodiment the
glycome
comrise substantial amounts of glycans from at least three groups, more
preferably from all
four groups.
Major subtypes of N-glycans in N-linked glycomes
The invention revealed certain structural groups present in N-linked glycomes.
The grouping
is based on structural features of glycan groups obtained by classification
based on the
monosaccharide compositions and structural analysis of the structurel groups.
The glycans
were analysed by NMR, specific binding reagents including lectins and
antibodies and
specific glycosidases releasing monosaccharide residues from glycans. The
glycomes are
preferably analysed as neutral and acidic glycomes
The major neutral glycan types
The neutral glycomes mean glycomes comprising no acidic monosaccharide
residues such as
sialic acids (especially NeuNAc and NeuGc), HexA (especially G1cA, glucuronic
acid) and
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acid modification groups such as phosphate and/or sulphate esters. There are
four major types
of neutral N-linked glycomes which all share the common N-glycan core
structure: High-
mannose N-glycans, low-mannose N-glycans, hydrid type and complex type N-
glycans.
These have characteristic monosaccharide compositions and specific
substructures. The
5 complex and hybrid type glycans may include certain glycans comprising
monoantennary
glycans.
The groups of complex and hybrid type glycans can be further analysed with
regard to the
presence of one or more fucose residues. Glycans containing at least one
fucose units are
classified as fucosylated. Glycans containing at least two fucose residues are
considered as
10 glycans with complex fucosylation indicating that other fucose linkages, in
addition to the
a1,6-linkage in the N-glycan core, are present in the structure. Such linkages
include a1,2-,
al,3-, and al,4-linkage.
Furthermore the complex type N-glycans may be classified based on the
relations of HexNAc
(typically G1cNAc or Ga1NAc) and Hex residues (typically Man, Gal). Terminal
HexNAc
15 glycans comprise at least three HexNAc units and at least two Hexose units
so that the
number of Hex Nac residues is at least larger or equal to the number of hexose
units, with the
provisiont that for non branched, monoantennary glycans the number of HexNAcs
is larger
than number of hexoses.
This consideration is based on presence of two G1cNAc units in the core of N-
glycan and
20 need of at least two Mannose units to for a single complex type N-glycan
branch and three
mannose to form a trimannosyl core structure for most complex type structures.
A specific
group of HexNAc N-Glycans contains the same number of HexNAcs and Hex units,
when
the number is at least 5.
Preferred Mannose type structures
The invention is forther directed to glycans comprosing terminal Mannose such
as Ma6-
residue or both Mana6- and Mana3-residues, respectively, can additionally
substitute other
Ma2/3/6 units to form a Mannose- type structures including hydrid, low-Man and
High-Man
structures according to the invention.
Preferred high- and low mannose type structures with GN2-core structure are
according to the
Formula M2:
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[Ma2]i1 [Ma3 ]õ2{ [Ma2] i3 [M(x6)]õ4 }[Ma6]õ5 { [Ma2] i6[Ma2]õ7[M(x3 ]õ8
)M(34GN(34[ {Fuca6 }]n,GNyR2
wherein p, nl, n2, n3, n4, n5, n6, n7, n8, and m are either independently 0 or
1; with the
proviso that when n2 is 0, also nl is 0; when n4 is 0, also n3 is 0; when n5
is 0, also nl, n2,
n3, and n4 are 0; when n7 is 0, also n6 is 0; when n8 is 0, also n6 and n7 are
0;
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacid and/or peptides derived from protein;
[] indicates determinant either being present or absent depending on the value
of nl, n2, n3,
n4, n5, n6, n7, n8, and m; and
{} indicates a branch in the structure.
Preferred yR2-structures include [(3-N-Asn]p, wherein p is either 0 or 1.
Preferred Mannose type glycomes comprising GN1-core structures
As described above a preferred variant of N-glycomes comprising only single
G1cNAc-
residue in the core. Such structures are especially preferred as glycomes
produced by endo-N-
acetylglucosaminidase enzymes and Soluble glycomes. Preferred Mannose type
glycomesnclude structures according to the
Formula M2
[Ma2]i1[Ma3]õ2 {[Ma2]i3[M(x6)]õ4 }[Ma6]õ5 {[Ma2]i6[Ma2]õ7[M(x3]õ8 )M(34GNyR2
Fucosylated high-mannose N-glycans according to the invention have molecular
compositions Man5_9G1cNAc2Fuc1. For the fucosylated high-mannose glycans
according to
the formula, the sum of nl, n2, n3, n4, n5, n6, n7, and n8 is an integer from
4 to 8 and m is 0.
The low -mannose structures have molecular compositions Man1_4G1cNAc2Fuco_l.
They
consist of two subgroups based on the number of Fuc residues: 1)
nonfucosylated low -
mannose structures have molecular compositions Man1_4GIcNAc2 and 2)
fucosylated low -
mannose structures have molecular compositions Man1_4G1cNAc2Fuc1. For the low
mannose
glycans the sum of nl, n2, n3, n4, n5, n6, n7, and n8 is less than or equal to
(m + 3); and
preferably nl, n3, n6, and n7 are 0 when m is 0.
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Low mannose glycans
The invention revealed a very unusual group glycans in N-glycomes of invention
defined here
as low mannose N-glycans. These are not clearly linked to regular biosynthesis
of N-glycans,
but may represent unusual biosynthetic midproducts or degradation products.
The low
mannose glycans are especially characteristics changing during the changes of
cell status, the
differentiation and other changes according to the invention, for examples
changes associated
with differentiation status of cells and their differentiated products and
control cell materials.
The invention is especially directed to recognizing low amounts of low-mannose
type
glycans in cell types, such as with low degree of differentiation.
The invention revealed large differences between the low mannose glycan
expression in the
cell and tissue glycomes and material from tissue secretions such as human
serum.
The invention is especially directed to the use of specific low mannose glycan
comprising
glycomes for analysis of tissues and cells, preferably cultivated cells..
The invention further revealed specific mannose directed recognition methods
useful for
recognizing the preferred glycomes according to the invention. The invention
is especially
directed to combination of glycome analysis and recognition by specific
binding agents, most
preferred binding agent include enzymes and theis derivatives. The invention
further revealed
that specific low mannose glycans of the low mannose part of the glycomes can
be recognized
by degradation by specific a-mannosidase (Man2_4G1cNAc2Fuc0_1) or (3-
mannosidase
(Man1GlcNAc2Fuc0_1) enzymes and optionally further recognition of small low
mannose
structures, even more preferably low mannose structures comprising terminal
Man^4-
structures according to the invention.
The low mannose N-glycans, and preferred subgroups and individual structures
thereof, are
especially preferred as markers of the novel glycome compositions of the cells
according to
the invention useful for characterization of the cell types.
The low-mannose type glycans includes a specific group of a3- and/or a6-linked
mannose
type structures according to the invention including a preferred terminal and
core structure
types according to the invention.
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The inventions further revealed that low mannose N-glycans comprise a unique
individual
structural markers useful for characterization of the cells according to the
invention by
specific binding agents according to the invention or by combinations of
specific binding
agents according to the invention.
Neutral low-mannose type N-glycans comprise one to four or five terminal Man-
residues,
preferentially Mana structures; for example ManaO_3Man(34G1cNAc(34G1cNAc((3-N-
Asn) or
Manao_41VIan(34G1cNAc(34(Fuca6)G1cNAc((3-N-Asn).
Low-mannose N-glycans are smaller and more rare than the common high-mannose N-
glycans (Man5_9G1cNAc2). The low-mannose N-glycans detected in cell samples
fall into two
subgroups: 1) non-fucosylated, with composition MannGlcNAc2, where 1< n< 4,
and 2)
core-fucosylated, with composition MannGlcNAc2Fuc1, where 1< n < 5. The
largest of the
detected low-mannose structure structures is Man5GlcNAc2Fuc1 (m/z 1403 for the
sodium
adduct ion), which due to biosynthetic reasons most likely includes the
structure below (in the
figure the glycan is free oligosaccharide and (3-anomer; in glycoproteins in
tissues the glycan
is N-glycan and (3-anomer):
OH
HO O
HO HO
O
HO O OH
HO
HO O H C O OH
3
HO HO O OH OH 0
O
O 0
OH OH HO O O HO O HO OH
NH NH
HO HO
H3C O ~O
H3C
OH OH
Preferred general molecular structural features of low Man glycans
According to the present invention, low-mannose structures are preferentially
identified by
mass spectrometry, preferentially based on characteristic
Hexl_414exNAc2dHex0_1
monosaccharide composition. The low-mannose structures are further
preferentially identified
by sensitivity to exoglycosidase digestion, preferentially a-mannosidase
(Hex2_
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4HexNAc2dHexc0_1) or (3-mannosidase (Hex1HexNAc2dHexo_1) enzymes, and/or to
endoglycosidase digestion, preferentially N-glycosidase F detachment from
glycoproteins,
Endoglycosidase H detachment from glycoproteins (only Hex1_4I4exNAc2liberated
as Hexl_
4HexNAc1), and/or Endoglycosidase F2 digestion (only Hex1-4HexNAc2dHex1
digested to
Hex1-4HexNAc1). The low-mannose structures are further preferentially
identified in NMR
spectroscopy based on characteristic resonances of the Man(34G1cNAc(34G1cNAc N-
glycan
core structure and Mana residues attached to the Man(34 residue.
Several preferred low Man glycans described above can be presented in a single
Formula:
[Ma3]õ2{ [M(x6)]n4 }[Ma6]n5 {[Ma3]n8 }M(34GN(34[ {Fuca6 }]mGNyR2
wherein p, n2, n4, n5, n8, and m are either independently 0 or 1; with the
proviso that when
n2 is 0, also nl is 0; when n4 is 0, also n3 is 0; when n5 is 0, also nl, n2,
n3, and n4 are 0;
when n7 is 0, also n6 is 0; when n8 is 0, also n6 and n7 are 0; the sum of nl,
n2, n3, n4, n5,
n6, n7, and n8 is less than or equal to (m + 3); [] indicates determinant
either being present or
absent depending on the value of n2, n4, n5, n8, and m; and
{} indicates a branch in the structure;
y and R2 are as indicated above.
Preferred non-fucosylated low-mannose glycans are according to the formula:
[Ma3]õ2([M(x6)]n4)[Ma6]õ5 { [Ma3]n8 }M[34GN[34GNyR2
wherein p, n2, n4, n5, n8, and m are either independently 0 or 1,
with the provisio that when n5 is 0, also n2 and n4 are 0, and preferably
either n2 or n4 is 0,
[] indicates determinant either being present or absent
depending on the value of , n2, n4, n5, n8,
{} and O indicates a branch in the structure,
y and R2 are as indicated above.
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Preferred individual structures of non-fucosylated low-mannose glycans
Special small structures
Small non-fucosylated low-mannose structures are especially unsual among known
N-linked
glycans and characteristic glycans group useful for separation of cells
according to the present
5 invention. These include:
M(34GN(34GNyR2
Ma6M(34GN(34GNyR2
Ma3M(34GN(34GNyR2 and
Ma6 {Ma3 }M(34GN(34GNyR2.
10 M(34GN(34GNyR2 trisaccharide epitope is a preferred common structure alone
and together with its
mono-mannose derivatives Ma6M(34GN(34GNyR2 and/or Ma3M(34GN(34GNyR2, because
these
are characteristic structures commonly present in glycomes according to the
invention. The invention
is specifically directed to the glycomes comprising one or several of the
small non-fucosylated low-
mannose structures. The tetrasaccharides are in a specific embodiment
preferred for specific
15 recognition directed to a-linked, preferably a3/6-linked Mannoses as
preferred terminal recognition
element.
Special larQe structures
The invention further revealed large non-fucosylated low-mannose structures
that are unsual
20 among known N-linked glycans and have special characteristic expression
features among the
preferred cells according to the invention. The preferred large structures
include
[Ma3]õ2([Ma6]n4)Ma6 {Ma3 }M(34GN(34GNyR2
more specifically
Ma6Ma6 {Ma3 }M(34GN(34GNyR2
25 Ma3Ma6 {Ma3 }M(34GN(34GNyR2 and
Ma3(M(x6)Ma6 {Ma3 }M(34GN(34GNyR2.
The hexasaccharide epitopes are preferred in a specific embodiment as rare and
characteristic
structures in preferred cell types and as structures with preferred terminal
epitopes. The
heptasaccharide is also preferred as structure comprising a preferred unusual
terminal epitope
30 Ma3(M(x6)Ma useful for analysis of cells according to the invention.
Preferred fucosylated low-mannose glycans are derived according to the
formula:
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[Ma3]õ2{[Ma6]n4} [Ma6]n5{[Ma3]n8}Mp4GNP4(Fuca6)GNyR2
wherein p, n2, n4, n5, n8, and m are either independently 0 or 1,with the
provisio that when
n5 is 0, also n2 and n4 are 0, [] indicates determinant either being present
or absent
depending on the value of nl, n2, n3, n4, ( indicates a branch in the
structure;
and wherein nl, n2, n3, n4 and m are either independently 0 or 1,
with the provisio that when n3 is 0, also nl and n2 are 0,
[] indicates determinant either being present or absent
depending on the value of nl, n2, n3, n4 and m,
{} and O indicate a branch in the structure.
Preferred individual structures of fucosylated low-mannose Qlycans
Small fucosylated low-mannose structures are especially unusual among known N-
linked
glycans and form a characteristic glycan group useful for separation of cells
according to the
present invention.. These include:
M(34GN(34(Fuc(x6)GNyR2
Ma6M(34GN(34(Fuc(x6)GNyR2
Ma3M(34GN(34(Fuc(x6)GNyR2 and
Ma6 {Ma3 }M(34GN(34(Fuc(x6)GNyR2.
M(34GN(34(Fuc(x6)GNyR2 tetrasaccharide epitope is a preferred common structure
alone and
together with its mono-mannose derivatives Ma6M(34GN(34(Fuc(x6)GNyR2 and/or
Ma3M(34GN(34(Fuc(x6)GNyR2, because these are commonly present characteristics
structures in
glycomes according to the invention. The invention is specifically directed to
the glycomes comprising
one or several of the small non-fucosylated low-mannose structures. The
tetrasaccharides are in a
specific embodiment preferred for specific recognition directed to a-linked,
preferably a3/6-linked
Mannoses as preferred terminal recognition element.
Special larQe structures
The invention further revealed large fucosylated low-mannose structures are
unsual among
known N-linked glycans and have special characteristic expression features
among the
preferred cells according to the invention. The preferred large structure
includes
[Ma3]õ2([Ma6]n4)Ma6 {Ma3 }M(34GN(34(Fuc(x6)GNyR2
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more specifically
Ma6Ma6 {Ma3 }M(34GN(34(Fuc(x6)GNyR2
Ma3Ma6 {Ma3 }M(34GN(34(Fuc(x6)GNyR2 and
Ma3(M(x6)Ma6 {Ma3 }M(34GN(34(Fuc(x6)GNyR2.
The heptasaccharide epitopes are preferred in a specific embodiment as rare
and characteristic
structures in preferred cell types and as structures with preferred terminal
epitopes. The octasaccharide
is also preferred as structure comprising a preferred unusual terminal epitope
Ma3(M(x6)Ma useful
for analysis of cells according to the invention.
Preferred non-reducing end terminal Mannose-epitopes
The inventors revealed that mannose-structures can be labeled and/or otherwise
specifically
recognized on cell surfaces or cell derived fractions/matrials of specific
cell types. The
present invention is directed to the recognition of specific mannose epitopes
on cell surfaces
by reagents binding to specific mannose structures from cell surfaces.
The preferred reagents for recognition of any structures according to the
invention include
specific antibodies and other carbohydrate recognizing binding molecules. It
is known that
antibodies can be produced for the specific structures by various immunization
and/or library
technologies such as phage display methods representing variable domains of
antibodies.
Similarily with antibody library technologies, including aptamer technologies
and including
phage display for peptides, exist for synthesis of library molecules such as
polyamide
molecules including peptides, especially cyclic peptides, or nucleotide type
molecules such as
aptamer molecules.
The invention is specifically directed to specific recognition high-mannose
and low-mannose
structures according to the invention. The invention is specifically directed
to recognition of
non-reducing end terminal Mana-epitopes, preferably at least disaccharide
epitopes,
according to the formula:
[Ma2]ml [Max]m2[Ma6],,,3 { { [Ma2]m9[Ma2]m8[Ma3]m7}mlo(M[34[GN]m4)m5} m6yR2
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wherein ml, m 2, m3, m4, m5, m6, m7, m8, m9 and mlO are independently either 0
or 1; with
the proviso that when m3 is 0, then ml is 0 and, when m7 is 0 then either ml-5
are 0 and m8
and m9 are 1 forming Ma2Ma2 -disaccharide or both m8 and m9 are 0
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
R2 is reducing end hydroxyl, chemical reducing end derivative
and x is linkage position 3 or 6 or both 3 and 6 forming branched structure,
{} indicates a branch in the structure.
The invention is further directed to terminal Ma2-containing glycans containg
at least one
Ma2-group and preferably Ma2-group on each, branch so that ml and at least one
of m8 or
m9 is 1. The invention is further directed to terminal Ma3 and/or Ma6-epitopes
without
terminal Ma2-groups, when all ml, m8 and m9 are 1.
The invention is further directed in a preferred embodiment to the terminal
epitopes linked to
a M(3-residue and for application directed to larger epitopes. The invention
is especially
directed to M(34GN-comprising reducing end terminal epitopes.
The preferred terminal epitopes comprise typically 2-5 monosaccharide residues
in a linear
chain. According to the invention short epitopes comprising at least 2
monosaccharide
residues can be recognized under suitable background conditions and the
invention is
specifically directed to epitopes comprising 2 to 4 monosaccharide units and
more preferably
2-3 monosaccharide units, even more preferred epitopes include linear
disaccharide units
and/or branched trisaccharide non-reducing residue with natural anomeric
linkage structures
at reducing end. The shorter epitopes may be preferred for specific
applications due to
practical reasons including effective production of control molecules for
potential binding
reagents aimed for recognition of the structures.
The shorter epitopes such as Ma2M-may is often more abundant on target cell
surface as it is
present on multiple arms of several common structures according to the
invention.
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Preferred disaccharide epitopes includes
Mana2Man, Mana3Man, Mana6Man, and more preferred anomeric forms Mana2Mana,
Mana3Man(3, Mana6Man(3, Mana3Mana and Mana6Mana.
Preferred branched trisaccharides includes Mana3(Man(x6)Man,
Mana3(Man(X6)Man(3, and
Mana3(Man(x6)Mana.
The invention is specifically directed to the specific recognition of non-
reducing terminal
Man(x2-structures especially in context of high-mannose structures.
The invention is specifically directed to following linear terminal mannose
epitopes:
a) preferred terminal Mana2-epitopes including following oligosaccharide
sequences:
Mana2Man,
Mana2Mana,
Mana2Mana2Man, Mana2Mana3Man, Mana2Mana6Man,
Mana2Mana2Mana, Mana2Mana3Man(3, Mana2Mana6Mana,
Mana2Mana2Mana3Man, Mana2Mana3Mana6Man, Mana2Mana6Mana6Man
Mana2Mana2Mana3Man(3, Mana2Mana3Mana6Man(3, Mana2Mana6Mana6Man(3;
The invention is further directed to recognition of and methods directed to
non-reducing end
terminal Man(x3- and/or Man(x6-comprising target structures, which are
characteristic
features of specifically important low-mannose glycans according to the
invention. The
preferred structural groups includes linear epitopes according to b) and
branched epitopes
according to the c3) especially depending on the status of the target matrial.
b) preferred terminal Mana3- and/or Mana6-epitopes including following
oligosaccharide
sequences:
Mana3Man, Mana6Man, Mana3Man(3, Mana6Man(3, Mana3Mana, Mana6Mana,
Mana3Mana6Man, Mana6Mana6Man, Mana3Mana6Man(3, Mana6Man(x6Man(3
and to following
c) branched terminal mannose epitopes, are preferred as characteristic
structures of especially
high.mannose structures (cl and c2) and low-mannose structures (c3), The
preferred
branched epitopes include:
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c 1) branched terminal Mana2-epitopes
Mana2Mana3(Mana2Mana6)Man, Mana2Mana3(Mana2Mana6)Mana,
Mana2Mana3(Mana2Mana6)Mana6Man, Mana2Mana3(Mana2Mana6)Mana6Man(3,
5 Mana2Mana3(Mana2Mana6)Mana6(Mana2Mana3)Man,
Mana2Mana3(Mana2Mana6)Mana6(Mana2Mana2Mana3)Man,
Mana2Mana3 (Mana2Mana6)Mana6(Mana2Mana3 )Man(3
Mana2Mana3 (Mana2Mana6)Mana6(ManaMana2Mana3 )Man(3
10 c2) branched terminal Mana2- and Mana3 or Mana6-epitopes
according to formula when ml and/or m8 and/m9 is 1 and the molecule comprise
at least one
nonreducing end terminal Mana3 or Man(x6-epitope
c3) branched terminal Mana3 or Mana6-epitopes
15 Mana3(Man(x6)Man, Mana3(Man(x6)Man(3, Mana3(Man(x6)Mana,
Mana3(Man(x6)Mana6Man, Mana3(Man(x6)Mana6Man(3,
Mana3(Man(x6)Mana6(Man(x3)Man, Mana3(Man(x6)Mana6(Man(x3)Man(3
The present invention is further directed to increase of selectivity and
sensitivity in
20 recognition of
Target glycans by combining recognition methods for terminal Mana2 and Mana3
and/or
Mana6-comprising structures. Such methods would be especially useful in
context of cell
material according to the invention comprising both high-mannose and low-
mannose glycans.
Complex type N-glycans
According to the present invention, complex-type structures are preferentially
identified by
mass spectrometry, preferentially based on characteristic monosaccharide
compositions,
wherein HexNAc>4 and Hex>3. In a more preferred embodiment of the present
invention,
4<HexNAc<20 and 3<Hex<21, and in an even more preferred embodiment of the
present
invention, 4<HexNAc<10 and 3<Hex<11. The complex-type structures are further
preferentially identified by sensitivity to endoglycosidase digestion,
preferentially N-
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glycosidase F detachment from glycoproteins. The complex-type structures are
further
preferentially identified in NMR spectroscopy based on characteristic
resonances of the
Mana3(Mana6)Man(34G1cNAc(34G1cNAc N-glycan core structure and G1cNAc residues
attached to the Mana3 and/or Mana6 residues.
Beside Mannose-type glycans the preferred N-linked glycomes include G1cNAc(32-
type
glycans including Complex type glycans comprising only G1cNAc(32-branches and
Hydrid
type glycan comprising both Mannose-type branch and G1cNAc(32-branch.
G1cNAc02-type glycans
The invention revealed G1cNAc(32Man structures in the glycomes according to
the invention.
Preferably G1cNAc(32Man-structures comprise one or several of G1cNAc(32Mana -
structures,
more preferably G1cNAc(32Mana3 or G1cNAc(32Mana6-structure.
The Complex type glycans of the invention comprise preferably two
G1cNAc(32Mana structures, which are preferably G1cNAc(32Mana3 and
G1cNAc(32Mana6-.
The Hybrid type glycans comprise preferably G1cNAc(32Mana3-structure.
The present invention is directed to at least one of natural oligosaccharide
sequence structures
and structures truncated from the reducing end of the N-glycan according to
the Formula GN(32
[R1GNR2]nl [Ma3]õ2{ [R3]n3[GN(32]n4Ma6 }n5M(34GNXyR2,
with optionally one or two or three additional branches according to formula
[RXGN(3z]õx linked to Ma6-, Ma3-, or M(34 and RX may be different in each
branch
wherein nl, n2, n3, n4, n5 and nx, are either 0 or 1, independently,
with the proviso that when n2 is 0 then nl is 0 and when n3 is 1 or/and n4 is
1 then n5 is also
1, and at least nl or n4 is 1, or n3 is 1,
when n4 is 0 and n3 is 1 then R3 is a mannose type substituent or nothing and
wherein X is glycosidically linked disaccharide epitope (34(Fuc(x6)nGN,
wherein n is 0 or 1,
or X is nothing and
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
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Rl, RX and R3 indicate independently one, two or three, natural substituents
linked to the core
structure,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacids and/or peptides derived from protein.
[] indicate groups either present or absent in a linear sequence. {}indicates
branching which
may be also present or absent.
Elongation of G1cNAc(32-type structures, complex/hydrid type structures
The substituents Rl, RX and R3 may form elongated structures. In the elongated
structures Rl,
and RX represent substituents of G1cNAc (GN) and R3 is either substituent of
G1cNAc or
when n4 is 0 and n3 is 1 then R3 is a mannose type substituent linked to
mannosea6-branch
forming a Hybrid type structure. The substituents of GN are monosaccharide
Gal, Ga1NAc, or
Fuc or and acidic residue such as sialic acid or sulfate or fosfate ester.
G1cNAc or GN may be elongated to N-acetyllactosaminyl also marked as Ga1(3GN
or di-N-
acetyllactosdiaminyl Ga1NAc(3G1cNAc preferably Ga1NAc(34G1cNAc. LN(32M can be
further
elongated and/or branched with one or several other monosaccharide residues
such as by
galactose, fucose, SA or LN-unit(s) which may be further substituted by SAa-
strutures,
and/or Ma6 residue and/or Ma3 residues can be further substituted one or two
(36-, and/or (34-
linked additional branches according to the formula,
and/or either of Ma6 residue or Ma3 residue may be absent
and/or Ma6- residue can be additionally substitutes other Mana units to form a
hybrid type
structures
and/or Man(34 can be further substituted by GN(34,
and/or SA may include natural substituents of sialic acid and/or it may be
substituted by other
SA-residues preferably by 0- or a9-linkages.
The SAa-groups are linked to either 3- or 6- position of neighboring Gal
residue or on 6-
position of G1cNAc, preferably 3- or 6- position of neighboring Gal residue.
In separately
preferred embodiments the invention is directed structures comprising solely 3-
linked SA or
6- linked SA, or mixtures thereof.
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Hybrid type structures
According to the present invention, hybrid-type or monoantennary structures
are
preferentially identified by mass spectrometry, preferentially based on
characteristic
monosaccharide compositions, wherein HexNAc=3 and Hex>2. In a more preferred
embodiment of the present invention 2<Hex<11, and in an even more preferred
embodiment
of the present invention 2<Hex<9. The hybrid-type structures are further
preferentially
identified by sensitivity to exoglycosidase digestion, preferentially a-
mannosidase digestion
when the structures contain non-reducing terminal a-mannose residues and
Hex>3, or even
more preferably when Hex>4, and to endoglycosidase digestion, preferentially N-
glycosidase
F detachment from glycoproteins. The hybrid-type structures are further
preferentially
identified in NMR spectroscopy based on characteristic resonances of the
Mana3(Mana6)Man(34G1cNAc(34G1cNAc N-glycan core structure, a G1cNAc(3 residue
attached to a Mana residue in the N-glycan core, and the presence of
characteristic resonances
of non-reducing terminal a-mannose residue or residues.
The monoantennary structures are further preferentially identified by
insensitivity to a-
mannosidase digestion and by sensitivity to endoglycosidase digestion,
preferentially N-
glycosidase F detachment from glycoproteins. The monoantennary structures are
further
preferentially identified in NMR spectroscopy based on characteristic
resonances of the
Mana3Man(34G1cNAc(34G1cNAc N-glycan core structure, a G1cNAc(3 residue
attached to a
Mana residue in the N-glycan core, and the absence of characteristic
resonances of further
non-reducing terminal a-mannose residues apart from those arising from a
terminal a-
mannose residue present in a ManaMan(3 sequence of the N-glycan core.
The present invention is directed to at least one of natural oligosaccharide
sequence structures
and structures truncated from the reducing end of the N-glycan according to
the Formula HY1
R1GN(32Ma3{[R3]n3Ma6}M(34GNXyR2,
wherein n3, is either 0 or 1, independently,
and
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wherein X is glycosidically linked disaccharide epitope (34(Fuc(x6)nGN,
wherein n is 0 or 1,
or X is nothing and
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
Rl indicate nothing or substituent or substituents linked to G1cNAc, R3
indicates nothing or
Mannose-substituent(s) linked to mannose residue, so that each of Rl, and R3
may correspond
to one, two or three, more preferably one or two, and most preferably at least
one natural
substituents linked to the core structure,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacids and/or peptides derived from protein.
[] indicate groups either present or absent in a linear sequence. {}indicates
branching which
may be also present or absent.
Preferred hybrid type structures
The preferred hydrid type structures include one or two additional mannose
residues on the
preferred core stucture.
Formula HY2
R1GN(32Ma3{[Ma3]ml([Ma6])m2Ma6}M(34GNXyR2,
wherein n3, is either 0 or 1,and ml and m2 are either 0 or 1, independently,
{} and ( ) indicates branching which may be also present or absent,
other variables are as described in Formula HY1.
Furthermore the invention is directed to structures comprising additional
lactosamine type
structures on GN(32- branch. The preferred lactosamine type elongation
structures includes N-
acetyllactosamines and derivatives, galactose, Ga1NAc, G1cNAc, sialic acid and
fucose.
Preferred structures according to the formula HY2 include:
Structures containing non-reducing end terminal G1cNAc
As a specific preferred group of glycans
GN(32Ma3 {Ma3Ma6 }M(34GNXyR2,
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GN(32Ma3 {Ma6Ma6 }M(34GNXyR2,
GN(32Ma3 {Ma3(Ma6)Ma6 }M(34GNXyR2,
and/or elongated variants thereof
R1GN(32Ma3 {Ma3Ma6 }M(34GNXyR2,
5 R1GN(32Ma3 {Ma6Ma6 }M(34GNXyR2,
R1GN(32Ma3 {Ma3(Ma6)Ma6 }M(34GNXyR2,
Formula HY3
10 [R1Ga1[NAc]o2(3z]o1GN(32Ma3 {[Ma3]ml [(M(x6)]m2Ma6 }n5M(34GNXyR2,
wherein nl, n2, n3, n5, ml, m2, 01 and o2 are either 0 or 1, independently,
z is linkage position to GN being 3 or 4, ? in a preferred embodiment 4,
Rl indicates on or two a N-acetyllactosamine type elongation groups or
nothing,
{} and ( ) indicates branching which may be also present or absent,
15 other variables are as described in Formula HY1.
Preferred structures according to the formula HY3 include especially
structures containing non-reducing end terminal Gal(3, preferably Ga1(33/4
forming a terminal
N-acetyllactosamine structure. These are preferred as a special group of
Hybrid type
20 structures, preferred as a group of specific value in characterization of
balance of Complex N-
glycan glycome and High mannose glycome:
Gal(3zGN(32Ma3 {Ma3Ma6 }M(34GNXyR2, Gal(3zGN(32Ma3 {Ma6Ma6 }M(34GNXyR2,
Gal(3zGN(32Ma3 {Ma3(M(x6)Ma6 }M(34GNXyR2,
and/or elongated variants thereof preferred for carrying additional
characteristic terminal
25 structures useful for characterization of glycan materials
R1Ga1(3zGN(32Ma3 {Ma3Ma6 }M(34GNXyR2,
R1Ga1(3zGN(32Ma3 {Ma6Ma6 }M(34GNXyR2,
R1Ga1(3zGN(32Ma3 {Ma3(M(x6)Ma6 }M(34GNXyR2. Preferred elongated materials
include
structures wherein Rl is a sialic acid, more preferably NeuNAc or NeuGc.
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Complex N-glycan structures
The present invention is directed to at least one of natural oligosaccharide
sequence structures
and structures truncated from the reducing end of the N-glycan according to
the Formula CO1
[R1GN(32]n1 [Ma3]õ2{ [R3GN(32]n4Ma6 }õsM(34GNXyR2
with optionally one or two or three additional branches according to formula
[RXGN(3z]õx linked to Ma6-, Ma3-, or M(34 and RX may be different in each
branch
wherein nl, n2, n4, n5 and nx, are either 0 or 1, independently,
with the proviso that when n2 is 0 then nl is 0 and when n4 is 1 then n5 is
also 1, and at least
nl is 1 or n4 is 1,and at least either of nl and n4 is 1
and
wherein X is glycosidically linked disaccharide epitope (34(Fuc(x6)nGN,
wherein n is 0 or 1,
or X is nothing and
y is anomeric linkage structure a and/or (3 or linkage from derivatized
anomeric carbon, and
Rl, RX and R3 indicate independently one, two or three, natural substituents
linked to the core
structure,
R2 is reducing end hydroxyl, chemical reducing end derivative or natural
asparagine N-
glycoside derivative such as asparagine N-glycosides including asparagines N-
glycoside
aminoacids and/or peptides derived from protein.
[] indicate groups either present or absent in a linear sequence. {}indicates
branching which
may be also present or absent.
Preferred Complex type structures
Incomplete monoantennary N-Qlycans
The present invention revealed incomplete Complex monoantennary N-glycans,
which are
unusual and useful for characterization of glycomes according to the
invention. The most of
the in complete monoantennary structures indicate potential degradation of
biantennary N-
glycan structures and are thus preferred as indicators of cellular status. The
incomplete
Complex type monoantennary glycans comprise only one GN(32-structure.
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The invention is specifically directed to structures are according to the
Formula CO1 above
when only nl is 1 or n4 is one and mixtures of such structures.
The preferred mixtures comprise at least one monoantennary complex type
glycans
A ) with single branches from a likely degradative biosynthetic process:
R1GN(32M(x3(34GNXyR2
R3GN(32M(x6M(34GNXyR2 and
B) with two branches comprising mannose branches
B 1) Rl GN(32Ma3 {M(x6 }n5M(34GNXyR2
B2) Ma3 {R3GN(32Ma6 }n5M(34GNXyR2
The structure B2 is preferred with A structures as product of degradative
biosynthesis, it is
especially preferred in context of lower degradation of Mana3-structures. The
structure B 1 is
useful for indication of either degradative biosynthesis or delay of
biosynthetic process
Biantennary and multiantennary structures
The inventor revealed a major group of biantennary and multiantennary N-
glycans from cells
according to the invention, the preferred biantennary and multiantennary
structures comprise
two GN(32 structures.
These are preferred as an additional characteristics group of glycomes
according to the
invention and are represented according to the Formula C02:
R1GN(32Ma3 {R3GN(32Ma6 }M(34GNXyR2
with optionally one or two or three additional branches according to formula
[RXGN(3z]õx linked to Ma6-, Ma3-, or M(34 and RX may be different in each
branch
wherein nx is either 0 or 1,
and other variables are according to the Formula CO1.
Preferred biantennary structure
A biantennary structure comprising two terminal GN(3-epitopes is preferred as
a potential
indicator of degradative biosynthesis and/or delay of biosynthetic process.
The more preferred
structures are according to the Formula C02 when Rl and R3 are nothing.
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Elongated structures
The invention revealed specific elongated complex type glycans comprising Gal
and/or
Ga1NAc-structures and elongated variants thereof. Such structures are
especially preferred as
informative structures because the terminal epitopes include multiple
informative
modifications of lactosamine type, which characterize cell types according to
the invention.
The present invention is directed to at least one of natural oligosaccharide
sequence structure
or group of structures and corresponding structure(s) truncated from the
reducing end of the
N-glycan according to
the Formula C03
[R1Ga1[NAc]o2(3z2]o1GN(32Ma3 {[R1Ga1[NAc]o4(3z2]o3GN(32Ma6 }M(34GNXyR2,
with optionally one or two or three additional branches according to formula
[RXGN(3z1]õx linked to Ma6-, Ma3-, or M(34 and RX may be different in each
branch
wherein nx, ol, o2, o3, and o4 are either 0 or 1, independently,
with the provisio that at least o1 or o3 is 1, in a preferred embodiment both
are 1
z2 is linkage position to GN being 3 or 4, in a preferred embodiment 4,
zl is linkage position of the additional branches.
Rl, Rx and R3 indicate one or two a N-acetyllactosamine type elongation groups
or nothing,
{} and ( ) indicates branching which may be also present or absent,
other variables are as described in Formula CO1.
Galactosylated structures
The inventors characterized especially directed to digalactosylated structure
Gal(3zGN(32Ma3 {Gal(3zGN(32Ma6 }M(34GNXyR2,
and monogalactosylated structures:
Gal(3zGN(32Ma3 {GN(32Ma6 }M(34GNXyR2,
GN(32Ma3 {Gal(3zGN(32Ma6 }M(34GNXyR2,
and/or elongated variants thereof preferred for carrying additional
characteristic terminal
structures useful for characterization of glycan materials
R1Ga1(3zGN(32Ma3 {R3Ga1(3zGN(32Ma6 }M(34GNXyR2
R1Ga1(3zGN(32Ma3 {GN(32Ma6 }M(34GNXyR2, and
GN(32Ma3 {R3Ga1(3zGN(32Ma6 }M(34GNXyR2.
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Preferred elongated materials include structures wherein Rl is a sialic acid,
more preferably
NeuNAc or NeuGc.
LacdiNAc-structure comprising N-glycans
The present invention revealed for the first time LacdiNAc, GalNacbGlcNAc
structures from
the cell according to the invention. Preferred N-glycan lacdiNAc structures
are included in
structures according to the Formula CO1, when at least one the variable o2 and
o4 is 1.
The major acidic glycan types
The acidic glycomes mean glycomes comprising at least one acidic
monosaccharide residue
such as sialic acids (especially NeuNAc and NeuGc) forming sialylated glycome,
HexA
(especially G1cA, glucuronic acid) and/or acid modification groups such as
phosphate and/or
sulphate esters.
According to the present invention, presence of phosphate and/or sulphate
ester (SP) groups
in acidic glycan structures is preferentially indicated by characteristic
monosaccharide
compositions containing one or more SP groups. The preferred compositions
containing SP
groups include those formed by adding one or more SP groups into non-SP group
containing
glycan compositions, while the most preferential compositions containing SP
groups
according to the present invention are selected from the compositions
described in the acidic
N-glycan fraction glycan group tables. The presence of phosphate and/or
sulphate ester
groups in acidic glycan structures is preferentially further indicated by the
characteristic
fragments observed in fragmentation mass spectrometry corresponding to loss of
one or more
SP groups, the insensitivity of the glycans carrying SP groups to sialidase
digestion. The
presence of phosphate and/or sulphate ester groups in acidic glycan structures
is preferentially
also indicated in positive ion mode mass spectrometry by the tendency of such
glycans to
form salts such as sodium salts as described in the Examples of the present
invention.
Sulphate and phosphate ester groups are further preferentially identified
based on their
sensitivity to specific sulphatase and phosphatase enzyme treatments,
respectively, and/or
specific complexes they form with cationic probes in analytical techniques
such as mass
spectrometry.
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Complex N-glycan glycomes, sialylated
The present invention is directed to at least one of natural oligosaccharide
sequence structures
and structures truncated from the reducing end of the N-glycan according to
the Formula
5
[{SAa3/6}s1LN(32]riMa3 {({SAa3/6}s2LN(32) r2Ma6}r8
{M[(34GN[(34{Fuca6}r3GN1r4]r5}r6
(I)
with optionally one or two or three additional branches according to formula
{SAa3/6}s3LN(3, (IIb)
wherein rl, r2, r3, r4, r5, r6, r7 and r8 are either 0 or 1, independently,
wherein sl, s2 and s3 are either 0 or 1, independently,
with the proviso that at least rl is 1 or r2 is 1, and at least one of s1, s2
or s3 is 1.
LN is N-acetyllactosaminyl also marked as Ga1(3GN or di-N-acetyllactosdiaminyl
GaINAc(3GIcNAc preferably GaINAc(34GIcNAc, GN is GIcNAc, M is mannosyl-,
with the proviso LN(32M or GN(32M can be further elongated and/or branched
with one or
several other monosaccharide residues such as by galactose, fucose, SA or LN-
unit(s) which
may be further substituted by SAa-strutures,
and/or one LN(3 can be truncated to GN(3
and/or Ma6 residue and/or Ma3 residues can be further substituted one or two
(36-, and/or (34-
Iinked additional branches according to the formula,
and/or either of Ma6 residue or Ma3 residue may be absent
and/or Ma6- residue can be additionally substitutes other Mana units to form a
hybrid type
structures
and/or Man(34 can be further substituted by GN(34,
and/or SA may include natural substituents of sialic acid and/or it may be
substituted by other
SA-residues preferably by 0- or a9-Iinkages.
O, {}, [] and [] indicate groups either present or absent in a linear
sequence. {}indicates
branching which may be also present or absent.
The SAa-groups are linked to either 3- or 6- position of neighboring Gal
residue or on 6-
position of GIcNAc, preferably 3- or 6- position of neighboring Gal residue.
In separately
preferred embodiments the invention is directed structures comprising solely 3-
linked SA or
6- linked SA, or mixtures thereof. In a preferred embodiment the invention is
directed to
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glycans wherein r6 is 1 and r5 is 0, corresponding to N-glycans lacking the
reducing end
G1cNAc structure.
The LN unit with its various substituents can in a preferred general
embodiment represented
by the formula:
[Gal(NAc)nla3]õ2{Fuca2 }n3Ga1(NAc)n4(33/4 {Fuca4/3 }n5G1cNAc(3
wherein nl, n2, n3, n4, and n5 are independently either 1 or 0,
with the provisio that
the substituents defined by n2 and n3 are alternative to presence of SA at the
non-reducing
end terminal
the reducing end G1cNAc -unit can be further (33- and/or (36-linked to another
similar LN-
structure forming a poly-N-acetyllactosamine structure
with the provision that for this LN-unit n2, n3 and n4 are 0,
the Gal(NAc)(3 and G1cNAc(3 units can be ester linked a sulphate ester group,
(), and [] indicate groups either present or absent in a linear sequence;
{}indicates branching
which may be also present or absent.
LN unit is preferably Ga1(34GN and/or Ga1(33GN. The inventors revealed that
early human
cells can express both types of N-acetyllactosamine, the invention is
especially directed to
mixtures of both structures. Furthermore the invention is directed to special
relatively rear
type 1 N-acetyllactosamines, Ga1(33GN, without any non-reducing end/site
modification, also
called lewis c-structures, and substituted derivatives thereof, as novel
markers of early human
cells.
Uses of glycan structure grouping and analysis
In the present invention, glycan signals with preferential monosaccharide
compositions can be
grouped into structure groups based on classification rules described in the
present invention.
The present invention includes parallel and overlapping classification systems
that are used
for the classification of the glycan structure groups.
Glycan signals isolated from the N-glycan fractions from the tissue material
types studied in
the present invention are grouped into glycan structure groups based on their
preferential
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monosaccharide compositions according to the invention, in Table 6 for neutral
glycan
fractions and Table 7 for acidic glycan fractions. Taken together, the
analyses revealed that
all the structure groups according to the invention are present in the studied
tissue material
types. In another aspect of the present invention, the glycan structure
grouping is used to
compare different tissue materials and characterize their specific
glycosylation features.
According to the present invention the discovered and analyzed differencies
between the
glycan signals within the glycan signal groups between different tissue
material samples are
used for comparison and characterization.
The quantitative glycan profiling combined with glycan structural
classification is used
according to the present invention to characterize and identify glycosylation
features
occurring in tissue materials, glycosylation features specific for certain
tissue materials as
well as differencies between different tissue materials. According to the
present invention, the
classification is used to characterize and compare glycosylation features of
different tissues,
of normal and diseased tissues, preferentially cancerous tissues, and solid
tissues such as lung
tissue and fluid tissues such as blood and/or serum. In another aspect of the
present invention,
the glycan structure grouping is used to compare different tissue materials
and characterize
their specific glycosylation features. According to the present invention
differencies between
relative proportions of glycan signal structure groups are used to compare
different tissue
material samples.
In a further aspect of the present invention, analysis of the glycan structure
groups,
preferentially including terminal HexNAc and/or low-mannose and optionally
other groups
separately or in combination, is used to differentiate between different
tissue materials or
different stages of tissue materials, preferentially to identify human disease
and more
preferentially human cancer. In a futher preferred form the present method is
used to
differentiate between benign and malignant tumors. According to the present
invention
analysis of human serum glycan groups or combinations thereof according to the
present
invention can be used to identify the presence of other tissue materials in
blood or serum
samples, more preferably to identify disease and preferably malignant cancer.
Integrated glycome analysis technology
The invention is directed to analysis of present cell materials based on
single or several
glycans (glycome profile) of cell materials according to the invention. The
analysis of
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multiple glycans is preferably performed by physical analysis methods such as
mass
spectrometry and/or NMR.
The invention is specifically directed to integrated analysis process for
glycomes, such as total
glycomes and cell surface glycomes. The integrated process represent various
novel aspects in
each part of the process. The methods are especially directed to analysis of
low amounts of
cells. The integrated analysis process includes
A) preferred preparation of substrate cell materials for analysis, including
one or several of
the methods: use of a chemical buffer solution, use of detergents, chemical
reagents and/or
enzymes.
B) release of glycome(s), including various subglycome type based on glycan
core, charge
and other structural features, use of controlled reagents in the process
C) purification of glycomes and various subglycomes from complex mixtures
D) preferred glycome analysis, including profiling methods such as mass
spectrometry and/or
NMR spectroscopy
E) data processing and analysis, especially comparative methods between
different sample
types and quantitative analysis of the glycome data.
A. Preparation of cell materials
Cell substrate material and its preparation for total and cell surface glycome
analysis. The
integrated glycome analysis includes preferably a cell preparation step to
increase the
availability of cell surface glycans. The cell preparation step preferably
degrades either total
cell materials or cell surface to yield a glycome for more effective glycan
release. The
degradation step preferably includes methods of physical degradation and/or
chemical
degradation. In a preferred embodiment at least one physical and one chemical
degradation
methods are combined, more preferably at least one physical method is combined
with at
least two chemical methods, even more preferably with at least three chemical
methods.
The physical degration include degration by energy including thermal and/or
mechanical
energy directed to the cells to degrade cell structures such as heating,
freezing, sonication, and
pressure. The chemical degradation include use of chemicals and specific
concentrations of
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chemicals for distruption distruption of cells preferably detergents including
ionic and neutral
detergents, chaotropic salts, denaturing chemicals such as urea, and non-
physiological salt
concentrations for distruption of the cells.
The glycome analysis according to the invention is divided to two methods
including Total
cell glycomes, and Cell surface glycomes. The production of Total cell
glycomes involves
degradation of cells by physical and/or chemical degradation methods,
preferably at least by
chemical methods, more preferably by physical and chemical methods. The Cell
surface
glycomes is preferably released from cell surface preserving cell membranes
intact or as intact
as possible, such methods involve preferably at least one chemical method,
preferably
enzymatic method. The cell surface glycomes may be alternatively released from
isolated cell
membranes, this method involves typically chemical and/or physical methods
similarily as
production of total cell glycomes, preferably at least use of detergents.
a. Total Cell glycomes
The present invention revealed special methods for effective purification of
released glycans
from total cell derived materials so that free oligosaccharides can be
obtained. In a preferred
embodiment a total glycome is produced from a cell sample, which is degraded
to form more
available for release of glycans. A preferred degraded form of cells is
detergent lysed cells
optionally involving physical distruption of cell materials.
Preferred detergents and reaction conditions include,
al) ionic detergents, preferably SDS type anionic detergent comprising an
anionic group such
as sulfate and an alkyl chain of 8-16 carbon atoms, more preferably the
anionic detergent
comprise 10-14 carbon atoms and it is most preferably sodium dodecyl sulfate
(SDS), and/or
a2) non-ionic detergents such as alkylglycosides comprising a hexose and 4-12
carbon alkyl
chain more preferably the alkyl chain comprises a hexoses being galactose,
glucose, and/or
mannose, more preferably glucose and/or mannose and the alkyl comprises 6-10
carbon
atoms, preferably the non-ionic detergent is octylglucoside .
It is realized that various detergent combinations may be produced and
optimized. The
combined use of an ionic, preferably anionic, and non-ionic detergents
according to the
invention is especially preferred.
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Preferred cell preparation methods for production of Total cell gl cy ome
The preferred methods of cell degration for Total cell glycomes include
physical degration
including at least heat treatment heat and chemical degration by a detergent
method or by a
non-detergent method preferably enzymatic degradation, preferably heat
treatment. Preferably
5 two physical degradation methods are included.
A preferred non-detergent method includes
A non-detergent method is preferred for avoiding detergent in later
purification. The preferred
non-detergent method involves physical degradation of cells preferably
pressure and or by
10 heat and a chemical degradation by protease. A preferred non-detergent
method includes:
i)cell degradation by physical methods, for example by pressure methods such
as by French
press.
The treatment is preferably performed quickly in cold temperatures, preferably
at 0-2 degrees
of Celsius, and more preferably at about 0 or 1 degree of celsius and/or in
the presence of
15 glycosidase inhibitors.
ii) The degraded cells are further treated with chemical degradation,
preferably by effective
general protease, more preferably trypsin is used for the treatment. Preferred
trypsin
preparation according to the invention does not cause glycan contamination to
the sample/
does not contain glycans releasable under the reaction conditions.
20 iii) optionally the physical degradation and chemical degradation are
repeated.
iv) At the end of protease treatment the sample is boiled for further
denaturing the sample and
the protease. The boling is performed at temperature denaturing/degrading
further the sample
and the protease activity (conditions thus depend on the protease used)
preferably about 100
degrees Celsius for time enough for denaturing protease activity preferably
about 10-20
25 minutes for trypsin, more preferably about 15 minutes.
Preferred detergent method for production of total glycomes
The invention is in another preferred embodiment directed to detergent based
method for
lysing cells. The invention includes effective methods for removal of
detergents in later
30 purification steps. The detergent methods are especially preferred for
denaturing proteins,
which may bind or degrade glycans, and for degrading cell membranes to
increase the
accessibility of intracellular glycans.
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For the detergent method the cell sample is preferably a cell pellet produced
at cold
temperature by centrifuging cells but avoiding distruption of the cells,
optionally stored frozed
and melted on ice. Optionally glycosidase inhibitors are used during the
process.
The method includes following steps:
i) production of cell pellet preferably by centrifugation,
ii) lysis by detergent on ice, the detergent is preferably an anionic
detergent according to the
invention, more preferably SDS. The concentration of the detergent is
preferably between
about 0.1 % and 5 %, more preferably between 0.5 %- 3 %, even more preferably
between
0.5- 1.5% and most preferably about 1% and the detergent is SDS (or between
0.9- 1.1%).
the solution is preferably produced in ultrapure water,
iii) mixing by effective degradation of cells, preferably mixing by a Vortex-
mixer as physical
degradation step,
iv) boiling on water bath, preferebly for 3- 10 min, most preferably about 5
min (4-6 min) as
second physical degradation step, it is realized that even longer boiling may
be performed for
example up to 30 min or 15 min, but it is not optimal because of evaporation
sample
v)adding one volume of non-ionic detergent, preferably alkyl-glycoside
detergent according
to the invention, most preferably n-octyl-(3-D-glucoside, the preferred amount
of the detergent
is about 5-15 % as water solution, preferably about 10% of octyl-glucoside.
The non-ionic
detergent is especially preferred in case an enzyme sensitive to SDS, such as
a N-glycosidase,
is to be used in the next reaction step.
and
vi)incubation at room temperature for about 5 min to about 1-4 hours, more
preferably less
than half an hour, and most preferably about 15 min.
Preferred amount of detergents in the detergent method
Preferably the anionic detergent and cationic detergent solutions are used in
equal volumes.
Preferably the solutions are about 1% SDS and about 10 % octyl-glucoside. The
preferred
amounts of the solutions are preferably from 0.1 1 to about 2 1, more
preferably 0.15 1 to
about 1.5 1 per and most preferably from 0.16 1 to 1 1 per 100 000 cells of
each solution.
Lower amounts of the detergents are preferred if possible for reduction of the
amount of
detergent in later purification, highest amounts in relation to the cell
amounts are used for
practical reasons with lowest volumes. It is further realized that
corresponding weight
amounts of the detergents may be used in volumes of about 10% to about 1000%,
or from
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about 20 % to about 500 % and even more effectively in volumes from 30 % to
about 300 %
and most preferably in volumes of range from 50 % to about 150 % of that
described. It is
realized that critical micellar concentration based effects may reduce the
effect of detergents
at lowest concentrations.
In a preferred embodiment a practical methods using tip columns as described
in the invention
uses about 1-3 gl of each detergent solution, more preferably 1.5-2.5 gl, and
most preferably
about 2 g1 of the preferred detergent solutions or corresponding detergent
amounts are used
for about 200 000 or less cells (preferably between 2000 and about 250 000
cells, more
preferably from 50 000 to about 250 000 cells and most preferably from 100 000
to about 200
000 cells). Another practical method uses uses about 2-10 g1 of each detergent
solution, more
preferably 4-8 gl, and most preferably about 5 g1(preferably between 4 and 6
g1 and more
preferably between 4.5 and 5.5 gl) of detergent solutions or corresponding
amount of the
detergents for lysis of cell of a cell amount from about 200 000 - 3 million
cells (preferred
more exact ranges include 200 000- 3.5 million, 200 000 to 3 million and 200
000 to 2,5
million cells), preferably a fixed amount (specific amount of microliters
preferably with the
accuracy of at least 0.1 microliter) in a preferred range such as of 5.0 gl is
used for the wider
range of cells 200 000 - 3 million. It was invented that is possible to handle
similarily wider
range of materials. It is further realized that the method can be optimized so
that exact amount
of detergent, preferably within the ranges described, is used for exact amount
of cells, such
method is preferably an automized when there is possible variation in amounts
of sample
cells.
b. Cell surface glycomes
In another preferred embodiment the invention is directed to release of
glycans from intact
cells and analysis of released cell surface glycomes. The present invention is
directed to
specific buffer and enzymatic cell pre-modification conditions that would
allow the efficient
use of enzymes for release and optionally modification and release of glycans.
B. The glycan release methods
The invention is directed to various enzymatic and chemical methods to release
glycomes.
The release step is not needed for soluble glycomes according to the
invention. The invention
further revealed soluble glycome components which can be isolated from the
cells using
methods according to the invention.
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C. Purification of glycans from cell derived materialsThe purification of
glycome materials
form cell derived molecules is a difficult task. It is especially difficult to
purify glycomes to
obtain picomol or low nanomol samples for glycome profiling by mass
spectrometry or
NMR-spectrometry. The invention is especially directed to production of
material allowing
quantitative analysis over a wide mass range. The invention is specifically
directed to the
purification of non-derivatized or reducing end derivatized glycomes according
to the
invention and glycomes containing specific structural characteristics
according to the
invention. The structural characteristics were evaluated by the preferred
methods according to
the invention to produce reproducible and quantitative purified glycomes.
Glycan purification process steps
The glycan purification method according to the present invention consists of
at least one of
purification options, preferably in specific combinations described below,
including one or
several of following the following purification process steps in varying
order:
6) Precipitation-extraction;
7) Ion-exchange;
8) Hydrophobic interaction;
9) Hydrophilic interaction; and
10) Affinity to carbon materials especially graphitized carbon.
Prepurification and purification process steps
In general the purification steps may be divided to two major categories:
Prepurification steps to remove major contaminations and purification steps
usually directed
to specific binding and optionally fractionation of glycomes
a)Prepurification to remove non-carbohydrate impurities
The need for prepurification depends on the type and amounts of the samples
and the amounts
of impurities present. Certain samples it is possible to omit all or part of
the prepurification
steps. The prepurification steps are aimed for removal of major non-
carbohydrate impurities
by separating the impurity and the glycome fraction(s) to be purified to
different phases by
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precipitation/extraction or binding to chromatography matrix and the
separating the impurities
from the glycome fraction(s).
The prepurification steps include one, two or three of following major steps:
Precipitation-extraction, Ion-exchange, Hydrophobic interaction.
The precipitation and/or extraction is based on the high hydrophilic nature of
glycome
compositions and components, which is useful for separation from different
cellular
components and chemicals. The prepurification ion exchange chromatography is
directed to
removal of classes molecules with different charge than the preferred glycome
or glycome
fraction to be studied. This includes removal of salt ions and aminoacids, and
peptides etc.
The glycome may comprise only negative charges or in more rare case also only
positive
charges and the same charge is selected for the chromatography matrix for
removal of the
impurities for the same charge without binding the glycome at prepurification.
In a preferred embodiment the invention is directed to removal of cationic
impurities from
glycomes glycomes containing neutral and/or negatively charged glycans. The
invention is
further directed to use both anion and cation exchange for removal of charged
impurities from
non-charged glycomes. The preferred ion exchange and cation exhange materials
includes
polystyrene resins such as Dowex resins.
The hydrophilic chromatography is preferably aimed for removal of hydrophobic
materials
such as lipids detergents and hydrophobic protein materials. The preferred
hydrophobic
chromatography materials includes.
It is realized that different combinations of the prepurification are usuful
depending on the
cell preparation and sample type. Preferred combinations of the
prepurification steps include:
Precipitation-extraction and Ion-exchange; Precipitation-extraction and
Hydrophobic
interaction; and Ion-exchange and Hydrophobic interaction. The two
prepurification steps are
preferably performed in the given order.
Purification steps including binding and optionally fractionation of glycomes
The purification steps utilize two major concepts for binding to carbohydrates
and
combinations thereof: a) Hydrophilic interactions and b) Ion exhange
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a) Hydrophilic interactions
The present invention is specifically directed to use of matrices with
repeating polar groups
with affinity for carbohydrates for purification of glycome materials
according to the
invention in processes according ot the invention. The hydrophilic interaction
material may
include additional ion exchange properties.
The preferred hydrophilic interaction materials includes carbohydrate
materials such as
carbohydrate polymers in presence of non-polar organic solvents. A especially
preferred
hydrophilic interaction chromatography matrix is cellulose.
A specific hydrophilic interaction material includes graphitized carbon. The
graphitized
carbon separates non-charged carbohydrate materials based mainly on the size
on the glycan.
There is also possible ion exchange effects. In a preferred embodiment the
invention is
directed to graphitized carbon chromatography of prepurified samples after
desalting and
removal of detergents.
The invention is specifically directed to purification of non-derivatized
glycomes and neutral
glycomes by cellulose chromatography. The invention is further directed to
purification of
non-derivatized glycomes and neutral glycomes by graphitized carbon
chromatography. In a
preferred embodiment the purification according to the invention includes both
cellulose and
graphitized carbon chromatography.
b) Ion exchange
The glycome may comprise only negative charges or in more rare case also only
positive
charges. At purification stage the ion exchange material is selected to
contain opposite charge
than the glycome or glycome fraction for binding the glycome. The invention is
especially
directed to the use of anion exchange materials for binding of negatively
charged Preferred
ion exchange materials includes ion exchange and especially anion exhange
materials
includes polystyrene resins such as Dowex-resins , preferably quatemary amine
resins anion
exchange or sulfonic acid cation exchange resins
It was further revealed that even graphitized carbon can be used for binding
of negatively
charged glycomes and the materials can be eluted from the carbon separately
from the neutral
glycomes or glycome fractions according to the invention.
The invention is specifically directed to purification of anionic glycomes by
anion exchange
chromatography.
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The invention is specifically directed to purification of anionic glycomes by
anion exchange
chromatography.
The invention is further directed to purification of anionic glycomes by
cellulose
chromatography. The preferred anionic glycomes comprise sialic acid and/or
sulfo/fosfo
esters, more preferably both sialic acid and sulfo/fosfo esters. A preferred
class of
sulfo/fosfoester glycomes are complex type N-glycans comprising sulfate
esters.
Prepurification and purification steps in detail
1) Precipitation-extraction may include precipitation of glycans or
precipitation of
contaminants away from the glycans. Preferred precipitation methods include:
1. Glycan material precipitation, for example acetone precipitation of
glycoproteins,
oligosaccharides, glycopeptides, and glycans in aqueous acetone,
preferentially ice-
cold over 80 % (v/v) aqueous acetone; optionally combined with extraction of
glycans
from the precipitate, and/or extraction of contaminating materials from the
precipitate;
2. Protein precipitation, for example by organic solvents or trichloroacetic
acid,
optionally combined with extraction of glycans from the precipitate, and/or
extraction
of contaminating materials from the precipitate;
3. Precipitation of contaminating materials, for example precipitation with
trichloroacetic
acid or organic solvents such as aqueous methanol, preferentially about 2/3
aqueous
methanol for selective precipitation of proteins and other non-soluble
materials while
leaving glycans in solution;
2) Ion-exchange may include ion-exchange purification or enrichment of glycans
or removal
of contaminants away from the glycans. Preferred ion-exchange methods include:
1. Cation exchange, preferably for removal of contaminants such as salts,
polypeptides,
or other cationizable molecules from the glycans; and
2. Anion exchange, preferably either for enrichment of acidic glycans such as
sialylated
glycans or removal of charged contaminants from neutral glycans, and also
preferably
for separation of acidic and neutral glycans into different fractions.
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3) Hydrophilic interaction may include purification or enrichment of glycans
due to their
hydrophilicity or specific adsorption to hydrophilic materials, or removal of
contaminants
such as salts away from the glycans. Preferred hydrophilic interaction methods
include:
1. Hydrophilic interaction chromatography with specific organic or inorganic
polar
interaction materials, preferably for purification or enrichment of glycans
and/or
glycopeptides;
2. Preferably adsorption of glycans to carbohydrate materials, preferably to
cellulose in
hydrophobic solvents for their purification or enrichment, preferably to
microcrystalline cellulose, and elution by polar solvents such as water and or
alchol,
which is preferably ethanol or methanol. The solvent system for absorption
comprise
preferably
i) a hydrophobic alcohol comprising about three to five carbon atoms,
including
propanols, butanols, and pentanols, more preferably being n-butanol;
ii) a hydrophilic alcohol such as methanol or ethanol, more preferably
methanol, or a
hydrophilic weak organic acid, preferably acetic acid and;
iii) water. The hydrophobic alcohol being the major constituent of the mixture
with
multifold exess to other components. The absorbtion composition is preferably
using an n-
butanol:methanol:water or similar solvent system for adsorption and washing
the adsorbed
glycans, in most preferred system n-butanol:methanol:water in relative volumes
of 10:1:2.
The preferred polar solvents for elution of the glycomes are water or
water:ethanol or
similar solvent system for elution of purified glycans from cellulose.
Fractionation is
possible by using first less polar elution solvent to elute a fraction of
glycome
compositions and the eluting rest by more polar solvent such as water
3.Affinity to carbon may include purification or enrichment of glycans due to
their affinity or
specific adsorption to specific carbon materials preferably graphitized
carbon, or removal of
contaminants away from the glycans. Preferred graphitized carbon affinity
methods includes
porous graphitized carbon chromatography.
Preferred purification methods according to the invention include combinations
of one or
more prepurification and/or purification steps. The preferred method include
preferably at
least two and more preferably at least three prepurification steps according
to the invention.
The preferred method include preferably at least one and more preferably at
least two
purification steps according to the invention. It is further realized that one
prepurification step
may be performed after a purification step or one purification step may be
performed after a
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prepurification step. The method is preferably adjusted based on the amount of
sample and
impurities present in samples. Examples of the preferred combinations include
the following
combinations:
For neutral underivatized glycan purification:
A. 1. precipitation and/or extraction 2. cation exchange of contaminants, 3.
hydrophobic
adsorption of contaminants, and 4. hydrophilic purification, preferably by
carbon, preferably
graphitized carbon, and/or carbohydrate affinity purification of glycans.
B. 1. precipitation and/or extraction ,2. hydrophobic adsorption of
contaminants 3. cation
exchange of contaminants, 4. hydrophilic purification by carbon, preferably
graphitized
carbon, and/or carbohydrate affinity purification of glycans
The preferred method variants further includes preferred variants when
1. both carbon and carbohydrate chromatography is performed in step 4,
2. only carbon chromatography is performed in step 4
3. only carbohydrate chromatography is performed in step 4
4. order steps three and four is exchanged
5. both precipitation and extraction are performed in prepurification step
Further preferred method variants include the preceding methods A. or B. when:
6. step 1 is omitted
7. step 2 is omitted
8. steps 1 and 2 are omitted
2) For sialylated/acidic underivatized glycan purification: The same methods
are preferred but
preferably both carbon and carbohydrate chromatography is performed in step 4.
The
carbohydrate affinity chromatography is especially preferred for acidic
and/sialylated glycans.
In a preferred embodiment for additional purification one or two last
chromatograpy methods
are repeated.
In a further preferred method for sialylated underivatized glycan
purification, glycan sample
containing sialylated glycans is derivatized at the sialic acid carboxylic
groups to produce
neutral sialylated glycans as described in the present invention. Thereafter
the neutral glycan
sample containing neutral sialylated glycans is efficiently purified like
neutral glycans
described above.
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D. Analysis of the glycomes
The present invention is specifically directed to detection various component
in glycomes by
specific methods for recognition of such components. The methods includes
binding of the
glycome components by specific binding agents according to the invention such
as antibodies
and/or enzymes, these methods peferebly include labeling or immobilization of
the glycomes.
For effective analysis of the glycome a large panel of the binding agents are
needed.
The invention is specifically directed to physicochemical profiling methods
for exact analysis
of glycomes. The preferred methods includes mass spectrometry and NMR-
spectroscopy,
which give simultaneously information of numerous components of the glycomes.
In a
preferred embodiment the mass spectrometryand NMR-spectroscopy methods are
used in a
combination.
E. Quantitative and qualitative analysis of glycome data
The invention revealed methods to create reproducible and quantitative
profiles of glycomes
over large mass ranges and degrees of polymerization of glycans. The invention
further
reveals novel methods for quantitative analysis of the glycomics data produced
by mass
spectrometry. The invention is specifically directed to the analysis of non-
derivatized or
reducing end derivatized glycomes according to the invention and the glycomes
containing
specific structureal characteristics according ot the invention.
The invention revealed effective means of comparision of glycome profiles from
different cell
types or cells with difference in cell status or cell types. The invention is
especially directed to
the quantitative comparision of relative amount of individual glycan signal or
groups of
glycan signals described by the invention.
The invention is especially directed to
i)calculating average value and variance values of signal or signals, which
have obtained from
several experiments/samples and which correspond to an individual glycan or
glycan group
according to the invention for a first cell sample and for a second cell
sample
ii)comparing these with values derived for the corresponding signal(s)
iii) optionally calculating statistic value for testing the probability of
similarity of difference
of the values obtained for the cell types or
estimating the similarity or difference based on the difference of the average
and optionally
also based on the variance values.
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iv) preferably repeating the comparision one or more signals or signal groups,
and further
preferably performing combined statistical analysis to estimate the similarity
and/or
differences between the data set or estimating the difference or similarity
v) preferably use of the data for estimating the differences between the first
and second cell
samples indicationg difference in cell status and/or cell type
The invention is further directed to combining information of several
quantitative
comparisions of between corresponding signals by method of
i)calculating differences between quantitative values of corresponding most
different glycan
signals or glycan group signals, changing negative values to corresponding
positive values,
optionally multiplying selected signals by selected factors to adjust the
weight of the signals
in the calculation
ii) adding the positive difference values to a sum value
iii) comparing the sum values as indicators of cell status or type.
It was further revealed that there is characteric signals that are present in
certain cell types
according to the invention but absent or practically absent in other cell
types. The invention is
therefore directed to the qualitative comparision of relative amount of
individual glycan signal
or groups of glycan signals described by the invention and observing signals
present or
absent/practically absent in a cell type. The invention is further directed to
selection of a cut
off value used for selecting absent or practically absent signals from a mass
spectrometric
data, for example the preferred cut off value may be selected in range of 0-3
% of relative
amount, preferably the cut off value is less than 2 %, or less than 1% or less
than 0.5 %. In a
preferred embodiment the cut off value is adjusted or defined based on quality
of the mass
spectrum obtained, preferably based on the signal intensities and/or based on
the number of
signals observable.
The invention is furher directed to automized qualitative and/or quantitative
comparisions of
data from corresponding signals from different samples by computer and
computer programs
prosessing glycome data produced according to the invention. The invention is
further
directed to raw data based analysis and neural network based Iearning system
analysis as
methods for revealing differences between the glycome data according to the
invention.
Identification and classification of differences in glycan datasets
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The present invention is specifically directed to analyzing glycan datasets
and glycan profiles
for comparison and characterization of different tissue materials. In one
embodiment of the
invention, glycan signals or signal groups associated with given tissue
material are selected
from the whole glycan datasets or profiles and indifferent glycan signals are
removed. The
resulting selected signal groups have reduced background and less observation
points, but the
glycan signals most important to the resolving power are included in the
selection. Such
selected signal groups and their patterns in different sample types serve as a
signature for the
identification of the cell type and/or glycan types or biosynthetic groups
that are typical to it.
By evaluating multiple samples from the same tissue material, glycan signals
that have
individual i.e. cell line specific variation can be excluded from the
selection. Moreover,
glycan signals can be identified that do not differ between tissue materials,
including major
glycans that can be considered as housekeeping glycans.
To systematically analyze the data and to find the major glycan signals
associated with given
tissue material according to the invention, difference-indicating variables
can be calculated
for the comparison of glycan signals in the glycan datasets. Preferential
variables between two
samples include variables for absolute and relative difference of given glycan
signal between
the datasets from two tissue materials. Most preferential variables according
to the invention
are:
1. absolute difference A = (S2 - Sl), and
2. relative difference R = A / Sl,
wherein Sl and S2 are relative abundances of a given glycan signal in cell
types 1 and 2,
respectively.
It is realized that other mathematical solutions exist to express the idea of
absolute and
relative difference between glycan datasets, and the above equations do not
limit the scope of
the present invention. According to the present invention, after A and R are
calculated for the
glycan profile datasets of the two tissue materials, the glycan signals are
thereafter sorted
according to the values of A and R to identify the most significant differing
glycan signals.
High value of A or R indicates association with tissue material 2, and vice
versa. In the list of
glycan data sorted independently by R and A, the tissue material specific
glycans occur at the
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top and the bottom of the lists. More preferentially, if a given signal has
high values of both A
and R, it is more significant.
Preferred representation of the dataset when comparing two tissue materials
The present invention is specifically directed to the comparative presentation
of the
quantitative glycome dataset as multidimensional graphs comparing the paraller
data or as
other three dimensional presentations or for example as two dimensional matrix
showing the
quantities with a quantitative code, preferably by a quantitative color code.
Methods for low sample amounts
The present invention is specifically directed to methods for analysis of low
amounts of
samples.
The invention further revealed that it is possible to use the methods
according to the invention
for analysis of low sample amounts. It is realized that the cell materials are
scarce and
difficult to obtain from natural sources. The effective analysis methods would
spare important
cell materials. Under certain circumstances such as in context of cell culture
the materials may
be available from large scale. The required sample scale depends on the
relative abundancy of
the characteristic components of glycome in comparision to total amount of
carbohydrates. It
is further realized that the amount of glycans to be measured depend on the
size and glycan
content of the cell type to be measured and analysis including multiple
enzymatic digestions
of the samples would likely require more material. The present invention
revealed especially
effective methods for free released glycans.
The picoscale samples comprise preferably at least about 1000 cells, more
preferably at least
about 50 000 cells, even more more preferably at least 100 000 cells, and most
preferably at
least about 500 000 cells. The invention is further directed to analysis of
about 1 000 000
cells. The preferred picoscale samples contain from at least about 1000 cells
to about 10 000
000 cells according to the invention. The useful range of amounts of cells is
between 50 000
and 5 000 000, even more preferred range of of cells is between 100 000 and 3
000 000 cells.
A preferred practical range for free oligosaccharide glycoomes is between
about 500 000 and
about 2 000 000 cells. It is realized that cell counting may have variation of
less than 20 %,
more preferably 10 % and most preferably 5 %, depending on cell counting
methods and cell
sample, these variations may be used instead of term about. It is further
understood that the
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methods according to the present invention can be upscaled to much larger
amounts of
material and the pico/nanoscale analysis is a specific application of the
technology.
The invention is specifically directed to use of microcolumn technologies
according to the
invention for the analysis of the preferred picoscale and low amount samples
according to the
invention,
The invention is specifically directed to purification to level, which would
allow production
of high quality mass spectrum covering the broad size range of glycans of
glycome
compositions according to the invention.
GI,~~preparation and purification for glycome analysis of cell materials
according to the
invention, especially for mass spectrometric methods
Use of microfluidistic methods including microcolumn chromatography
The present invention is especially directed to use microfluidistic methods
involving low
sample volumes in handling of the glycomes in low volume cell preparation, low
volume
glycan release and various chromatographic steps. The invention is further
directed to
integrated cell preparation, glycan release, and purification and analysis
steps to reduce loss of
material and material based contaminations. It is further realized that
special cleaning of
materials is required for optimal results.
Low volume reaction in cell preparation and glycan release
The invention is directed to reactions of volume of 1-100 microliters,
preferably about 2-50
microliters and even more preferably 3-20 microliters, most preferably 4-10
microliter. The
most preferred reaction volumes includes 5-8 microliters+/- 1 microliters. The
minimum
volumes are preferred to get optimally concentrated sample for purification.
The amount of
material depend on number of experiment in analysis and larger amounts may be
produced
preferably when multiple structural analysis experiments are needed.
It is realized that numerous low volume chromatographic technologies may be
applied, such
low volume column and for example disc based microfluidistic systems.
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The inventors found that the most effective methods are microcolumns. Small
colomn can be
produced with desired volume. Preferred volumes of microcolumns are from about
2
Microliters to about 500 microliters, more preferably for rutine sample sizes
from about 5
microliter to about 100 microliters depending on the matrix and size of the
sample.
Preferred microcolumn volumes for graphitised carbon, cellulose chromatography
and other
tip-columns are from 2 to 20 gl, more preferably from 3 to 15 g1, even more
preferably from
4 to 10 g1, For the microcolumn technologies in general the samples are from
about 10 000
to about million cells. The methods are useful for production of picomol
amounts of total
glycome mixtures from cells according to the invention.
In a preferred embodiment microcolumns are produced in regular disposable
usually plastic
pipette tips used for example in regular "Finnpipette"-type air-piston
pipettes. The pipette-tip
microcolumn contain the preferred chromatographic matrix. In a preferred
embodiment the
microcolumn contains two chromatographic matrixes such as an anion and cation
exchange
matrix or a hydrophilic and hydrophobic chromatography matrix.
The pipette tips may be chosen to be a commercial tip contain a filter. In a
preferred
embodiment the microcolumn is produced by narrowing a thin tip from lower half
so that the
preferred matrix is retained in the tip. The narrowed tip is useful as the
volume of filter can be
omitted from washing steps
The invention is especially directed to plastic pipette tips containing the
cellulose matrix, and
in an other embodiment to the pipette tip microclumns when the matrix is
graphitised carbon
matrix. The invention is further directed to the preferred tip columns when
the columns are
narorrowed tips, more preferably with column volumes of 1 microliter to 100
microliters.
The invention is further directed to the use of the tip columns containing any
of the preferred
chromatographic matrixes according to the invention for the purification of
glycomes
according to the invention, more preferably matrixes for ion exchange,
especially polystyrene
anion exchangers and cation exchangers according to the invention; hydrophilic
chromatographic matrixes according to the invention, especially carbohydrate
matrixes and
most cellulose matrixes.
Preferred combinations of glycan purification and analysis methods
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The present invention is especially directed to method for glycan
purification. The
purification consists of the steps of 1) contamination removal including
hybrophobic affinity
absorption of contaminants and 2) glycan isolation glycans including
hydrophilic affinity
chromatography.
Preferably step 1) includes also cation-exchange step. More preferably, cation-
exchange and
hydrophobic chromatography media are used sequentially, and even more
preferably they are
packed together in a single column.
Preferably the hydrophilic affinity step in step 2) is carbon affinity, more
preferably
graphitized carbon affinity.
The inventors realized that improved purification of small sample amounts
and/or purification
of glycans from complex biological matrices are especially improved using both
miniaturized
liquid and miniaturized chromatography media volumes as well as connecting the
purification
steps directly into series. This is further exemplified in the Examples of the
invention,
including Examples 20 and 21.
The present invention is especially directed to analysis of small glycan
amounts
corresponding to glycans of 1000 cells - 1 million cells, with steps 1) and 2)
preferentially
consisting of a total of 0.1 l - 1 ml bed volume chromatography media each;
total liquid
volume in sample loading into step 1) of 0.2 l - 0.5 ml; and total liquid
volume in sample
eluting from step 2) of 0.2 l - 2.5 ml.
In a more preferred embodiment, the present invention is directed to analysis
of small glycan
amounts corresponding to glycans of 1000 - 200 000 cells, with steps 1) and 2)
preferentially
consisting of a total of 0.1 - 5 l bed volume chromatography media each;
total liquid volume
in sample loading into step 1) of 0.2 - 5 jil; and total liquid volume in
sample eluting from
step 2) of 0.2 l - 20 l.
In a further preferred embodiment, the present invention is directed to on-
line analytical
method of small glycan amounts corresponding to glycans of 1000 - 10 000
cells, with steps
1) and 2) preferentially consisting of a total of 0.1 - 0.5 111 bed volume
chromatography media
each; total liquid volume in sample loading into step 1) of 0.2 - 1 l; and
total liquid volume
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in sample eluting from step 2) of 0.2 1- 1 1; with the sample directly
eluted to the analytical
method.
Preferably, the analysis method is mass spectrometry, more preferably MALDI-
TOF mass
spectrometry.
In a further embodiment of the invention, the steps 1) and 2) are connected by
eluting the
sample directly from step 1) into step 2).
In a further embodiment of the invention, acidic glycans are further purified
after steps 1) and
2) by another hydrophilic chromatography step as described in the present
invention,
preferably using cellulose adsorption.
The inventors found that analysis sensistivity of glycan profiles and signal
detection can be
improved by data analysis, preferentially using quantitative analysis of
glycan signal profile
datasets. In another embodiment of the present invention, glycan analysis is
combined with
quantitative data analysis according to the present invention, preferably with
correction and
normalization of data according to the invention.
Glycan purification device and/or apparatus
The inventors were able to demonstrate efficient purification method for
glycans, essentially
forming a device for glycan purification from samples with varying volumes and
matrices.
Further, the inventors were able to standardize such purification method to
essentially form a
programmed method of using such device. The present invention is specifically
directed to a
device for glycan purification, the device consisting of:
a) contamination removing cartridge
b) glycan isolation cartridge
c) sample inlet going through a) and b)
d) washing and elution inlet going through b)
e) outlet leading from b) to either waste, sample collection, or analysis;
and optionally the device further consists of one or more of the following:
f) switch for changing inlet between c) and d)
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g) switch for changing outlet between waste, sample collection, or analysis
h) device for generating liquid flow to operate the abovementioned device
i) switch for changing inlet between sample, washing, and elution liquids.
Optionally, a) and b) are operated independently and c) only transiently
connects a) and b).
Optionally, the glycan purification device is partly or fully automated and
operated by a
programmed liquid handling device and/or a computer.
The device is operated by liquid flow through the device, optionally using h),
changing the
composition of the liquid flowing through the device, optionally using i), and
changing the
inlet and outlet destinations, optionally using f) and/or g), respectively.
The operation is done
in the following order:
1) liquid containing glycan sample goes to c) and outlet e) goes to waste
2) washing liquid goes to d) and outlet e) goes to waste
3) elution liquid goes to d) and outlet e) goes to either sample collection or
analysis.
Preferentially, a) incorporates one or more chromatography media useful for
contamination
removal according to the present invention, and b) incorporates one or more
chromatography
media useful for glycan absorption or adsorption according to the present
invention; they are
used according to the glycan purification methods described in the present
invention. More
preferentially, media in a) are selected from cation exchange resin and/or
hydrophobic affinity
resin, and media in b) are selected from hydrophilic affinity chromatography
media according
to the invention, more preferentially from graphitized carbon, hydrophilic
affinity resin,
and/or cellulose. In a further preferred embodiment of the present invention,
a) contains cation
exchange and hydrophobic affinity resin and b) contains graphitized carbon.
Preferentially the liquid, inlet, outlet, sample, washing, elution,
chromatography media,
cartridge, and sample collection volumes are minimized as described in the
present invention,
more preferentially using microfluidistics according to the present invention.
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The binding methods for recognition of structures from cell surfaces
Recognition of structures from glycome materials and on cell surfaces by
binding
methods
The present invention revealed that beside the physicochemical analysis by NMR
and/or mass
spectrometry several methods are useful for the analysis of the structures.
The invention is
especially directed to two methods:
i) Recognition by enzymes involvingbinding and alteration of structures.
This method alters specific glycan structures by enzymes cabable of altering
the glycan
structures. The preferred enzymes includes
a) glycosidase-type enzymes capable of releasing monosaccharide units from
glycans
b) glycosyltransferring enzymes, including transglycosylating enzymes and
glycosyltransferases
c) glycan modifying enzymes including sulfate and or fosfate modifying
enzymes
ii) Recognition by molecules binding glycans referred as the binders
These molecules bind glycans and include property allowing observation of the
binding
such as a label linked to the binder. The preferred binders include
a) Proteins such as antibodies, lectins and enzymes
b) Peptides such as binding domains and sites of proteins, and synthetic
library
derived analogs such as phage display peptides
c) Other polymers or organic scaffold molecules mimicking the peptide
materials
The peptides and proteins are preferably recombinant proteins or corresponding
carbohydrate
recognition domains derived therereof, when the proteins are selected from the
group
monoclonal antibody, glycosidase, glycosyl transferring enzyme, plant lectin,
animal lectin or
a peptide mimetic thereof, and wherein the binder includes a detectable label
structure..
Preferred binder molecules
The present invention revealed various types of binder molecules useful for
characterization
of cells according to the invention and more specifically the preferred cell
groups and cell
types according to the invention. The preferred binder molecules are
classified based on the
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binding specificity with regard to specific structures or structural features
on carbohydrates of
cell surface. The preferred binders recognize specifically more than single
monosaccharide
residue.
It is realized that most of the current binder molecules such as all or most
of the plant lectins
are not optimal in their specificity and usually recognize roughly one or
several
monosaccharides with various linkages. Furthermore the specificities of the
lectins are usually
not well characterized with several glycans of human types.
The preferred high specificity binders recognize
A) at least one monosaccharide residue and a specific bond structure between
those to
another monosaccharides next monosaccharide residue referred as MS1B1-binder,
B) more preferably recognizing at least part of the second monosaccharide
residue
referred as MS2B 1-binder,
C) even more preferably recognizing second bond structure and or at least part
of third
mono saccharide residue, referred as MS3B2-binder, preferably the MS3B2
recognizes a specific complete trisaccharide structure.
D) most preferably the binding structure recognizes at least partially a
tetrasaccharide
with three bond structures, referred as MS4B3-binder, preferably the binder
recognizes complete tetrasaccharide sequences.
The preferred binders includes natural human and or animal, or other proteins
developed for
specific recognition of glycans. The preferred high specificity binder
proteins are specific
antibodies preferably monoclonal antibodies; lectins, preferably mammalian or
animal lectins;
or specific glycosyltransferring enzymes more preferably glycosidase type
enzymes,
glycosyltransferases or transglycosylating enzymes.
Target structures for specific binders and examples of the binding molecules
Combination of terminal structures in combination with specifi'c Qlycan core
structures
It is realized that part of the structural elements are specifically
associated with specific
glycan core structure. The recognition of terminal structures linked to
specific core structures
are especially preferred, such high specificity reagents have capacity of
recognition almost
complete individual glycans to the level of physicochemical characterization
according to the
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invention. For example many specific mannose structures according to the
invention are in
general quite characteristic for N-glycan glycomes according to the invention.
The present
invention is especially directed to recognition terminal epitopes.
Common terminal structures on several Qlycan core structures
The present invention revealed that there are certain common structural
features on several
glycan types and that it is possible to recognize certain common epitopes on
different glycan
structures by specific reagents when specificity of the reagent is limited to
the terminal
without specificity for the core structure. The invention especially revealed
characteristic
terminal features for specific cell types according to the invention. The
invention realized that
the common epitopes increase the effect of the recognition. The common
terminal structures
are especially useful for recognition in the context with possible other cell
types or material,
which do not contain the common terminal structure in substancial amount.
Specifac preferred structural Qroups
The present invention is directed to recognition of oligosaccharide sequences
comprising
specific terminal monosaccharide types, optionally further including a
specific core structure.
The preferred oligosaccharide sequences classified based on the terminal
monosaccharide
structures.
1. Structures with terminal Mannose monosaccharide
Preferred mannose-type target structures have been specifically classified by
the invention.
These include various types of high and low-mannose structures and hybrid type
structures
according to the invention.
Low or uncharacterised specificity binders
preferred for recognition of terminal mannose structures includes mannose-
monosaccharide
binding plant lectins.
Preferred high specific high specificity binders
include
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i) Specific mannose residue releasing enzymes such as linkage specific
mannosidases, more
preferably an a-mannosidase or (3-mannosidase.
Preferred a-mannosidases includes linkage specific a-mannosidases such as a-
Mannosidases
cleaving preferably non-reducing end terminal
a2-linked mannose residues specifically or more effectively than other
linkages, more
preferably cleaving specifically Mana2-structures; or
a6-linked mannose residues specifically or more effectively than other
linkages, more
preferably cleaving specifically Man(x6-structures;
Preferred (3-mannosidases includes (3-mannosidases capable of cleaving (34-
linked mannose
from non-reducing end terminal of N-glycan core Man(34G1cNAc-structure without
cleaving
other (3-linked monosaccharides in the glycomes.
ii) Specific binding proteins recognizing preferred mannose structures
according to the
invention. The preferred reagents include antibodies and binding domains of
antibodies (Fab-
fragments and like), and other engineered carbohydrate binding proteins. The
invention is
directed to antibodies recognizing MS2B1 and more preferably MS3B2-structures
2. Structures with terminal Gal- monosaccharide
Preferred galactose-type target structures have been specifically classified
by the invention.
These include various types of N-acetyllactosamine structures according to the
invention.
Low or uncharacterised specificity binders for terminal Gal
Prereferred for recognition of terminal galactose structures includes plant
lectins such as ricin
lectin (ricinus communis agglutinin RCA), and peanut lectin(/agglutinin PNA).
Preferred high specific hiQh specificity binders include
i) Specific galactose residue releasing enzymes such as linkage specific
galactosidases, more
preferably a-galactosidase or(3-galactosidase.
Preferred a-galactosidases include linkage galactosidases capable of cleaving
Gala3Ga1-
structures revealed from specific cell preparations
Preferred (3-galactosidases includes (3- galactosidases capable of cleaving
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(34-linked galactose from non-reducing end terminal Gal(34G1cNAc-structure
without cleaving
other (3-linked monosaccharides in the glycomes and
(33-linked galactose from non-reducing end terminal Gal(33G1cNAc-structure
without cleaving
other (3-linked monosaccharides in the glycomes
ii)Specific binding proteins recognizing preferred galactose structures
according to the
invention. The preferred reagents include antibodies and binding domains of
antibodies (Fab-
fragments and like), and other engineered carbohydrate binding proteins and
animal lectins
such as galectins.
3. Structures with terminal GaINAc- monosaccharide
Preferred Ga1NAc-type target structures have been specifically revealed by the
invention.
These include especially LacdiNAc, Ga1NAc(3G1cNAc-type structures according to
the
invention.
Low or uncharacterised specificity binders for terminal GaINAc
Several plant lectins has been reported for recognition of terminal Ga1NAc. It
is realized that
some Ga1NAc-recognizing lectins may be selected for low specificity reconition
of the
preferred LacdiNAc-structures.
Preferred hiQh specific hiQh specificity binders include
i) The invention revealed that (3-linked Ga1NAc can be recognized by specific
(3-N-
acetylhexosaminidase enzyme in combination with (3-N-acetylhexosaminidase
enzyme.
This combination indicates the terminal monosaccharide and at least part of
the linkage
structure.
Preferred (3-N-acetylehexosaminidase, includes enzyme capable of cleaving (3-
linked Ga1NAc
from non-reducing end terminal Ga1NAc(34/3-structures without cleaving (x-
linked HexNAc
in the glycomes; preferred N-acetylglucosaminidases include enzyme capable of
cleaving (3-
linked G1cNAc but not Ga1NAc.
ii) Specific binding proteins recognizing preferred Ga1NAc(34, more preferably
Ga1NAc(34G1cNAc, structures according to the invention. The preferred reagents
include
antibodies and binding domains of antibodies (Fab-fragments and like), and
other engineered
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carbohydrate binding proteins, and a special plant lectin WFA (Wisteria
floribunda
agglutinin).
4. Structures with terminal GIcNAc- monosaccharide
Preferred G1cNAc-type target structures have been specifically revealed by the
invention.
These include especially G1cNAc(3-type structures according to the invention.
Low or uncharacterised specificity binders for terminal GIcNAc
Several plant lectins has been reported for recognition of terminal G1cNAc. It
is realized that
some G1cNAc-recognizing lectins may be selected for low specificity reconition
of the
preferred G1cNAc-structures.
Preferred high specific high specificity binders include
i) The invention revealed that (3-linked G1cNAc can be recognized by specific
(3-
N-acetylglucosaminidase enzyme.
Preferred (3-N-acetylglucosaminidase includes enzyme capable of cleaving (3-
linked G1cNAc
from non-reducing end terminal G1cNAc(32/3/6-structures without cleaving (3-
linked Ga1NAc
or (Ainked HexNAc in the glycomes;
ii) Specific binding proteins recognizing preferred G1cNAc(32/3/6, more
preferably
G1cNAc(32Mana, structures according to the invention. The preferred reagents
include
antibodies and binding domains of antibodies (Fab-fragments and like), and
other engineered
carbohydrate binding proteins.
5. Structures with terminal Fucose- monosaccharide
Preferred fucose-type target structures have been specifically classified by
the invention.
These include various types of N-acetyllactosamine structures according to the
invention.
Low or uncharacterised specificity binders for terminal Fuc
Prereferred for recognition of terminal fucose structures includes fucose
monosaccharide
binding plant lectins. Lectins of Ulex europeaus and Lotus tetragonolobus has
been reported
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to recognize for example terminal Fucoses with some specificity binding for a2-
linked
structures, and branching a3-fucose, respectively.
Preferred high specific high specificity binders include
i) Specific fucose residue releasing enzymes such as linkage fucosidases, more
preferably a-
fucosidase.
Preferred a-fucosidases include linkage fucosidases capable of cleaving
Fuca2Ga1-, and
Ga1(34/3(Fuc(x3/4)G1cNAc-structures revealed from specific cell preparations.
ii)Specific binding proteins recognizing preferred fucose structures according
to the invention.
The preferred reagents include antibodies and binding domains of antibodies
(Fab-fragments
and like), and other engineered carbohydrate binding proteins and animal
lectins such as
selectins recognizing especially Lewis type structures such as Lewis x,
Ga1(34(Fuc(x3)G1cNAc, and sialyl-Lewis x, SAa3Ga1(34(Fuca3)G1cNAc.
The preferred antibodies includes antibodies recognizing specifically Lewis
type structures
such as Lewis x, and sialyl-Lewis x. More preferably the Lewis x-antibody is
not classic
SSEA-1 antibody, but the antibody recognizes specific protein linked Lewis x
structures such
as Ga1(34(Fuc(x3)G1cNAc(32Man(x-linked to N-glycan core.
6. Structures with terminal Sialic acid- monosaccharide
Preferred sialic acid-type target structures have been specifically classified
by the invention.
Low or uncharacterised specificity binders for terminal Fuc
Preferred for recognition of terminal sialic acid structures includes sialic
acid monosaccharide
binding plant lectins.
Preferred high specific high specificity binders include
i) Specific sialic acid residue releasing enzymes such as linkage sialidases,
more preferably a-
sialidases.
Preferred a-sialidases include linkage sialidases capable of cleaving SAa3Ga1-
and SAa6Ga1
-structures revealed from specific cell preparations by the invention.
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Preferred lectins, with linkage specificity include the lectins, that are
specific for SAa3Ga1-
structures, preferably being Maackia amurensis lectin and/or lectins specific
for SA(x6Ga1-
structures, preferably being Sambucus nigra agglutinin.
ii)Specific binding proteins recognizing preferred sialic acid oligosaccharide
sequence
structures according to the invention. The preferred reagents include
antibodies and binding
domains of antibodies (Fab-fragments and like), and other engineered
carbohydrate binding
proteins and animal lectins such as selectins recognizing especially Lewis
type structures such
as sialyl-Lewis x, SAa3Ga1(34(Fuc(x3)G1cNAc or sialic acid recognizing Siglec-
proteins.
The preferred antibodies includes antibodies recognizing specifically sialyl-N-
acetyllactosamines, and sialyl-Lewis x.
Preferred antibodies for NeuGc-structures includes antibodies recognizes a
structure
NeuGca3Ga1(34Glc(NAc)o or 1 and/or Ga1NAc(34[NeuGca3]Gal(34G1c(NAc)o or 1,
wherein []
indicates branch in the structure and Oo or 1 a structure being either present
or absent. In a
preferred embodiment the invention is directed recognition of the N-glycolyl-
Neuraminic acid
structures by antibody, preferably by a monoclonal antibody or human/humanized
monoclonal antibody. A preferred antibody contains the variable domains of P3-
antibody.
Binder-label conjugates
The present invention is specifically directed to the binding of the
structures according to the
present invention, when the binder is conjugated with "a label structure". The
label structure
means a molecule observable in a assay such as for example a fluorescent
molecule, a
radioactive molecule, a detectable enzyme such as horse radish peroxidase or
biotin/streptavidin/avidin. When the labelled binding molecule is contacted
with the cells
according to the invention, the cells can be monitored, observed and/or sorted
based on the
presence of the label on the cell surface. Monitoring and observation may
occur by regular
methods for observing labels such as fluorescence measuring devices,
microscopes,
scintillation counters and other devices for measuring radioactivity.
Use of binder and labelled binder-conjugates for cell sorting
The invention is specifically directed to use of the binders and their
labelled cojugates for
sorting or selecting cells from biological materials or samples including cell
materials
comprising other cell types. The preferred cell types includes cultivated
cells and associated
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cells such as feeder cells. The labels can be used for sorting cell types
according to invention
from other similar cells. In another embodiment the cells are sorted from
different cell types
such as blood cells or in context of cultured cells preferably feeder cells,
for example in
context of complex cell cultures corresponding feeder cells such as human or
mouse feeder
cells. A preferred cell sorting method is FACS sorting. Another sorting
methods utilized
immobilized binder structures and removal of unbound cells for separation of
bound and
unbound cells.
Use of immobilized binder structures
In a preferred embodiment the binder structure is conjugated to a solid phase.
The cells are
contacted with the solid phase, and part of the material is bound to surface.
This method may
be used to separation of cells and analysis of cell surface structures, or
study cell biological
changes of cells due to immobilization. In the analytics involving method the
cells are
preferably tagged with or labelled with a reagent for the detection of the
cells bound to the
solid phase through a binder structure on the solid phase. The methods
preferably further
include one or more steps of washing to remove unbound cells.
Preferred solid phases include cell suitable plastic materials used in
contacting cells such as
cell cultivation bottles, petri dishes and microtiter wells; fermentor surface
materials
Specific recognition between preferred tissue materials and contaminating
materials
The invention is further directed to methods of recognizing different tissue
materials,
preferably human tissues and more preferably human excretions or serum. It is
further
realized, that the present reagents can be used for purification of tissue
materials by any
fractionation method using the specific binding reagents.
Preferred fractionation methods includes fluorecense activated cell sorting
(FACS), affmity
chromatography methods, and bead methods such as magnetic bead methods.
The invention is further directed to positive selection methods including
specific binding to
the tissue material but not to contaminating tissue materials. The invention
is further directed
to target selection methods including specific binding to the contaminating
tissue material but
not to the target tissue materials. In yet another embodiment of recognition
of tissue materials
the tissue material is recognized together with a homogenous reference sample,
preferably
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when separation of other materials is needed. It is realized that a reagent
for positive selection
can be selected so that it binds tissue materials as in the present invention
and not to the
contaminating tissue materials and a reagent for negative selection by
selecting opposite
specificity. In case of tissue material type according to the invention is to
be selected amongst
novel tissue materials not studied in the present invention, the binding
molecules according to
the invention maybe used when verified to have suitable specificity with
regard to the novel
tissue material (binding or not binding). The invention is specifically
directed to analysis of
such binding specificity for development of a new binding or selection method
according to
the invention.
The preferred specificities according to the invention include recognition of:
i) mannose type structures, especially alpha-Man structures like lectin PAA
ii) sialylated structures similarily as by MAA-lectin
iii) GaUGa1NAc binding specificity, preferably Gall-3/Ga1NAc1-3 binding
specificity, more preferably Gal(31-3/Ga1NAc(31-3 binding specificity similar
to
PNA
Preferred cell population to be produced by glycomodification according to the
present
invention
The present invention is directed to specific cell populations comprising in
vitro
enzymatically altered glycosylations according to the present invention. It is
realized that
special structures revealed on cell surfaces have specific targeting, and
immune recognition
properties with regard to cells carrying the structures. It is realized that
sialylated and
fucosylated terminal structures such as sialyl-lewis x structures target cells
to selectins
involved in bone marrow homing of cells and invention is directed to methods
to produce
such structures on cells surfaces. It is further realized that mannose and
galactose terminal
structures revealed by the invention target cells to liver and/or to immune
recognition, which
in most cases are harmful for effective cell therapy, unless liver is not
targeted by the cells.
NeuGc is target for immune recognition and has harmful effects for survival of
cells
expressing the glycans.
The invention revealed glycosidase methods for removal of the structures from
cell surface
while keeping the cells intact.. The invention is especially directed to
sialyltransferase
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methods for modification of terminal galactoses. The invention further
revealed novel method
to remove mannose residues from intact cells by alpha-manosidase.
The invention is further directed to metabolic regulation of glycosylation to
alter the
glycosylation for reduction of potentially harmful structures.
The present invention is directed to specific cell populations comprising in
vitro
enzymatically altered sialylation according to the present invention. The
preferred cell
population includes cells with decreased amount of sialic acids on the cell
surfaces, preferably
decreased from the preferred structures according to the present invention.
The altered cell
population contains in a preferred embodiment decreased amounts of a3-Iinked
sialic acids.
The present invention is preferably directed to the cell populations when the
cell populations
are produced by the processes according to the present invention.
Cell populations with altered sialylated structures
The invention is further directed to novel cell populations produced from the
preferred cell
populations according to the invention when the cell population comprises
altered sialylation
as described by the invention. The invention is specifically directed to cell
populations
comprising decreased sialylation as described by the invention. The invention
is specifically
directed to cell populations comprising increased sialylation of specific
glycan structures as
described by the invention. Furthermore invention is specifically directed to
cell populations
of specifically altered 0- and or a6- sialylation as described by the
invention These cells are
useful for studies of biological functions of the cell populations and role of
sialylated, linkage
specifically sialylated and non-sialylated structures in the biological
activity of the cells.
Preferred cell populations with decreased sialylation
The preferred cell population includes cells with decreased amount of sialic
acids on the cell
surfaces, preferably decreased from the preferred structures according to the
present
invention. The altered cell population contains in a preferred embodiment
decreased amounts
of a3-Iinked sialic or a6-Iinked sialic acid. In a preferred embodiment the
cell populations
comprise practically only a3-sialic acid, and in another embodiment only a6-
Iinked sialic
acids, preferably on the preferred structures according to the invention, most
preferably on the
preferred N-glycan structures according to the invention. The present
invention is preferably
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directed to the cell populations when the cell populations are produced by the
processes
according to the present invention. The cell populations with altered
sialylation are preferably
cultivated human or animal cell populations according to the invention.
Preferred cell populations with increased sialylation
The preferred cell population includes cells with increased amount of sialic
acids on the cell
surfaces, preferably decreased from the preferred structures according to the
present
invention. The altered cell population contains in preferred embodiments
increased amounts
of a3--Iinked sialic or a6-Iinked sialic acid. In a preferred embodiment the
cell populations
comprise practically only a3-sialic acid, and in another embodiment only a6-
Iinked sialic
acids, preferably on the preferred structures according to the invention, most
preferably on the
preferred N-glycan structures according to the invention. The present
invention is preferably
directed to the cell populations when the cell populations are produced by the
processes
according to the present invention. The cell populations with altered
sialylation are preferably
cultivatedcells or tissue derived cell populations according to the invention.
Preferred cell populations with altered sialylation
The preferred cell population includes cells with altered linkage structures
of sialic acids on
the cell surfaces, preferably decreased from the preferred structures
according to the present
invention. The altered cell population contains in a preferred embodiments
altered amount of
a3-Iinked sialic and/or a6-Iinked sialic acid. The invention is specifically
directed to cell
populations having a sialylation level similar to the original cells but the
linkages of structures
are altered to a3-Iinkages and in another embodiment the linkages of
structures are altered to
a6-structures. In a preferred embodiment the cell populations comprise
practically only a3-
sialic acid, and in another embodiment only a6-Iinked sialic acids, preferably
on the preferred
structures according to the invention, most preferably on the preferred N-
glycan structures
according to the invention. The present invention is preferably directed to
the cell populations
when the cell populations are produced by the processes according to the
present invention.
The cell populations with altered sialylation are preferably cultivated cells
or tissue derived
cell populations according to the invention.
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Cell populations comprising preferred cell populations with preferred sialic
acid types
The preferred cell population includes cells with altered types of sialic
acids on the cell
surfaces, preferably on the preferred structures according to the present
invention. The altered
cell population contains in a preferred embodiment altered amounts of NeuAc
and/or
NeuGc sialic acid. The invention is specifically directed to cell populations
having sialylation
levels similar to original cells but the sialic acid structures altered to
NeuAc and in another
embodiment the sialic acid type structures altered to NeuGc. In a preferred
embodiment the
cell populations comprise practically only NeuAc, and in another embodiment
only NeuGc
sialic acids, preferably on the preferred structures according to the
invention, most preferably
on the preferred N-glycan structures according to the invention. The present
invention is
preferably directed to the cell populations when the cell populations are
produced by the
processes according to the present invention. The cell populations with
altered sialylation are
preferably cultivated or tissue derived cell populations according to the
invention.
Methods to alter (remove or reduce or change) glycosylation of cells
Analysis and degradative removal of the harmful glycan structure
The present invention is further directed to degradative removal of specific
harmful glycan
structures from cell, preferably from desired cell populations according to
the invention.
The removal of the glycans or parts thereof occur preferably by enzymes such
as glycosidase
enzymes.
In some cases the removal of carbohydrate structure may reveal another harmful
structure. In
another preferred embodiment the present invention is directed to replacement
of the removed
structure by less harmful or better tolerated structure more optimal for the
desired use.
Desialylation methods
Preferred special target cell type
Effective and specific desialylation methods for the specific cell populations
were developed.
The invention is specifically directed to desialylation methods for
modification of human
tissue and cell culture cells. The present invention is further directed to
desialylation
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modifications of any human cell or tissue cell subpopulation according to the
invention.
Sialylation modifications of cultivated cells have not been studied previously
in detail.
The present invention is specifically directed to methods for desialylation of
the preferred
structures according to the present invention from the surfaces of preferred
cells. The present
invention is further directed to preferred methods for the quantitative
verification of the
desialylation by the preferred analysis methods according to the present
invention. The
present invention is further directed to linkage specific desialylation and
analysis of the
linkage specific sialylation on the preferred carbohydrate structures using
analytical methods
according to the present invention.
The invention is preferably directed to linkage specific a3-desialylation of
the preferred
structures according to the invention without interfering with the other
sialylated structures
according to the present invention. The invention is further directed to
simultaneous
desialylation 0- and a6-sialylated structures according to the present
invention.
Furthermore the present invention is directed to desialylation when both NeuAc
and NeuGc
are quantitatively removed from cell surface, preferably from the preferred
structures
according to the present invention. The present invention is specifically
directed to the
removal of NeuGc from preferred cell populations, most preferably cultivated
or tissue
derived populations and from the preferred structures according to the present
invention. The
invention is further directed to preferred methods according to the present
invention for
verification of removal of NeuGc, preferably quantitative verification and
more preferably
verification performed by mass spectrometry.
Modification of cell surfaces of the preferred cells by glycosyltransferases
The inventors revealed that it is possible to produce controlled cell surface
glycosylation
modifications on the preferred cells according to the invention. The present
invention is
specifically directed to glycosyltransferase catalysed modifications of N-
linked glycans on the
surfaces of cells, preferably blood cells, more preferably leukocytes or
cultivated cells or
more preferably the preferred cell or tissue materials according to the
present invention.
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The present invention is directed to cell modifications by sialyltransferases
and
fucosyltransferases. Two most preferred transfer reactions according to the
invention are a3-
modification reactions such as a3-sialylation and a3-fucosylations. When
combined these
reactions can be used to produce important cell adhesion structures which are
sialylated and
fucosylated N-acetyllactosamines such as sialyl-Lewis x (sLex).
Sialylation
Possible a6-sialylation has been implied in bone marrow cells and in
peripheral blood CD34+
cells released from bone marrow to circulation by growth factor
administration, cultivated or
other cell types have not been investigated.Furthermore, the previous study
utilized an
artificial sialic acid modification method, which may affect the specificity
of the
sialyltransferase enzyme and, in addition the actual result of the enzyme
reaction is not
known as the reaction products were not analysed by the investigators. The
reactions are
likely to have been very much limited by the specificity of the a6-
sialyltransferase used and
cannot be considered prior art in respect to the present invention.
The inventors of the present invention further revealed effective modification
of the preferred
cells according to the present inventions by sialylation, in a preferred
embodiment by a3-
sialylation.
The prior art data cited above does not indicate the specific modifications
according to the
present invention to cultivated cells, preferably cultivated or tissue derived
cells. The present
invention is specifically directed to sialyltransferase reactions towards
these cell types. The
invention is directed to sialyltransferase catalyzed transfer of a natural
sialic acid, preferably
NeuAc, NeuGc or Neu-O-Ac, from CMP-sialic acid to target cells.
Sialyltransferase catalyzed reaction according to Formula:
CMP-SA + target cell 4 SA-target cell + CMP,
Wherein SA is a sialic acid, preferably a natural sialic acid,
preferably NeuAc, NeuGc or Neu-O-Ac and
the reaction is catalysed by a sialyltransferase enzyme preferably by an
a3-sialyltransferase
and
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the target cell is a cultured cell or tissue derived cell.
Preferably the sialic acid is transferred to at least one N-glycan structure
on the
cell surface, preferably to form a preferred sialylated structure according to
the invention
Fucosyltransferase reactions
In the prior art fucosyltransferase reactions towards unspecified cell surface
structures has
been studied
The prior art indicates that human cord blood cell populations may be be a3-
fucosylated by
human fucosyltransferase VI and such modified cell populations may be directed
to bone
marrow due to interactions with selectins.
DirectinQ cells and selectin ligands
The present invention describes reactions effectively modifying blood cells or
cultivated cells
in vitro by fucosyltransferases, especially in order to produce sialylated and
fucosylated N-
acetyllactosamines on cell surfaces, preferably sLex and related structures.
The present
invention is further directed to the use of the increased sialylated and/or
fucosylated structures
on the cell surfaces for targeting the cells, in a preferred embodiment for
selectin directed
targeting of the cells.
The invention is further directed to 0- and/or a4-fucosylation of cultured
cells, tissue cells..
Fucosylation of human peripheral blood mononuclear cell populations
In a specific embodiment the present invention is directed to a3-fucosylation
of the total
mononuclear cell populations from human peripheral blood. Preferably the
modification is
directed to at least to one protein linked glycan, more preferably to a N-
linked glycan. The
prior art reactions reported about cord blood did not describe reactions in
such cell
populations and the effect of possible reaction cannot be known. The invention
is further
directed to combined increased a3-sialylation and fucosylation, preferably a3-
sialylation of
human peripheral blood leukocytes. It is realized that the structures on the
peripheral blood
leukocytes can be used for targeting the peripheral blood leukocytes,
preferably to selecting
expressing sites such as selectin expressing malignant tissues.
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Methods for combined increased a3-sialylation and a3-fucosylation
The invention is specifically directed to selection of a cell population from
the preferred cell
population according to the present invention, when the cell population
demonstrate increased
amount of a3-sialylation when compared with the baseline cell populations.
Use of selected cultured a3-sialic acid expressing cell populations
The inventors revealed that specific subpopulations of native cells express
increased amounts
of a3-linked sialic acidFurthermore it was found that cultured cells according
to the invention
have a high tendency to express a3-sialic acid instead to a6-linked sialic
acids. The present
invention is preferably directed to cultured cell lines, tissue cells
expressing increased
amounts of a3-linked sialic acid
Fucosylation of a3-sialylated cells
The present invention is preferably directed to fucosylation after a3-
sialylation of cells,
preferably the preferred cells according to the invention. The invention
describes for the first
time combined reaction by two glycosyltransferases for the production of
specific terminal
epitopes comprising two different monosaccharide types on cell surfaces.
Fucosylation of desialylated and a3-sialylated cells
The present invention is preferably directed to fucosylation after
desialylation and a3-
sialylation of cells, preferably the preferred cells according to the
invention. The invention
describes for the first time combined reaction by two glycosyltransferases and
a glycosidase
for the production of specific terminal epitopes comprised of two different
monosaccharide
types on cell surfaces.
Sialylation methods
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Preferred special target cell type
Cultivated cells and tissues
Effective specific sialylation methods for the specific cell populations were
developed. The
invention is specifically directed to sialylation methods for modification of
human cultivated
cells and subpopulations thereof as cell lines and human tisues.
Production of preferred sialylated structures
Present invention is specifically directed to methods for sialylation to
produce preferred
structures according to the present invention from the surfaces of preferred
cells. The present
invention is specifically directed to production preferred NeuGc- and NeuAc-
structures. The
invention is directed to production of potentially in vivo harmful structures
on cells surfaces,
e.g. for control materials with regard to cell labelling. The invention is
further directed to
production of specific preferred terminal structure types, preferably a3-and
a6-sialylated
structures, and specifically NeuAc- and NeuGc-structures for studies of
biological activities
of the cells.
The present invention is further directed to preferred methods for the
quantitative verification
of the sialylation by the preferred analysis methods according to the present
invention. The
present invention is further directed to linkage specific sialylation and
analysis of the linkage
specific sialylation on the preferred carbohydrate structures using analytical
methods
according to the present invention.
The invention is preferably directed to linkage specific a3-sialylation of the
preferred
structures according to the invention without interfering with the other
sialylated structures
according to the present invention. The invention is preferably directed to
linkage specific a6-
sialylation of the preferred structures according to the invention without
interfering with the
other sialylated structures according to the present invention.
The invention is further directed to simultaneous sialylation 0- and a6-
sialylated structures
according to the present invention. The present invention is further directed
for the production
of preferred relation of 0- and a6-sialylated structures, preferably in single
reaction with two
sialyl-transferases.
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Furthermore the present invention is directed to sialylation when either NeuAc
or NeuGc are
quantitatively synthesized to the cell surface, preferably on the preferred
structures according
to the present invention. Furthermore the invention is directed to sialylation
when both NeuAc
and NeuGc are, preferably quantitatively, transferred to acceptor sites on the
cell surface.
The present invention is specifically directed to the removal of NeuGc from
preferred cell
populations, most preferably cultivated cell populations and from the
preferred structures
according to the present invention, and resialylation with NeuAc.
The invention is further directed to preferred methods according to the
present invention for
verification of removal of NeuGc, and resialylation with NeuAc, preferably
quantitative
verification and more preferably verification performed by mass spectrometry
with regard to
the preferred structures.
Controlled cell modification
The present invention is further directed to cell modification according to
the invention,
preferably desialylation or sialylation of the cells according to the
invention, when the
sialidase reagent is a controlled reagent with regard of presence of
carbohydrate material.
Purification of cells with regard to modification enzyme
The preferred processes according to the invention comprise of the step of
removal of the
enzymes from the cell preparations, preferably the sialyl modification enzymes
according to
the invention. Most preferably the enzymes are removed from a cell population
aimed for
therapeutic use. The enzyme proteins are usually antigenic, especially when
these are from
non-mammalian origin. If the material is not of human origin its glycosylation
likely increases
the antigenicity of the material. This is particularily the case when the
glycosylation has major
differences with human glycosylation, preferred examples of largely different
glycosylations
includes: procaryotic glycosylation, plant type glycosylation, yeast or fungal
glycosylation,
mammalian/animal glycosylation with Gala3Ga1(34GIcNAc-structures, animal
glycosylations
with NeuGc structures. The glycosylation of a recombinant enzyme depends on
the
glycosylation in the production cell line, these produce partially non-
physiological glycan
structures. The enzymes are preferably removed from any cell populations aimed
for culture
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or storage or therapeutic use. The presence of enzymes which have affinity
with regard to cell
surface may otherwise alter the cells as detectable by carbohydrate binding
reagents or mass
spectrometric or other analysis according to the invention and cause adverse
immunological
responses.
Under separate embodiment the cell population is cultured or stored in the
presence of the
modification enzyme to maintain the change in the cell surface structure, when
the cell
surface structures are recovering from storage especially at temperatures
closer physiological
or culture temperatures of the cells. Preferably the cells are then purified
from trace amounts
of the modification enzyme before use.
The invention is furthermore directed to methods of removal of the
modification reagents
from cell preparations, preferably the modification reagents are desialylation
or resialylation
reagents. It is realized that soluble enzymes can be washed from the modified
cell
populations. Preferably the cell material to be washed is immobilized on a
matrix or
centrifuged to remove the enzyme, more preferably immobilized on a magnetic
bead matrix.
However, extraneous washing causes at least partial destruction of cells and
their decreased
viability. Furthermore, the enzymes have affinity with regard to the cell
surface. Therefore the
invention is specifically directed to methods for affinity removal of the
enzymes. The
preferred method includes a step of contacting the modified cells with an
affinity matrix
binding the enzyme after modification of the cells.
Under specific embodiment the invention is directed to methods of tagging the
enzyme to be
removed from the cell population. The tagging step is performed before
contacting the
enzyme with the cells. The tagging group is designed to bind preferably
covalently to the
enzyme surface, without reduction or without major reduction of the enzyme
activity. The
invention is further directed to the removal of the tagged enzyme by binding
the tag to a
matrix, which can be separated from the cells. Preferably the matrix comprises
at least one
matrix material selected from the group: polymers, beads, magnetic beads, or
solid phase
surface
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Enzymes acceptable for humans for modification of reagents or cells
Under specific embodiment the invention is directed to the use for
modification of the cells
according to the invention, or in a separate embodiment reagents for processes
according to
the invention, of a human acceptable enzyme, preferably a glycosidase
according to the
invention or in preferred embodiment sialidase or sialyltransferase, which is
acceptable at
least in certain amounts to human beings without causing harmful allergic or
immune
reactions. It is realized that the human acceptable enzymes may not be needed
to be removed
from reaction mixtures or less washing steps are needed for desirable level of
the removal.
The human acceptable enzyme is in preferred embodiment a human
glycosyltransferase or
glycosidase. The present invention is separately directed to human acceptable
enzyme which
is a sialyltransferase. The present invention is separately directed to human
acceptable
enzyme which is a sialidase, the invention is more preferably directed to
human sialidase
which can remove specific type of sialic acid from cells.
In a preferred embodiment the human acceptable enzyme is purified from human
material,
preferably from human serum, urine or milk. In another preferred embodiment
the enzyme is
recombinant enzyme corresponding to natural human enzyme. More preferably the
enzyme
corresponds to human natural enzyme corresponds to natural cell surface or a
secreted from of
the enzyme, more preferably serum or urine or human milk form of the enzyme.
Even more
preferably the present invention is directed to human acceptable enzyme which
corresponds to
a secreted form of a human sialyltransferase or sialidase, more preferably
secreted
serum/blood form of the human enzyme. In a preferred embodiment the human
acceptable
enzyme, more preferably recombinant human acceptable enzyme, is a controlled
reagent with
regard to potential harmful glycan structures, preferably NeuGc-structures
according to the
invention. The recombinant proteins may contain harmful glycosylation
structures and
inventors revealed that these kinds of structures are also present on
recombinant
glycosyltransferases, even on secreted (truncated) recombinant
glycosyltransferases.
mRNA corresponding to glycosylation enzymes
The present invention is further directed to correlation of specific messenger
mRNA
molecules with the preferred glycan structures according to the present
invention. It is
realized that glycosylation can be controlled in multiple levels and one of
them is
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transcription. The presence of glycosylated structures may in some case
correlate with
mRNAs involved in the synthesis of the structures.
The present invention is especially directed to analysis of mRNA-species
having correlation
with expressed fucosylated glycan structures and "terminal HexNAc" containing
structures
preferred according to the present invention. The preferred mRNA-species
includes mRNA
corresponding to fucosyltransferases and N-acetylglucosaminyltransferases.
Observation of glycan binding structures, lectins, corresponding mRNA-markers
The invention further revealed changes in mRNA-expression of glycosylation
recognizing
lectins such as galectins. The cells were further revealed to contain
lactosamine receptors for
lectins. The invention is especially directed to analysis of expression levels
of human lectins
and lactosamine galectin receptors, preferably analysis of galectins and
selectins more
preferably galectins for analysis of status of cells according to the present
invention.
The invention specifically revealed novel NeuGc(N-glycolylneuraminic acid)-
binding lectin
activity from human cells. The lectin lectin recognizes polyvalent NeuGc but
does not bind
effectively to polyvalent NeuNAc. The present invention is especially directed
to labelling
cells according to the invention by carbohydrate conjugates binding cells
according to the
invention, preferably labelled conjugates of NeuGc. The invention is further
directed to
sorting and selecting cells, and cell derived materials and purifying proteins
from cells, using
labelled carbohydrate conjugates, pereferably, conjugates of NeuGc.
NMR-analysis of glycomes
The present invention is directed to analysis of released glycomes by
spectrometric method
useful for characterization of the glycomes from tissue specimens or cells.
The invention is
directed to NMR spectroscopic analysis of the mixtures of released glycans.
The invention is especially directed to methods of producing NMR from specific
subglycomes, preferably N-linked glycome, 0-linked glycome, glycosaminoglycan
glycome
and/or glycolipid glycome. The NMR-profiling according to the invention is
further directed
to the analysis of the novel and rare structure groups revealed from cell
glycomes according to
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the invention. The general information about complex cell glycome material
directed NMR-
methods are limited.
Preferably the NMR-analysis is performed from an isolated subglycome. The
preferred
isolated subglycomes include acidic glycomes and neutral glycomes.
NMR-glycome analysis of larger tissue specimens or larger amounts of cells
It is realized that numerous methods have been desribed for purification of
oligosaccharide
mixtures useful for NMR from various materials, including usually purified
individual
proteins. It is realized that present methods are useful for NMR-profiling
even for larger tissue
specimens or higher amounts of cells according to the invention, especially in
combination
with NMR-profiling according to the invention and/or when directed to the
analysis specific
and preferred structure groups according to the invention. The preferred
purification methods
are effective and form an optimised process for purification of glycomes from
even larger
amounts of cells and tissues than described for nanoscale methods below. The
methods are
preferred also for any larger amount of cells.
Purification method for low amount nanoscale NMR-profiling of samples
Moreover, when purification methods for larger amounts of carbohydrate
materials exists, but
very low and complex carbohydrate materials with very complex impurities such
as cell-
derived materials have been less studied as low amounts, especially when
purity useful for
NMR-analysis is needed.
Preferred sample amounts allowing effective NMR analysis of cell glycomes
The invention is directed to analysis of NMR-samples that can be produced from
very low
amounts of cells according to the invention. Preferred sample amounts of cells
or
corresponding amount of tissue material for a one-dimensional proton-NMR
profiling are
from about 2 million to 100 million cells, more preferably 10-50 million
cells. It is further
realized that good quality NMR data can be obtained from samples containing at
least about
10-20 million cells.
The preferred analysis methods is directed to high resolution NMR observing
oligosaccharide/saccharide conjugate mixture from an amount of at least 4
nmo1, more
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preferably at least 1 nmol and the cell amount yielding the preferred amount
of saccharide
mixture. For nanoscale analysis according to the invention cell material is
selected so that it
would yield at least about 50 nmol of oligosaccharide mixture, more preferably
at least about
nmol and most preferably at least about 1 nmol of oligosaccharide mixture.
Preferred
5 amounts of major components in glycomes to be observed effectively by the
methods
according to the invention include yield at least about 10 nmol of
oligosaccharide component,
more preferably at least about 1 nmol and most preferably at least about 0.2
nmol of
oligosaccharide component.
The preferred cell amount for analysis of a subglycome from a cell type is
preferably
optimised by measuring the amounts of glycans produced from the cell amounts
of preferred
ranges.
It is realized that depending on the cell and subglycome type the required
yield of glycans and
the heterogeneity of the materials vary yielding different amounts of major
components.
Preferred purification methods
For the production of sample for nanoscale NMR, the methods described for
preparation of
cell samples and release of oligosaccharides for mass spectrometric profiling
according to the
invention may be applied.
For the purification of sample for nanoscale NMR the methods described for
purification
mass spectrometry profiling samples according to the invention may be applied.
The preferred purification method for nanoscale NMR- profiling according to
the invention
include following general purification process steps:
1) Precipitation/extraction;
2) Hydrophobic interaction;
3) Affinity to carbon material, especially graphitized carbon.
4) Gel filtration chromatography
The more preferred purification process includes precipitation/extraction
aimed for removal
of major non-carbohydrate impurities by separating the impurity and the
glycome fraction(s)
to be purified to different phases. Hydrophobic interaction step aims to
purify the glycome
components from more hydrophobic impurities as these are bound to hydrophobic
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chromatography matrix and the glycome components are not retained.
Chromatography on
graphitized carbon may include purification or enrichment of glycans due to
their affmity or
specific adsorption to graphitized carbon, or removal of contaminants from the
glycans. The
glycome components obtained by the aforementioned steps are then subjected to
gel filtration
chromatography, separating molecules according to their hydrodynamic volume,
i.e. size in
solution. The gel filtration chromatography step allows detection and
quantitation of glycome
components by absorption at low wavelenghts (205-214 nm).
The most preferred purification process includes precipitation/extraction and
hydrophobic
interaction steps aimed for removal of major non-carbohydrate impurities and
more
hydrophobic impurities. Chromatography on graphitized carbon is used for
removal of
contaminants from the glycans, and to devide the glycome components to
fractions of neutral
glycome components and acidic glycome components. The neutral and acidic
glycome
component fractions are then subjected to gel filtration chromatography, which
separates
molecules according to their size. Preferably, a high-performance liquid
chromatography
(HPLC) type gel filtration column is used. The neutral glycome component
fraction is
preferebly chromatographed in water and the acidic glycome component fraction
is
chromatographed in 50-200 mM aqueous ammonium bicarbonate solution. Fractions
are
collected and evaporated prior to further analyses. The gel filtration
chromatography step
allows detection and quantitation of glycome components by absorption at low
wavelenghts
(205-214 nm). Quantitation is performed against external standards. The
standards are
preferably N-acetylglucosamine, N-acetylneuraminic acid, or oligosaccharides
containing the
same. Fractions showing absorbance are subjected to MALDI-TOF mass
spectrometry.
Preferably, the neutral glycome components are analyzed in the positive-ion
mode and the
acidic glycome components in the negative-ion mode in a delayed-extraction
MALDI-TOF
mass spectrometer.
Preferred methods for producing enriched glycome fractions for NMR
The fractionation can be used to enrich components of low abundance. It is
realized that
enrichment would enhance the detection of rare components. The fractionation
methods may
be used for larger amounts of cell material. In a preferred embodiment the
glycome is
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fractionated based on the molecular weight, charge or binding to carbohydrate
binding agents
such as lectins and/or other binding agents according to the invention.
These methods have been found useful for specific analysis of specific
subglycomes and
enrichment more rare components. The present invention is in a preferred
embodiment
directed to charge based separation of neutral and acidic glycans. This method
gives for
analysis method, preferably mass spectroscopy material of reduced complexity
and it is useful
for analysis as neutral molecules in positive mode mass spectrometry and
negative mode mass
spectrometry for acidic glycans.
It is realized that preferred amounts of enriched glycome oligosacccharide
mixtures and major
component comprising fractions can be produced from larger glycome
preparations.
In a preferred embodiment the invention is directed to size based
fractionation methods for
effective analysis of preferred classes of glycans in glycomes. The invention
is especially
directed to analysis of lower abundance components with lower and higher
molecular weight
than the glycomes according to the invention. The preferred method for size
based
fractionation is gel filtration. The invention is especially directed to
analysis of enriched
group glycans of N-linked glycomes preferably including lower molecular weight
fraction
including low-mannose glycans, and one or several preferred low mannose glycan
groups
according to the invention.
Preferred NMR-methods
In a preferred embodiment the NMR-analysis of the glycome is one-dimensional
proton-
NMR analysis showing structural reporter groups of the major components in the
glycome.
The invention is further directed to specific two- and multidimensional NMR-
experiments of
the glycomes when enough sample is available. It is realized that two-
dimensional NMR-
experiments require about a ten-fold increase in sample amount compared to
proton-NMR
analyses.
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Combination of NMR- and mass spectrome , for glycome analysis
The present invention is further directed to combination of the mass
spectrometric and NMR
glycome analyses. The preferred method include production of any mass
spectrometric profile
from any glycome according to the invention from a cell sample according to
the invention,
optionally characterizing the glycome by other methods like glycosidase
digestion,
fragmentation mass spectrometry, specific binding agents, and production of
NMR-profile
from the same sample glycome or glycomes to compare these profiles.
Specific characteristic marker structures and glycome marker
components/compositions
The N-glycan analysis of total profiles of released N-glycans revealed beside
the glycans
above, which were verified to comprise
1) complex biantennary N-glycans, such as
Gal(34G1cNAc(32Mana3 (Gal(34G1cNAc(32Man(x6)Man(34G1cNAc(34(Fuc(x6)o_ 1
G1cNAc(3-,
wherein the reminal N-acetyllactosamines can be elongated from Gal with
NeuNAca3
aand/or NeuNAc(x6 and
2) terminal mannose containing N-glycans such as High-mannose glycans with
formula Hex5_
9HexNAc2 and degradation products thereof comprising low number of mannose
residues
(Low mannose glycans) Hex1-4HexNAc2.
The specific N-glycan core marker structure
The glycan share common core structure according to the Formula:
[Mana3]nl(Mana6) õ2Man(34G1cNAc(34(Fuca6)0_1G1cNAc(3Asn,
wherein nl and n2 are integers 0 or 1, independently indicating the presence
or
absence of the terminal Man-residue, and
wherein the non-reducing end terminal Mana3/Mana6- residues can be elongated
to the
complex type, especially biantennary structures or to mannose type (high-Man
and/or low
Man) or to hybrid type structures as desribed in examples.
It was further analyzed that the N-glycan compositios contained only very
minor amounts of
glycans with additional HexNAx in comparison to monosaccharide compositions of
the
complex type glycan above, which could indicate presence of no or very low
amounts of the
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N-glycan core linked G1cNAc-residues described by Stanley PM and Raju TS (JBC-
(1998)
273 (23) 14090-8; JBC (1996) 271 (13) 7484-93) and/or bisecting G1cNAc. is
realized that
part of the terminal HexNAc-type structures appear to represent bisecting
G1cNAc-type type
glycans, and quite low or non-existent amounts of the GIcNAca6-branching and
also low
amounts of G1cNAc(32-branch of Man(34 described by Stanley and colleagues.
Here,
essentially devoid of indicates less than 10 % of all the protein linked N-
glycans, more
preferably the additional HexNAc units are present in less than 8 % of the
tissue material N-
glycans by mass spectrometric analysis.
The invention thus describes the major core structure of N-glycans in human
tissue materials
verified by NMR-spectroscopy and by specific glycosidase digestions and was
further
quantitated to comprise a characteristic smaller structural group glycans
comprising specific
terminal HexNAc group and/or bisecting G1cNAc-type structures, which
additionally modify
part of the core structure. The invention further reveals that the core
structure is a useful target
structure for analysis of tissue materials.
The characteristic monosaccharide composition of the core structure will allow
recognition of
the major types of N-glycan structure groups present as additional
modification of the core
structure. Furthermore composition of the core structure is characteristic in
mass
spectrometric analysis of N-glycan and allow immediate recognition for example
from
HexXHexNAcl -type (preferentially ManXGIcNAc1) glycans also present in total
glycome
compostion.
Low-molecular weight glycan marker structures and tissue material glycome
components
The invention describes novel low-molecular weight acidic glycan components
within the
acidic N-glycan and/or soluble glycan fractions with characteristic
monosaccharide
compositions SAXHexI_2HexNAc1_2, wherein x indicates that the corresponding
glycans are
preferentially sialylated with one or more sialic acid residues. The inventors
realized that such
glycans are novel and unusual with respect to N-glycan biosynthesis and
described
mammalian cell glycan components, as reveal also by the fact that they are
classified as
"other (N-)glycan types" in N-glycan classification scheme of the present
invention. The
invention is directed to analyzing, isolating, modifying, and/or binding to
these novel glycan
components according to the methods and uses of the present invention, and
further to other
uses of specific marker glycans as described here. As demonstrated in the
Examples of the
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present invention, such glycan components were specific parts of total
glycomes of certain
tissue materials and preferentially to certain tissue material types, making
their analysis and
use beneficial with regard to tissue materials. The invention is further
directed to tissue
material glycomes and subglycomes containing these glycan components.
Preferred glycomes
The present invention is specifically directed to tissue material glycomes,
which are
essentially pure glycan mixtures comprising various glycans as described in
the invention
preferably in proportions shown by the invention. The essentially pure glycan
mixtures
comprise the key glycan components in proportions which are characteristics to
tissue
material glycomes. The preferred glycomes are obtained from human tissue
materials
according to the invention.
The invention is further directed to glycomes as products of purification
process and
variations thereof according to the invention. The products purified from
tissue materials by
the simple, quantitative and effective methods according to the invention are
essentially pure.
The essentially pure means that the mixtures are essentially devoid of
contaminations
disturbing analysis by MALDI mass spectrometry, preferably by MALDI-TOF mass
spectrometry. The mass spectra produced by the present methods from the
essentially pure
glycomes reveal that there is essentially no non-carbohydrate impurities with
weight larger
than trisaccharide and very low amount of lower molecular weight impurities so
that
crystallization of MALDI matric is possible and the glycan signals can be
observed for broad
glycomes with large variations of monosaccharide compositions and ranges of
molecular
weight as described by the invention. It is realized that the purification of
the materials from
low amounts of tissue materials comprising very broad range of cellular
materials is very
challenging task and the present invention has accomplished this.
Combination compositions of the preferred glycome mixtures with matrix for
analysis
Mass spectrometric matrix
The invention further revealed that it is possible to combine the glycomes
with matrix useful
for a mass spectrometric analysis and to obtain combination mixture useful for
spectrometric
analysis. The preferred mass spectrometric matrix is matrix for MALDI (matrix
assisted laser
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desorption ionization mass spectrometry) with mass spectrometric analysis
(abbreviated as
MALDI matrix), MALDI is preferably performed with TOF (time of flight)
detection.
Preferred MALDI matrices include aromatic preferably benzene ring structure
comprising
molecules with following characteristic. The benzene ring structure molecules
preferably
comprises 1-4 substituents such as hydroxyl, carboxylic acid or ketone groups.
Known
MALDI matrixes have been reviewed in Harvey, Mass. Spec. Rev. 18, 349 (1999).
The
present invention is especially and separately directed to specific matrixes
for analysis in
negative ion mode of MALDI mass spectrometry, preferred for analysis of
negatively charged
(acidic, such as sialylated and/or sulfated and/or phosphorylated) subglycome,
and in positive
ion mode of MALDI mass spectrometry (preferred for analysis of neutral
glycomes). It is
realized that the matrices can be optimized for negative ion mode and positive
ion mode.
The present invention is especially directed to glycome matrix composition
optimized for the
use in positive ion mode, and to the use of the MALDI-TOF matrix and matrix
glycome
composition, that is optimized for the use in the analysis in positive ion
mode, for the analysis
of glycome, preferably neutral glycome. The preferred matrices for positive
ion mode are
aromatic matrices, e.g. 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid/2-
hydroxy-5-
methoxybenzoic acid, 2,4,6-trihydroxyacetophenone or 6-aza-2-thiothymine, more
preferably
2,5-dihydroxybenzoic acid. The present invention is especially directed to
glycome matrix
composition optimized for the use in negative ion mode, and to the use of the
MALDI-TOF
matrix and the matrix glycome compositions, that is optimized for the negative
ion mode, for
the analysis of glycome, preferably acidic glycome. The preferred matrices for
negative ion
mode are aromatic matrices, e.g. 2,4,6-trihydroxyacetophenone, 3-
hydroxypicolinic acid, 2,5-
dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid/2-hydroxy-5-methoxybenzoic
acid, or 6-
aza-2-thiothymine, more preferably 2,4,6-trihydroxyacetophenone. The invention
is further
directed to analysis method and glycome-matrix compostion for the analysis of
glycome
compositions, wherein the glycome composition comprises both negative and
neutral
glycome components. Preferred matrices for analysis of negative and neutral
glycome
components comprising glycome are aromatic matrices, e.g. 2,4,6-
trihydroxyacetophenone, 3-
hydroxypicolinic acid, 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid/2-
hydroxy-5-
methoxybenzoic acid, or 6-aza-2-thiothymine, more preferably 2,4,6-
trihydroxyacetophenone.
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The MALDI-matrix is a molecule capable of
1) Specifically and effectively co-crystallizing with glycome composition with
the matrix,
crystallizing meaning here forming a solid mixture composition allowing
analysis of glycome
involving two steps below
2) absorbing UV-light typically provided by a laser in MALDI-TOF instrument,
preferred
wavelength of the light is 337 nm as defined by the manuals of MALDI-TOF
methods
3) transferring energy to the glycome compostion so that these will ionize and
be analyzable
by the MALDI-TOF mass spectrometry. The present invention is especially
directed to
compositions of glycomes in complex with MALDI mass spectrometry matrix.
The present invention is specifically directed to methods of searching novel
MALDI-matrixes
with the above characteristic, preferably useful for analysis by the method
below. The method
for searching novel MALDI-matrixes using the method in the next paragraph.
The present invention is specifically directed to methods of analysis of
glycomes by MALDI-
TOF including the steps:
1) Specifically and effectively co-crystallizing the glycome composition with
the MALDI-
TOF-matrix, crystallizing meaning here forming a solid mixture composition
allowing
analysis of glycome involving two steps below
2) Providing UV linght to crystalline sample by a laser in MALDI-TOF
instrument allowing
the ionization of sample
3) Analysis of the ions produced by the MALDI mass spectrometer, preferably by
TOF
analysis. The invention is further directed to the combination of glycome
purification methods
and/or quantitative and qualitative data analysis methods according to the
invention.
Crystalline compositions of glycomes
The present invention is further directed to essentially pure glycome
compositions in solid co-
crystalline form with MALDI matrix. The invention is preferably a neutral
and/or acidic
glycome as complex with a matrix optimized for analysis of the specific
glycome type,
preferably analysis in negative ion mode with a matrix such as 2,4,6-
trihydroxyacetophenone.
The invention is preferably a neutral (or non-acidic) glycome as complex with
a matrix
optimized for analysis in positive ion mode such as 2,5-dihydroxybenzoic acid.
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The invention revealed that it is possible to analyze glycomes using very low
amount of
sample. The preferred crystalline glycome composition comprises between 0.1 -
100 pmol,
more preferably 0.5- 10 pmol, more preferably 0.5- 5 pmol and more preferably
about 0.5-3
pmol, more preferably about 0.5 - 2 pmol of sample co-crystallized with
optimized amount of
matrix preferably about 10-200 nmo1, more preferably 30-150 nmo1, and more
preferably
about 50-120 nmol and most preferably between 60-90 nmols of the matrix,
preferably when
the matrix is 2,5-dihydroxybenzoic acid. The matrix and analyte amounts are
optimized for a
round analysis spot with radius of about 1 mm and area of about 0.8 mm2. It is
realized that
the amount of materials can be changed in proportion of the area of the spot,
smaller amount
for smaller spot. Examples of preferred amounts per area of spot are 0.1-100
pmol/0.8 mm2
and 10-200 pmol/3 mm2. Preferred molar excess of matrix is about 5000-1000000
fold, more
preferably about 10000-500000 fold and more preferably about 15000 to 200 000
fold and
most preferably about 20000 to 100000 fold excess when the matrix is 2,5-
dihydroxybenzoic
acid.
It is realized that the amount and relative amount of new matrix is optimized
based on
forming suitable crystals and depend on chemical structure of the matrix. The
formation of
crystals is observed by microscope and further tested by performing test
analysis by MALDI
mass spectrometry.
The invention is further directed to specific methods for crystallizing MALDI-
matrix with
glycome. Preferred method for crystallization in positive ion mode includes
steps: (1)
optionally, elimination of impurities, like salts and detergents, which
interfere with the
crystallization, (2) providing solution of glycome in H20 or other suitable
solvent in the
preferred concentration, (3) mixing the glycome with the matrix in solution or
depositing the
glycome in solution on a precrystallized matrix layer and (4) drying the
solution preferably by
a gentle stream of air.
Preferred method for crystallization in negative ion mode includes steps: (1)
optionally,
elimination of impurities, like salts and detergents, which interfere with the
crystallization, (2)
providing solution of glycome in H20 or other suitable solvent in the
preferred concentration,
(3) mixing the glycome with the matrix in solution or depositing the glycome
in solution on a
precrystallized matrix layer and (4) drying the solution preferably by vacuum.
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Other preferred glycome analysis compostions
Binder glycome compositions
The invention is further directed to compositions of essentially pure glycome
composition
with specific glycan binding molecules such as lectins, glycosidases or
glycosyltransferases
and other glycosyl modifying enzymes such as sulfateses and/or phosphatases
and antibodies.
It is realized that these composition are especially useful for analysis of
glycomes.
The present invention revealed that the complex glycome compositions can be
effectively and
even quantitatively modified by glycosidases even in very low amounts. It was
revealed that
the numerous glycan structures similar to target structures of the enzymes do
not prevent the
degradation by competive inhibition, especially by the enzymes used. The
invention is
specifically directed to preferred amounts directed to MALDI analysis for use
in composition
with a glycosylmodifying enzyme, preferably present in low amounts. Preferred
enzymes
suitable for analysis include enzymes according to the Examples.
The invention is further directed to binding of specific component of glycome
in solution with
a specific binder. The preferred method further includes affinity
chromatography step for
purification of the bound component or analysis of the non-bound fraction and
comparing it to
the glycome solution without the binding substance. Preferred binders include
lectins
engineered to be lectins by removal of catalytic amino acids (methods
published by Roger
Laine, Anomeric, Inc., USA, and Prof. Jukka Finne, Turku, Finland), lectins
and antibodies or
antibody fragments or minimal binding domains of the proteins.
Additional data analysis and related methods
The present invention is especially directed to the use of glycome data for
production of
mathematical formulas, or algorithms, for specific recognition or
identification of specific
tissue materials. Data analysis methods are presented e.g. in Example 17.
The invention is especially directed to selecting specific "structural
features" such as mass
spectrometric signals (such as individual mass spectrometric signal
corresponding to one or
several monosaccharide compositions and/or glycan structures), or signal
groups or
subglycomes or signals corresponding to specific glycan classes, which are
preferably
according to the invention, preferably the signal groups (preferably defined
as specific
structure group by the invention), from quantitative glycome data, preferably
from
quantitative glycome data according to the invention, for the analysis of
status of tissue
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materials. The invention is furthermore directed to the methods of analysis of
the tissue
materials by the methods involving the use of the specific signals or signal
groups and a
mathematical algorithm for analysis of the status of a tissue material.
Preferred algorithm includes use of proportion (such as %-proportion) of the
specific signals
from total signals as specific values (structural features) and creating a
"glycan score", which
is algorithm showing characteristics/status of a tissue material based on the
specific
proportional signal intensities (or quantitative presence of glycan structures
measured by any
quantitation method such as specific binding proteins or quantitative
chromatographic or
electrophoresis analysis such as HPLC analysis). Preferably signals which are,
preferably
most specifically, upregulated in specific tissue materials and signals which
are, preferably
most specifically, downregulated in the tissue material in comparison to
control tissue
materials are selected to for the glycan score. In a preferred embodiment
value(s) of
downregulated signals are subtracted from upregulated signals when glycan
score is
calculated. The method yields largest score values for a specific tissue
material type or types
selected to be differentiated from other tissue materials.
The invention is specifically directed to methods for searching characteristic
structural
features (values) from glycome profiling data, preferably quantitative or
qualitative glycome
profiling data. The preferred methods include methods for comparing the
glycome data sets
obtained from different samples, or from average data sets obtained from a
group of similar
samples such as paraller samples from same or similar tissue material
preparations. Methods
for searching characteristic features are briefly described in the section:
identification and
classification of differences in glycan datasets. The comparison of datasets
of the glycome
data according to the invention preferably includes calculation of relative
and/or absolute
differences of signals, preferably each signal between two data sets, and in
another preferred
embodiment between three or more datasets. The method preferably further
includes step of
selecting the differing signals, or part thereof, for calculating glycan
score.
It is further realized that the analyzed glycome data has other uses preferred
by the invention
such as use of the selected characteristic signals and corresponding glycan
material:
1) for targets for structural analysis of glycans (preferably chemically by
glycosidases,
fragmentation mass spectrometry and/or NMR spectroscopy as shown by the
present
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invention and/or structural analysis based on the presence of other signals
and knowledge of
biosynthesis of glycans). The preferred use for targets includes estimation of
chemical
characteristics of potential corresponding glycans for complete or partial
purification/separation of the specific glycan(s). The preferred chemical
characteristics to be
analysed preferably include one or several of following properties: a) acidity
(e.g. by
presence of acidic residues such as sialic acid and/or sulfate and/or
phosphate) for charge
based separation, b) molecular weight or hydrodymanamic volume affecting
chromatographic
separation, e.g. estimation of the elution volume in gel filtration methods
(the effect of acidic
residue can be estimated from effects of similar structures and the "size" of
HexNAc
(Ga1NAc/G1cNAc) is in general twice the size of Hex (such as Gal, Man or Glc),
c) estimation
(e.g. based on composition and biosynthetic knowledge of glycans) of presence
of epitopes
for specific binding reagents for labelling identification in a mixture or for
affinity
purification, d) estimation of presence of target epitopes for specific
glycosylmodifying
enzymes including glycosidases and/or glycosyltransferases (types of binding
reagents) or for
specific chemical modification reagents (such as periodate for specific
oxidation or acid for
specific acid hydrolysis), for modification of glycans and recognition of the
modification by
potential chemical change such as incorporation of radioactive label or by
change of mass
spectrometric signal of the glycan for labelling identification in a mixture.
2) use of the signals or partially or fully analysed glycan structures
corresponding to the
signals for searching specific binding reagents for recognition of tissue
materials which are
preferably selected as described by the present invention (especially as
described above) and
in the methods for identification and classification of differences in glycan
datasets and/or
signals selected and/or tested by glycan score methods, are preferably
selected for targets for
structural analysis of glycans (preferably by glycosidases, fragmentation mass
spectrometry
and/or NMR spectroscopy as shown by the present invention) and/or for use of
the signals or
partially or fully analysed glycan structures corresponding to the signals for
searching specific
binding reagents for recognition of tissue materials.
The preferred method includes the step of comparing the values, and preferably
presenting the
score values in graphs such as ones shown in Fig. 36 (example 23), and
preferably evaluating
the statistic significance of the result by a statistic analysis methods such
as a mathematical
test for statistic significance. tissue material type refers here to tissue
materials with specific
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status and/or identity, e.g. malignancy, with possible individual variability,
e.g. between
individual patients.
It is realized that to differentiate a tissue materials type from other(s)
different characteristic
signals may be selected than for another tissue material type. The invention
however revealed
that for tissue materials and especially for human cancer patients preferred
characteristic
signals include ones selected in the Examples as described above. It is
realized that a glycan
score can be also created with less characteristic signals or with only part
of signals and still
relevant results can be obtained. The invention is further directed to methods
for optimisation
of glycan score algorithms and methods for selecting signals for glycan
scores.
In case the specific proportion (value) of a characteristic signal is low in
comparision to other
values a specific factor can be selected for increase the relative "weight" of
the value in the
glycan scores to be calculated for the cell populations.
The preferred statuses of tissue materials, to be analysed by mathematical
methods such as
algorithms using quantitative glycome profiling data according to the
invention include
differentiation status, individual characteristics and mutation, cell culture
or storage
conditions related status, effects of chemicals or biochemicals on cells, and
other statuses
described by the invention.
Preferred structures of O-glycan glycomes of tissue materials
The present invention is especially directed to following 0-glycan marker
structures of tissue
materials:
Core 1 type 0-glycan structures following the marker composition
NeuAc2Hex1HexNAc1,
preferably including structures SAa3Ga1(33Ga1NAc and/or
SAa3Ga1(33(Saa6)Ga1NAc;
and Core 2 type 0-glycan structures following the marker composition NeuAco_
2Hex2HexNAc2dHexo_l, more preferentially further including the glycan series
NeuAco_
2Hex2+nHexNAc2+ndHexO_1, wherein n is either 1, 2, or 3 and more
preferentially n is 1 or 2,
and even more preferentially n is 1;
more specifically preferably including R1Ga1(34(R3)G1cNAc(36(R2Ga1(33)Ga1NAc,
wherein Rl and R2 are independently either nothing or sialic acid residue,
preferably a2,3-
linked sialic acid residue, or an elongation with HexnHexNAcn, wherein n is
independently an
integer at least 1, preferably between 1-3, most preferably between 1-2, and
most preferably
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1, and the elongation may terminate in sialic acid residue, preferably a2,3-
linked sialic acid
residue; and
R3 is independently either nothing or fucose residue, preferably a1,3-linked
fucose residue.
It is realized that these structures correlate with expression of 06G1cNAc-
transferases
synthesizing core 2 structures.
Preferred qualitative and quantitative complete N-glycomes of tissue materials
High-mannose type and glucosylated N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of high-mannose type and glucosylated N-glycans according to the formula:
Hexn3HexNAcn4,
wherein n3 is 5, 6, 7, 8, 9, 10, 11, or 12, and n4 = 2.
According to the present invention, within total N-glycomes of tissue
materials the major
high-mannose type and glucosylated N-glycan signals preferentially include the
compositions
with 5< n3 < 10: Hex5HexNAc2 (1257), Hex6HexNAc2 (1419), Hex7HexNAc2 (1581),
Hex8HexNAc2 (1743), Hex9HexNAc2 (1905), and HexlOHexNAc2 (2067);
and more preferably with 5< n3 < 9: Hex5HexNAc2 (1257), Hex6HexNAc2 (1419),
Hex7HexNAc2 (1581), Hex8HexNAc2 (1743), and Hex9HexNAc2 (1905).
Low-mannose type N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of low-mannose type N-glycans according to the formula:
Hexn3HexNAcn4dHexn5,
wherein n3 is 1, 2, 3, or 4, n4 = 2, and n5 is 0 or 1.
According to the present invention, within total N-glycomes of tissue
materials the major low-
mannose type N-glycan signals preferably include the compositions with 2< n3 <
4:
Hex2HexNAc2 (771), Hex3HexNAc2 (933), Hex4HexNAc2 (1095), Hex2HexNAc2dHex
(917), Hex3HexNAc2dHex (1079), and Hex4HexNAc2dHex (1241); and more preferably
when n5 is 0: Hex2HexNAc2 (771), Hex3HexNAc2 (933), and Hex4HexNAc2 (1095).
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As demonstrated in the present invention by glycan structure analysis of
tissue materials,
preferably this glycan group in tissue materials includes the molecular
structures:
(Mana)1_3Man(34G1cNAc(34(Fuca6)0_1G1cNAc within the glycan signals 771, 917,
933, 1079,
1095, and 1095, and
the preferred low-Man structures includes structures common all tissue
material types, tri-
Man and tetra-Man structures according to the Examples,
(Mana)0_1Mana6(Mana3)Man(34G1cNAc(34(Fuc(x6)0_1G1cNAc, more preferably the tri-
Man
structures:
Mana6(Mana3)Man(34G1cNAc(34(Fuc(x6)0_1G1cNAc
even more preferably the abundant molecular structure:
Mana6(Mana3)Man(34G1cNAc(34G1cNAc within the glycan signa1933.
Quantitative analysis directed to the low-Man components
Beside the qualitative variations the low-Man glycans have specific value in
quantitative
analysis of tissue materials. The present invention revealed that the low-Man
glycans are
especially useful for the analysis of status of the cells. For example the
analysis in the
Examples revealed that the amounts of the glycans vary between total tissue
profiles and
specific organelles, preferably lysosomes.
The group of low-Man glycans form a characteristic group among glycome
compositions. The
relative total amount of neutral glycans is notable in average human tissues.
The glycan group
was revealed also to be characteristic in cancerous tissues and tumorsa with
total relative
amount of neutral glycomes increased. The difference is more pronounced within
lysosomal
organelle-specific glycome, wherein low-Man structures accounted nearly 50% of
the neutral
protein-linked glycome. Glycome analysis of tissue materials is especially
useful for methods
for development of binder reagents for separation of different tissue
materials.
The invention is directed to analysis of relative amounts of low-Man glycans,
and to the
specific quantitative glycome compositions, especially neutral glycan
compositions,
comprising about 0 to 50 % of low-Man glycans, more preferably between about 1
to 50 % of
solid tissue glycomes, for the analysis of tissue materials according to the
invention, and use
of the composition for the analysis of tissue materials.
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Fucosylated high-mannose type N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of fucosylated high-mannose type N-glycans according to the formula:
Hexn3HexNAcn4dHexn5,
wherein n3 is 5, 6, 7, 8, or 9, n4 = 2, and n5 = 1.
According to the present invention, within total N-glycomes of tissue
materials the major
fucosylated high-mannose type N-glycan signal preferentially is the
composition
Hex5HexNAc2dHex (1403).
Soluble glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of neutral soluble N-glycan type glycans according to the formula:
Hexn3HexNAcn4,
wherein n3 is 1, 2, 3, 4, 5, 6, 7, 8, or 9, and n4 = 1.
Within total N-glycomes of tissue materials the major soluble N-glycan signals
include the
compositions with 4< n3 < 8, more preferably 4< n3 < 7: Hex4HexNAc (892),
Hex5HexNAc (1054), Hex6HexNAc (1216), Hex7HexNAc (1378). In the most preferred
embodiment of the present invention, the major glycan signal in this group
within total neutral
glycomes of tissue materials is Hex5HexNAc (1054).
Neutral monoantennary or hybrid-type N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of neutral monoantennary or hybrid-type N-glycans according to the formula:
Hexn3HexNAcn4dHexn5,
wherein n3 is an integer greater or equal to 2, n4 = 3, and n5 is an integer
greater or equal to
0.
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According to the present invention, within total N-glycomes of tissue
materials the major
neutral monoantennary or hybrid-type N-glycan signals preferentially include
the
compositions with 2< n3 < 8 and 0< n5 < 2, more preferentially compositions
with 3< n3 <
6 and 0< n5 < 1, with the proviso that when n3 = 6 also n5 = 0: preferentially
Hex4HexNAc3
(1298), Hex4HexNAc3dHex (1444), Hex5HexNAc3 (1460), and Hex6HexNAc3 (1622).
Neutral complex-type N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of neutral complex-type N-glycans according to the formula:
Hexn3HexNAcn4dHexn5,
wherein n3 is an integer greater or equal to 3, n4 is an integer greater or
equal to 4, and n5 is
an integer greater or equal to 0.
Within the total N-glycomes of tissue materials the major neutral complex-type
N-glycan
signals preferentially include the compositions with 3< n3 < 8, 4< n4 < 7, and
0< n5 < 4,
more preferentially the compositions with 3< n3 < 5, n4 = 4, and 0< n5 < 1,
with the proviso
that when n3 is 3 or 4, then n5 = 1: Hex3HexNAc4dHex (1485), Hex4HexNAc4dHex
(1647),
Hex5HexNAc4 (1663), Hex5HexNAc4dHex (1809); and even more preferentially also
including the composition Hex3HexNAc5dHex (1688).
In another embodiment of the present invention, the N-glycan signal
Hex3HexNAc4dHex
(1485) contains non-reducing terminal GIcNAc(3, and more preferentially the
total N-glycome
includes the structure:
G1cNAc(32Mana3(G1cNAc(32Mana6)Man(34G1cNAc(34(Fuca6)G1cNAc (1485).
In yet another embodiment of the present invention, within the total N-glycome
of tissue
materials, the N-glycan signal Hex5HexNAc4dHex (1809), more preferentially
also
Hex5HexNAc4 (1663), contain non-reducing terminal 01,4-Gal. Even more
preferentially the
total N-glycome includes the structure:
Gal(34G1cNAc(32Mana3(Gal(34G1cNAc(32Mana6)Man(34G1cNAc(34G1cNAc (1663); and in
a
further preferred embodiment the total N-glycome includes the structure:
Gal(34G1cNAc(32Mana3(Gal(34G1cNAc(32Mana6)Man(34G1cNAc(34(Fuca6)G1cNAc (1809).
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Neutral fucosylated N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of neutral fucosylated N-glycans according to the formula:
Hexn3HexNAcn4dHexn5,
wherein n5 is an integer greater than or equal to 1.
Within the total N-glycomes of tissue materials the major neutral fucosylated
N-glycan
signals preferentially include glycan compositions wherein 1< n5 < 4, more
preferentially 1<
n5 < 3, even more preferentially 1< n5 < 2, and further more preferentially
compositions
Hex3HexNAc2dHex (1079), more preferentially also Hex2HexNAc2dHex (917), and
even
more preferentially also Hex5HexNAc4dHex (1809).
The inventors further found that within the total N-glycomes of tissue
materials a major
fucosylation form is N-glycan core a1,6-fucosylation. In a preferred
embodiment of the
present invention, major fucosylated N-glycan signals contain
GIcNAc(34(Fuca6)GIcNAc
reducing end sequence.
Neutral N-glycans with non-reducing terminal HexNAc
The present invention is especially directed to glycan compositions
(structures) and analysis
of neutral N-glycans with non-reducing terminal HexNAc according to the
formula:
Hexn3HexNAcn4dHexn5,
wherein n4 > n3.
Preferably these glycan signals include Hex3HexNAc4dHex (1485) in all tissue
materials.
Acidic hybrid-type or monoantennary N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of acidic hybrid-type or monoantennary N-glycans according to the formula:
NeuAcn1NeuGcõ2Hexn3HexNAcn4dHexn5SPn6,
wherein nl and n2 are either independently 1, 2, or 3; n3 is an integer
between 3-9; n4 is 3; n5
is an integer between 0-3; and n6 is an integer between 0-2; with the proviso
that the sum
nl+n2+n6 is at least 1.
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Within the total N-glycomes of tissue materials the major acidic hybrid-type
or
monoantennary N-glycan signals preferentially include glycan compositions
wherein 3< n3 <
6, more preferentially 3< n5 < 5, and further more preferentially composition
NeuAcHex4HexNAc3dHex (1711).
Acidic complex-type N-glycans
The present invention is especially directed to glycan compositions
(structures) and analysis
of acidic complex-type N-glycans according to the formula:
NeuAcn1NeuGcõ2HexoHexNAcn4dHexn5SPn6,
wherein nl and n2 are either independently 1, 2, 3, or 4; n3 is an integer
between 3-10; n4 is
an integer between 4-9; n5 is an integer between 0-5; and n6 is an integer
between 0-2; with
the proviso that the sum nl+n2+n6 is at least 1.
Within the total N-glycomes of tissue materials the major acidic complex-type
N-glycan
signals preferentially include glycan compositions wherein 4< n4 < 8, more
preferentially 4 <
n4 < 6, more preferentially 4< n4 < 5, and further more preferentially
compositions
NeuAcHex5HexNAc4 (1930), NeuAcHex5HexNAc4dHex (2076), NeuAc2Hex5HexNAc4
(2221), NeuAcHex5HexNAc4dHex2 (2222), and NeuAc2Hex5HexNAc4dHex (2367).
Modified glycan types
The inventors found that tissue material total N-glycomes; and soluble+N-
glycomes further
contain characteristic modified glycan signals, including sialylated
fucosylated N-glycans,
multifucosylated glycans, sialylated N-glycans with terminal HexNAc (the N>H
and N=H
subclasses), and sulphated or phosphorylated N-glycans, which are subclasses
of the
abovementioned glycan classes. According to the present invention, their
quantitative
proportions in different tissue materials have characteristic values as
described in Tables 8
and 13.
Phosphorylated and sulphated glycans
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Specifically, major phosphorylated glycans typical to tissue materials, more
preferentially to
lysosomal organelle glycomes, include Hex5HexNAc2(HPO3) (1313),
Hex6HexNAc2(HPO3)
(1475), and Hex7HexNAc2(HPO3) (1637).
Preferred combinations of glycan types in complete glycomes
The preferred complete glycomes of tissue materials include low-mannose type,
hybrid-type
or monoantennary, hybrid, and complex-type N-glycans,
which more preferentially contain fucosylated glycans, even more
preferentially also
sialylated glycans, and further more preferentially also sulphated and/or
phosphorylated
glycans;
and most preferentially also including soluble glycans as described in the
present invention.
In a preferred embodiment of the present invention the tissue material total N-
glycome
contains the three glycan types: 1) high-mannose type, 2) hybrid-type or
monoantennary, and
3) complex-type N-glycans; and more preferably, in the case of solid tissues
or cells also 4)
low-mannose type N-glycans; and further more preferably, in the case of solid
tissues or cells
additionally 5) soluble glycans.
In a preferred embodiment of the preferred glycan type combinations within the
tissue
material complete glycomes, their relative abundances are as described in
Tables 8 and 13.
Characterization of 0-linked and glycolipid glycomes see Example 19
Glycolipid glycomes
The invention revealed that the present method is useful for the
characteization of 0- glycans
and glycolipids. It is realized that the key glycolipid structures easily
analyzable are based on
common Iactosylceramide backbone structure cleavable by ceramide glycanase.
The key
structural families present varyingly on cells are
1) Neolacto- and Lacto families. The Iactoriasyl(GIcNAc(33Ga1(34GIc(3Cer)
based
lactosamine (Ga1(33GIcNAc and/or Ga1(34GIcNAc) were present in all cell types.
2) Ganglioseries structures comprising core structures
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[Ga1NAc(34],n[(SA(x3)]nGal(34G1c(3Cer, wherein m and n are integers 0 or 1,
and SA is
sialic acid preferably Neu5Ac, the structures can be further elongated from
the
terminal Ga1NAc(34 and/or SAa3, preferably both m and n are 1.
3) Globoseries. The globotriasyl (Gal(xGal(34G1c(3Cer) based structures.
4) Fucosyllactosyl and sialyllactosyl ceramide, with Fuca2'LacCer, and
SAa'LacCer
based structures.
Exoglycosidase digestions and mass spectrometric fragmentation analyses
suggested the
presence of various glycolipid types including these glycolipid classes: lacto-
, neolacto-,
ganglio-, globo-, and isoglobo-type structures. By use of relative
quantitation methods,
quantitative data of these glycan classes could be compared within cell types
and between cell
types, as demonstrated in the Examples of the present invention. Cell types
were found to
differ both in qualitative and in quantitative expression of these glycan
classes.
Quantitative and qualitative analysis of cell lipid glycomes
The Table "Examples of glycosphingolipid glycan classification" reveals
quantitative
differences in various classes of glycolipids. The glycolipid classes are in
the example
produced based on monosaccharide compositions. The Hex2-group is corresponds
to
lactosylceramide, the trisaccahride was associated mainly to
Lactotriasylceramide
G1cNAc(33LacCer (especially preferred s hESC-marker). The Gb-group was
characterised to
contain the Gal(33isogloboside, Gal(33Ga1NAc(3Gala3Ga1(34Glc(3Cer in the hESC,
glycolipids
(see Example about fragmentation mass spectrometry of glycolipids). The L1
glycan groups
with characteristic monosaccharide compositions were analysed to contain lacto-
, and/or
neolacto-glycolipids such as Gal(33G1cNAc(33Ga1(34Glc(3Cer and
Gal(34G1cNAc(33Ga1(34Glc(3Cer respectively in all analyzed cell types and for
hESC cells
further comprise Gal(33Ga1NAc(34(SA(x3)Gal(34G1c(3Cer (see Example about
fragmentation
mass spectrometry of glycolipids), the groups. Fractions L2-L3 comprise
elongated variants
of the previous structures. The invention is further directed to qunatitation
of subspecies
within the groups and providing quantitative comparisions of the different
glycolipid family
structures for the cells
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The glycosidase analysis performed reveal presence of terminal Gal(34G1cNAc
and
Gal(33G1cNAc structures in the cells types indicating Neolacto- and Lacto-
glycolipid series
according ot the invention. These appear to be characteristic ot cell types.
The invention is directed to the use of the present glycomics analysis methods
for
oligosaccharides released from glycolipids of a cell type according to the
invention. The
invention is further directed to quantitative mass spectrometric analysis by
the present
methods, when the signal profile is quantitated according to the invention and
combination of
the glycolipid glycome analysis with a protein glycome analysis, preferably
with analysis of
0-glycans and/or N-glycans. The quantitative data is compared between cell or
tissue types
The preferred classification for the quantitative analysis by mass
spectrometry is represented
in the Table "Examples of glycosphingolipid glycan classification". The
invention is
preferably directed to glycolipid glycomes of the cells comprising the signals
in the %-
amounts in the ranges defined by smallest and largest numbers (preferably 5 %
is added to
largest and 5% is subtracted from the smallest if possible to allow
experimental variation)
according to the Table.
The invention is directed to glycolipid oligosaccharide compostions isolated
from cells
according to invention and compositions thereof in complex with analysis
matrix.
0-glycan glycome analysis
The Table "Examples of 0-linked glycan classification" reveals quantitative
differences in
various classes of glycolipids. The 0-glycan classes are in the example
produced based on
monosaccharide compositions. The 01-group correspond to characteristic core 1
structures
and 02 group core 2 glycans, the fucosylation and sialyaltion produce
characteristic changes
in the quantitative 0-glycan glycomes.
A preferred classification for the quantitative analysis by mass spectrometry
is represented in
the Table "Examples of 0-linked glycan classification". The invention is
preferably directed
to glycolipid glycomes of the cells comprising the signals in the %-amounts in
the ranges
defined by smallest and largest numbers (preferably 5 % is added to largest
and 5% is
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subtracted from the smallest if possible to allow experimental variation)
according to the
Table. The invention is directed to 0-glycome oligosaccharide compostions
isolated from
cells according to invention and compositions thereof in complex with analysis
matrix.
The high relative amounts neutral fucosylated 0-glycans are preferably
characteristic for a
cell type and low amounts are preferred markers for other cells cells. The
invention is further
directed to quantitation of sulphated or fosforylated glycolipids.
The invention is directed to the use of the present glycomics analysis methods
for
oligosaccharides released from 0-glycans of a cell type according to the
invention. The
invention is further directed to quantitative mass spectrometric analysis by
the present
methods, when the signal profile is quantitated according to the invention and
combination of
the 0-glycan glycome analysis with a with analysis of glycolipid
oligosaccharide and/or N-
glycan glycomes. The quantitative data is compared between cell or tissue
types
Comparision of the glycomes
Comparison of qualitative and quantitative glycan data from N- and/or 0-linked
glycans
revealed that cell types express specific modulation of the terminal epitopes
for example by
fucosylation and/or sialylation. The inventors further found that these
different glycan types
were differently modified by terminal epitopes both within cell type, such as
fucosylation and
type 1 and type 2 chain expression that were found to differ between 0-glycans
and
glycolipids of different cell types. This characterized more completely useful
glycan epitope
selections in each cell type for their identification and manipulation
according to the methods
of the present invention.
The present invention is specifically directed to representing glycan
structures in cells as
combined relatively quantitative representation of glycan type, glycan class,
and/or glycan
epitope expression. In another embodiment of the present invention, glycans
are selected
based on their cell type specific qualitative or quantitative expression, such
as cell type
specificity, abundancy, or functionality. The present invention is further
directed to using such
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selected glycan structures in cells for identification and/or manipulation of
cells according to
the methods of the present invention.
Comparision of at least two glycomes from specific cell preparations
The invention is directed to characterization to the method wherein at least
two glycomes,
selected from the group of conjugated glycomes: N-glycome, 0-glycome, and
lipid linked
glycome; are determined from at least two cell populations according to the
invention and the
data from both or all glycomes are compared between the cells quantitatively.
In a preferred
embodiment all three glycomes are analyzed.
In a preferred embodiment the two glycomes comprise at least one protein
linked glycome,
preferably N-linked glycome, which is convienient to analyze. In another
preferred
embodiment a glycolipid glycome is compared together with comparision a
protein linked
glycome. In another preferred embodiment two protein linked glycomes, N-
glycome and 0-
glycome, are determined according to theinvention. In a preferred embodiment
the glycomes
to be compared includes both acidic and neutral glycans, in another embodiment
neutral
glycomes are compared or acidic glycomes are compared or both acid and neutral
glycomes
are compared only for part of conjugated glycomes and acidic or netral
glycomes are
compared for the rest. It is realized that neutral glycomes are often easier
to analyze and
compare a wealth of data.
The invention is further directed to use of the glycome analysis together with
specific binder
analysis especially analysis of terminal epitopes..
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EXAMPLE 1. Glycan isolation and analysis.
EXAMPLES OF GLYCAN ISOLATION METHODS
Glycan isolation. N-linked glycans are preferentially detached from cellular
glycoproteins by
F. meningosepticum N-glycosidase F digestion (Calbiochem, USA) essentially as
described
previously (Nyman et al., 1998), after which the released glycans are
preferentially purified
for analysis by solid-phase extraction methods, including ion exchange
separation, and
divided into sialylated and non-sialylated fractions. For 0-glycan analysis,
glycoproteins are
preferentially subjected to reducing alkaline 0-elimination essentially as
described previously
(Nyman et al., 1998), after which sialylated and neutral glycan alditol
fractions are isolated as
described above. Free glycans are preferentially isolated by extracting them
from the sample
with water.
Example of a glycan purification method. Isolated oligosaccharides can be
purified from
complex biological matrices as follows, for example for MALDI-TOF mass
spectrometric
analysis. Optionally, contaminations are removed by precipitating glycans with
80-90 % (v/v)
aqueous acetone at -20 C, after which the glycans are extracted from the
precipitate with 60
% (v/v) ice-cold methanol. After glycan isolation, the glycan preparate is
passed in water
through a strong cation-exchange resin, and then through C18 silica resin. The
glycan
preparate can be further purified by subjecting it to chromatography on
graphitized carbon
material, such as porous graphitized carbon (Davies, 1992). To increase
purification
efficiency, the column can be washed with aqueous solutions. Neutral glycans
can be washed
from the column and separated from sialylated glycans by elution with aqueous
organic
solvent, such as 25 % (v/v) acetonitrile. Sialylated glycans can be eluted
from the column by
elution with aqueous organic solvent with added acid, such as 0.05 % (v/v)
trifluoroacetic
acid in 25 % (v/v) acetonitrile, which elutes both neutral and sialylated
glycans. A glycan
preparation containing sialylated glycans can be further purified by
subjecting it to
chromatography on microcrystalline cellulose in n-butanol:ethanol:water
(10:1:2, v/v) and
eluted by aqueous solvent, preferentially 50 % ethanol:water (v/v).
Preferentially, glycans
isolated from small sample amounts are purified on miniaturized chromatography
columns
and small elution and handling volumes. An efficient purification method
comprises most of
the abovementioned purification steps. In an efficient purification sequence,
neutral glycan
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fractions from small samples are purified with methods including carbon
chromatography and
separate elution of the neutral glycan fraction, and glycan fractions
containing sialylated
glycans are purified with methods including both carbon chromatography and
cellulose
chromatography.
MALDI-TOF mass spectrometry. MALDI-TOF mass spectrometry is performed with a
Voyager-DE STR BioSpectrometry Workstation or a Bruker Ultraflex TOF/TOF
instrument,
essentially as described previously (Saarinen et al., 1999; Harvey et al.,
1993). Relative molar
abundancies of both neutral (Naven & Harvey, 1996) and sialylated (Papac et
al., 1996)
glycan components are assigned based on their relative signal intensities. The
mass
spectrometric fragmentation analysis is done with the Bruker Ultraflex TOF/TOF
instrument
according to manufacturer's instructions.
RESULTS
Examples of analysis sensitivity. Protein-linked and free glycans, including N-
and 0-glycans,
are typically isolated from as little as about 5 x 104 cells in their natual
biological matrix and
analyzed by MALDI-TOF mass spectrometry.
Examples of analysis reproducibility and accuracy. The present glycan analysis
methods have
been validated for example by subjecting a single biological sample,
containing human cells
in their natural biological matrix, to analysis by five different laboratory
personnel. The
results were highly comparable, especially by the terms of detection of
individual glycan
signals and their relative signal intensities, indicating that the reliability
of the present
methods in accurately describing glycan profiles of biological samples
including cells is
excellent. Each glycan isolation and purification phase has been controlled by
its
reproducibility and found to be very reproducible. The mass spectrometric
analysis method
has been validated by synthetic oligosaccharide mixtures to reproduce their
molar proportions
in a manner suitable for analysis of complex glycan mixtures and especially
for accurate
comparison of glycan profiles from two or more samples. The analysis method
has also been
successfully transferred from one mass spectrometer to another and found to
reproduce the
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analysis results from complex glycan profiles accurately by means of
calibration of the
analysis.
Examples of biological samples and matrices for successful glycan analysis.
The method has
been successfully implied on analysis of e.g. blood cells, cell membranes,
aldehyde-fixated
cells, glycans isolated from glycolipids and glycoproteins, free cellular
glycans, and free
glycans present in biological matrices such as blood. The experience indicates
that the method
is especially useful for analysis of oligosaccharide and similar molecule
mixtures and their
optional and optimal purification into suitable form for analysis.
EXAMPLE 2. Glycan profiling.
Generation of glycan profiles from mass spectrometric data. Figure 1A shows a
MALDI-
TOF mass spectrum recorded in positive ion mode from a sample of neutral N-
glycans. The
profile includes multiple signals that interfere with the interpretation of
the original sample's
glycosylation, including non-glycan signals and multiple signals arising from
single glycan
signals. According to the present invention, the mass spectrometric data is
transformed into a
glycan profile (Fig. 1B), which represents better the original glycan profile
of the sample. An
exemplary procedure is briefly as follows, and it includes following steps: 1)
The mass
spectrometric signals are first assigned to proposed monosaccharide
compositions e.g.
according to Table 1. 2) The mass spectrometric signals of ions in the
molecular weight are of
glycan signals typically show isotopic patterns, which can be calculated based
on natural
abundancies of the isotopes of the elements in the Earth's crust. The relative
signal intensities
of mass spectrometric signals near each other can be overestimated or
underestimated, if their
isotopic patterns are not taken into account. According to the present method,
the isotopic
patterns are calculated for glycan signals near each other, and relative
intensities of glycan
signals corrected based on the calculations. 3) Glycan ions are predominantly
present as
[M+Na]+ ions in positive ion mode, but also as other adduct ions such as
[M+K]+. The
proportion of relative signal intensities of [M+Na]+ to [M+K]+ ions is deduced
from several
signals in the spectrum, and the proportion is used to remove the effect of
[M+K]+ adduct
ions from the spectrum. 4) Other contaminating mass spectrometric signals not
arising from
the original glycans in the sample can optionally be removed from the profile,
such as known
contaminants, products of elimination of water, or in a case of permethylated
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oligosaccharides, undermethylated glycan signals. 5) The resulting glycan
signals in the
profile are normalized, for example to 100 %, for allowing comparison between
samples.
Figure 2A shows a MALDI-TOF mass spectrum recorded in negative ion mode from a
sample of neutral N-glycans. The profile includes multiple signals that
interfere with the
interpretation of the original sample's glycosylation, including non-glycan
signals and
multiple signals arising from single glycan signals. According to the present
invention, the
mass spectrometric data is transformed into a glycan profile (Fig. 2B), which
represents better
the original glycan profile of the sample. An exemplary procedure is briefly
as follows, and it
includes following steps: 1) The mass spectrometric signals are first assigned
to proposed
monosaccharide compositions e.g. according to Table 2. 2) The mass
spectrometric signals of
ions in the molecular weight are of glycan signals typically show isotopic
patterns, which can
be calculated based on natural abundancies of the isotopes of the elements in
the Earth's crust.
The relative signal intensities of mass spectrometric signals near each other
can be
overestimated or underestimated, if their isotopic patterns are not taken into
account.
According to the present method, the isotopic patterns are calculated for
glycan signals near
each other, and relative intensities of glycan signals corrected based on the
calculations. 3)
Glycan ions are predominantly present as [M-H]- ions in negative ion mode, but
also as ions
such as [M-2H+Na]- or [M-2H+K]-. The proportion of relative signal intensities
of e.g. [M-
H]- to [M-2H+Na]- and [M-2H+K]- ions is deduced from several signals in the
spectrum, and
the proportion is used to remove the effect of e.g. these adduct ions from the
spectrum. 4)
Other contaminating mass spectrometric signals not arising from the original
glycans in the
sample can optionally be removed from the profile, such as known contaminants
or products
of elimination of water. 5) The resulting glycan signals in the profile are
normalized, for
example to 100 %, for allowing comparison between samples.
EXAMPLE 3. Glycoprotein-linked glycans of human serum.
EXPERIMENTATION AND RESULTS
Glycoprotein-linked glycans in human serum. Protein-linked glycans,
corresponding to both
N- and 0-glycans, were isolated as described above from glycoproteins
precipitated from
serum of one donor, and analyzed by MALDI-TOF mass spectrometry. Major glycans
that
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were detected included 1) in the neutral glycan fraction, biantennary neutral
N-glycans (IgG
type), and Hex5_9HexNAc2 N-glycans (high-mannose type), and 2) in the
sialylated N-glycan
fraction, biantennary and larger sialylated N-glycans (orosomucoid type). The
obtained serum
protein-linked glycomes serve as a control database against which changes in
serum
glycoprotein glycans can be detected. It was noted that the neutral N-glycan
fraction isolated
according to the present invention could be detected by the present method
from significantly
smaller sample amounts than total N-glycan glycomes or sialylated N-glycan
glycomes. It is
suggested that the neutral glycan fraction isolated from human serum
glycoproteins allows
very sensitive detection of changes in serum protein-linked glycan profiles.
EXAMPLE 4. Profiling of human blood cell glycosylation.
EXPERIMENTATION AND RESULTS
Isolation and analysis of protein-linked glycans from human blood cells.
Mononuclear cells
and red blood cells are isolated from human blood for example by gradient
centrifugation in
solution containing sodium citrate (Vacutainer CPT, BD) and washed with
phosphate
buffered saline. Total cellular glycoproteins are precipitated and washed with
organic
solvents, such as aqueous solutions of acetone and ethanol. N-glycans and 0-
glycans are
isolated from the precipitated glycoproteins, divided into sialylated and
neutral glycan
fractions, and analyzed by MALDI-TOF mass spectrometry as described above.
White blood cell N-glycan profiles. The isolated neutral N-glycans included
glycan signals
corresponding to glycan groups according to the present invention: high-
mannose type, low-
mannose type, hybrid-type/monoantennary, and complex N-glycans, as well as
monosaccharide compositions Hex1_9HexNAc1, the latter possibly being free
glycans and not
protein-linked glycans. The isolated sialylated N-glycans included glycan
signals
corresponding to glycan groups according to the present invention: hybrid-
type,
monoantennary, and complex-type N-glycans. The resulting profiles differed
from both serum
glycoprotein glycan profiles and human tissue protein-linked glycans.
EXAMPLE 5. Profiling of human blood cell glycosylation after in vitro cell
culture.
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EXAMPLES OF CELL MATERIAL PRODUCTION
A lymphocyte subpopulation was isolated from human blood using Ficoll
isolation
(Amersham Pharmacia Biotech) and differentiation marker affinity purification.
Cells were
cultured for one week in a synthetic cell culture medium supplemented with 1%
human
serum.
EXPERIMENTATION AND RESULTS
N- and O-glycan profiling analysis. Glycans are isolated from cells, purified,
and analyzed by
MALDI-TOF mass spectrometry as described above. Typically, at least 25 and
more
preferentially 50 sialylated N-linked glycan signals, at least 10 and more
preferentially over
neutral N-linked glycan signals, at least 5 sialylated 0-glycan signals, and
at least 2 neutral
15 0-glycan signals can be detected and their relative abundancies determined
according to the
present method. Changes in glycan profiles and specific glycan structures upon
in vitro cell
culture can be detected by comparing the glycosylation data before and after
in vitro culture.
Examples of such analyses are presented below.
Glycan profile analysis. Protein-linked glycans were isolated from a human
lymphocyte
subpopulation grown in cell culture conditions for one week. In glycan
profiling analysis it
was observed that the relative amounts of five major neutral N-glycans at m/z
1257, 1419,
1581, 1743, and 1905, corresponding to [M+Na]+ ions of high-mannose type N-
glycans Hex5_
9HexNAc2, were significantly more abundant than the major sialylated N-
glycans. This is in
contrast to native human cells.
Sialic acid linkage analysis. The isolated sialylated N- and 0-glycans were
treated with
recombinant S. pneumoniae a2,3-sialidase essentially as desribed previously
(Saarinen et al.,
1999). The vast majority of the sialylated N-glycans were susceptible to
hydrolysis by the
enzyme, indicating that nearly all sialic acids in the sialylated N-glycans
were a2,3-linked.
This is in contrast to native human cells. a2,3-linkage was also predominant
in the 0-glycans.
Fucosylation analysis. By sequential digestions with specific glycosidases
performed
essentially as described previously (Hemmerich et al., 1995), except that
analysis of digestion
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results was performed by MALDI-TOF mass spectrometry according to the present
invention,
it was shown that fucose residues in 0-glycans occurred in the sialyl-Lewis x
epitope,
Neu5Aca2-3Ga1(31-4(Fucal-3)G1cNAc(3. In contrast, fucose residues were shown
by similar
sequential digestions to reside mainly in the N-glycan core sequence. The
latter result is in
accordance with the initial glycan grouping according to the present
invention, because
sialylated N-glycans in the original sample did not include significant
amounts of glycans
with proposed monosaccharide compositions with more than one deoxyhexose
residue, which
was not indicative of other fucose linkages apart from a1,6-Fuc of the N-
glycan core
sequence.
CONCLUSIONS
The present results demonstrate that cell glycosylation profiles can change
significantly upon
in vitro cell culture even in a relatively short time. In particular, the
present results indicate
that cell cultuvation can change the relative amounts of high-mannose N-
glycans compared to
other glycan types, N-glycan sialic acid linkages, and the overall
glycosylation profile. It was
also demonstrated that these glycosylation changes can be detected and
characterized
according to the present method. It was also shown that N- and 0-glycan
specific fucosylation
can be characterized by the present method.
EXAMPLE 6. Effect of culture conditions and cell line age on protein-linked
glycosylation
of SW 480 cells.
EXPERIMENTAL PROCEDURES
Cell culture. SW 480 cells (human colon adenocarcinoma cell line) were
cultured in 5% CO2
atmosphere in RPMI medium containing 10% fetal calf serum (FCS). Cells were
split twice a
week. The bigger the passage was, more rapidly the cells grew. For starvation
cells were
washed twice with RPMI containing 0.2 % FCS and incubated in the same medium
for 14
hours. Normally cells were approximately 70 % confluent, except for sample
"confluent"
which was 100 % confluent.
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Membrane protein isolation. Membrane protein isolation was performed at +0 -
+4 C. Cells
were washed with phosphate buffered saline and collected by centrifugation.
The cells were
incubated in hypotonic buffer (25 mM Tris-HC1 pH 8.5), broken by
homogenisation, and
brought back to isotonic buffer by the addition of NaC1 to 150 mM. The
homogenate was
centrifuged at 40,000 g in order to recover the cell membranes. The crude
membrane pellet
was homogenized in detergent buffer containing 25 mM Tris-HC1 pH 7.5, 150 mM
NaC1, and
1%(w/v) (3-octylglucoside. After incubation, the preparate was centrifuged at
100,000 g and
the supernatant that contained the detergent extracted proteins was collected.
Buffer salts and
the detergent were removed by cold acetone precipitation as described
previously (Verostek et
al., 2000).
RESULTS AND DISCUSSION
Glycan profiling of cultured cells in different cell culture phases. Figure 3
shows the neutral
N-glycan profile of SW 480 cells in growth phase, and the sialylated glycan
fraction is shown
in Figure 6. Figure 4 shows the relative abundancies of sialylated and neutral
glycans after
desialylation of the sialylated glycan fraction. Cell condition dependent
changes in neutral
glycan profiles are depicted in Figure 5. The present results indicate that
starvated, confluent,
and old cells resemble each other and are different from young cells with
respect to relative
abundancies of their glycan biosynthetic groups. It is indicated that high-
mannose type N-
glycans are significantly more abundant when compared to e.g. low-mannose type
N-glycans
or complex-type N-glycans including sialylated N-glycans, when the cells are
both young and
in growth phase. Also other changes, both in individual glycan signals and in
the overall
glycan profiles, are evident as presented in the Figures.
General change in cultured human cell lines. Similar phenomena were observed
in also other
human cell lines studied, including over 10 cancer cell lines as well as cell
lines derived from
normal human tissues. The major change occurring in the cells when they are
cultured in
vitro, is that high-mannose type N-glycans are significantly more abundant
when compared to
e.g. low-mannose type N-glycans or complex-type N-glycans including sialylated
N-glycans.
EXAMPLE 7. Analysis of antibody glycosylation and its modification in vitro.
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EXPERIMENTATION AND RESULTS
Analysis of antibody glycosylation. Monoclonal antibodies (mAbs) are typically
produced as
recombinant proteins in mammalian cell lines or in plants, and polyclonal
antibodies can be
purified from e.g. serum by methods known in the art. IgG is a glycoprotein
with two N-
glycosylation sites per molecule (one per heavy chain), but occasionally also
two (or 2n) extra
N-glycosylation sites per molecule (one per variable region, or n). The
structures of the
conserved N-glycans are highly conserved (REF1), but they can change due to
e.g.
disturbances in the recombinant protein production. The present method as
described above
allows for detection of these abnormalities, as demonstrated by model mAbs
analyzed by the
present method, as described below. The glycan profiles were analyzed from
glycan pools
isolated by N-glycosidase F, or N-glycosidase A (from almonds; Calbiochem,
USA) in the
case of plant glycoproteins, and analyzed as described in the preceding
Examples.
In an example where antibody molecules contain abnormal N-glycans in the
conserved site,
glycan signals arising from the abnormal glycans are observed in increased
amounts
compared to the normal glycan signals that correspond to the monosaccharide
compositions
Hex3_5HexNAc4dHex1. Easily detected abnormal structures include mannose type N-
glycans
corresponding to the monosaccharide compositions Hex2_9HexNAc2.
In an example where antibody molecules contain normal N-glycans in the
conserved site, but
in abnormal relative amounts compared to each other, the abnormality can be
observed from
the glycan profile according to the present invention. Normal glycan profiles
of IgG
molecules are described in the literature (e.g. Raju et al., 2000). An example
of an abnormal
profile of normal glycans is such that the relative amounts of the
Hex3HexNAc4d14ex1
glycoforms are significantly increased compared to the Hex4_5HexNAc4dHex1
glycoforms,
which can give rise to side effects in the potential use of the antibody
molecule, such as
affinity towards receptors of the innate immunity and/or serum clearance
systems. Observed
examples (1. and 2.) of differential proportions of the glycoforms
Hex3HexNAc4dHex1 :
Hex3HexNAc4dHex1 : Hex3HexNAc4dHex1 are presented below together with examples
from
the literature (3. and 4.). The present results suggest that the method of
glycan analysis
according to the present invention is useful in the characterization of
recombinant antibodies.
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No. Proportions IgG source Glycosylation state
1. 3: 2 :1 Preparate of human serum IgG Abnormal
2. 10 : 5 :1 Recombinant glycoprotein Abnormal
3. 1: 1,8 : 1 Human serum, Raju et al. (2000) Normal
4. 1: 2 :1 Recombinant glycoprotein, Normal
Sheeley et al. (1997)
In an example where antibody molecules contain normal complex-type N-glycans
in the
conserved sites and abnormal glycan types in an extra N-glycosylation site,
the abnormality
can be observed in the glycan profile according to the present invention: in
this case the
abnormal variable region N-glycans and the normal conserved N-glycans occur in
approximate molar proportions of 1:1. For example, high-mannose type N-glycans
observed
in an extra variable region N-glycosylation site can give rise to side effects
in the potential use
of the antibody molecule, such as affinity towards receptors of the innate
immunity and/or
serum clearance systems.
In an example where antibody molecules are produced in plants, the glycan
profiles can differ
significantly from animal-type glycan profiles. For evaluating suitability of
glycosylation for
antibodies, the relationship of Hex3HexNAc4dHex1, less preferentially
Hex3HexNAc4, even
less preferentially Hex3HexNAc2dHexo_l, monosaccharide compositions, to other
glycan
signals is calculated, with the proviso that the deoxyhexose-containing N-
glycans can be
liberated by N-glycosidase F as well as N-glycosidase A enzymes, indicating
that they do not
contain a1,3-Iinked fucose in the N-glycan core. Generally, glycan profiles
generated by N-
glycosidase F and N-glycosidase A enzymes should not differ at all, if the
antibody is suited
for human use. As another means of evaluating the suitability of glycosylation
for antibodies,
non-animal type glycans should not appear in the glycan profile. These include
all glycan
signals corresponding to monosaccharide compositions containing pentose or
more than one
deoxyhexose. For reasons listed above, also high-mannose or low-mannose type N-
glycans
are not preferred in recombinant antibodies. In conclusion, the present method
allows for
effective and rapid evaluation of recombinant protein glycosylation, when they
are produced
in plants or other non-animal systems.
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In vitro modification of native preparations - galactosylation. It was
observed that an
antibody preparation had the relative amounts of the Hex3HexNAc4dHex0_1
glycoforms
significantly increased compared to the Hex4_5HexNAc4dHex0_1 glycoforms,
possibly causing
side effects in the use of the antibody. A normal glycoform profile of the
preparation was
restored by in vitro incubation with 0 1,4-galactosyltransferase, uridine
diphospho-galactose,
divalent cations, and suitable buffer and temperature as known in the art,
without denaturing
the antibody.
In vitro modification of native preparations - demannosylation. It was
observed that an
antibody preparation had abnormal variable region N-glycans, since its N-
glycan profile
showed the normal conserved N-glycans and high-mannose type N-glycans
occurring in
approximate molar proportions of 1:1 in the profile. High-mannose glycoforms
were removed
from the glycan profile by in vitro incubation with either a-mannosidase from
Jack beans (C.
ensiformis; Sigma, USA) or Endoglycosidase H (Calbiochem, USA) in suitable
reaction
buffers and temperature as known in the art, without denaturing the antibody.
In these
reactions, the characterized reaction products were antibody preparations
containing Hexl
5HexNAc2 (Manal_41VIan(31-4GIcNAc(31-4GIcNAc) and GIcNAc variable region extra
N-
glycans, respectively.
EXAMPLE 8. Novel oligosaccharides of human milk and methods for production and
analysis.
EXPERIMENTATION AND RESULTS
Isolation and fractionation of human milk oligosaccharides. Samples of human
milk were
defatted by centrifugation and deproteinized by precipitation with 68 % (v/v)
aqueous ethanol.
The supernatant was dried and the residue was extracted with water. The water-
soluble
oligosaccharides were subjected to gel filtration chromatography in water.
High-molecular
weight neutral oligosaccharides were separated from sialylated
oligosaccharides at the void
volume by porous graphitized carbon chromatography as described above. The gel
filtration
chromatography phase resulted in enrichment of high-molecular weight neutral
and sialylated
oligosaccharides, as described below. Furthermore, from a sample of Lea"b"
milk, neutral
oligosaccharide fraction corresponding approximately to lacto-N-octaoses was
further
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fractionated by normal-phase high-performance liquid chromatography (HPLC) on
amino-
bonded silica column and porous graphitized carbon HPLC (I4ypercarb), with
absorbance
detection at 214 nm and either descending (normal-phase) or ascending
(Hypercarb)
acetonitrile gradient in aqueous mild ammonia solution. The enhanced
separation capabilities
resulted in isolation of a heptasaccharide not previously described in human
milk, as
described below.
Novel high-molecular weight oligosaccharides in human milk. By MALDI-TOF mass
spectrometry, novel neutral and acidic oligosaccharides were detected in high-
molecular
weight fractions of the gel filtration chromatography step. The detected
neutral and sialylated
oligosaccharides had molecular masses up to nearly 3700 Da and nearly 2600 Da,
respectively. Overall, the detected neutral oligosaccharides had apparent
monosaccharide
compositions Hexm+2HexNAcmdHexn, corresponding to (GaImGIcNAcmFucn)Ga1(31-
4GIc,
where (n < m) and (1 < m < 8), or even (m < 9) when (n = 0). Overall, the
detected sialylated
oligosaccharides had apparent monosaccharide compositions
NeuAcoHexm+2HexNAcmdHexn,
corresponding to (GaImGIcNAcmFucnNeuAco)GaI(31-4GIc, where (n < m) and (o < m)
and (1
< m < 4), or even (m < 5) when (n = 0) and (o = 1).
Novel fucosylated heptasaccharide in human milk. From the Lea"b" milk sample,
the following
isomeric neutral heptasaccharides eluting into the same fraction in normal-
phase HPLC, were
isolated and characterized: (I) Ga1(31-4(Fucal-3)GIcNAc(31-6(Ga1(31-4GIcNAc(31-
3)Ga1(31-
4GIc, (II) Ga1(31-4(Fucal-3)GIcNAc(31-6(Ga1(31-3GIcNAc(31-3)Ga1(31-4GIc, (III)
Ga1(31-
4(Fucal-3)GIcNAc(31-3(Ga1(31-4GIcNAc(31-6)Ga1(31-4GIc, (IV) Fucal-2GaI(31-
3GIcNAc(31-
3(GaI(31-4GIcNAc(31-6)GaI(31-4GIc, (V) Ga1(31-4GIcNAc(31-3Ga1(31-4(Fucal-
3)GIcNAc(31-
3Ga1(31-4GIc, and (VI) Ga1(31-3GIcNAc(31-3Ga1(31-4(Fucal-3)GIcNAc(31-3Ga1(31-
4GIc. The
structures were verified by sequential exoglycosidase digestions, mild acid
hydrolysis, 1D 1H-
NMR against known standard molecules, chromatographic coelution with known
standard
molecules, and MALDI-TOF mass spectrometry. The oligosaccharide III has not
been
previously described in human milk.
EXAMPLE 9. Oligosaccharide and glycoprotein compositions of bovine milk and
methods
for analysis.
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EXPERIMENTATION AND RESULTS
Analysis of bovine milk glycoprotein fractions. Neutral N-glycans were
analyzed from
samples of delipidated bovine milk and milk powder as described in the
preceding Examples.
Lactoferrin was also isolated from the same samples by cation exchange
chromatography and
the purified Iactoferrin was analyzed similarly as the total milk sample.
Figure 15. shows the
analysis results. It is evident from the glycan profiles that the
glycosylation of total milk
glycoproteins (Fig. 15A) and a single glycoprotein, Iactoferrin (Fig. 15B),
isolated among
them can differ significantly. Similar glycans occur also in the total milk
glycoprotein
fraction, but some they are enriched in the Iactoferrin fraction. Bovine
Iactoferrin has been
determined to contain significant amounts of complex N-glycans previously, but
it is evident
that there is significant sample-to-sample variation in Iactoferrin glycan
structures.
Analysis of human digestive tract tissue samples. Figure 16. shows the neutral
protein-linked
glycan analysis results obtained from human stomach (Fig. 16A) and colon (Fig.
16B). Both
these tissues contain similar glycan structures than Iactoferrin described
above. It is concluded
that there occurs in bovine milk neutral N-glycans that resemble human
digestive tract N-
glycans, and that a specific fraction of bovine milk can be selected to
resemble more closely
the human glycosylation in specific organs.
EXAMPLE 10. Protein-linked glycan profiling of human tissues.
EXPERIMENTAL PROCEDURES
Figures 7. through 14. show neutral protein-linked glycan analyses of protein-
linked
glycans, performed on paraffin-embedded and formalin-fixed archival human
tissue samples,
performed after deparaffinisation essentially as described above in the
preceding Examples.
The glycan isolation was however done by non-reductive alkaline elimination
essentially as
described by Huang et al. (2000). The m/z values in the Figures refer to the
Tables in the
present invention. Sialylated glycans were analyzed similarly
RESULTS AND DISCUSSION
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Tissue-specific glycosylation information and it use. It is seen that each
tissue depicted in the
Figures differs from another by 1) differences in overall glycan profiles, 2)
differences in
individual glycan signals, and/or 3) relative abundancies of individual glycan
signals or
glycan signal groups according to the present invention. The tissues can be
recognized based
on the glycan profiles and individual glycan signals that correspond to tissue-
specific
expression of glycans. The sialylated glycan fractions also contain similar
specific
information that can be combinated with the neutral glycan fraction
information to gain more
specificity and resolving power.
Individual differences in tissue glycosylation. Figures 11, 12, and 13 show
neutral protein-
linked glycan profiles of tissue samples from human stomach. The results are
presented as
groups 1-3 according to distinct blood-group specific glycan structures that
are present in each
donor group. Any future stomach sample can be grouped accordingly in its blood-
group
specific group. Also other individual differencies can be observed among
tissues from
different donors. It is therefore indicated that the present method can detect
individual
differencies in tissue glycosylation.
Disease-specific differences in glycosylation. Figure 14. shows the comparison
of neutral
glycan profiles from healthy lung tissue and tumor tissue from a patient with
non-small cell
lung cancer. It is seen that the disease state can be differentiated from the
healthy state
according to the methods of the present invention. Numerous cancer-associated
glycan signals
in the present patient that are changed in cancer are indicative of cancer
also in other patients.
Figure 9. shows a neutral protein-linked glycan profile of human ovary with
abnormal
growth. There are clear differences in the overall glycan profiles,
EXAMPLE 11. N-glycan profiling of rat liver.
RESULTS AND DISCUSSION
Total N-glycans were isolated and analyzed as described above from formalin-
fixed rat liver.
The major difference between the glycan profiles of rat and humans was in the
sialylated
glycan fraction, namely the occurrence of acetylated sialylated glycans at m/z
1972, 2263, and
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2305, corresponding to Ac1NeuAc1Hex5HexNAc4, Ac1NeuAc2Hex5HexNAc4, and
Ac2NeuAc2Hex5HexNAc4, respectively, as major sialylated N-glycans in rat
liver. It is
concluded that the present method is well suited to finding species-specific
differences in
glycosylation.
EXAMPLE 12. MALDI-TOF mass spectrometric glycolipid glycan profiling of
peripheral
blood mononuclear cells.
EXPERIMENTAL PROCEDURES AND RESULTS
Glycolipid and glycan isolation. Glycolipids were isolated from peripheral
blood
mononuclear cells essentially as described in (Karlsson et al., 2000).
Sphingoglycolipids were
detached by digestion with endoglycoceramidase from Macrobdella decora
(Calbiochem,
USA). After the reaction, liberated glycans were purified, fractionated into
sialylated and
neutral glycan fractions, and analyzed by MALDI-TOF mass spectrometry as
described in the
preceding Examples.
Glycolipid glycan profiles. Table 3 describes the detected glycan signals and
their proposed
monosaccharide compositions. The monosaccharide compositions correlate with
known
glycolipid core structures, such as gangliosides, lacto- and
neolactoglycolipids, and
globosides, and extensions of the core structures, such as poly-N-
acetyllactosamine chains.
Several glycans show fucosylation and/or sialylation of the core and extended
structures.
EXAMPLE 13. MALDI-TOF mass spectrometric profiling of cell surface glycans.
EPREIMENTAL PROCEDURES AND RESULTS
Cells, Mononuclear cells were isolated from human peripheral blood by Ficoll-
Hypaque
density gradient (Amersham Biosciences, Piscataway, USA) essentially as
described. The
surface glycoprotein glycans were liberated by mild trypsin treatment (80
micrograms/m1 in
PBS) at +37 degrees Celsius for 2 hours. The intact cells were harvested by
centrifugation,
and the supernatant containing the liberated glycans (at this stage as cell
surface glycoprotein
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glycopeptides) was taken for further analyses. The harvested cells and the
supematant were
subjected to Glycan profiling by protein N-glycosidase as described in the
preceding
examples. The N-glycan profiles of the supematant containing the cell surface
glycoprotein
glycopeptides, were compared against N-glycan profiles of the cells harvested
from the
trypsin treatment.
RESULTS
N-Glycan analyses of HMC cell surface glycopeptide glycomes. HMC were isolated
from
peripheral blood, treated with trypsin to release the surface glycoprotein
glycopeptides,
followed by release of glycopeptide glycans, and subjected to glycome
profiling as described
under Experimental procedures. In MALDI-TOF mass spectrometry of the
sialylated N-
glycan fractions, several glycon signals were detected in these samples. When
the resulting
glycome profile was compared to a corresponding glycome isolated from the
trypsin treated
cells, it could be observed that many sialylated components were enriched in
the surface
glycoprotein glycopeptide fraction, whereas some structures appeared to have
more
intracellular localization. Examples or the former structures are
(monosaccharide
compositions in parenthesis): m/z [M-H]" 1930 (SaHex5HexNAc4), 2221
(Sa2Hex5HexNAc4), 2222 (SaHex5HexNAc4dHex2), 2367 (Sa2Hex5HexNAc4dHex),
2368(SaHex5HexNAc4dHex3), 2587 (SaHex6HexNAc5dHex2), and 3024
(Sa3Hex6HexNAc5dHex). Examples of the latter are m/z 1873(SaHex5HexNAc3dHex),
and
203 5( S aHexHexNAc3 dHex).
EXAMPLE 14. Analyses of human tissue material and cell protein-linked glycan
structures.
EXPERIMENTAL PROCEDURES
Protein-linked glycans were isolated by non-reductive alkaline elimination
essentially as
described by Huang et al. (2000), or by N-glycosidase digestion to
specifically retrieve N-
glycans as described in the preceding Examples.
RESULTS AND DISCUSSION
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Tissue-specific glycosylation analyses and comparison of glycan profiles
between tissues.
Human tissue protein-linked glycan profiles were analyzed from lung, breast,
kidney,
stomach, pancreas, lymph nodes, liver, colon, larynx, ovaries, and blood cells
and serum. In
addition, cultured human cells were analyzed similarly. Tables 6 and 7 show
neutral and
acidic protein-linked glycan signals, respectively, observed in these human
tissues and cells
together with their classification into glycan structure groups. However, the
individual glycan
signals in each structure group varied from sample type to sample type,
reflecting tissue
material and cell type specific glycosylation. Importantly, in analyses of
multiple samples,
such as 10 samples from an individual human tissue type, glycan group feature
proportions
remain relatively constant with respect to variation in the occurrence of
individual glycan
signals.
Furthermore, it was observed that each tissue demonstrated a specific glycan
profile that
could be distinguished from the other tissues, cells, or blood or serum
samples by comparison
of glycan profiles according to the methods described in the present
invention. It was also
found out that glycan profile difference could be quantitated by comparing the
difference
between two glycan profiles, for example according to the Equation (resulting
in difference
expressed in %):
1 ra
difference = -I p,,a - p,,b
2 ,_1
wherein p is the relative abundance (%) of glycan signal i in profile a or b,
and n is the total
number of glycan signals. For example, the Equation reveals that human lung
and ovary tissue
protein-linked glycan profiles differ from each other significantly more than
human lung and
kidney tissue protein-linked glycan profiles differ from each other. Each
tissue or cell type
could be compared in this manner.
Comparison of glycosylation features between human tissue materials. Table 8
shows how
glycan signal structural classification according to the present invention was
applied to the
comparison of quantitative differences in glycan structural features in glycan
profiles between
human tissue materials. The results show that each sample type was different
from each other
with respect to the quantitative glycan grouping and classification.
Specifically, normal
human lung and lung cancer tissues were different from each other both in the
neutral glycan
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and sialylated glycan fractions with respect to the quantitative glycan
structure grouping. In
particular, lung cancer showed increased amounts of glycan signals classified
into terminal
HexNAc containing glycans. In analysis of individual glycan signals by 0-
glucosaminidase
digestion, it was found that lung cancer associated glycan signals, such as
Hex3HexNAc4dHex1, contained terminal(3-linked GIcNAc residues, correlating
with the
classification of these glycan signals into the terminal HexNAc (N>H and/or
N=H) glycan
groups. Furthermore, the human serum protein-linked glycan profile showed
significantly
lower amounts of high-mannose and especially low-mannose type N-glycan
signals. It is
concluded that the glycan grouping profile of human serum is significantly
different from the
corresponding profiles of solid tissues, and the present methods are suitable
for identification
of normal and diseased human tissue materials and blood or serum typical
glycan profiles
from each other.
Disease- and tissue-specific differences in glycan structure groups. Fig. 9
shows a neutral
protein-linked glycan profile of human ovary with abnormal growth. As
described above,
there are clear differences in the overall glycan profiles of Fig. 9 and other
human tissue
samples. In analyses of multiple samples of ovarian tissues, it was found that
benign abnormal
growth of the ovary is especially characterized by increased amounts of glycan
signals
classified as terminal HexNAc (N>H). In structural analyses by fragmentation
mass
spectrometry and combined 0-hexosaminidase and 0-glucosaminidase digestions,
the
corresponding terminal HexNAc glycan signals were found to include structures
with terminal
and sialylated (3-GaINAc, more specifically terminal and sialylated
Ga1NAc(34GIcNAc(3
(LacdiNAc) structures. According to the glycan structure classification, the
protein-linked
glycan profiles of normal ovarian tissue also contain increased amounts of
terminal HexNAc
glycans compared to other human tissues studied in the present invention, and
normal human
ovary preferentially also contains higher amounts of terminal and/or
sialylated LacdiNAc
structures than other human tissues on average. However, in malignant
transformation the
proportion of LacdiNAc structures among the protein-linked glycans of the
ovary are
decreased, and this is also reflected in the glycan grouping classification of
malignant ovarian
glycan profiles.
The analysis of protein-linked glycan profiles of human tissues revealed also
that tissues with
abundant epithelial structures, such as stomach, colon, and pancreas, contain
increased
amounts of small glycan structures, preferentially mucin-type glycans, and
fucosylated glycan
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structures compared to the other glycan structure groups in structure
classification. Similarly
as epithelial tissues, mucinous carcinomas were differentiated from other
carcinoma types
based on analysis of their protein-linked glycan profiles and structure groups
according to the
methods of the present invention.
EXAMPLE 15. Proton-NMR analysis of glycan fractions
Glycan material is liberated from biological material by enzymatic or chemical
means. To
obtain a less complex sample, glycans are fractionated into neutral and acidic
glycan fractions
by chromatography on a graphitized carbon as described above. A useful
purification step
prior to NMR analysis is gel filtration high-performance liquid chromatography
(HPLC). For
glycans of glycoprotein or glycolipid origin, a Superdex Peptide HR10/300
column
(Amersham Pharmacia) may be used. For larger glycans, chromatography on a
Superdex 75
HR10/300 column may yield superior results. Superdex columns are eluted at a
flow rate of 1
ml per minute with water or with 50-200 mM ammonium bicarbonate for the
neutral and
acidic glycan fractions, respectively, and absorbance at 205-214 nm is
recorded. Fractions are
collected (typically 0.5 - 1 ml) and dried. Repeated dissolving in water and
evaporation may
be necessary to remove residual ammonium bicarbonate salts in the fractions.
The fractions
are subjected to MALDI-TOF mass spectrometry and all fractions containing
glycans are
pooled.
Prior to NMR analysis, the pooled fractions are dissolved in deuterium oxide
and evaporated.
With glycan preparations containing about 100 nmol or more material, the
sample is finally
dissolved in 600 microliters of high-quality deuterium oxide (99.9-99.996%)
and transferred
to a NMR analysis tube. A roughly equimolar amount of an internal standard,
e.g. acetone, is
commonly added to the solution. With glycan preparations derived from small
tissue
specimens or from a small number of cells (5-25 million cells), the sample is
preferably
evaporated from very high quality deuterium oxide (99.996%) twice or more to
eliminate H20
as efficiently as possible, and then finally dissolved in 99.996% deuterium
oxide. These low-
material samples are preferebly analyzed by more sensitive NMR techniques. For
example,
NMR analysis tubes of smaller volumes can be used to obtain higher
concentration of
glycans. This kind of tubes include e.g. nanotubes (Varian) in which sample is
typically
dissolved in a volume of 37 microliters. Alternatively, higher sensitivity is
achieved by
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analyzing the sample in a cryo-NMR instrument, which increases the analysis
sensitivity
through low electronic noise. The latter techniques allow gathering of good
quality proton-
NMR data from glycan samples containing about 1-5 nmol of glycan material.
It is realized that numerous studies have shown that proton-NMR data has the
ability to
indicate the presence of several structural features in the glycan sample. In
addition, by
careful integration of the spectra, the relative abundancies of these
structural features in the
glycan sample can be obtained.
For example, the proton bound to monosaccharide carbon-1, i.e. H-1, yields a
distinctive
signal at the lower field, well separated from the other protons of sugar
residues. Most
monosaccharide residues e.g. in N-glycans are identified by their H-1 signals
(see Tables 4
and 5 for representative examples). In addition, the H-2 signals of mannose
residues are
indicative of their linkages.
Sialic acids do not possess a H-1, but their H-3 signals (H-3 axial and H-3
equatorial) reside
well separated from other protons of sugar residues. Moreover, differently
bound sialic acids
may be identified by their H-3 signals. For example, the Neu5Ac H-3 signals of
Neu5Aca2-
3Gal structure are found at 1.797 ppm (axial) and 2.756 ppm (equatorial). On
the other hand,
the Neu5Ac H-3 signals of Neu5Aca2-6Gal structure are found at 1.719 ppm
(axial) and
2.668 ppm (equatorial). By comparing the integrated areas of these signals,
the molar ratio of
these structural features is obtained.
Other structural reporter signals are commonly known and those familiar with
the art use the
extensive literature for reference in glycan NMR assignments.
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NMR References:
Fu D., Chen L. and O'Neill R.A. (1994) Carbohydr. Res. 261, 173-186
Hard K., Mekking A., Kamerling J.P., Dacremont G.A.A. and Vliegenthart J.F.G.
(1991)
Glycoconjugate J. 8, 17-28
Hard K., Van Zadelhoff G., Moonen P., Kamerling J.P. and Vliegenthart J.F.G.
(1992) Eur. J.
Biochem. 209, 895-915
Helin J., Maaheimo H., Seppo A., Keane A. and Renkonen O. (1995) Carbohydr.
Res. 266,
191-209
EXAMPLE 16. Lysosomal organelle-specific N-glycosylation.
EXPERIMENTAL PROCEDURES
Lysosomal protein sample including human myeloperoxidase was chosen to
represent
lysosomal organelle glycoproteins. The sample was digested with N-glycosidase
F to isolate
N-glycans, and they were purified for MALDI-TOF mass spectrometric analysis as
described
in the preceding Examples.
Alkaline phosphatase digestion was performed essentially according to
manufacturer's
instructions. After the digestion glycans were purified for MALDI-TOF mass
spectrometric
analysis as above.
RESULTS AND DISCUSSION
Neutral N-glycan profiles. The neutral N-glycan profile is presented in Figure
19 (upper
panel). The profile is dominated by low-mannose type and high-mannose-type N-
glycan
signals, comprising 49% and 46% of the total signal intensity, respectively.
Especially the
high proportion of low-mannose type N-glycans is characteristic to the sample
(Table 9,
upper panel).
Acidic N-glycan profiles. The acidic N-glycan profile is presented in Figure
19 (lower panel).
The profile is dominated by three glycan signal groups: 1) sulphated or
phosphorylated low-
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mannose type and high-mannose type N-glycans (Hex3_8HexNAc2SP), 2) fucosylated
hybrid-
type or monoantennary N-glycans (NeuAc1Hex3_4HexNAc3dHex1), and 3) fucosylated
complex-type N-glycans (NeuAc1Hex4_5HexNAc4dHex1_2). Unusual features of the
sample are
the high proportion of hybrid-type or monoantennary N-glycans (Table 9, lower
panel), high
fucosylation rate of hybrid-type, monoantennary, and complex-type N-glycans,
and the high
proportion of the characteristic sulphated or phosphorylated low-mannose type
and high-
mannose type N-glycans.
Phosphorylated N-glycans. Major glycan signals with phosphate or sulphate
ester (SP) in
their monosaccharide compositions were Hex5HexNAc2SP (1313), Hex6HexNAc2SP
(1475),
and Hex7HexNAc2SP (1637). When the acidic glycan fraction was subjected to
alkaline
phosphatase digestion, these major signals were specifically digested and
disappeared from
the acidic glycan spectrum as detected by MALDI-TOF mass spectrometry (data
not shown).
In contrast, the major glycan signals with sialic acids in their
monosaccharide compositions
were not digested, including NeuAc1Hex3HexNAc3d4ex1 (1549). This indicates
that the three
original glycan signals corresponded to phosphorylated N-glycans
(PO3H)Hex5HexNAc2,
(PO3H)Hex6HexNAc2, and (PO3H)Hex7HexNAc2, respectively, wherein PO3H denotes
phosphate ester.
The data further indicated that the present organelle-specific N-glycan
profile included
phosphorylated low-mannose type and high-mannose type N-glycans
(PO3H)Hex3HexNAc2
(989), (PO3H)Hex4HexNAc2 (1151), (PO3H)Hex5HexNAc2 (1313), (PO3H)Hex6HexNAc2
(1475), (PO3H)Hex7HexNAc2 (1637), and (PO3H)Hex8HexNAc2 (1799). In this glycan
profile the phosphorylated glycan residues are preferentially mannose
residues, more
preferentially a-mannose residues, and most preferentially 6-phospho-a-mannose
residues i.e.
(PO3H-6Mana).
EXAMPLE 17. Identification of specific glycosylation signatures from glycan
profiles of
malignant and normal human tissue samples based on quantitative glycomics.
EXPERIMENTAL PROCEDURES
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Normal lung (Sample I) and malignant lung tumor samples (Sample II) were
archival
formalin-fixed and paraffin-embedded tissue sections from cancer patients with
small cell
lung cancer. Protein-linked glycans were isolated from the representative
samples by non-
reductive 0-elimination, purified, and analyzed by MALDI-TOF mass spectrometry
as
described in the preceding Examples. In the present analysis, the total
desialylated protein-
linked glycomes from each sample were used.
To analyze the data and to find the major glycan signals associated with
either the normal
state or the disease, two variables were calculated for the comparison of
glycan signals
between the two samples:
1. absolute difference A=(SII - SI), and
2. relative difference R = A / SI,
wherein SI and SII are relative abundances of a given glycan signal in Sample
I(normal
human lung tissue) and Sample II (small cell lung cancer), respectively.
The glycan signals were further classified into structure classes by a one
letter code:
a b c d,
wherein a is either N (neutral) or S (sialylated); b is either L(Iow-mannose
type), M (high-
mannose type), H (hybrid-type or monoantennary), C (complex-type), S
(soluble), or 0
(other); c is either - (nothing), F (fucosylated), or E (multifucosylated);
and d is either -
(nothing), T (terminal HexNAc, N>H), or B (terminal HexNAc, N=H); as described
in the
present invention.
RESULTS
To identify protein-linked glycan signals correlating with malignant tumors in
total tissue
glycomes from cancer patient, major signals specific to either normal lung
tissue or malignant
small cell lung cancer tumors were selected based on their relative
abundances. When A and R
were calculated for the glycan profile datasets of the two samples, and the
glycan signals
thereafter sorted according to the values of A and R, the most significant
differing glycan
signals between the two samples could be identified (Table 10). Among the most
abundant
protein-linked glycan signals in the data, the following three signals had
emerged in II (new in
Table x): 1955, 2685, and 2905, corresponding to fucosylated complex-type N-
glycans. The
absolute differences of these signals were among the ten most large in the
data, indicating that
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they were significant. The signals that experienced the highest relative
increase in R were: 771
(R = 2.4, corresponding to 3.4-fold increase), 1905 (R = 2.2, corresponding to
3.2 fold
increase), and 1485 (R = 1.3, corresponding to 2.3 fold increase). The latter
signal
corresponded to complex-type N-glycans with terminal HexNAc. Significantly,
its +2Hex
counterpart 1809 was the most drastically reduced glycan signal in II with A =
-8.9 and R=-
0.4 (corresponding to 40% decrease in II), indicating a large change in
terminal HexNAc
expression. Moreover, the data easily shows that the glycan signals 1704,
1866, 1136, and
755 were not present in II.
Further, the obtained results, especially the identified major glycan signals
indicative of either
Sample II (high A and R) or Sample I(Iow A and R) were used to compile two
alternative
algorithms to produce glycan score with which lung cancer sample could be
identified from
normal lung sample based on the glycan signal values of the quantitative
glycome data:
1. glycan score = 1(1485) - 1(1809),
wherein I(1485) is the relative abundance of glycan signal 1485 and 1(1809) is
the relative
abundance of glycan signal 1809;
and alternatively:
2. glycan score = 1(1485) / 1(1809)
These glycan score algorithms yield high numerical value when applied to lung
cancer sample
and low numerical value when applied to normal lung sample.
DISCUSSION
The present identification analysis produced selected glycan signal groups,
from where
indifferent glycan signals have been removed and that have reduced noise or
background and
less observation points, but have the resolving power of the initially
obtained glycan profiles.
Such selected signal groups and their patterns in different sample types can
serve as a
signature for the identification of for example 1) normal human glycosylation,
2) tissue-
specific glycosylation, 3) disease states affecting tissue glycosylation, 4)
malignant cancer, 5)
malignancy in comparison to benign tumors, and grade of malignancy, or 6)
glycan signals
that have individual variation. Moreover, glycan signals can be identified
that do not change
between samples, including major glycans that can be considered as invariant
or
housekeeping glycans.
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The present data analysis identified potential glycan marker signals for
future identification of
either the normal lung of the lung tumor glycome profiles. Further, glycan
classes that are
associated with e.g. disease state in humans can be identified. Specifically,
the analysis
revealed that within the total complex-type N-glycan structure class in the
tissue glycomes,
terminal HexNAc (N>H) were typical to small cell lung cancer.
The method also allows identification of major glycans or major changes within
glycan
structure classes. For example, the proportion of multifucosylated glycans
within the total
tissue glycome profile was increased in II (1.1%) compared to I(0.3%). The
data analysis
identified this change predominantly to the appearance of glycan signals 1955
and 2685 in II.
EXAMPLE 18. Glycosphingolipid glycans of human cells.
EXPERIMENTAL PROCEDURES
Samples from human leukocytes were analyzed. Neutral and acidic
glycosphingolipid
fractions were isolated from the cells essentially as described (Miller-
Podraza et al., 2000;
Karlsson et al., 2000). Glycans were detached by Macrobdella decora
endoglycoceramidase
digestion (Calbiochem, USA) essentially according to manuacturer's
instructions, yielding the
total glycan oligosaccharide fractions from the samples. The oligosaccharides
were purified
and analyzed by MALDI-TOF mass spectrometry as described in the preceding
Examples for
the protein-linked oligosaccharide fractions. Proposed compositions for the
oligosaccharides
and signal nomenclature are presented in Tables 11 and 12 for the neutral and
acidic glycan
fractions, respectively.
RESULTS AND DISCUSSION
Leukocyte neutral lipid glycans. The analyzed mass spectrometric profile of
the
glycosphingolipid neutral glycan fraction is shown in Figure 20.
Structural analysis of the major neutral lipid glycans. The four major glycan
signals, together
comprising more than 75% of the total glycan signal intensity, corresponded to
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monosaccharide compositions Hex3HexNAc1 (730), Hex2HexNAc1 (568), Hex4HexNAc2
(1095), and Hex3HexNAc1dHex1 (876).
Acidic lipid glycans. The analyzed mass spectrometric profile of the hESC
glycosphingolipid
sialylated glycan fraction is shown in Figure 21. The four major glycan
signals, together
comprising more than 90% of the total glycan signal intensity, corresponded to
monosaccharide compositions NeuAc1Hex3HexNAc1 (997), NeuAc1Hex4HexNAc2 (1362),
NeuAc1Hex5HexNAc3 (1727), and NeuAc1Hex5HexNAc3dHex1 (1873).
Terniinal glycan epitopes that were demonstrated in the present experiments in
leukocyte
glycosphingolipid glycans include, as demonstrated by 01,4-galactosidase,
a1,3/4-fucosidase,
a1,2-fucosidase, and a2,3-sialidase digestions:
Gal
Ga1(34GIc (Lac)
Ga1(34GIcNAc (LacNAc type 2)
Non-reducing terminal HexNAc
Fuc
al,3-Fuc
Neu5Ac
Neu5Aca2,3
Neu5Aca2,6
EXAMPLE 19. Evaluation of glycan classes and epitopes in tissue cell types.
EXPERIMENTAL PROCEDURES
Human leukocytes from peripheral blood and cultured human cell types were
produced
essentially as described in the preceding Examples. Glycosphingolipid glycans
were isolated
from glycolipid fractions isolated from these cells by endoglycoceramidase
digestion; 0-
glycans were isolated by non-reductive alkaline 0-elimination with
concentrated ammonia in
saturated ammonium carbonate; all glycan fractions were isolated with
miniaturized solid-
phase extraction steps; glycans were analyzed by MALDI-TOF mass spectrometry;
terminal
glycan epitopes were analyzed by specific exoglycosidase enzymatic digestions
combined
with analysis by mass spectrometry; the analysis steps were performed as
described in the
present invention.
RESULTS AND DISCUSSION
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Mass spectrometric profiles providing relative quantitative information about
glycan signals
and specific exoglycosidase digestions, together with antibody, lectin, and
biochemical
characterization of the cell types as described above, was used to further
characterize tissue
cell types and compare them with different cell types, here cultured cells.
Table 14 describe
examples of combinatiorial characterization of glycan types associated with
each cell type.
Average values from cultured human cell types were used in the present
comparisons.
Analysis of glycolipid and/or 0-glycan structures and classes in addition to N-
glycan
structures and classes yielded a more complete characterization of the cell
types, revealed
further differences between cell types, and provided more glycan epitopes and
classes
associated with each cell type. In conclusion, combination of analysis of
different glycan
types and epitopes was useful in analysis and identification of cell types.
EXAMPLE 20. Purification methods.
Example of detergent method for preparation of cells for total cell glycome
analysis
Cell sample is preferably a cell pellet produced at cold temperature by
centrifuging cells but
avoiding distruption of the cells, optionally stored frozed and melted on ice.
The cells are
lysed on ice by the detergent 1% SDS, mixed by Vortex and optionally by
pipette tip as
physical degradation step, boiled on water bath for 5min and one volume of 10%
n-octyl-(3-
D-glucoside is added after which the sample is incubated at room temperature
for 15 min.
Detergents are used in amounts of 5 g1 for 200 000 - 3 million cells and 2 g1
for 200 000
cells.
Example of N-glycosidase F reaction
Na-phosphate buffer pH 7.3 is added to octyl-glycoside preparete from
detergent method so
that the added volume is 8 times the volume of the added SDS (or
octylglycoside solution),
for example 16 g1 for reaction containing of 2 g1 of each detergent, the final
concentration of
the phosphate buffer is 20 mM. 2.5 U of NGF (U=1 nmoUmin, Calbiochem) is added
and the
and the reaction is incubated overnight at 37 degrees of Celsius.
Preferred prepurification step in context of N-glycosidase reaction is acetone
precipitation. In
the prepurification the reaction vessel is cooled on ice. 9 volumes of ice
cold acetone is added
and reaction is mixed carefully and incubated for 15 min. at - 20 degrees of
Celsius. The
sample is centrifuged at 13 000 by desk eppendorf tube centrifuge. The
supernatant is
removed and the sample is mixed in 60 % (aq, vol/vol) ice cold methanol, 200
g160 % ice
cold methanol is added, the solution is mixed by Vortex and centrifuged as
above. The
supernatant is collected and the pellet is washed again with methanol as above
and
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supernatant is collected. Methanol is evaporated from the sample and glycan
fraction is
subjected to further purification.
Example of purification steps
Hydrophobic chromatography by C 18-alkyl-matrix
Commercial "Bond Elut" C 18-column of 100 mg of silica matrix can be used for
cell up to a
few million cells (up to 2-4 million, more preferably less than 2 million
cells). 500 mg
cartidge is preferred for cell amounts about 10 million or more. The sample
volumes of 100 -
500 g1 can be used. The elution volume is about 1 ml and eluent/washing
solution is ultrapure
water.
Carbon chromatography
Commercial "Alltech Carbograph" graphite column can be used, 150 mg graphite
is useful for
cell ranges of 200 000-to about 10 million cells, for amounts over 10 million
cells (up to at
least about 20 million cells) corresponding graphite column containing 300 mg
graphite is
preferred. The carbon may be also used in tip column format for amounts of
1000 - 200 000
cells or less the tip column is preferred. The columns separate neutral and
acidic glycans. The
invention is in specific embodiment directed a carbon column method involving
washing the
column with ammonium carbonate, this method specifically aimed for effective
removal ionic
organic type impurities remaining in the column.
The method is useful after the hydrophobic chromatography or in other method
for
concentration of larger sample volumes. Preferred volumes to be used in the
process for 150
mg column include elution of neutral glycans by 2.5 ml of 25% aqueous ACN
(acetonitrile)
and acidic glycans by 25% ACN - 0.05 % TFA, for 300 mg column the volumes are
5 ml and
for tip-column 40 g1.
Carbon tip column
A practical range tip column for about 1000 - 200 000 cells or less is build
in a Eppendorf
GELoader tip by narrowing the tip close to its narrower end (tip of of the
tip). The narrowing
may be produced by pressing the tip from two sides of the tip. In a preferred
embodiment the
narrowing is produced during the production of the disposable pipette tip and
more preferably
the column materials according to the invention for the uses according to the
invention are
prepacked in the column by the manufacturer. The narrowing is so thin that the
carbon
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particles (preferably graphitized carbon 120/400 mesh) remain in the column.
10 gl of carbon
suspension in 50 % ACN is poured into the tip column so that a bed 5 g1 bed of
carbon is
formed above the narrowing. Preferred practical tip sizes includes carbon
volumes of about
0.1 g1 to about 100 gl, more preferably from about 1 g1 to about 50 g1. The
preferred sample
volumes for practical type columns of about 5 to 20 g1 varies from about 10 to
100 g1. For
elution of neutral oligosaccharides from 5 g1 column 40 g1 of 25 % aqueous ANC
is used as
above and for acidic glycans corresponding amount of TFA-solution as above.
Optionally combined cation exchange and hydrophobic chromatography
Millipore Ziptip _08 tip is packed with 10 g1 washed H+ resin (preferably for
eaxample
BioRad AG50W-X8) in ethanol forming about 5 g1 bed of the resin. The column is
especially
useful combined method for both cationic and hydrophobic impurities. Practical
sample
volume for 5 g1 column is 10 g1 and elution volume 20 g1. Alternative resins
includes NH4+
counterion resins and combination of cation and anion exhange resins
preferably H+/Ac" -
resins, preferably so that H+resin is palced above the Ac resin. Preferred
resin volumes
include about 0.1 g1 to about 100 g1, more preferably from about 1 g1 to about
20 g1 and most
preferably from about 2 gX io a(3ovi 10 g1, for large volumes larger tip may
be used.
Cellulose chromatography in a tip column
10 g1 suspension containing cellulose (5 g1) mixed with 50 % ethanol in poured
in StarLab
TipOne filter tip (tip size 0.5-10 g1). Practical sample volume for 5 g1
column is produced as
follows: sample is soluted 10 or 20 gl,of water, and then mixed with 55 or 100
g1 of solvent A
(BuOH: EtOH in mixture of 10:1), column is washed by 5 x 60 g1 of solvent B
(BuOH:
EtOH:H2O in mixture of 10:1:2 vol/vol/vol) and column is eluted by 100 g1 of
50 % aq EtOH.
Preferred cellulose volumes include about 0.1 g1 to about 100 g1, more
preferably from about
1 g1 to about 50 g1 and most preferably from about 2 g1 to about 30 g1 for
large volumes
larger tip may be used.
EXAMPLE 21. Glycan purification with purification device.
Glycan samples: Detached N-glycan samples were separately prepared by N-
glycosidase
digestion of human cell material prepurified by precipitation by cold acetone,
extraction by
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cold methanol-water solution, and drying as described in the preceding
Examples; and N-
glycosidase digestion of glycoprotein.
Use of purification device: Thereafter, sample was applied in water to a
purification device
formed from interconnected miniaturized solid-phase extraction columns as
described in the
following.The sample was applied in water to a combined bed (1:1) of strong H+
form cation-
exchange resin (Bio-Rad) and C18-bonded silica (ZipTip), and the flowthrough
was eluted
with water directly to graphitized carbon column (Carbograph), wherein the
glycans were
concentrated. The carbon column was separated from the first column before
washing and
elution. The carbon column was washed with water, neutral N-glycans were
eluted with 25%
acetonitrile, and acidic N-glycans were eluted with 25% acetonitrile in 0.05%
trifluoroacetic
acid.
Analysis of purification efficiency: The eluate was dried and applied onto a
MALDI-TOF
mass spectrometry plate with MALDI matrix and mass spectrometry and data
analysis were
performed as described in the preceding Examples. The mass spectrum
demonstrated a profile
of efficiently purified N-glycans, demonstrating that direct coupling of
purification columns
produced efficient device for glycan purification.
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Table 1. Preferred neutral glycan compositions. Calculated mass-to-charge
ratios (calc. m/z) refer
to the first isotope signal of [M+Na]+ ion.
Proposed composition caic. m/z HexHexNAc5dHex 1364,51
HexHexNAc 406,13 Hex3HexNAc2dHex3 1371,49
Hex3 527,16 Hex7HexNAc 1378,45
HexHexNAcdHex 552,19 Hex4HexNAc2dHex2 1387,49
Hex2HexNAc 568,19 Hex2HexNAc5 1380,50
HexHexNAc2 609,21 Hex5NexNAc2dHex 1403,48
Hex4 689,21 Hex2HexNAc3dHex3 1412,52
Hex2HexNAcdHex 714,24 Hex6HexNAc2 1419,48
Hex3HexNAc 730,24 HexHexNAc6 1421,53
HexHexNAc2dHex 755,27 Hex3HexNAc3dHex2 1428,51
Hex2HexNAc2 771,26 Hex4HexNAc3dHex 1444,51
HexHexNAc3 812,29 HexHexNAc4dHex3 1453,54
Hex5 851,26 Hex5HexNAc3 1460,50
Hex2HexNAcdHex2 860,30 Hex2HexNAc4dHex2 1469,54
Hex4HexNAc 892,29 Hex3HexNAc4dHex 1485,53
HexHexNAc2dHex2 901,33 Hex9 1499,48
Hex2HexNAc2dHex 917,32 Hex4HexNAc4 1501,53
Hex3HexNAc2 933,32 HexHexNAc5dHex2 1510,57
HexHexNAc3dHex 958,35 Hex3HexNAc2dHex4 1517,55
Hex2HexNAc3 974,34 Hex2HexNAc5dHex 1526,56
Hex2HexNAcdHex3 1006,36 Hex4HexNAc2dHex3 1533,54
Hex6 1013,32 Hex8HexNAc 1540,50
HexHexNAc4 1015,37 Hex3HexNAc5 1542,56
Hex3HexNAcdHex2 1022,35 Hex5HexNAc2dHex2 1549,54
Hex5HexNAc 1054,34 Hex6HexNAc2dHex 1565,53
Hex2HexNAc2dHex2 1063,38 Hex3HexNAc3dHex3 1574,57
Hex3HexNAc2dHex 1079,38 Hex7HexNAc2 1581,53
Hex4HexNAc2 1095,37 Hex2HexNAc6 1583,58
HexHexNAc3dHex2 1104,41 Hex4HexNAc3dHex2 1590,57
Hex2HexNAc3dHex 1120,40 Hex5HexNAc3dHex 1606,56
Hex3HexNAc3 1136,40 Hex2HexNAc4dHex3 1615,60
Hex2HexNAcdHex4 1152,42 Hex6HexNAc3 1622,56
HexHexNAc4dHex 1161,43 Hex3HexNAc4dHex2 1631,59
Hex7 1175,37 Hex4HexNAc4dHex 1647,59
Hex2HexNAc4 1177,42 HexlO 1661,53
Hex2HexNAc2dHex3 1209,44 Hex5HexNAc4 1663,58
Hex6HexNAc 1216,40 Hex2HexNAc5dHex2 1672,62
HexHexNAc5 1218,45 Hex3HexNAc5dHex 1688,61
Hex3HexNAc2dHex2 1225,43 Hex5HexNAc2dHex3 1695,60
Hex4HexNAc2dHex 1241,43 Hex9HexNAc 1702,56
Hex5HexNAc2 1257,42 Hex4HexNAx5 1704,61
Hex2HexNAc3dHex2 1266,46 Hex6HexNAc2dHex2 1711,59
Hex3HexNAc3dHex 1282,45 Hex3HexNAc3dHex4 1720,63
Hex4HexNAc3 1298,45 Hex7HexNAc2dHex 1727,59
HexHexNAc4dHex2 1307,49 Hex2HexNAc6dHex 1729,64
Hex2HexNAc4dHex 1323,48 Hex4HexNAc3dHex3 1736,62
Hex8 1337,42 Hex8HexNAc2 1743,58
Hex3HexNAc4 1339,48 Hex3HexNAc6 1745,64
Hex2HexNAc2dHex4 1355,50 Hex5HexNAc3dHex2 1752,62
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Hex6HexNAc3dHex 1768,61 Hex4HexNAc7 2110,77
Hex3HexNAc4dHex3 1777,65 Hex6HexNAc4dHex2 2117,75
Hex7HexNAc3 1784,61 Hex3HexNAc5dHex4 2126,79
Hex4HexNAc4dHex2 1793,64 Hex7HexNAc4dHex 2133,75
Hex5HexNAc4dHex 1809,64 Hex4HexNAc5dHex3 2142,78
Hex2HexNAc5dHex3 1818,68 Hex13 2147,69
Hex11 1823,58 Hex8HexNAc4 2149,74
Hex6HexNAc4 1825,63 Hex5HexNAc5dHex2 2158,78
Hex3HexNAc5dHex2 1834,67 Hex6HexNAc5dHex 2174,77
Hex4HexNAc5dHex 1850,67 Hex8HexNAc2dHex3 2181,76
Hex6HexNAc2dHex3 1857,65 Hex3HexNAc6dHex3 2183,81
Hex10HexNAc 1864,61 Hexl2HexNac 2188,71
Hex5HexNAc5 1866,66 Hex7HexNAc5 2190,77
Hex7HexNAc2dHex2 1873,64 Hex4HexNAc6dHex2 2199,80
Hex2HexNAc6dHex2 1875,70 Hex5HexNAc6dHex 2215,80
Hex4HexNAc3dHex4 1882,68 Hex7HexNAc3dHex3 2222,78
Hex8HexNAc2dHex 1889,64 Hex2HexNAc7dHex3 2224,84
Hex3HexNAc6dHex 1891,69 Hex11HexNAc2 2229,74
Hex5HexNAc3dHex3 1898,68 Hex6HexNAc6 2231,79
Hex9HexNAc2 1905,63 Hex8HexNAc3dHex2 2238,78
Hex4HexNAc6 1907,69 Hex3HexNAc7dHex2 2240,83
Hex6HexNAc3dHex2 1914,67 Hex5HexNAc4dHex4 2247,81
Hex3HexNAc4dHex4 1923,71 Hex4HexNAc7dHex 2256,83
Hex7HexNAc3dHex 1930,67 Hex6HexNAc4dHex3 2263,81
Hex2HexNAc7dHex 1932,72 Hex5HexNAc7 2272,82
Hex4HexNAc4dHex3 1939,70 Hex7HexNAc4dHex2 2279,80
Hex8HexNAc3 1946,66 Hex4HexNAc5dHex4 2288,84
Hex5HexNAc4dHex2 1955,70 Hex5HexNAc5dHex3 2304,84
Hex6HexNAc4dHex 1971,69 Hex14 2309,74
Hex3HexNAc5dHex3 1980,73 Hex9HexNAc4 2311,79
Hex12 1985,63 Hex6HexNAc5dHex2 2320,83
Hex7HexNAc4 1987,69 Hex7HexNAc5dHex 2336,82
Hex4HexNAc5dHex2 1996,72 Hex4HexNAc6dHex3 2345,86
Hex5HexNAc5dHex 2012,72 Hex8HexNAc5 2352,82
Hex7HexNAc2dHex3 2019,70 Hex5HexNAc6dHex2 2361,86
Hex2HexNAc6dHex3 2021,76 Hex6HexNAc6dHex 2377,85
Hex11 HexNAc 2026,66 Hex8HexNAc3dHex3 2384,83
Hex6HexNAc5 2028,71 Hex3HexNAc7dHex3 2386,89
Hex8HexNAc2dHex2 2035,70 Hex12HexNac2 2391,79
Hex3HexNAc6dHex2 2037,75 Hex7HexNAc6 2393,85
Hex5HexNAc3dHex4 2044,73 Hex4HexNAc7dHex2 2402,88
Hex4HexNAc6dHex 2053,75 Hex6HexNAc4dHex4 2409,87
Hex6HexNAc3dHex3 2060,73 Hex5HexNAc7dHex 2418,88
HexlOHexNAc2 2067,69 Hex7HexNAc4dHex3 2425,86
Hex5HexNAc6 2069,74 Hex6HexNAc7 2434,87
Hex7HexNAc3dHex2 2076,72 Hex5HexNAc5dHex4 2450,89
Hex2HexNAc7dHex2 2078,78 Hex6HexNAc5dHex3 2466,89
Hex4HexNAc4dHex4 2085,76 Hex15 2471,79
Hex8HexNAc3dHex 2092,72 Hex7HexNAc5dHex2 2482,88
Hex3HexNAc7dHex 2094,77 Hex8HexNAc5dHex 2498,88
Hex5HexNAc4dHex3 2101,76 Hex5HexNAc6dHex3 2507,91
Hex9HexNAc3 2108,71 Hex6HexNAc6dHex2 2523,91
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Hex7HexNAc6dHex 2539,90 Hex15HexNAc2 2877,95
Hex4HexNAc7dHex3 2548,94 Hex8HexNAc7dHex 2905,04
Hex13HexNAc2 2553,85 Hex8Hexnac5dHex4 2937,05
Hex8HexNAc6 2555,90 Hex18 2957,95
Hex5HexNAc7dHex2 2564,94 Hex7HexNAc6dHex4 2978,08
Hex6HexNAc7dHex 2580,93 Hex17HexNAc 2998,98
Hex6HexNAc5dHex4 2612,95 Hex8HexNAc7dHex2 3051,09
Hex7HexNAc5dHex3 2628,94 Hex9HexNAc8 3124,11
Hex16 2633,85 Hex8HexNAc6dHex4 3140,13
Hex8HexNAc5dHex2 2644,94 Hex8HexNAc7dHex3 3197,15
Hex6HexNAc6dHex3 2669,97 Hex9HexNAc8dHex /
Hex7HexNAc6dHex2 2685,96 Hex7HexNAc6dHex6 3270,17
Hex5HexNAc7dHex3 2710,99 Hex9HexNAc6dHex4 3302,18
Hex14HexNAc2 2715,90 Hex8HexNAc7dHex4 3343,21
Hex6HexNAc7dHex2 2726,99 Hex9HexNAc8dHex2 3416,23
Hex7HexNAc7dHex 2742,98 HexlOHexNAc6dHex4 3464,24
Hex8HexNAc7 2758,98 HexlOHexNAc9 3489,24
Hex7Hexnac5dHex4 2775,00 Hex9HexNAc8dHex3 3562,28
Hex8HexNAc5dHex3 2790,99 Hex11 HexNAc6dHex4 3626,29
Hexl7 2795,90 HexlOHexNAc9dHex 3635,30
Hex7HexNAc6dHex3 2832,02 Hex9HexNAc8dHex4 3708,34
HexlOHexNAc9dHex2
/
Hex16HexNAc 2836,92 Hex8HexNAc7dHex7 3781,36
Hex9HexNAc6dHex 2864,01 Hex9HexNAc8dHex5 /
Hex6HexNAc7dHex3 2873,05 Hex7HexNAc6dHex10 3854,40
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Table 2. Preferred acidic glycan compositions. Calculated mass-to-charge
ratios (calc. m/z) refer to
the first isotope signal of [M-H]- ion.
Proposed composition caic. m/z NeuAcHex6HexNAc / 1483,49 /
NeuAcHexHexNAc 673,23 NeuAcHex3HexNAc3SP 1483,45
NeuAcHexHexNAcdHex 819,29 NeuAc2Hex3HexNAc2 1491,51
NeuAcHex2HexNAc 835,28 NeuAcHex3HexNAc2dHex2 1492,53
NeuAcHexHexNAc2 876,31 Hex4HexNAc3dHexSP 1500,47
NeuAc2HexHexNAc 964,33 NeuAcHex4HexNAc2dHex 1508,53
NeuAcHexHexNAcdHex2 965,35 NeuAc2HexHexNAc3dHex / 1516,54 /
Hex5HexNAc3SP 1516,46
NeuAcHex2HexNAcdHex 981,34 NeuAcHex5HexNAc2 1524,52
Hex3HexNAc2SP 989,28 NeuAc2Hex2HexNAc3 1532,54
NeuAcHex3HexNAc 997 ~ NeuAcHex2HexNAc3dHex2 1533,56
NeuAcHexHexNAc2dHex 1022,37 NeuAcHex3HexNAc3dHex 1549,55
NeuAcHex2HexNAc2 1038,36 NeuAc2Hex2HexNAc2dHexSP 1555,47
NeuAcHexHexNAc3 1079,39 Hex4HexNAc4SP 1557,49
NeuAc2HexHexNAcdHex 1110,38 NeuAcHex3HexNAc3 SP 2 1563,41
NeuAc2Hex2HexNAc 1126,38 NeuAcHex4HexNAc3 1565,55
NeuAcHex2HexNAcdHex2 1127,40 NeuAc2HexHexNAc4 1573,56
NeuAcHex3HexNAcdHex 1143,39 NeuGcHex4HexNAc3 1581,54
Hex4HexNAc2SP 1151,33 NeuAcHex2HexNac4dHex 1590,58
NeuAcHex4HexNAc 1159,39 NeuAc2Hex4HexNAcdHex 1596,54
NeuAc2HexHexNAc2 1167,41 NeuAcHex3HexNAc4 1606,57
NeuAcHexHexNAc2dHex2 1168,43 NeuAc2Hex2HexNAc2dHex2 / 1621,57 /
NeuAcHex2HexNAc2dHex 1184,42 Hex6HexNAc2dHexSP 1621,49
Hex3HexNAc3SP 1192,36 NeuAc2Hex3HexNAc2dHex 1637,57
NeuAcHex3HexNAc2 / NeuAcHex4HexNAc3SP 1645,50
NeuGcHex2HexNAc2dHex 1200,42 NeuAcHex2HexNAc5 1647,60
NeuGcHex3HexNAc2 1216,41 NeuAcHex4HexNAc2dHex2 1654,58
NeuAcHexHexNAc3dHex 1225,45 Hex5HexNAc3dHexSP 1662,52
NeuAcHex2HexNAc3 1241,44 NeuAcHex5HexNAc2dHex 1670,58
NeuAc2Hex2HexNAcdHex 1272,44 NeuAc2Hex2HexNAc3dHex 1678,60
NeuAcHexHexNAc4 1282,47 NeuAcHex2HexNAc3dHex3 1679,62
NeuAc2Hex3HexNAc 1288,43 NeuAcHex6HexNAc2 1686,57
NeuAcHex4HexNAcdHex 1305,45 NeuAc2Hex3HexNAc3 1694,59
NeuAc2HexHexNAc2dHex 1313,46 Hex4HexNAc4dHexSP 1703,55
NeuAcHex5HexNAc / 1321,44 /
NeuAcHex2HexNAc3SP 1321,40 NeuAcHex3HexNAc3dHex SP 2 1709,47
NeuAc2Hex2HexNAc2 / NeuGcNeuAcHex3HexNAc3 1710,59
NeuGcNeuAcHexHexNAc2dHex 1329,46 NeuAcHex4HexNAc3dHex 1711,61
NeuAcHex2HexNAc2dHex2 1330,48 Hex5HexNAc4SP 1719,54
Hex3HexNAc3dHexSP 1338,41 NeuAcHex4HexNAc3 SP 2 1725,46
NeuAcHex3HexNAc2dHex 1346,47 Hex4HexNAc3dHex2(SP)2 / 1726,48 /
Hex4HexNAc3SP 1354,41 NeuGc2Hex3HexNAc3 1726,58
NeuAcHex4HexNAc2 1362,47 NeuAcHex5HexNAc3 /
NeuGcHex4HexNAc3dHex 1727,60
NeuAc2HexHexNAc3 1370,48 NeuAc2Hex2HexNAc4 1735,62
NeuAcHex2HexNAc3dHex 1387,50 NeuAcHex2HexNAc4dHex2 1736,64
NeuAcHex3HexNAc3 1403,49 NeuGcHex5HexNAc3 1743,60
NeuGcHex3HexNAc3 1419,49 NeuAcHex3HexNAc4dHex 1752,63
NeuAcHexHexNAc4dHex 1428,53 NeuAc2Hex2HexNAc3dHexSP 1758,55
NeuAc2Hex3HexNAcdHex 1434,49 NeuAcHex3HexNAc4(SP)2 / 1766,49 /
NeuAcHex2HexNAc4 1444,52 NeuAcHex6HexNAc2SP 1766,53
NeuAcHex3HexNAc3Ac 1445,51 Hex6HexNAc2dHex2SP /
NeuAc2Hex4HexNAc 1450,48 Hex3HexNAc4dHex2(SP)2 / 1767,55 /
Hex5HexNAc2dHexSP 1459,44 NeuAc2Hex2HexNAc2dHex3 1767,51
NeuAc2Hex2HexNAc2dHex 1475,52 NeuAcHex4HexNAc4 1768,63
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NeuAc2Hex6HexNAc / 1774,59 / Hex4HexNAc5dHex2SP 2052,68
NeuAc2Hex3HexNAc3SP 1774,55 NeuAc2Hex4HexNAc4 2059,72
Hex7HexNAc2dHexSP 1783,55 NeuAcHex4HexNAc4dHex2 2060,74
NeuGcHex4HexNac4 1784,62 Hex5HexNAc5dHexSP 2068,68
NeuAcHex4HexNAc3dHexSP 1791,56 NeuAcHex4HexNAc4dHex SP 2 2074,60
NeuAcHex2HexNAc5dHex 1793,66 NeuAcHex5HexNAc4dHex 2076,74
NeuAc2Hex4HexNAc2dHex/ NeuAc2Hex4HexNAc3dHexSP 2082,66
Hex5HexNAc4 SP 2 1799,62
NeuAcHex3HexNac5 1809,65 NeuGc2Hex4HexNAc4 2091,71
NeuAcHex6HexNAc4/
NeuAc2Hex5HexNAc2
NeuAc2Hex2HexNAc4SP 1815,62 NeuGcHex5HexNAc4dHex 2092,73
NeuAcHex5HexNAc2dHex2/ NeuAc2Hex5HexNAc3SP/
NeuAcHex2HexNAc4dHex2SP 1816,64 NeuGcNeuAcHex4HexNAc3dHexSP 2098,65
NeuAcHexSHexNAc3dHex2SP
Hex6NexNAc3dHexSP 1824,57 /
NeuGcHex4HexNAc3dHex3SP 2099,67
NeuGcHex3HexNAc5 1825,65 NeuAc2Hex3HexNAc5 2100,75
NeuAcHex6HexNAc2dHex 1832,63 NeuAcHex3HexNAc5dHex2 / 2101,77 /
NeuAc2Hex3HexNAc3dHex 1840,65 NeuAc2Hex4HexNAc4Ac 2101,73
NeuAcHex3HexNAc3dHex3 1841,67 NeuAcHex6HexNAc3dHexSP 2115,67
NeuAc2Hex4HexNAc3 1856,64 NeuAcHex4HexNAc5dHex 2117,76
NeuAcHex4HexNAc3dHex2 1857,66 Hex7HexNAc3dHex2SP / 2132,68 /
Hex5HexNAc4dHexSP 1865,60 NeuAc2Hex3HexNAc3dHex3 2132,76
NeuAcHex4HexNAc3dHex SP 2 1871,52 NeuAcHex5HexNAc5 2133,76
NeuAcHex5HexNAc3dHex/ Hex8HexNAc3dHexSP/
NeuGcHex4HexNAc3dHex2 1873,66 NeuAc2Hex4HexNAc3dHex2 2148,68
Hex6HexNAc4SP 1881,65 NeuAcHex8Hexnac2dHex / 2156,74 /
NeuAcHex5HexNAc4dHexSP 2156,69
NeuAcHex5HexNAC3 SP 2 1887,51 Hex5HexNAC4dHex3SP 2157,71
NeuAcHex6HexNAc3 1889,65 NeuAc2Hex5HexNAc3dHex 2164,75
NeuAcHex3HexNAc4dHex2 1898,69 NeuAcHex5HexNAc3dHex3 2165,77
Hex4HexNAc5dHexSP 1906,63 NeuAcHex9HexNAc2 /
NeuAcHex6HexNAc2dHexSP / NeuAcHex6HexNAc4SP / 2172,73 /
NeuAcHex3HexNAc4dHex SP 2 1912,59 NeuGcHex5HexNAc4dHexSP 2172,69
NeuAcHex4HexNAc4dHex 1914,68 NeuAcHex4Hexnac6 2174,79
NeuAc2Hex3HexNAc3dHexSP 1920,60 NeuAc2Hex6HexNAc3
Hex5HexNAc5SP 1922,62 NeuGc2Hex4HexNAc3dHex2 2180,75
NeuAcHex4HexNAc4 SP 2 1928,54 NeuAcHex6HexNAc3dHex2 2181,77
NeuAcHex5HexNAc4 1930,68 NeuAc3Hex3HexNAc4/
NeuGcHex5HexNAc4 1946,67 NeuGcHex6HexNAc4SP / 2188,76 /
NeuAc2NeuGcHex2HexNAc4dHex 2188,68
NeuAcHex5HexNAc3dHexSP 1953,62 NeuAc2Hex3HexNAc4dHex2 / 2189,79 /
NeuAcHex3HexNAc5dHex 1955,71 Hex7HexNAc4dHexSP 2189,70
NeuAc2Hex5HexNAc2dHex / 1961,67 / NeuAcHex3HexNAc4dHex4 2190,81
Hex6HexNAc4 SP 2 1961,55 NeuGcNeuAcHex6HexNAc3 /
NeuAcHex4HexNAc5 1971,71 NeuGc2Hex5HexNAc3dHex 2196,74
NeuAcHex5HexNAc4Ac 1972,69 Hex4HexNAc5dHex3SP 2198,74
NeuAcHex6HexNAc2dHex2 / 1978,69 / NeuAc2Hex4HexNAc4dHex 2205,78
NeuAcHex3HexNAc4dHex2SP 1978,65 NeuAcHex4HexNAc4dHex3 2206,80
NeuAc2Hex4HexNAc3dHex / 2002,70 /
Hex8HexNAc3SP 2002,62 NeuAc2Hex4HexNAc4 SP 2 2219,64
NeuAcHex4HexNAc3dHex3 2003,72 NeuAc2Hex5HexNAc4 2221,78
NeuAcHex5HexNAc4SP 2010,64 NeuAcHex5HexNAc4dHex2 2222,80
Hex5HexNAc4dHex2SP 2011,66 Hex6HexNAc5dHexSP 2230,73
NeuAc2Hex5HexNAc3/ NeuGcNeuAcHex5HexNAc4 2237,77
NeuGcNeuAcHex4HexNAc3dHex 2018,70 NeuAcHex6HexNAc4dHex/
NeuAcHex5HexNAc3dHex2 2019,72 NeuGcHex5HexNAc4dHex2 2238,79
NeuGcHex5HexNAc4SP 2026,63 NeuAc2Hex3HexNAc5dHex 2246,81
Hex6HexNAc4dHexSP 2027,65 NeuAcHex3HexNAc5dHex3 2247,83
NeuAcHex6HexNAc3dHex 2035,71 NeuGc2Hex5Hexnac4 2253,76
NeuAc2Hex3HexNAc4dHex/ 2043,73/ NeuAcHex7HexNAc4/
Hex7HexNAc4SP 2043,65 NeuGcHex6HexNAc4dHex 2254,79
NeuAcHex7HexNAc3 2051,71 NeuAc2Hex4HexNAc5 2262,80
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NeuAcHex4HexNAc5dHex2 / 2263,82 / NeuAcHex3HexNAc6dHex3 2450,91
NeuAc2Hex5HexNAc4Ac 2263,79 NeuAc2Hex5HexNAc4dHexAc2 2451,85
NeuAcHex5HexNAc5dHex 2279,82 NeuAc2Hex5HexNAc3dHex3 2456,87
NeuAc2Hex4HexNAc4dHexSP 2285,74 NeuAcHex7HexNAc5 2457,86
NeuAcHex4HexNAc4dHex3SP 2286,76 NeuAcHex5HexNAc5dHex2Ac 2467,89
NeuAcHex8HexNAc3SP / 2293,72 / NeuAc2Hex6HexNAc3dHex2 2472,86
NeuAc3Hex4HexNAc3dHex 2293,80 NeuAcHex6HexNAc3dHex4 /
NeuAc2Hex4HexNAc3dHex3 2294,82 NeuGcHex7HexNAc5 2473,88
NeuAcHex6HexNAc5 2295,81 NeuAcHex5HexNAc6dHex 2482,90
NeuAc2Hex5HexNAc4SP 2301,73 NeuAcHex6HexNAc5Ac 2483,88
NeuAcHex5HexNAc4dHex2SP 2302,75 NeuAc2Hex7HexNAc3dHex 2488,86
NeuAc2Hex5HexNAc4Ac2 2305,80 NeuAcHex7HexNAc3dHex3 2489,88
NeuAc2Hex5HexNAc3dHex2 / NeuAcHex6HexNAc6 /
NeuGcNeuAcHex4HexNAc3dHex3 2310,81 NeuGcHex5hexNAc6dHex 2498,89
NeuAcHex5HexNAc3dHex4/ NeuAc3Hex5HexNAc4 2512,87
NeuGcHex6HexNAc5 2311,83
NeuAcHex6HexNAc4dHexSP 2318,75 NeuAc2Hex5HexNAc4dHex2 2513,89
Hex6HexNAc4dHex3SP / NeuAcHex5HexNAc4dHex4 2514,91
NeuGcNeuAcHex3HexNAc6 2319,77 NeuAcHex6HexNAc5dHexSP/
NeuAcHex4HexNAc6dHex 2320,84 NeuAcHex9HexNAc3dHex / 2521,83 /
NeuAc3Hex2HexNAc5dHex2 2521,87
NeuAcHex5HexNAc5dHexAc 2321,83 Hex6HexNAc5dHex3SP 2522,85
NeuAc2Hex6HexNAc3dHex 2326,81 NeuGcNeuAc2Hex5HexNAc4 2528,87
NeuAcHex6HexNAc3dHex3 2327,83 NeuAc2Hex6HexNAc4dHex/
NeuAcHex7HexNAc4SP/ NeuGcNeuAcHex5HexNAc4dHex2 2529,89
NeuGcHex6HexNAc4dHexSP / 2334,74 / NeuAcHex6HexNAc4dHex3 2530,91
NeuAcHex10HexNAc2 2334,79 NeuAc3Hex3HexNAc5dHex/
NeuAcHex5HexNAc6 2336,84 NeuGcHex6HexNAc5dHexSP / 2537,90 /
NeuAc3Hex4HexNac4 2350,82 NeuAcHex7HexNAc5SP 2537,82
NeuAc2Hex4HexNAc4dHex2 / 2351,84 / NeuAc2Hex3HexNAc5dHex3 2538,92
Hex8HexNAc4dHexSP 2351,76 NeuAcHex5HexNAc7/
NeuGcNeuAc2Hex4HexNAc4 2366,81 NeuAcHex3HexNAc5dHex5 2539,92
NeuAc2Hex5HexNAc4dHex 2367,83 NeuGc2NeuAcHex5HexNAc4 2544,86
NeuAcHex5HexNAc4dHex3 2368,85 NeuGc2Hex5Hexnac4dHex2 /
NeuAcHex5HexNAc4dHex2 SP 2 2382,71 NeuGcNeuAcHex6HexNAc4dHex 2545,88
NeuAc2Hex6HexNAc4/ NeuAc3Hex4HexNAc5 2553,90
NeuGcNeuAcHex5HexNAc4dHex 2383,83 NeuAc2Hex4HexNAc5dHex2 2554,92
NeuAcHex6HexNAc4dHex2/ NeuAcHex4HexNAc5dHex4 2555,94
NeuGcHex5HexNAc4dHex3 2384,85 NeuGc3Hex5HexNAc4 2560,86
NeuAc3Hex5HexNAc3SP / 2389,75 /
NeuAc2Hex5HexNAc4Ac4 2389,82 NeuAc2Hex5HexNAc5dHex 2570,91
NeuAc2Hex5HexNAc3dHex2SP 2390,77 NeuAcHex5HexNAc5dHex3 2571,93
NeuAcHex5HexNAc3dHex4SP / 2391,79 / NeuAc2Hex6HexNAc5 2586,91
NeuAc3Hex3HexNAc5 2391,84 NeuAcHex6HexNAc5dHex2 2587,93
NeuAc2Hex3HexNAc5dHex2 2392,86 Hex7HexNAc6dHexSP 2595,86
NeuAcHex3HexNAc5dHex4 2393,89 NeuGcNeuAcHex6HexNAc5 2602,90
NeuGc2Hex5HexNAc4dHex 2399,82 NeuAcHex7HexNAc5dHex/ 2603,92/
Hex4HexNAc6dHex3SP 2401,82 NeuGcHex6HexNAc5dHex2 603,92
NeuAc2Hex6HexNAc3dHexSP 2406,76 NeuGc2Hex6HexNac5 2618,90
NeuAc2Hex4HexNAc5dHex 2408,86 NeuAcHex8HexNAc5/
NeuAcHex4HexNAc5dHex3 / 2409,88 / NeuGcHex7HexNAc5dHex 2619,92
NeuAc2Hex5HexNAc4dHexAc 2409,84 NeuAc2Hex5HexNAc6 2627,93
NeuAc2Hex5HexNAc5 2424,85 NeuAcHex5HexNAc6dHex2 2628,95
NeuAcHex5HexNAc5dHex2 2425,87 NeuGcHex8HexNAc5 / 2635,91 /
NeuAcHex8HexNAc3dHexSP / NeuAcHex4HexNAc5dHex4SP 2635,89
NeuAc3Hex4HexNAc3dHex2 2439,77 NeuAcHex6HexNAc6dHex 2644,95
NeuAcHex6HexNAc5dHex 2441,87 NeuAc2Hex5HexNAc5dHexSP 2650,87
NeuAc2Hex8HexNAc2dHex/ 2447,83/ NeuAc2Hex5HexNAc4dHex3 2659,95
NeuAc2Hex5HexNAc4dHexSP 2447,79 NeuAcHex7HexNAc6 2660,94
NeuAcHex8HexNAc2dHex3/ 2448,85/ NeuGcNeuAc2Hex5HexNAc4dHex
NeuAcHex5HexNAc4dHex3SP 2448,81 NeuAc3Hex6HexNAc4 2674,92
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NeuGcHex6HexNAc5dHexSP / NeuAcHex5HexNAc6dHex4 2921,07
NeuAcHex7HexNAc5dHexSP 2683,88 NeuGc3Hex6HexNAc5 2925,99
NeuAcHex5HexNAc7dHex 2685,98 NeuGcNeuAc2Hex5HexNAc6 2935,02
NeuAc2Hex7HexNAc4dHex 2691,94 NeuAc2Hex6HexNAc6dHex/
NeuAcHex7HexNAc4dHex3 2692,96 NeuGcNeuAcHex5HexNAc6dHex2 2936,04
NeuAc2Hex4HexNAc5dHex2 SP 2 2714,83 NeuAcHex6HexNAc6dHex3 2937,07
NeuAcHex4HexNAc5dHex4(SP)2 / 2715,85 / NeuGc2NeuAcHex5HexNAc6 / 2951,02 /
NeuAc3Hex5HexNAc5 2715,95 NeuAc3Hex5HexNAc4dHex3 2951,04
NeuAc2Hex5HexNAc5dHex2 2716,97 NeuAc2Hex7HexNAc6 2952,04
NeuAcHex5HexNAc5dHex4 2717,99 NeuAcHex7HexNAc6dHex2 2953,06
NeuAc2Hex6HexNAc5dHex 2732,97 NeuAc2Hex6HexNAc5dHex2SP 2958,98
NeuAcHex6HexNAc5dHex3 2733,99 NeuAcHex6HexNAc5dHex4SP 2960,00
NeuAcHex6HexNAc5dHex2 SP 2 2747,84 NeuAc2Hex4HexNAc7dHex2 2961,08
NeuGcNeuAcHex6HexNAc5dHex 2748,96 NeuAcHex4HexNAc7dHex4 2962,10
NeuAc3Hex4HexNAc6 2756,98 NeuAcHex6HexNAc7dHex2 2994,09
NeuAc2Hex4HexNAc6dHex2 2758,00 NeuAcHex7HexNAc7dHex 3010,08
NeuAcHex4HexNAc6dHex4 2759,02 NeuAc3Hex6HexNAc5dHex 3024,06
NeuAc3Hex6HexNAc3dHex2 2763,96 NeuAc2Hex6HexNAc5dHex3 3025,08
NeuAc2Hex6HexNAc3dHex4/ NeuAcHex8HexNAc7 3026,08
NeuGc2Hex6HexNAc5dHex / 2764,98 / NeuAc3Hex5HexNAc6dHex 3065,09
NeuGcHex7HexNAc5 2764,96 NeuAc2Hex5HexNAc6dHex3 3066,11
NeuAcHex8HexNAc5dHex 2765,98 NeuAcHex7HexNAc8 3067,10
NeuAc2Hex5HexNAc6dHex 2773,99 NeuAc3Hex6HexNAc6 3081,08
NeuAcHex5HexNAc6dHex3 2775,01 NeuAc2Hex6HexNAc6dHex2 3082,10
NeuGc2Hex7HexNAc5 2780,95 NeuAc2Hex7HexNAc6dHex 3098,10
NeuGcHex8HexNAc5dHex /
NeuAcHex9HexNac5 2781,97 NeuAcHex7HexNAc6dHex3 3099,12
NeuAc2Hex6HexNAc6 2789,99 NeuAc3Hex6HexNAc5dHexSP 3104,02
NeuAcHex6HexNAc6dHex2 2791,01 NeuAc2Hex6HexNAc5dHex3SP 3105,04
NeuAc4Hex5HexNAc4 2803,97 NeuAcHex8HexNAc7SP / 3106,03 /
NeuAc3Hex5HexNAc4dHex2 / 2804,99 / NeuAc3Hex4HexNAc7dHex 3106,11
NeuAcHex6HexNAc6dHex SP 2 2804,86 Hex8HexNAc7dHex2SP / 3107,05 /
Hex6HexNAc6dHex3SP2 2805,88 NeuAc2Hex4HexNAc7dHex3 3107,13
NeuAc2Hex5HexNAc4dHex4 2806,01 NeuAcHex7HexNAc7dHex2 3156,14
NeuAcHex7Hexnac6dHex 2807,00 NeuAc3Hex6HexNAc5dHex2 3170,12
NeuAc2Hex6HexNAc5dHexSP 2812,92 NeuAc2Hex6HexNAc5dHex4 3171,14
NeuAcHex6HexNAc5dHex3SP 2813,94 NeuAcHex8HexNAc7dHex 3172,13
NeuGcNeuAc3Hex5HexNAc4 2819,96 NeuAc2Hex7HexNAc6dHexSP 3178,05
NeuAc3Hex6HexNAc4dHex/ NeuAc3Hex6HexNAc6dHex 3227,14
NeuGcNeuAc2Hex5HexNAc4dHex2 2820,98 NeuAc2Hex6HexNAc6dHex3 3228,16
NeuAc2Hex6HexNAc4dHex3 2822,00 NeuAcHex8HexNAc8 3229,16
NeuAcHex8HexNAc6 2823,00 NeuAc3Hex7HexNAc6 3243,13
NeuGc2NeuAc2Hex5HexNAc4 2835,96 NeuAc2Hex7HexNAc6dHex2 3244,16
NeuGc2NeuAcHex5HexNAc4dHex2 2836,98 NeuAcHex7HexNAc6dHex4 3245,18
NeuAc3Hex6HexNAc5 2878,00 NeuAc2Hex8HexNAc6dHex/
NeuGcNeuAcHex7HexNAc6dHex2 3260,15
NeuAc2Hex6HexNAc5dHex2 2879,02 NeuAcHex8HexNAc6dHex3 /
NeuAcHex6HexNAc5dHex4 288,04 NeuGcHex7HexNAc6dHex4 3261,17
NeuAcHex7HexNAc6dHexSP / 2886,96 / NeuAc3Hex7HexNAc5dHexSP /
NeuAcHex10HexNAc4dHex 2887,00 NeuGcNeuAc2Hex6HexNAc5dHex2SP 3266,07
NeuGcNeuAc2Hex6HexNAc5 2894,00 NeuAc3Hex5HexNAc7dHex/ 3268,17/
NeuAc2Hex7HexNAc5dHex / NeuGcHex8HexNAc7dHexSP 3268,09
NeuGcNeuAcHex6HexNAc5dHex2 2895,02 NeuAc2Hex5HexNAc7dHex3 3269,19
NeuAc3Hex6HexNAc4dHexSP/ NeuAcHex7HexNAc9 3270,18
NeuGcNeuAc2Hex5HexNAc4dHex2SP 2900,94 NeuGc2Hex7HexNAc6dHex2 3276,15
NeuGc2NeuAcHex6HexNAc5 2909,99 NeuAc4Hex4HexNAc5dHex2 SP 2 3297,02
NeuGc2Hex6HexNAc5dHex2 2911,01 NeuAc3Hex4HexNAc5dHex4 SP 2 3298,04
NeuAc3Hex5HexNAc6 2919,03 NeuAc2Hex7HexNAc7dHex 3301,18
NeuAc2Hex5HexNAc6dHex2 2920,05 NeuAcHex7HexNAc7dHex3 3302,20
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NeuAc3Hex6HexNAc5dHex3 3316,18 NeuAcHex8HexNAc10dHex 3781,37
NeuAc2Hex8HexNAc7 3317,17 NeuAc4Hex7HexNAc6dHex2 3826,35
NeuAcHex8HexNAc7dHex2 3318,19 NeuAc3Hex7Hexnac6dHex4 3827,37
NeuAc3Hex7HexNAc6dHex 3389,19 NeuAc2Hex9HexNAc8dHex 3828,36
NeuAc2Hex7HexNAc6dHex3 3390,21 NeuAcHex9HexNAc8dHex3 3829,38
NeuAcHex7HexNAc6dHex5/ NeuAc4Hex8HexNAc7 3899,36
NeuAcHex9HexNAc8 3391,23 NeuAc3Hex8HexNAc7dHex2 3900,38
NeuAc3Hex5HexNAc7dHex2 3414,22 NeuAc2Hex8HexNAc7dHex4 3901,40
NeuAc2Hex5HexNAc7dHex4 3415,24 NeuAcHex10HexNAc9dHex 3902,40
NeuAcHex7HexNAc9dHex 3416,24 NeuAc4Hex6HexNAc8dHex 3924,39
NeuAc3Hex6HexNAc7dHex 3430,22 NeuAc3Hex6HexNAc8dHex3 3925,41
NeuAc2Hex6HexNAc7dHex3 3431,24 NeuAc2Hex8HexNAclO 3926,41
NeuAcHex8HexNAc9 3432,24 NeuAcHex8HexNAc10dHex2 3927,43
NeuAc2Hex8Hexnac7dHex 3463,23 NeuAc3Hex9HexNAc8 3973,40
NeuAcHex8HexNAc7dHex3 3464,25 NeuAc2Hex9HexNAc8dHex2 3974,42
NeuAc3Hex7HexNAc6dHexSP 3469,15 NeuAcHex9HexNAc8dHex4 3975,44
NeuAc2Hex7HexNAc6dHex3SP 3470,17 NeuAc4Hex8HexNAc7dHex 4045,42
NeuAc3Hex5HexNAc8dHex 3471,25 NeuAc3Hex8HexNAc7dHex3 4046,44
NeuAc2Hex5HexNAc8dHex3 3472,27 NeuAc2Hex10HexNAc9/
NeuAcHex7HexNAc10 3473,26 NeuAc2Hex8HexNAc7dHex5 4047,44
NeuAc4Hex7HexNAc6 3534,23 NeuAcHex10HexNAc9dHex2 4048,46
NeuAc3Hex7HexNAc6dHex2 3535,25 NeuAc3Hex9HexNAc8dHex 4119,46
NeuAc2Hex7HexNAc6dHex4 3536,27 NeuAc2Hex9HexNAc8dHex3 4120,48
NeuAcHex9HexNAc8dHex 3537,27 NeuAcHex11 HexNAc10 /
NeuAc4Hex5HexNAc7dHex 3559,26 NeuAcHex9HexNAc8dHex5 4121,47
NeuAc3Hex5HexNAc7dHex3 3560,28 NeuAc2Hex10HexNAc9dHex2 4339,55
NeuAc2Hex7HexNAc9 3561,28 NeuAcHex10HexNAc9dHex4 4340,57
NeuAcHex7HexNAc9dHex2 3562,30 NeuAc2Hex10HexNAc9dHex3 4485,61
NeuAc3Hex7HexNac7dHex 3592,27
NeuAc2Hex7HexNAc7dHex3 3593,29
NeuAcHex9HexNAc9 3594,29
NeuAc3Hex8HexNAc7 3608,27
NeuAc2Hex8HexNac7dHex2 3609,29
NeuAcHex8HexNac7dHex4 3610,31
NeuAc3Hex5HexNAc8dHex2 3617,30
NeuAc2Hex5HexNAc8dHex4 3618,32
NeuAcHex7HexNAc10dHex 3619,32
NeuAc3Hex6HexNAc8dHex 3633,30
NeuAc4Hex7HexNAc6dHex 3680,29
NeuAc3Hex7HexNAc6dHex3 3681,31
NeuAc2Hex9HexNAc8 3682,30
NeuAcHex9HexNAc8dHex2 3683,32
NeuAc4Hex6HexNAc7dHex 3721,31
NeuAc3Hex6HexNAc7dHex3 3722,34
NeuAc2Hex8HexNAc9 3723,33
NeuAcHex8HexNAc9dHex2 3724,35
NeuAc3Hex7HexNac7dHex2 3738,33
NeuAc2Hex7HexNAc7dHex4 3739,35
NeuAcHex9HexNAc9dHex 3740,35
NeuAc3Hex8HexNAc7dHex 3754,33
NeuAc2Hex8HexNAc7dHex3 3755,35
NeuAcHex10HexNAc9/
NeuAcHex8HexNAc7dHex5 3756,34
NeuAc4Hex6HexNAc8 3778,34
NeuAc3Hex6HexNAc8dHex2 3779,36
NeuAc2Hex6HexNAc8dHex4 3780,38
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Table 3. MALDI-TOF mass spectrometric analysis of endoglycoceramidase-released
peripheral
blood mononuclear cell glycolipid glycans.
A. Neutral oligosaccharides detected from glycolipids of peripheral blood
mononuclear cells. Five
major peaks are bolded.
Proposed composition calc. m/z exp. m/z
Hex2HexNAc 568,18 568,09
Hex3HexNAc 730,24 730,18
Hex3HexNAcdHex 876,30 876,27
Hex4HexNAc 892,29 892,27
Hex3HexNAc2 933,31 933,30
Hex5HexNAc 1054,34 1054,33
Hex4HexNAc2 1095,37 1095,36
Hex4HexNAc2dHex 1241,43 1241,42
Hex4HexNAc2dHex2 1387,49 1387,48
Hex6HexNAc2 1419,48 1419,47
Hex5HexNAc3 1460,50 1460,49
Hex5HexNAc4dHex 1606,56 1606,55
Hex5HexNac3dHex2 1752,62 752,60
Hex6HexNAc4dHex2 2117,75 2117,71
Hex6HexNAc4dHex3 2263,81 2263,76
B. Acidic oligosaccharides detected from glycolipids of peripheral blood
mononuclear cells. Five
major peaks are bolded.
Proposed composition calc. m/z exp. m/z
NeuAcHexHexNAc 673,23 673,95
NeuAcHex2HexNAc 835,28 835,31
NeuAcHex3HexNAc 997,34 997,52
NeuAcHex3HexNAc2 1200,42 1200,62
NeuAcHex4HexNAc2 1362,47 1362,80
NeuAcHex4HexNAc2dHex 1508,53 1508,89
NeuAcHex2HexNAc3 dHex2 1533,56 1533,66
NeuAc2Hex2HexNAc2dHexSP 1555,47 1555,68
NeuAcHex5HexNAc3 1727,60 1728,01
NeuAcHex5HexNAc3dHex 1873,66 1874,07
NeuAc2Hex3HexNAc3dHexSP 1920,60 1920,87
NeuAcHex3HexNAc5dHex3 2247,83 2247,99
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Table 4.
Glycan A1) B C D
residue linkage proton ppm ppm ppm ppm
D-G1cNAc H-la 5.191 5.187 5.187 5.188
H-1(3 4.690 4.693 4.693 4.695
NAc 2.042 2.037 2.037 2.038
(3-D-G1cNAc 4 H-1 4.596 4.586 4.586 4.600
NAc 2.072 2.063 2.063 2.064
(3-D-Man 4,4 H-1 4.775 4.771 4.771 4.780
H-2 4.238 4.234 4.234 4.240
a-D-Man 6,4,4 H-1 4.869 4.870 4.870 4.870
H-2 4.149 4.149 4.149 4.150
a-D-Man 6,6,4,4 H-1 5.153 5.151 5.151 5.143
H-2 4.025 4.021 4.021 4.020
a-D-Man 2,6,6,4,4 H-1 5.047 5.042 5.042 5.041
H-2 4.074 4.069 4.069 4.070
a-D-Man 3,6,4,4 H-1 5.414 5.085 5.415 5.092
H-2 4.108 4.069 4.099 4.070
a-D-Man 2,3,6,4,4 H-1 5.047 - 5.042 -
H-2 4.074 - 4.069 -
a-D-Man 3,4,4 H-1 5.343 5.341 5.341 5.345
H-2 4.108 4.099 4.099 4.120
a-D-Man 2,3,4,4 H-1 5.317 5.309 5.050 5.055
H-2 4.108 4.099 4.069 4.070
a-D-Man 2,2,3,4,4 H-1 5.047 5.042 - -
H-2 4.074 4.069 - -
See Fig. 17 for structures.
Chemical shift values obtained from Fu et al., 1994 and Hard et al., 1991.
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Table 5.
Glycan Ai) B C D E
residue linkage proton ppm ppm ppm ppm ppm
D-G1cNAc H-l(x 5.180 5.188 5.189 5.181 5.189
H-10 4.692 n.a. 2) 4.695 n.a. 4.694
NAc 2.038 2.038 2.038 2.039 2.038
a-L-Fuc 6 H-la 4.890 - 3) - 4.892 -
H-10 4.897 - - 4.900 -
H-5a 4.098 - - 4.10 -
H-50 4.134 - - n.a. -
CH3a 1.209 - - 1.211 -
CH3 (3 1.220 - - 1.223 -
(3-D-G1cNAc 4 H-la 4.664 4.612 4.614 4.663 4.613
H-10 4.669 4.604 4.606 n.a. 4.604
NAc 2.097 2.081 2.081 2.096/ 2.084
((X/(3) 2.093
(3-D-Man 4,4 H-1 4.772 n.a. n.a. n.a. n.a.
H-2 4.257 4.246 4.253 4.248 4.258
a-D-Man 6,4,4 H-1 4.929 4.928 4.930 4.922 4.948
H-2 4.111 4.11 4.112 4.11 4.117
(3-D-G1cpNAc 2,6,4,4 H-1 4.583 4.581 4.582 4.573 4.604
NAc 2.048 2.047 2.047 2.043 2.066
(3-D-Ga1 4,2,6,4,4 H-1 4.544 4.473 4.473 4.550 4.447
H-3 n.a. n.a. n.a. 4.119 n.a.
H-4 4.185 n.a. n.a. n.a. n.a.
a-D-Galp 3,4,2,6,4,4 H-1 5.149 - - - -
a-D-Neup5Ac 3,4,2,6,4,4 H-3a - - - 1.800 -
H-3e - - - 2.758 -
NAc - - - 2.031 -
a-D-Neup5Ac 6,4,2,6,4,4 H-3a - - - - 1.719
H-3e - - - - 2.673
NAc - - - - 2.029
a-D-Man 3,4,4 H-1 5.135 5.118 5.135 5.116 5.133
H-2 4.195 4.190 4.196 4.189 4.197
(3-D-G1cpNAc 2,3,4,4 H-1 4.605 4.573 4.606 4.573 4.604
NAc 2.069 2.047 2.069 2.048 2.070
(3-D-Ga1p 4,2,3,4,4 H-1 4.445 4.545 4.445 4.544 4.443
H-3 n.a. 4.113 n.a. 4.113 n.a.
(x-D-Neup5Ac 6,4,2,3,4,4 H-3a 1.722 - 1.719 - 1.719
H-3e 2.666 - 2.668 - 2.667
NAc 2.029 - 2.030 - 2.029
(x-D-Neup5Ac 3,4,2,3,4,4 H-3a - 1.797 - 1.797 -
H-3e - 2.756 - 2.758 -
NAc - 2.030 - 2.031 -
See Fig. 18 for structures
2) n.a., not assigned.
3) -, not present.
Chemical shift values obtained from Hard et al., 1992 and Helin et al., 1995.
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Table 6. HexNAcZ4 and HexZ3
(including complex-type N-
E E_
glycans
Hex5.9HexNAc2 )
(including high-mannose type _ Proposed composition m/z
N-glycans) E = Hex3HexNAc4 1339 + +
Hex3HexNAc4dHex 1485 + +
Proposed composition m/z Hex4HexNAc4 1501 + +
Hex5HexNAc2 1257 + + Hex3HexNAc5 1542 + +
Hex6HexNAc2 1419 + + Hex4HexNAc4dHex 1647 + +
Hex7HexNAc2 1581 + + Hex5HexNAc4 1663 + +
Hex8HexNAc2 1743 + + Hex3HexNAc5dHex 1688 + +
Hex9HexNAc2 1905 + + Hex4HexNAx5 1704 + +
Hex4HexNAc4dHex2 1793 + +
Hex5HexNAc4dHex 1809 + +
Hexl.4HexNAc2dHexo-+ Hex6HexNAc4 1825 + +
(including low-mannose type Hex4HexNAc5dHex 1850 + +
E u) E = =
N-glycans) 21) Hex5HexNAc5 1866 +
Hex3HexNAc6dHex 1891 + +
Proposed composition m/z Hex5HexNAc4dHex2 1955 + +
HexHexNAc2 609 + Hex6HexNAc4dHex 1971 + +
HexHexNAc2dHex 755 + Hex7HexNAc4 1987 + +
Hex2HexNAc2 771 + + Hex4HexNAc5dHex2 1996 + +
Hex2HexNAc2dHex 917 + + Hex5HexNAc5dHex 2012 +
Hex3HexNAc2 933 + + Hex6HexNAc5 2028 + +
Hex3HexNAc2dHex 1079 + + Hex5HexNAc4dHex3 2101 + +
Hex4HexNAc2 1095 + + Hex6HexNAc4dHex2 2117 +
Hex4HexNAc2dHex 1241 + + Hex7HexNAc4dHex 2133 + +
Hex4HexNAc5dHex3 2142 +
Hex8HexNAc4 2149 + +
HexIo.12HexNAc2 Hex5HexNAc5dHex2 2158 +
(including glucosylated high- m R= Hex6HexNAc5dHex 2174 + +
mannose type N-glycans) E E= Hex7HexNAc5 2190 + +
Hex5HexNAc6dHex 2215 + +
Proposed composition m/z Hex6HexNAc6 2231 +
Hex10HexNAc2 2067 + + Hex5HexNAc4dHex4 2247 + +
Hex11HexNAc2 2229 + Hex7HexNAc4dHex2 2279 +
Hex12HexNAc2 2391 + Hex5HexNAc5dHex3 2304 + +
Hex6HexNAc5dHex2 2320 + +
Hex7HexNAc5dHex 2336 +
Hex5.9HexNAc2dHexj Hex8HexNAc5 2352 + +
(including fucosylated high- m 0 = Hex7HexNAc6 2393 + +
mannose type N-glycans) s" E Hex7HexNAc4dHex3 2425 +
Hex6HexNAc5dHex3 2466 +
Proposed composition m/z Hex8HexNAc5dHex 2498 +
Hex5HexNAc2dHex 1403 + + Hex7HexNAc6dHex 2539 + +
Hex6HexNAc2dHex 1565 + + Hex6HexNAc5dHex4 2612 + +
Hex8HexNAc7 2758 +
Hex7Hexnac5dHex4 2775 + +
HexNAc=3 and HexZ2 Hex8HexNAc5dHex4 2937 + +
(including hybrid-type and RU) R= Hex8HexNAc6dHex4 3140 + +
monoantennary N-glycans) ~ = Hex9HexNAc6dHex4 3302 + +
_ = Hex10HexNAc6dHex4 3464 + +
Pro osedcom osition m/z Hex11HexNAc6dHex4 3626 + +
Hex2HexNAc3 974 +
Hex2HexNAc3dHex 1120 +
Hex3HexNAc3 1136 + + Hexl.9HexNAc,
R w R_=
Hex2HexNAc3dHex2 1266 + (including soluble glycans)
Hex3HexNAc3dHex 1282 + + E ~ _
Hex4HexNAc3 1298 + + _ ~
Hex3HexNAc3dHex2 1428 + Proposed composition m/z
Hex4HexNAc3dHex 1444 + + Hex2HexNAc 568 +
Hex5HexNAc3 1460 + + Hex3HexNAc 730 + +
Hex4HexNAc3dHex2 1590 + + Hex4HexNAc 892 +
Hex5HexNAc3dHex 1606 + + Hex5HexNAc 1054 + +
Hex6HexNAc3 1622 + + Hex6HexNAc 1216 + +
Hex5HexNAc3dHex2 1752 + + Hex7HexNAc 1378 + +
Hex6HexNAc3dHex 1768 + + Hex8HexNAc 1540 + +
Hex7HexNAc3 1784 + + Hex9HexNAc 1702 +
Hex7HexNAc3dHex 1930 + +
Hex8HexNAc3 1946 +
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HexNAcZ3 and dHexZ1
(including fucosylated HexNAcZ3 and dHexZ2
hYbrid/monoant. N-glycans) E = _
~ (including multifucosylated m ~+ 0 _
e- e=
Pro osed composition m/z hybrid/monoant. N-glycans)
Hex2HexNAc3dHex 1120 + Proposed composition m/z
Hex2HexNAc3dHex2 1266 + Hex2HexNAc3dHex2 1266 +
Hex3HexNAc3dHex 1282 + + Hex3HexNAc3dHex2 1428 +
Hex3HexNAc3dHex2 1428 + Hex4HexNAc3dHex2 1590 + +
Hex4HexNAc3dHex 1444 + + Hex5HexNAc3dHex2 1752 + +
Hex4HexNAc3dHex2 1590 + + Hex4HexNAc4dHex2 1793 + +
Hex5HexNAc3dHex 1606 + + Hex5HexNAc4dHex2 1955 + +
Hex5HexNAc3dHex2 1752 + + Hex4HexNAc5dHex2 1996 + +
Hex6HexNAc3dHex 1768 + + Hex5HexNAc4dHex3 2101 + +
Hex7HexNAc3dHex 1930 + + Hex6HexNAc4dHex2 2117 +
Hex3HexNAc4dHex 1485 + + Hex4HexNAc5dHex3 2142 +
Hex4HexNAc4dHex 1647 + + Hex5HexNAc5dHex2 2158 +
Hex3HexNAc5dHex 1688 + + Hex5HexNAc4dHex4 2247 + +
Hex4HexNAc4dHex2 1793 + + Hex7HexNAc4dHex2 2279 +
Hex5HexNAc4dHex 1809 + + Hex5HexNAc5dHex3 2304 + +
Hex4HexNAc5dHex 1850 + +
Hex3HexNAc6dHex 1891 + + Hex6HexNAc5dHex2 2320 + +
Hex5HexNAc4dHex2 1955 + + Hex7HexNAc4dHex3 2425 +
Hex6HexNAc4dHex 1971 + + Hex6HexNAc5dHex3 2466 +
Hex4HexNAc5dHex2 1996 + + Hex6HexNAc5dHex4 2612 + +
Hex5HexNAc5dHex 2012 + Hex7Hexnac5dHex4 2775 + +
Hex5HexNAc4dHex3 2101 + + Hex8HexNAc5dHex4 2937 + +
Hex6HexNAc4dHex2 2117 + Hex8HexNAc6dHex4 3140 + +
Hex7HexNAc4dHex 2133 + + Hex9HexNAc6dHex4 3302 + +
Hex4HexNAc5dHex3 2142 + Hex10HexNAc6dHex4 3464 + +
Hex5HexNAc5dHex2 2158 + Hex11HexNAc6dHex4 3626 + +
Hex6HexNAc5dHex 2174 + +
Hex5HexNAc6dHex 2215 + + HexNAc>HexZ2
Hex5HexNAc4dHex4 2247 + + c c d
Hex7HexNAc4dHex2 2279 + (terminal HexNAc, N>H)
Hex5HexNAc5dHex3 2304 + + E E
Hex6HexNAc5dHex2 2320 + + Proposed composition m/z
Hex7HexNAc5dHex 2336 +
Hex7HexNAc4dHex3 2425 + Hex2HexNAc3 974 +
Hex6HexNAc5dHex3 2466 + Hex2HexNAc3dHex 1120 +
Hex8HexNAc5dHex 2498 + Hex2HexNAc3dHex2 1266 +
Hex7HexNAc6dHex 2539 + + Hex3HexNAc4 1339 + +
Hex6HexNAc5dHex4 2612 + + Hex3HexNAc4dHex 1485 + +
Hex7Hexnac5dHex4 2775 + + Hex3HexNAc5 1542 + +
Hex8HexNAc5dHex4 2937 + + Hex3HexNAc5dHex 1688 + +
Hex8HexNAc6dHex4 3140 + + Hex4HexNAx5 1704 + +
Hex9HexNAc6dHex4 3302 + + Hex4HexNAc5dHex 1850 + +
Hex10HexNAc6dHex4 3464 + + Hex3HexNAc6dHex 1891 + +
Hex11HexNAc6dHex4 3626 + + Hex4HexNAc5dHex2 1996 + +
Hex4HexNAc5dHex3 2142 +
Hex5HexNAc6dHex 2215 + +
HexNAc=HexZ5
(terminal HexNAc, N=H)
Proposed composition m/z
Hex5HexNAc5 1866 +
Hex5HexNAc5dHex 2012 +
Hex5HexNAc5dHex2 2158 +
Hex6HexNAc6 2231 +
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Table 7. HexNAcZ4 and HexZ3
(including complex-type N-
E E_
glycans)
and HexZ2 )
(including hybrid-type and R w _= Proposed composition m/z
monoantennary N-glycans) _ E Hex4HexNAc4SP 1557 + +
NeuAcHex3HexNAc4 1606 +
Proposed composition m/z Hex4HexNAc4SP2 1637 +
Hex3HexNAc3SP 1192 + + Hex4HexNAc4dHexSP 1703 + +
Hex3HexNAc3dHexSP 1338 + + Hex4HexNAc4SP3 and/or 1717 +
Hex4HexNAc3SP 1354 + + Hex7HexNAc2SP2
NeuAcHex3HexNAc3 1403 + + Hex5HexNAc4SP 1719 + +
NeuGcHex3HexNAc3 1419 + NeuAcHex4HexNAc4 1768 + +
Hex4HexNAc3dHexSP 1500 + + NeuGcHex4HexNac4 1784 +
Hex5HexNAc3SP 1516 + + Hex5HexNAc4SP2 and/or 1799 +
NeuAcHex3HexNAc3dHex 1549 + + Hex8HexNAc2SP
NeuAcHex3HexNAc3SP2 1563 + NeuAcHex3HexNac5 1809 +
NeuAcHex4HexNAc3 1565 + + NeuGcHex3HexNAc5 1825 + +
NeuGcHex4HexNAc3 1581 + + Hex5HexNAc4dHexSP 1865 + +
Hex4HexNAc3dHex2SP 1646 + Hex6HexNAc4SP 1881 + +
Hex5HexNAc3dHexSP 1662 + + Hex4HexNAc5dHexSP 1906 +
Hex6HexNAc3SP and/or NeuAcHex4HexNAc4dHex 1914 + +
NeuAc2Hex2HexNAc3dHex 1678 + + NeuAcHex4HexNAc4SP2 1928 +
NeuAc2Hex3HexNAc3 1694 + + NeuAcHex5HexNAc4 1930 + +
NeuAcHex3HexNAc3dHexSP2 1709 + NeuGcHex5HexNAc4 1946 + +
NeuAcHex4HexNAc3dHex 1711 + + NeuAcHex4HexNAc5 1971 +
NeuAcHex5HexNAc3 and/or NeuAcHex5HexNAc4Ac 1972 +
NeuGcHex4HexNAc3dHex 1727 + + Hex5HexNAc5SP2 2002 +
NeuGcHex5HexNAc3 1743 + + NeuAcHex5HexNAc4SP 2010 + +
NeuAcHex4HexNAc3dHexSP 1791 + Hex5HexNAc4dHex2SP 2011 +
Hex5HexNAc3dHex2SP 1808 + NeuGcHex5HexNAc4SP 2026 +
Hex6NexNAc3dHexSP 1824 + + Hex6HexNAc4dHexSP 2027 + +
NeuAc2Hex3HexNAc3dHex 1840 + + Hex7HexNAc4SP and/or
NeuAc2Hex4HexNAc3 1856 + Hex4HexNAc6SP2 and/or 2043 +
NeuAcHex4HexNAc3dHex2 1857 + NeuAc2Hex3HexNAc4dHex
NeuAcHex5HexNAc3dHex and/or NeuAcHex4HexNAc5SP 2051 +
NeuGcHex4HexNAc3dHex2 1873 + + Hex4HexNAc5dHex2SP 2052 +
NeuAcHex5HexNAc3SP2 1887 + NeuAc2Hex4HexNAc4 2059 +
NeuAcHex6HexNAc3 1889 + + NeuAcHex4HexNAc4dHex2 2060 + +
Hex8HexNAc3SP and/or NeuAcHex4HexNAc4dHexSP2 2074 +
NeuAc2Hex4HexNAc3dHex 2002 + + NeuAcHex5HexNAc4dHex 2076 + +
NeuAcHex4HexNAc3dHex3 2003 + NeuAcHex6HexNAc4 and/or 2092 + +
NeuAc2Hex5HexNAc3 and/or 2018 + + NeuGcHex5HexNAc4dHex
NeuGcNeuAcHex4HexNAc3dHex NeuAcHex3HexNAc5dHex2 and/or 2101 +
NeuAcHex5HexNAc3dHex2 2019 + + NeuAc2Hex4HexNAc4Ac
NeuGcNeuAcHex5HexNAc3 and/or NeuGcHex6HexNAc4 2108 +
NeuGc2Hex4HexNAc3dHex 2034 + NeuAcHex4HexNAc5dHex 2117 +
NeuAcHex6HexNAc3dHex 2035 + + Hex4HexNAc5dHex2SP2 2132 +
NeuGc2Hex5HexNAc3 2050 + NeuAcHex5HexNAc5 2133 + +
NeuAcHex7HexNAc3 2051 + + NeuAc2Hex4HexNAc4SP 2139 +
NeuAc2Hex4HexNAc3dHexSP and/or 2082 + NeuAcHex5HexNAc4dHexSP 2156 + +
Hex8HexNAc3SP2 Hex5HexNAc4dHex3SP 2157 +
NeuAcHex6HexNAc3dHexSP 2115 + Hex6HexNAc5SP2 2164 +
Hex8HexNAc3dHexSP and/or 2148 + NeuAcHex6HexNAc4SP and/or 2172 + +
NeuAc2Hex4HexNAc3dHex2 NeuGcHex5HexNAc4dHexSP
NeuAc2Hex5HexNAc3dHex and/or 2164 + + Hex6HexNAc4dHex2SP and/or 2173 +
Hex6HexNAc5SP2 Hex3HexNAc6dHex2SP2
NeuAcHex5HexNAc3dHex3 2165 + + NeuAcHex4HexNAc6 2174 +
NeuAcHex8HexNAc3SP and/or 2293 + NeuAc3Hex3HexNAc4 and/or
NeuAc3Hex4HexNAc3dHex NeuGcHex6HexNAc4SP and/or 2188 +
NeuAc2Hex5HexNAc3dHex2 and/or 2310 + NeuAc2NeuGcHex2HexNAc4dHex
NeuGcNeuAcHex4HexNAc3dHex3 NeuAc2Hex3HexNAc4dHex2 and/or
NeuAc3Hex5HexNAc3SP 2389 + Hex7HexNAc4dHexSP and/or 2189 + +
NeuAc2Hex5HexNAc3dHex2SP 2390 + + Hex4HexNAc6dHexSP2
NeuAc2Hex6HexNAc3dHexSP 2406 NeuAcHex3HexNAc4dHex4 2190 + +
NeuAcHex8HexNAc3dHexSP and/or 2439 Hex4HexNAc5dHex3SP 2198 + +
NeuAc3Hex4HexNAc3dHex2 NeuAc2Hex4HexNAc4dHex 2205 +
NeuAcHex9HexNAc3dHex 2521 + NeuAc2Hex4HexNAc4SP2 2219 +
NeuAc2Hex5HexNAc4 2221 + +
NeuAcHex5HexNAc4dHex2 2222 + +
Hex6HexNAc5dHexSP 2230 +
NeuGcNeuAcHex5HexNAc4 2237 +
NeuAcHex6HexNAc4dHex and/or 2238 + +
NeuGcHex5HexNAc4dHex2
NeuAc2Hex3HexNAc5dHex and/or 2246 +
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Hex7HexNAc5SP NeuAc2Hex5HexNAc6 2627 +
NeuGc2Hex5HexNAc4 2253 + NeuGcHex8HexNAc5 and/or 2635 +
NeuAcHex7HexNAc4 and/or NeuAcHex4HexNAc5dHex4SP
NeuGcHex6HexNAc4dHex 2254 + + NeuAcHex6HexNAc6dHex 2644 + +
NeuAc2Hex4HexNAc5 2262 + NeuAc2Hex5HexNAc4dHex3 2659 +
NeuAcHex4HexNAc5dHex2 and/or 2263 + NeuAcHex7HexNAc6 2660 + +
NeuAc2Hex5HexNAc4Ac NeuGcNeuAc2Hex5HexNAc4dHex 2674 +
Hex5HexNAc6dHexSP 2271 + and/orNeuAc3Hex6HexNAc4
NeuAcHex5HexNAc5dHex 2279 + + NeuAc2Hex4HexNAc5dHex2SP2 2714 +
NeuAc2Hex4HexNAc4dHexSP and/or 2285 + NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
Hex11HexNAc2SP NeuAc3Hex5HexNAc5
NeuAcHex6HexNAc5 2295 + + NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAc2Hex5HexNAc4SP 2301 + NeuAc2Hex6HexNAc5dHex 2732 + +
NeuAcHex5HexNAc4dHex2SP 2302 + NeuAcHex6HexNAc5dHex3 2733 + +
NeuAc2Hex5HexNAc4Ac2 2305 + NeuGcNeuAcHex6HexNAc5dHex 2748 +
NeuAcHex6HexNAc4dHexSP 2318 + + NeuAcHex8HexNAc5dHex 2765 + +
Hex6HexNAc4dHex3SP and/or 2319 + NeuGcHex8HexNAc5dHex and/or 2781 +
NeuGcNeuAcHex3HexNAc6 NeuAcHex9HexNAc5
NeuAcHex4HexNAc6dHex 2320 + NeuAcHex6HexNAc6dHex2 2791 +
NeuAcHex5HexNAc5dHexAc 2321 + NeuAc3Hex5HexNAc4dHex2 and/or 2804 +
Hex7HexNAc4dHex2SP and/or 2335 + NeuAcHex6HexNAc6dHexSP2
Hex4HexNAc6dHex2SP2 Hex6HexNAc6dHex3SP2 2805 +
NeuAcHex5HexNAc6 2336 + + NeuAcHex7HexNAc6dHex 2807 + +
NeuAc3Hex4HexNac4 2350 + NeuAc2Hex6HexNAc5dHexSP 2812 +
NeuAc2Hex4HexNAc4dHexSP 2365 + NeuAcHex6HexNAc5dHex3SP 2813 +
NeuAc2Hex5HexNAc4dHex 2367 + + NeuGcNeuAc3Hex5HexNAc4 2819 +
NeuAcHex5HexNAc4dHex3 2368 + + NeuAc3Hex6HexNAc4dHex and/or 2820 +
NeuAc2Hex6HexNAc4 and/or 2383 + + NeuGcNeuAc2Hex5HexNAc4dHex2
N euGcNeuAcH ex5HexNAc4d Hex NeuAc3Hex6HexNAc5 2878 + +
NeuAcHex6HexNAc4dHex2 and/or 2384 + + NeuAc2Hex6HexNAc5dHex2 2879 + +
NeuGcHex5HexNAc4dHex3 NeuAcHex6HexNAc5dHex4 2880 + +
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + + NeuGcNeuAc2Hex6HexNAc5 2894 +
Hex7HexNAc5dHexSP NeuAc2Hex7HexNAc5dHex and/or 2895 + +
NeuAcHex3HexNAc5dHex4 2393 + NeuGcNeuAcHex6HexNAc5dHex2
NeuGc2H ex5HexNAc4d Hex 2399 + NeuAc3Hex6HexNAc4dHexSP and/or 2900 +
NeuAcHex4HexNAc6dHexSP and/or NeuGcNeuAc2Hex5HexNAc4dHex2SP
NeuGcHex6HexNAc4dHex2 and/or 2400 + NeuGc2Hex6HexNAc5dHex2 2911 +
NeuAcHex7HexNAc4dHex NeuAc2Hex5HexNAc6dHex2 2920 +
Hex4HexNAc6dHex3SP 2401 + NeuGc3Hex6HexNAc5 2925 +
NeuAc2Hex4HexNAc5dHex 2408 + NeuGcNeuAc2Hex5HexNAc6 2935 +
NeuAcHex4HexNAc5dHex3 and/or 2409 + NeuAc2Hex6HexNAc6dHex and/or 2936 +
NeuAc2Hex5HexNAc4dHexAc NeuGcNeuAcHex5HexNAc6dHex2
NeuAc2Hex5HexNAc5 2424 + NeuAcHex6HexNAc6dHex3 2937 +
NeuAcHex5HexNAc5dHex2 2425 + + NeuGc2NeuAcHex5HexNAc6 and/or
NeuAcHex6HexNAc5dHex 2441 + + NeuAc3Hex5HexNAc4dHex3 2951 +
NeuAc2Hex5HexNAc4dHexSP 2447 + + NeuAc2Hex7HexNAc6 2952 +
NeuAcHex5HexNAc4dHex3SP 2448 + NeuAcHex7HexNAc6dHex2 2953 + +
NeuAcHex7HexNAc5 and/or 2457 + + Hex8HexNAc7dHexSP 2961 +
NeuGcHex6HexNAc5dHex NeuAc2Hex4HexNAc7dHex2 2961 +
NeuGcHex7HexNAc5 2473 + NeuAcHex7HexNAc7dHex 3010 +
NeuAcHex5HexNAc6dHex 2482 + NeuAc3Hex6HexNAc5dHex 3024 + +
NeuAcHex4HexNAc5dHex3SP 2489 + NeuAc2Hex6HexNAc5dHex3 3025 + +
Hex6HexNAc7SP 2490 + NeuAcHex8HexNAc7 3026 +
NeuAc3Hex5HexNAc4 2512 + NeuGc3Hex6HexNAc5dHex and/or
NeuAc2Hex5HexNAc4dHex2 2513 + + NeuGc2NeuAcHex7HexNAc5 3072 +
NeuAcHex5HexNAc4dHex4 2514 + NeuAc3Hex6HexNAc6 3081 + +
NeuAcHex6HexNAc5dHexSP and/or 2521 + NeuAc2Hex6HexNAc6dHex2 3082 +
NeuAc3Hex2HexNAc5dHex2 NeuAc2Hex7HexNAc6dHex 3098 + +
Hex6HexNAc5dHex3SP 2522 + NeuAcHex7HexNAc6dHex3 3099 + +
NeuGcNeuAc2Hex5HexNAc4 2528 + NeuAc3Hex6HexNAc5dHexSP 3104 +
NeuAc2Hex6HexNAc4dHex and/or 2529 + NeuAc2Hex6HexNAc5dHex3SP 3105 +
NeuGcNeuAcHex5HexNAc4dHex2 NeuAc3Hex6HexNAc5dHex2 3170 +
NeuGc2NeuAcHex5HexNAc4 2544 + NeuAc2Hex6HexNAc5dHex4 3171 +
NeuGc2Hex5HexNAc4dHex2 and/or 2545 + NeuAcHex8HexNAc7dHex 3172 +
NeuGcNeuAcHex6HexNAc4dHex NeuAc3Hex6HexNAc6dHex 3227 +
NeuGc3Hex5HexNAc4 2560 + NeuAc2Hex6HexNAc6dHex3 3228 +
NeuGc2Hex6HexNAc4dHex 2561 + NeuAc3Hex7HexNAc6 3243 +
NeuAc2Hex5HexNAc5dHex 2570 + + NeuAc2Hex7HexNAc6dHex2 3244 + +
NeuAcHex5HexNAc5dHex3 2571 + NeuAcHex7HexNAc6dHex4 3245 + +
NeuAc2Hex6HexNAc5 2586 + + NeuAc2Hex7HexNAc7dHex 3301 +
NeuAcHex6HexNAc5dHex2 2587 + + NeuAcHex7HexNAc7dHex3 3302 +
Hex7HexNAc6dHexSP 2595 + NeuAc2Hex8HexNAc7 3317 +
NeuGcNeuAcHex6HexNAc5 2602 + NeuAcHex8HexNAc7dHex2 3318 +
NeuAcHex7HexNAc5dHex and/or 2603 + + NeuAc3Hex7HexNAc6dHex 3389 + +
NeuGcHex6HexNAc5dHex2 NeuAc2Hex7HexNAc6dHex3 3390 + +
NeuAcHex8HexNAc5 and/or 2619 + NeuAcHex7HexNAc6dHex5 and/or
NeuGcHex7HexNAc5dHex NeuAcHex9HexNAcB 3391 +
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NeuAc2Hex8HexNAc7dHex 3463 + HexNAcZ3 and dHexZ1
NeuAcHex8HexNAc7dHex3 3464 + _ = d
NeuAc2Hex7HexNAc6dHex4 3536 + (including fucosylated N- ~ ~+ ~ __
E E NeuAcHex9HexNAc8dHex 3537 + glycans)
NeuAc3Hex8HexNAc7 3608 + Proposed composition m/z
NeuAc2Hex8HexNac7dHex2 3609 + Hex3HexNAc3dHexSP 1338 + +
NeuAcHex8HexNac7dHex4 3610 Hex4HexNAc3dHexSP 1500 + +
NeuAc4Hex7HexNAc6dHex 3680 + NeuAcHex3HexNAc3dHex 1549 + +
NeuAc3Hex7HexNAc6dHex3 3681 + Hex4HexNAc3dHex2SP 1646 +
NeuAc2Hex9HexNAc8 3682 + Hex5HexNAc3dHexSP 1662 + +
NeuAcHex9HexNAc8dHex2 3683 + Hex6HexNAc3SP and/or
NeuAc3Hex8HexNAc7dHex 3754 + NeuAc2Hex2HexNAc3dHex 1678 + +
NeuAc2Hex8HexNAc7dHex3 3755 + NeuAcHex3HexNAc3dHexSP2 1709 +
NeuAcHex10HexNAc9 and/or 3756 + NeuAcHex4HexNAc3dHex 1711 + +
NeuAcHex8HexNAc7dHex5 NeuAcHex5HexNAc3 and/or
NeuAc4Hex6HexNAc8 3778 + NeuGcHex4HexNAc3dHex 1727 + +
NeuAc3Hex7HexNAc6dHex4 3827 + NeuAcHex4HexNAc3dHexSP 1791 +
NeuAc2Hex9HexNAc8dHex 3828 + Hex5HexNAc3dHex2SP 1808 +
NeuAcHex9HexNAc8dHex3 3829 + Hex6NexNAc3dHexSP 1824 + +
NeuAc2Hex8HexNAc7dHex4 3901 + NeuAc2Hex3HexNAc3dHex 1840 + +
NeuAc2Hex9HexNAc8dHex2 3974 + NeuAcHex4HexNAc3dHex2 1857 +
NeuAcHex9HexNAc8dHex4 3975 + NeuAcHex5HexNAc3dHex and/or
NeuAc4Hex8HexNAc7dHex 4045 + NeuGcHex4HexNAc3dHex2 1873 + +
NeuAc3Hex8HexNAc7dHex3 4046 + Hex8HexNAc3SP and/or
NeuAc2Hex10HexNAc9 and/or 4047 + NeuAc2Hex4HexNAc3dHex 2002 + +
NeuAc2Hex8HexNAc7dHex5 NeuAcHex4HexNAc3dHex3 2003 +
NeuAc3Hex9HexNAcBdHex 4119 + NeuAc2Hex5HexNAc3 and/or
NeuAc2Hex9HexNAc8dHex3 4120 NeuGcNeuAcHex4HexNAc3dHex 2018 + +
NeuAcHex5HexNAc3dHex2 2019 + +
NeuGcNeuAcHex5HexNAc3 and/or 2034 +
NeuGc2Hex4HexNAc3dHex
NeuAcHex6HexNAc3dHex 2035 + +
NeuAc2Hex4HexNAc3dHexSP and/or 2082 +
Hex8HexNAc3SP2
NeuAcHex6HexNAc3dHexSP 2115 +
Hex8HexNAc3dHexSP and/or 2148 +
NeuAc2Hex4HexNAc3dHex2
NeuAc2Hex5HexNAc3dHex and/or 2164 + +
Hex6HexNAc5SP2
NeuAcHex5HexNAc3dHex3 2165 + +
NeuAcHex8HexNAc3SP and/or 2293 +
NeuAc3Hex4HexNAc3dHex
NeuAc2Hex5HexNAc3dHex2 and/or 2310 +
NeuGcNeuAcHex4HexNAc3dHex3
NeuAc2Hex5HexNAc3dHex2SP 2390 + +
NeuAc2Hex6HexNAc3dHexSP 2406 +
NeuAcHex8HexNAc3dHexSP and/or 2439 +
NeuAc3Hex4HexNAc3dHex2
NeuAcHex9HexNAc3dHex 2521 +
Hex4HexNAc4dHexSP 1703 + +
Hex5HexNAc4dHexSP 1865 + +
Hex4HexNAc5dHexSP 1906 +
NeuAcHex4HexNAc4dHex 1914 + +
Hex5HexNAc4dHex2SP 2011 +
Hex6HexNAc4dHexSP 2027 + +
Hex7HexNAc4SP and/or
Hex4HexNAc6SP2 and/or 2043 +
NeuAc2Hex3HexNAc4dHex
Hex4HexNAc5dHex2SP 2052 +
NeuAcHex4HexNAc4dHex2 2060 + +
NeuAcHex4HexNAc4dHexSP2 2074 +
NeuAcHex5HexNAc4dHex 2076 + +
NeuAcHex6HexNAc4 and/or 2092 + +
NeuGcHex5HexNAc4dHex
NeuAcHex3HexNAc5dHex2 and/or 2101 +
NeuAc2Hex4HexNAc4Ac
NeuAcHex4HexNAc5dHex 2117 +
Hex4HexNAc5dHex2SP2 2132 +
NeuAcHex5HexNAc4dHexSP 2156 + +
Hex5HexNAc4dHex3SP 2157 +
NeuAcHex6HexNAc4SP and/or 2172 + +
NeuGcHex5HexNAc4dHexSP
Hex6HexNAc4dHex2SP and/or 2173 +
Hex3HexNAc6dHex2SP2
NeuAc3Hex3HexNAc4 and/or
NeuGcHex6HexNAc4SP and/or 2188 +
NeuAc2NeuGcHex2HexNAc4dHex
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NeuAc2Hex3HexNAc4dHex2 and/or and/or NeuAc3Hex6HexNAc4
Hex7HexNAc4dHexSP and/or 2189 + + NeuAc2Hex4HexNAc5dHex2SP2 2714 +
Hex4HexNAc6dHexSP2 NeuAcHex4HexNAc5dHex4SP2 and/or
NeuAcHex3HexNAc4dHex4 2190 + + NeuAc3Hex5HexNAc5 2715 + +
Hex4HexNAc5dHex3SP 2198 + + NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAc2Hex4HexNAc4dHex 2205 + NeuAc2Hex6HexNAc5dHex 2732 + +
NeuAcHex5HexNAc4dHex2 2222 + + NeuAcHex6HexNAc5dHex3 2733 + +
Hex6HexNAc5dHexSP 2230 + NeuGcNeuAcHex6HexNAc5dHex 2748 +
NeuAcHex6HexNAc4dHex and/or 2238 + + NeuAcHex8HexNAc5dHex 2765 + +
NeuGcHex5HexNAc4dHex2 NeuGcHex8HexNAc5dHex and/or 2781 +
NeuAc2Hex3HexNAc5dHex and/or 2246 + NeuAcHex9HexNAc5
Hex7HexNAc5SP NeuAcHex6HexNAc6dHex2 2791 +
NeuAcHex7HexNAc4 and/or 2254 + + NeuAc3Hex5HexNAc4dHex2 and/or 2804 +
NeuGcHex6HexNAc4dHex NeuAcHex6HexNAc6dHexSP2
NeuAcHex4HexNAc5dHex2 and/or 2263 + Hex6HexNAc6dHex3SP2 2805 +
NeuAc2Hex5HexNAc4Ac NeuAcHex7HexNAc6dHex 2807 + +
Hex5HexNAc6dHexSP 2271 + NeuAc2Hex6HexNAc5dHexSP 2812 +
NeuAcHex5HexNAc5dHex 2279 + + NeuAcHex6HexNAc5dHex3SP 2813 +
NeuAc2Hex4HexNAc4dHexSP and/or 2285 + NeuAc3Hex6HexNAc4dHex and/or
Hex11HexNAc2SP NeuGcNeuAc2Hex5HexNAc4dHex2 2820 +
NeuAcHex5HexNAc4dHex2SP 2302 + NeuAc2Hex6HexNAc5dHex2 2879 + +
NeuAcHex6HexNAc4dHexSP 2318 + + NeuAcHex6HexNAc5dHex4 2880 + +
Hex6HexNAc4dHex3SP and/or 2319 + NeuAc2Hex7HexNAc5dHex and/or
NeuGcNeuAcHex3HexNAc6 NeuGcNeuAcHex6HexNAc5dHex2 2895 + +
NeuAcHex4HexNAc6dHex 2320 + NeuAc3Hex6HexNAc4dHexSP and/or
NeuAcHex5HexNAc5dHexAc 2321 + NeuGcNeuAc2Hex5HexNAc4dHex2SP 2900 +
Hex7HexNAc4dHex2SP and/or 2335 + NeuGc2Hex6HexNAc5dHex2 2911 +
Hex4HexNAc6dHex2SP2 NeuAc2Hex5HexNAc6dHex2 2920 +
NeuAc2Hex4HexNAc4dHexSP 2365 + NeuAc2Hex6HexNAc6dHex and/or
NeuAc2Hex5HexNAc4dHex 2367 + + NeuGcNeuAcHex5HexNAc6dHex2 2936 +
NeuAcHex5HexNAc4dHex3 2368 + + NeuAcHex6HexNAc6dHex3 2937 +
NeuAc2Hex6HexNAc4 and/or 2383 + + NeuGc2NeuAcHex5HexNAc6 and/or 2951 +
NeuGcNeuAcHex5HexNAc4dHex NeuAc3Hex5HexNAc4dHex3
NeuAcHex6HexNAc4dHex2 and/or 2384 + + NeuAcHex7HexNAc6dHex2 2953 + +
NeuGcHex5HexNAc4dHex3 Hex8HexNAc7dHexSP 2961 +
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + + NeuAc2Hex4HexNAc7dHex2 2961 +
Hex7HexNAc5dHexSP NeuAcHex7HexNAc7dHex 3010 +
NeuAcHex3HexNAc5dHex4 2393 + NeuAc3Hex6HexNAc5dHex 3024 + +
NeuGc2H ex5HexNAc4d Hex 2399 + NeuAc2Hex6HexNAc5dHex3 3025 + +
NeuAcHex4HexNAc6dHexSP and/or NeuGc3Hex6HexNAc5dHex and/or
NeuGcHex6HexNAc4dHex2 and/or 2400 + NeuGc2NeuAcHex7HexNAc5 3072 +
NeuAcHex7HexNAc4dHex NeuAc2Hex6HexNAc6dHex2 3082 +
Hex4HexNAc6dHex3SP 2401 + NeuAc2Hex7HexNAc6dHex 3098 + +
NeuAc2Hex4HexNAc5dHex 2408 + NeuAcHex7HexNAc6dHex3 3099 + +
NeuAcHex4HexNAc5dHex3 and/or 2409 + NeuAc3Hex6HexNAc5dHexSP 3104 +
NeuAc2Hex5HexNAc4dHexAc NeuAc2Hex6HexNAc5dHex3SP 3105 +
NeuAcHex5HexNAc5dHex2 2425 + + NeuAc3Hex6HexNAc5dHex2 3170 +
NeuAcHex6HexNAc5dHex 2441 + + NeuAc2Hex6HexNAc5dHex4 3171 +
NeuAc2Hex5HexNAc4dHexSP 2447 + + NeuAcHex8HexNAc7dHex 3172 +
NeuAcHex5HexNAc4dHex3SP 2448 + NeuAc3Hex6HexNAc6dHex 3227 +
NeuAcHex7HexNAc5 and/or 2457 + + NeuAc2Hex6HexNAc6dHex3 3228 +
NeuGcHex6HexNAc5dHex NeuAc2Hex7HexNAc6dHex2 3244 + +
NeuAcHex5HexNAc6dHex 2482 + NeuAcHex7HexNAc6dHex4 3245 + +
NeuAcHex4HexNAc5dHex3SP 2489 + NeuAc2Hex7HexNAc7dHex 3301 +
NeuAc2Hex5HexNAc4dHex2 2513 + + NeuAcHex7HexNAc7dHex3 3302 +
NeuAcHex5HexNAc4dHex4 2514 + NeuAcHex8HexNAc7dHex2 3318 +
NeuAcHex6HexNAc5dHexSP and/or 2521 + NeuAc3Hex7HexNAc6dHex 3389 + +
NeuAc3Hex2HexNAc5dHex2 NeuAc2Hex7HexNAc6dHex3 3390 + +
Hex6HexNAc5dHex3SP 2522 + NeuAcHex7HexNAc6dHex5 and/or
NeuAc2Hex6HexNAc4dHex and/or 2529 + NeuAcHex9HexNAc8 3391 +
NeuGcNeuAcHex5HexNAc4dHex2 NeuAc2Hex8HexNAc7dHex 3463 +
NeuGc2Hex5HexNAc4dHex2 and/or 2545 + NeuAcHex8HexNAc7dHex3 3464 +
NeuGcNeuAcHex6HexNAc4dHex NeuAc2Hex7HexNAc6dHex4 3536 +
NeuGc2Hex6HexNAc4dHex 2561 + NeuAcHex9HexNAcBdHex 3537 +
NeuAc2Hex5HexNAc5dHex 2570 + + NeuAc2Hex8HexNac7dHex2 3609 +
NeuAcHex5HexNAc5dHex3 2571 + NeuAcHex8HexNac7dHex4 3610 +
NeuAcHex6HexNAc5dHex2 2587 + + NeuAc4Hex7HexNAc6dHex 3680 +
Hex7HexNAc6dHexSP 2595 + NeuAc3Hex7HexNAc6dHex3 3681 +
NeuAcHex7HexNAc5dHex and/or 2603 + + NeuAcHex9HexNAcBdHex2 3683 +
NeuGcHex6HexNAc5dHex2 NeuAc3Hex8HexNAc7dHex 3754 +
NeuAcHex8HexNAc5 and/or 2619 +
NeuGcHex7HexNAc5dHex NeuAc2Hex8HexNAc7dHex3 3755 +
NeuGcHex8HexNAc5 and/or NeuAcHex10HexNAc9 and/or 3756 +
NeuAcHex4HexNAc5dHex4SP 2635 + NeuAcHex8HexNAc7dHex5
NeuAcHex6HexNAc6dHex 2644 + + NeuAc3Hex7HexNAc6dHex4 3827 +
NeuAc2Hex5HexNAc4dHex3 2659 + NeuAc2Hex9HexNAc8dHex 3828 +
NeuGcNeuAc2Hex5HexNAc4dHex 2674 + NeuAcHex9HexNAc8dHex3 3829 +
NeuAc2Hex8HexNAc7dHex4 3901 +
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NeuAc2Hex9HexNAc8dHex2 3974 + NeuAcHex6HexNAc5dHexSP and/or 2521 +
NeuAcHex9HexNAc8dHex4 3975 + NeuAc3Hex2HexNAc5dHex2
NeuAc4Hex8HexNAc7dHex 4045 + Hex6HexNAc5dHex3SP 2522 +
NeuAc3Hex8HexNAc7dHex3 4046 + NeuAc2Hex6HexNAc4dHex and/or 2529 +
NeuAc2Hex10HexNAc9 and/or NeuGcNeuAcHex5HexNAc4dHex2
NeuAc2Hex8HexNAc7dHex5 4047 + NeuGc2Hex5HexNAc4dHex2 and/or 2545 +
NeuAc3Hex9HexNAc8dHex 4119 + NeuGcNeuAcHex6HexNAc4dHex
NeuAc2Hex9HexNAc8dHex3 4120 + NeuAcHex5HexNAc5dHex3 2571 +
NeuAcHex6HexNAc5dHex2 2587 + +
HexNAcZ3 and dHexZ2 NeuGcHex6HexNAc5dHex2nd/or 2603 + +
(including multifucosylated N- = NeuGcHex8HexNAc5 and/or 2635 +
E E NeuAcHex4HexNAc5dHex4SP
glycans) _ _ E NeuAc2Hex5HexNAc4dHex3 2659 +
Proposed composition m/z NeuAc2Hex4HexNAc5dHex2SP2 2714 +
Hex4HexNAc3dHex2SP 1646 + NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAcHex3HexNAc3dHexSP2 1709 + NeuAc3Hex5HexNAc5
Hex5HexNAc3dHex2SP 1808 + NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAcHex4HexNAc3dHex2 1857 + NeuAcHex6HexNAc5dHex3 2733 + +
NeuAcHex5HexNAc3dHex and/or NeuAcHex6HexNAc6dHex2 2791 +
NeuGcHex4HexNAc3dHex2 1873 + + NeuAc3Hex5HexNAc4dHex2 and/or
NeuAcHex6HexNAc6dHexSP2 2804 +
NeuAcHex4HexNAc3dHex3 2003 +
NeuAcHex5HexNAc3dHex2 2019 + + Hex6HexNAc6dHex3SP2 2805 +
Hex8HexNAc3dHexSP and/or NeuAcHex6HexNAc5dHex3SP 2813 +
NeuAc2Hex4HexNAc3dHex2 2148 + NeuAc3Hex6HexNAc4dHex and/or
NeuGcNeuAc2Hex5HexNAc4dHex2 2820 +
NeuAcHex5HexNAc3dHex3 2165 + +
NeuAc2Hex5HexNAc3dHex2 and/or NeuAc2Hex6HexNAc5dHex2 2879 + +
NeuGcNeuAcHex4HexNAc3dHex3 2310 + NeuAcHex6HexNAc5dHex4 2880 + +
NeuAc2Hex5HexNAc3dHex2SP 2390 + + NeuAc2Hex7HexNAc5dHex and/or 2895 + +
NeuAcHex8HexNAc3dHexSP and/or NeuGcNeuAcHex6HexNAc5dHex2
NeuAc3Hex4HexNAc3dHex2 2439 + NeuAc3Hex6HexNAc4dHexSP and/or
NeuGcNeuAc2Hex5HexNAc4dHex2SP 2900 +
Hex5HexNAc4dHex2SP 2011 +
Hex4HexNAc5dHex2SP 2052 + NeuGc2Hex6HexNAc5dHex2 2911 +
NeuAcHex4HexNAc4dHex2 2060 + + NeuAc2Hex5HexNAc6dHex2 2920 +
NeuAcHex3HexNAc5dHex2 and/or NeuAc2Hex6HexNAc6dHex and/or 2936 +
NeuAc2Hex4HexNAc4Ac 2101 + NeuGcNeuAcHex5HexNAc6dHex2
Hex4HexNAc5dHex2SP2 2132 + NeuAcHex6HexNAc6dHex3 2937 +
Hex5HexNAc4dHex3SP 2157 + NeuGc2NeuAcHex5HexNAc6 and/or 2951 +
NeuAcHex6HexNAc4SP and/or NeuAc3Hex5HexNAc4dHex3
NeuGcHex5HexNAc4dHexSP 2172 + + NeuAcHex7HexNAc6dHex2 2953 + +
Hex6HexNAc4dHex2SP and/or NeuAc2Hex4HexNAc7dHex2 2961 +
Hex3HexNAc6dHex2SP2 2173 + NeuAc2Hex6HexNAc5dHex3 3025 + +
NeuAc2Hex3HexNAc4dHex2 and/or NeuAc2Hex6HexNAc6dHex2 3082 +
Hex7HexNAc4dHexSP and/or 2189 + + NeuAcHex7HexNAc6dHex3 3099 + +
Hex4HexNAc6dHexSP2 NeuAc3Hex6HexNAc5dHexSP 3104 +
NeuAcHex3HexNAc4dHex4 2190 + + NeuAc2Hex6HexNAc5dHex3SP 3105 +
Hex4HexNAc5dHex3SP 2198 + + NeuAc3Hex6HexNAc5dHex2 3170 +
NeuAcHex5HexNAc4dHex2 2222 + + NeuAc2Hex6HexNAc5dHex4 3171 +
NeuAcHex6HexNAc4dHex and/or NeuAc2Hex6HexNAc6dHex3 3228 +
NeuGcHex5HexNAc4dHex2 2238 + + NeuAc2Hex7HexNAc6dHex2 3244 + +
NeuAcHex4HexNAc5dHex2 and/or NeuAcHex7HexNAc6dHex4 3245 + +
NeuAc2Hex5HexNAc4Ac 2263 + NeuAcHex7HexNAc7dHex3 3302 +
NeuAc2Hex4HexNAc4dHexSP and/or 2285 + NeuAcHex8HexNAc7dHex2 3318 +
Hex11HexNAc2SP NeuAc2Hex7HexNAc6dHex3 3390 + +
NeuAcHex5HexNAc4dHex2SP 2302 + NeuAcHex7HexNAc6dHex5 and/or 3391 +
Hex6HexNAc4dHex3SP and/or 2319 + NeuAcHex9HexNAc8
NeuGcNeuAcHex3HexNAc6 NeuAcHex8HexNAc7dHex3 3464 +
Hex7HexNAc4dHex2SP and/or 2335 + NeuAc2Hex7HexNAc6dHex4 3536 +
Hex4HexNAc6dHex2SP2 NeuAc2Hex8HexNac7dHex2 3609 +
NeuAc2Hex4HexNAc4dHexSP 2365 + NeuAcHex8HexNac7dHex4 3610 +
NeuAcHex5HexNAc4dHex3 2368 + + NeuAc3Hex7HexNAc6dHex3 3681 +
NeuAcHex6HexNAc4dHex2 and/or 2384 + + NeuAcHex9HexNAcBdHex2 3683 +
NeuGcHex5HexNAc4dHex3 NeuAc2Hex8HexNAc7dHex3 3755 +
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + + NeuAcHex10HexNAc9 and/or
Hex7HexNAc5dHexSP NeuAcHex8HexNAc7dHex5 3756 +
NeuAcHex3HexNAc5dHex4 2393 + NeuAc3Hex7HexNAc6dHex4 3827 +
NeuAcHex4HexNAc6dHexSP and/or NeuAcHex9HexNAcBdHex3 3829 +
NeuGcHex6HexNAc4dHex2 and/or 2400 + NeuAc2Hex8HexNAc7dHex4 3901 +
NeuAcHex7HexNAc4dHex NeuAc2Hex9HexNAc8dHex2 3974 +
Hex4HexNAc6dHex3SP 2401 + NeuAcHex9HexNAcBdHex4 3975 +
NeuAcHex4HexNAc5dHex3 and/or 2409 + NeuAc3Hex8HexNAc7dHex3 4046 +
NeuAc2Hex5HexNAc4dHexAc NeuAc2Hex10HexNAc9 and/or
NeuAcHex5HexNAc5dHex2 2425 + + NeuAc2Hex8HexNAc7dHex5 4047 +
NeuAcHex5HexNAc4dHex3SP 2448 + NeuAc2Hex9HexNAc8dHex3 4120 +
NeuAcHex4HexNAc5dHex3SP 2489 +
NeuAc2Hex5HexNAc4dHex2 2513
NeuAcHex5HexNAc4dHex4 2514 +
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HexNAc>HexZ3 HexNAc=HexZ5
(terminal HexNAc, N>H) E ~ _ (terminal HexNAc, N=H) ~
x'`
Proposed composition m/z Proposed composition m/z
Hex6HexNAc3SP and/or 1678 + + Hex5HexNAc5SP2 2002 +
NeuAc2Hex2HexNAc3dHex NeuAcHex5HexNAc5 2133 + +
NeuAcHex3HexNAc4 1606 + NeuAcHex5HexNAc5dHex 2279 + +
NeuAcHex3HexNac5 1809 + NeuAcHex5HexNAc5dHexAc 2321 +
NeuGcHex3HexNAc5 1825 + + NeuAc2Hex5HexNAc5 2424 +
Hex4HexNAc5dHexSP 1906 + NeuAcHex5HexNAc5dHex2 2425 + +
NeuAcHex4HexNAc5 1971 + NeuAc2Hex5HexNAc5dHex 2570 + +
Hex7HexNAc4SP and/or NeuAcHex5HexNAc5dHex3 2571 +
Hex4HexNAc6SP2 and/or 2043 + NeuAcHex6HexNAc6dHex 2644 + +
NeuAc2Hex3HexNAc4dHex NeuAcHex4HexNAc5dHex4SP2 and/or
NeuAcHex4HexNAc5SP 2051 + NeuAc3Hex5HexNAc5 2715 + +
Hex4HexNAc5dHex2SP 2052 + NeuAc2Hex5HexNAc5dHex2 2716 +
NeuAcHex3HexNAc5dHex2 and/or 2101 + NeuAcHex6HexNAc6dHex2 2791 +
NeuAc2Hex4HexNAc4Ac Hex6HexNAc6dHex3SP2 2805 +
NeuAcHex4HexNAc5dHex 2117 + NeuAc2Hex6HexNAc6dHex and/or
Hex4HexNAc5dHex2SP2 2132 + NeuGcNeuAcHex5HexNAc6dHex2 2936 +
Hex6HexNAc4dHex2SP and/or 2173 + NeuAcHex6HexNAc6dHex3 2937 +
Hex3HexNAc6dHex2SP2 NeuAcHex7HexNAc7dHex 3010 +
NeuAcHex4HexNAc6 2174 + NeuAc3Hex6HexNAc6 3081 + +
NeuAc3Hex3HexNAc4 and/or NeuAc2Hex6HexNAc6dHex2 3082 +
NeuGcHex6HexNAc4SP and/or 2188 + NeuAc3Hex6HexNAc6dHex 3227 +
NeuAc2NeuGcHex2HexNAc4dHex NeuAc2Hex6HexNAc6dHex3 3228 +
NeuAc2Hex3HexNAc4dHex2 and/or NeuAc2Hex7HexNAc7dHex 3301 +
Hex7HexNAc4dHexSP and/or 2189 + + NeuAcHex7HexNAc7dHex3 3302 +
Hex4HexNAc6dHexSP2
NeuAcHex3HexNAc4dHex4 2190 + +
Hex4HexNAc5dHex3SP 2198 + +
NeuAc2Hex4HexNAc5 2262 +
NeuAcHex4HexNAc5dHex2 and/or 2263 +
NeuAc2Hex5HexNAc4Ac
Hex5HexNAc6dHexSP 2271 +
NeuAcHex4HexNAc6dHex 2320 +
Hex7HexNAc4dHex2SP and/or 2335 +
Hex4HexNAc6dHex2SP2
NeuAcHex5HexNAc6 2336 + +
NeuAc2Hex3HexNAc5dHex2 and/or 2392 + +
Hex7HexNAc5dHexSP
NeuAcHex3HexNAc5dHex4 2393 +
NeuAcHex4HexNAc6dHexSP and/or
NeuGcHex6HexNAc4dHex2 and/or 2400 +
NeuAcHex7HexNAc4dHex
Hex4HexNAc6dHex3SP 2401 +
NeuAc2Hex4HexNAc5dHex 2408 +
NeuAcHex4HexNAc5dHex3 and/or 2409 +
NeuAc2Hex5HexNAc4dHexAc
NeuAcHex5HexNAc6dHex 2482 +
NeuAcHex4HexNAc5dHex3SP 2489 +
Hex6HexNAc7SP 2490 +
NeuGc3Hex5HexNAc4 2560 +
NeuAc2Hex5HexNAc6 2627 +
NeuAc2Hex4HexNAc5dHex2SP2 2714 +
NeuAcHex4HexNAc5dHex4SP2 and/or 2715 + +
NeuAc3Hex5HexNAc5
NeuAc2Hex5HexNAc6dHex2 2920 +
NeuGcNeuAc2Hex5HexNAc6 2935 +
NeuAc2Hex4HexNAc7dHex2 2961 +
NeuAc4Hex6HexNAc8 3778 +
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NeuGcHex6HexNAc4SP and/or
SPZ1 NeuAc2NeuGcHex2HexNAc4dHex
NeuAc2Hex3HexNAc4dHex2 and/or
(including sulphated and/or R R_= Hex7HexNAc4dHexSP and/or 2189 + +
~' E = Hex4HexNAc6dHexSP2
phosphorylated glycans) E
_ = Hex4HexNAc5dHex3SP 2198 + +
Proposed composition m/z NeuAc2Hex4HexNAc4SP2 2219 +
Hex3HexNAc2SP 989 + Hex6HexNAc5dHexSP 2230 +
Hex3HexNAc2dHexSP 1135 + NeuAc2Hex3HexNAc5dHex and/or 2246 +
Hex4HexNAc2SP 1151 + Hex7HexNAc5SP
Hex3HexNAc3SP 1192 + + NeuAc2Hex4HexNAc4dHexSP and/or 2285 +
Hex5HexNAc2SP 1313 + Hex11HexNAc2SP
Hex3HexNAc3dHexSP 1338 + + NeuAcHex8HexNAc3SP and/or 2293 +
Hex4HexNAc3SP 1354 + + NeuAc3Hex4HexNAc3dHex
Hex5HexNAc2dHexSP 1459 + + NeuAc2Hex5HexNAc4SP 2301 +
Hex6HexNAc2SP 1475 + NeuAcHex5HexNAc4dHex2SP 2302 +
Hex4HexNAc3dHexSP 1500 + + NeuAcHex6HexNAc4dHexSP 2318 + +
Hex5HexNAc3SP 1516 + Hex6HexNAc4dHex3SP 2319 +
Hex6HexNAc2SP2 1555 + Hex7HexNAc4dHex2SP and/or 2335 +
Hex4HexNAc4SP 1557 + + Hex4HexNAc6dHex2SP2
NeuAcHex3HexNAc3SP2 1563 + NeuAc2Hex4HexNAc4dHexSP 2365 +
Hex6HexNAc2dHexSP 1621 + + NeuAc3Hex5HexNAc3SP and/or 2389 +
Hex4HexNAc4SP2 and/or NeuAc2Hex5HexNAc4Ac4
Hex7HexNAc2SP 1637 + NeuAc2Hex5HexNAc3dHex2SP 2390 + +
Hex4HexNAc3dHex2SP 1646 + NeuAc2Hex3HexNAc5dHex2 and/or 2392 + +
Hex5HexNAc3dHexSP 1662 + + Hex7HexNAc5dHexSP
Hex6HexNAc3SP 1678 + NeuAcHex4HexNAc6dHexSP and/or
Hex4HexNAc4dHexSP 1703 + + NeuGcHex6HexNAc4dHex2 and/or 2400 +
NeuAcHex3HexNAc3dHexSP2 1709 + NeuAcHex7HexNAc4dHex
Hex4HexNAc4SP3 and/or NeuAc2Hex6HexNAc3dHexSP 2406 +
Hex7HexNAc2SP2 1717 + NeuAcHex8HexNAc3dHexSP and/or 2439 +
Hex5HexNAc4SP 1719 + + NeuAc3Hex4HexNAc3dHex2
Hex7HexNAc2dHexSP 1783 + NeuAc2Hex5HexNAc4dHexSP and/or
NeuAcHex4HexNAc3dHexSP 1791 + NeuAc2Hex8HexNAc2dHex and/or 2447 + +
Hex5HexNAc4SP2 and/or Hex12HexNAc2SP
Hex8HexNAc2SP 1799 + NeuAcHex5HexNAc4dHex3SP and/or 2448 +
NeuAcHex8HexNAc2dHex3
Hex5HexNAc3dHex2SP 1808 +
NeuAc2Hex5HexNAc2 and/or NeuAcHex7HexNAc3dHex3 and/or 2489 +
NeuAc2Hex2HexNAc4SP 1815 + NeuAcHex4HexNAc5dHex3SP
Hex6HexNAc7SP
Hex6NexNAc3dHexSP 1824 + + 2490 +
Hex5HexNAc4dHexSP 1865 + + NeuAcHex6HexNAc5dHexSP and/or
NeuAcHex9HexNAc3dHex and/or 2521 +
Hex6HexNAc4SP 1881 + + NeuAc3Hex2HexNAc5dHex2
Hex4HexNAc5dHexSP 1906 + Hex6HexNAc5dHex3SP 2522 +
NeuAcHex6HexNAc2dHexSP and/or 1912 + Hex7HexNAc6dHexSP 2595 +
NeuAcHex3HexNAc4dHexSP2 NeuGcHex8HexNAc5 and/or
NeuAcHex4HexNAc4SP2 1928 + NeuAcHex4HexNAc5dHex4SP 2635 +
Hex8HexNAc3SP and/or NeuAc2Hex5HexNAc5dHexSP 2650 +
Hex5HexNAc5SP2 and/or 2002 + + Hex7HexNAc7SP 2652 +
NeuAc2Hex4HexNAc3dHex Hex6HexNAc5dHex4SP 2668 +
NeuAcHex5HexNAc4SP 2010 + + NeuGcHex6HexNAc5dHexSP and/or
Hex5HexNAc4dHex2SP 2011 + NeuAcHex7HexNAc5dHexSP 2683 +
NeuGcHex5HexNAc4SP 2026 + NeuAc2Hex4HexNAc5dHex2SP2 2714 +
Hex6HexNAc4dHexSP 2027 + + NeuAcHex4HexNAc5dHex4SP2 and/or
Hex7HexNAc4SP and/or NeuAc3Hex5HexNAc5 2715 +
Hex4HexNAc6SP2 and/or 2043 + Hex6HexNAc6dHex3SP 2725 +
NeuAc2Hex3HexNAc4dHex Hex7HexNAc6dHex2SP 2741 +
NeuAcHex7HexNAc3 and/or 2051 + + NeuAcHex6HexNAc5dHex2SP2 2747 +
NeuAcHex4HexNAc5SP NeuAc2Hex4HexNAc6dHex2 and/or
Hex4HexNAc5dHex2SP 2052 + Hex8HexNAc6dHexSP 2757 +
NeuAcHex4HexNAc4dHexSP2 2074 + Hex7HexNAc7dHexSP 2798 +
NeuAc2Hex4HexNAc3dHexSP and/or NeuAc3Hex5HexNAc4dHex2 and/or
Hex8HexNAc3SP2 and/or 2082 + NeuAcHex6HexNAc6dHexSP2 2804 +
Hex5HexNAc5SP3 Hex6HexNAc6dHex3SP2 2805 +
NeuAcHex6HexNAc3dHexSP 2115 + NeuAc2Hex6HexNAc5dHexSP 2812 +
Hex8HexNAc3dHexSP and/or 2148 + NeuAcHex6HexNAc5dHex3SP 2813 +
NeuAc2Hex4HexNAc3dHex2 Hex8HexNAc7SP 2814 +
NeuAcHex5HexNAc4dHexSP and/or 2156 + + Hex6HexNAc6dHex4SP 2871 +
NeuAcHex8HexNAc2dHex NeuAcHex7HexNAc6dHexSP and/or
Hex5HexNAc4dHex3SP 2157 + NeuAcHex10HexNAc4dHex 2887 +
NeuAc2Hex5HexNAc3dHex and/or 2164 + + Hex7HexNAc6dHex3SP 2887 +
Hex6HexNAc5SP2 NeuAc3Hex6HexNAc4dHexSP and/or
NeuAcHex6HexNAc4SP and/or NeuGcNeuAc2Hex5HexNAc4dHex2SP 2900 +
NeuGcHex5HexNAc4dHexSP and/or 2172 + +
NeuAcHex9HexNAc2 NeuAc3Hex4HexNAc6dHex and/or 2903 +
Hex6HexNAc4dHex2SP and/or NeuAcHex8HexNAc6SP
Hex3HexNAc6dHex2SP2 2173 + Hex7HexNAc7dHex2SP 2945 +
NeuAc3Hex3HexNAc4 and/or 2188 + NeuAc2Hex6HexNAc5dHex2SP 2958 +
NeuAcHex6HexNAc5dHex4SP 2960 +
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Hex8HexNAc7dHexSP 2961 +
Hex8HexNAc8SP 3018 +
Hex7HexNAc6dHex4SP 3034 +
Hex7HexNAc7dHex3SP 3091 +
NeuAc3Hex6HexNAc5dHexSP 3104 +
NeuAc2Hex6HexNAc5dHex3SP 3105 +
NeuAcHex8HexNAc7SP and/or 3106 +
NeuAc3Hex4HexNAc7dHex
Hex8HexNAc7dHex2SP and/or 3107 +
NeuAc2Hex4HexNAc7dHex3
NeuAc2Hex7HexNAc6dHexSP 3178 +
Hex7HexNAc7dHex4SP 3237 +
NeuAc3Hex7HexNAc5dHexSP and/or 3266 +
NeuGcNeuAc2Hex6HexNAc5dHex2SP
NeuAc3Hex5HexNAc7dHex and/or 3268 +
NeuGcHex8HexNAc7dHexSP
NeuAc4Hex4HexNAc5dHex2SP2 3297 +
NeuAc3Hex4HexNAc5dHex4SP2 3298 +
Hex8HexNAc8dHex3SP and/or 3456 +
NeuAc2Hex4HexNAc8dHex4
NeuAc3Hex7HexNAc6dHexSP 3469 +
NeuAc2Hex7HexNAc6dHex3SP 3470 +
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Table 8. Structural classification of neutral glycan fraction glycan signals
isolated from normal
human lung tissue (1. column), human lung cancer tissue (2. column), normal
human serum (5.
column), and a cultured human cell line (6. column). Acidic glycan fraction
glycans analyzed as
neutral desialylated glycan signals together with the corresponding neutral
glycan fraction are similarly
classified from the same human tissue samples (3. and 4. column, total normal
and total cancer).
Structural features of Neutral N 1 cans %
.'.,
structural feature proposed composition ~ ~ o 0
Hex5_9HexNAc2 high-mannose 47,0 46,0 17,8 22,3 25,7 53,7
Hex1_4HexNAc2dHexO_1 low-mannose 28,0 19,5 15,5 24,4 0,7 8,5
Hex10_12HexNAc2 high-mannose / Glc 0,0 0,0 0,0 0,0 0,0 1,9
Hex5_6HexNAc2dHex1 low-mannose + Fuc 0,7 0,0 0,3 0,2 0,0 1,0
nHe,a,iA. = 3 ja nHeX 2 hybrid / monoantennary 7,9 8,7 8,4 7,1 6,6 7,3
nxe,a,iA~ _ 4 ja nxeX 2 complex type 15,8 24,4 57,8 46,0 66,2 9,3
Hex1_9HexNAc soluble 0,7 0,5 0,0 0,0 0,8 11,3
other - 0,0 0,9 0,2 0,0 0,0 6,9
ndHeX >_ 1 fucosylation 19,4 33,6 42,8 34,6 50,5 13,9
ndHeX 2 a2/3/4- Fuc 0,0 0,8 0,3 1,1 0,0 1,3
nHex=rAc > nHeX ? 2 terminal HexNAc 3,9 17,8 3,8 7,1 21,8 4,2
nHex=rAc = nHeX ? 3 terminal HexNAc 6,9 8,2 8,2 5,0 31,4 1,9
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Table 9. N-glycan structural classification of lysosomal protein sample.
Neutral N 1 can structural features:
Glycan feature Proposed structure Proportion, %
Hex5_loHexNAc2 Hi h-mannose tyG1c1 46
Hex1_4HexNAc2dHex0_1 Low-mannose type 49
nHe,a,rA. = 3'a nHeX 2 H brid- e/ Monoantennary 2
nHe,a,rA~ 4'a nHeX 2 Com lex- e 0,6
Other - < 3
ndHeX 1 Fucosylation 29
ndHeX 2 a2/3/4-linked Fuc 0,8
nxeXrrAc > nHeX >- 2 Terminal HexNAc (N>H) 0,2
nxeXrrAc = nHeX >- 5 Terminal HexNAc =H -
Acidic N 1 can structural features:
Glycan feature Proposed structure Proportion, %
nHe,a,rAc = 3'a nHeX 3 H brid- e/ Monoantennary 46
nHe,a,rAc 4'a nHeX 3 Com lex- e 37
muut - 17
ndHeX 1 Fucos lation 80
ndHeX 2 a2/3/4-linked Fuc 10
nxeXrrAc > nHeX > 2 Terminal HexNAc (N>H) 0,1
nxeXrrAc = nHeX - 5 Terminal HexNAc (N=H) 0,4
+ 80 Da Sulphate or phosphate ester 17
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Table 10. Identification of disease-specific glycosylation by quantitative
glycome analysis.
Abs. Rel.
Composition m/z Class I II m/z Class differ. m/z Class differ.
HexlHexNAc2 609 NL 0,00 0,00 771 NL 12,8 1955 NCE new
Hex2HexNAcldHexl 714 NOF 0,00 0,00 1485 NCFT 3,5 2685 NCE new
Hex3HexNAcl 730 NS 0,00 0,00 1743 NM 2,1 2905 NCF new
Hex1HexNAc2dHex1 755 NLF 2,47 0,00 1905 NM 1,8 771 NL 2,4
Hex2HexNAc2 771 NL 5,44 18,25 1419 NM 1,4 1905 NM 2,2
Hex2HexNAc2dHexl 917 NLF 1,81 2,61 917 NLF 0,8 1485 NCFT 1,3
Hex3HexNAc2 933 NL 2,47 1,12 1581 NM 0,5 2394 NC 1,3
Hex2HexNAc3 974 NH-T 0,00 0,00 1955 NCE 0,4 1743 NM 1,2
Hex2HexNAc2dHex2 1063 NOE 0,00 0,00 2685 NCE 0,4 917 NLF 0,4
Hex3HexNAc2dHexl 1079 NLF 1,81 1,12 2905 NCF 0,4 1419 NM 0,4
Hex4HexNAc2 1095 NL 1,48 1,30 2539 NCF 0,3 2539 NCF 0,4
Hex2HexNAc3dHexl 1120 NHFT 0,00 0,00 2394 NC 0,2 1581 NM 0,2
Hex3HexNAc3 1136 NH 0,82 0,00 2175 NCF 0,2 1282 NHF 0,1
Hex2HexNAc2dHex3 1209 NOE 0,00 0,00 1622 NH 0,2 2012 NCFB 0,1
Hex3HexNAc2dHex2 1225 NOE 0,00 0,00 1282 NHF 0,1 1622 NH 0,1
Hex4HexNAc2dHexl 1241 NLF 0,00 0,00 2012 NCFB 0,1 1339 NH-T 0,1
Hex5HexNAc2 1257 NM 8,90 7,64 1339 NH-T 0,0 2320 NCE 0,1
Hex2HexNAc3dHex2 1266 NHET 0,00 0,00 2320 NCE 0,0 2175 NCF 0,0
Hex3HexNAc3dHexl 1282 NHF 0,82 0,93 609 NL 0,0 609 NL 0,0
Hex4HexNAc3 1298 NH 1,48 1,12 714 NOF 0,0 714 NOF 0,0
Hex3HexNAc4 1339 NH-T 0,33 0,37 730 NS 0,0 730 NS 0,0
Hex5HexNAc2dHex1 1403 NMF 0,33 0,19 974 NH-T 0,0 974 NH-T 0,0
Hex6HexNAc2 1419 NM 3,95 5,40 1063 NOE 0,0 1063 NOE 0,0
Hex3HexNAc3dHex2 1428 NHE 0,00 0,00 1120 NHFT 0,0 1120 NHFT 0,0
Hex4HexNAc3dHexl 1444 NHF 1,65 1,30 1209 NOE 0,0 1209 NOE 0,0
Hex5HexNAc3 1460 NH 2,47 2,42 1225 NOE 0,0 1225 NOE 0,0
Hex3HexNAc4dHexl 1485 NCFT 2,64 6,15 1241 NLF 0,0 1241 NLF 0,0
Hex4HexNAc4 1501 NC 1,32 0,93 1266 NHET 0,0 1266 NHET 0,0
Hex3HexNAc5 1542 NC-T 0,00 0,00 1428 NHE 0,0 1428 NHE 0,0
Hex7HexNAc2 1581 NM 2,31 2,79 1542 NC-T 0,0 1542 NC-T 0,0
Hex6HexNAc3 1622 NH 1,15 1,30 1688 NCFT 0,0 1688 NCFT 0,0
Hex4HexNAc4dHexl 1647 NCF 3,95 2,23 2028 NC 0,0 2028 NC 0,0
Hex5HexNAc4 1663 NC 17,63 13,97 1460 NH -0,1 1460 NH 0,0
Hex3HexNAc5dHex1 1688 NCFT 0,00 0,00 1850 NCFT -0,1 1095 NL -0,1
Hex4HexNAc5 1704 NC-T 0,16 0,00 1403 NMF -0,1 1257 NM -0,1
Hex8HexNAc2 1743 NM 1,81 3,91 1704 NC-T -0,2 1850 NCFT -0,2
Hex5HexNAc4dHex1 1809 NCF 20,59 11,73 1095 NL -0,2 1663 NC -0,2
Hex6HexNAc4 1825 NC 2,47 0,56 1444 NHF -0,3 1444 NHF -0,2
Hex4HexNAc5dHex1 1850 NCFT 0,66 0,56 1298 NH -0,4 1298 NH -0,2
Hex5HexNAc5 1866 NC-B 0,49 0,00 1501 NC -0,4 1501 NC -0,3
Hex9HexNAc2 1905 NM 0,82 2,61 1866 NC-B -0,5 1079 NLF -0,4
Hex5HexNAc4dHex2 1955 NCE 0,00 0,37 1079 NLF -0,7 1809 NCF -0,4
Hex5HexNAc5dHex1 2012 NCFB 0,82 0,93 1136 NH -0,8 1647 NCF -0,4
Hex6HexNAc5 2028 NC 1,32 1,30 1257 NM -1,3 1403 NMF -0,4
Hex6HexNAc5dHex1 2175 NCF 4,12 4,28 933 NL -1,4 933 NL -0,5
Hex6HexNAc5dHex2 2320 NCE 0,33 0,37 1647 NCF -1,7 1825 NC -0,8
Hex7HexNAc6 2394 NC 0,16 0,37 1825 NC -1,9 1704 NC-T gone
Hex7HexNAc6dHexl 2539 NCF 0,82 1,12 755 NLF -2,5 1866 NC-B gone
Hex7HexNAc6dHex2 2685 NCE 0,00 0,37 1663 NC -3,7 1136 NH gone
Hex8HexNAc7dHexl 2905 NCF 0,00 0,37 1809 NCF -8,9 755 NLF gone
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Table 11. Neutral glycan signal nomenclature for glycosphingolipid glycans.
Proposed composition m/z Proposed composition m/z
Hex2dHex 511,24 511 Hex7HexNAc 1378,45 1378
Hex3 527,15 527 Hex4HexNAc2dHex2 1387,49 1387
Hex2HexNAc 568,19 568 Hex5HexNAc2dHex 1403,48 1403
Hex2HexNAcdHex 714,24 714 Hex6HexNAc2 1419,48 1419
Hex3HexNAc 730,24 730 Hex3HexNAc3dHex2 1428,51 1428
Hex2HexNAc2 771,26 771 Hex4HexNAc3dHex 1444,51 1444
HexHexNAc3 812,29 812 Hex5HexNAc3 1460,50 1460
Hex3HexNAcdHex 876,30 876 Hex4HexNAc2dHex3 1533,54 1533
Hex4HexNAc 892,29 892 Hex8HexNAc 1540,5 1540
HexHexNAc2dHex2 901,33 901 Hex6HexNAc2dHex 1565,53 1565
Hex2HexNAc2dHex 917,32 917 Hex4HexNAc3dHex2 1590,57 1590
Hex3HexNAc2 933,31 933 Hex5HexNAc3dHex 1606,56 1606
Hex2HexNAc3 974,34 974 Hex6HexNAc3 1622,56 1622
Hex2HexNAcdHex3 1006,36 1006 Hex9HexNAc 1702,56 1702
Hex3HexNAcdHex2 1022,35 1022 Hex4HexNAc3dHex3 1736,62 1736
Hex5HexNAc 1054,34 1054 Hex5HexNAc3dHex2 1752,62 1752
Hex2HexNAc2dHex2 1063,38 1063 Hex4HexNAc5dHex 1850,67 1850
Hex2HexNAc2dHex 1079,38 1079 Hex10HexNAc 1864,61 1864
Hex4HexNAc2 1095,37 1095 Hex7HexNAc2dHex2 1873,64 1873
Hex3HexNAc3 1136,40 1136 Hex4HexNAc3dHex4 1882,68 1882
Hex6HexNAc 1216,40 1216 Hex5HexNAc3dHex3 1898,68 1898
Hex3HexNAc2dHex2 1225,43 1225 Hex5HexNAc4dHex2 1955,70 1955
Hex4HexNAc2dHex 1241,43 1241 Hex11HexNAc 2026,66 2026
Hex5HexNAc2 1257,42 1257 Hex5HexNAc4dHex3 2101,76 2101
Hex3HexNAc3dHex 1282,45 1282 Hex6HexNAc4dHex2 2117,75 2117
Hex4HexNAc3 1298,45 1298 Hex4HexNAc5dHex3 2142,78 2142
Hex2HexNAc4dHex 1323,48 1323 Hex12HexNAc 2188,71 2188
Hex3HexNAc2dHex3 1371,49 1371
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Table 12. Acidic glycan signal nomenclature for glycosphingolipid glycans.
Proposed composition m/z
NeuAcHexHexNAcdHex 819,29 819
NeuAcHex2HexNAc 835,28 835
NeuAc2Hex2 905,30 905
NeuAcHexHexNAcdHex2 965,35 965
NeuAcHex3HexNAc 997,34 997
NeuAc2Hex2HexNAc 1126,38 1126
NeuAcHex3HexNAcdHex 1143,39 1143
Hex4HexNAc2SP 1151,33 1151
NeuAcHex4HexNAc 1159,39 1159
NeuAcHexHexNAc2dHex2 1168,43 1168
NeuAcHex3HexNAc2 1200,42 1200
NeuGcHex3HexNAc2 1216,41 1216
Hex2HexNAc4SP 1233,38 1233
NeuAc2Hex3HexNAc 1288,43 1288
NeuAc2HexHexNAc2dHex 1313,46 1313
NeuAcHex2HexNAc2dHex2 1330,48 1330
NeuAcHex4HexNAc2 1362,47 1362
NeuAc2Hex4HexNAc / NeuAc2HexHexNAc3SP 1450,48 1450
NeuAcHex4HexNAc2dHex 1508,53 1508
NeuAcHex2HexNAc3dHex2 1533,56 1533
Hex6HexNAc2SP2 / NeuAc2Hex2HexNac2dHexSP 1555,47 / 1555,39 1555
NeuAcHex4HexNAc3 1565,55 1565
NeuAcHex5HexNAc3 1727,60 1727
NeuGcHex5HexNAc3 1743,60 1743
NeuAcHex5HexNAc3dHex 1873,66 1873
NeuAcHex6HexNAc3 1889,65 1889
NeuAcHex3HexNAc4dHex2 1898,69 1898
NeuAc2Hex3HexNac3dHexSP 1920,60 1920
NeuAc2Hex5HexNAc3 2018,70 2018
NeuAcHex6HexNAc3dHex 2035,71 2035
NeuAcHex6HexNAc4 2092,73 2092
NeuGcHex6HexNAc4 2108,73 2108
NeuAcHex4HexNAc4dHex3SP 2286,76 2286
NeuAc2Hex5HexNAc4SP 2301,73 2301
NeuGc3Hex4HexNAc4 2398,80 2398
NeuAcHex5HexNAc4dHex3SP / 2448,81 2448
NeuAcHex8HexNAc2dHex3
Hex7HexNAc6SP 2449,81 2449
NeuGc2Hex7HexNAc5 2780,95 2780
NeuGcHex8HexNAc5dHex/NeuAcHex9HexNAc5 2781,97 2781
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Table 13. Detected tissue material N-linked and soluble glycome compositions.
Neutral N 1 can structural features:
Proportion,
Glycan feature Proposed structure %
Hex5_10HexNAc2 High-mannose tyG1c1 10-60
Hex1_4HexNAc2dHexo_1 Low-mannose type 0-50
nH,NAc = 3'a nHeX 2 H brid- e/ Monoantennary 5-20
nHcxNAc 4'a nH, 2 Com lex- e 5-75
Hex1_9HexNAc1 Soluble 0-10
ndH, 1 Fucosylation 10-80
ndH, 2 a2/3/4-linked Fuc 0-40
nHxNAc > nHx >_ 2 Terminal HexNAc > 1-30
nHcxNAc = nH, _ 5 Terminal HexNAc = 1-40
Acidic N 1 can structural features: all
Proportion,
Glycan feature Proposed structure %
nH~NAc = 3'a nHex 3 H brid- e/ Monoantennary 5-60
nH,NAc > 4'a nH, 3 Com lex- e 40-95
ndH, _ 1 Fucosylation 20-90
ndH, _ 2 a2/3/4-linked Fuc 0-50
nH,xNAc > nHx >_ 2 Terminal HexNAc (N>H) 0-40
nHcxNAc = nHex >_ 5 Terminal HexNAc (N=H) 0-40
+ 80 Da Sulphate or phosphate ester 0-25
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Table 14.
Examples of glycosphingolipid glycan classification Neutral Sialylated
glycans glycans
Class Definition
Lac nH.. = 2 1 a) 1
Ltri nH, = 2 and nH~NAc = 1 25 14 25
Ll nH, = 3 and nHNAc = 1 39 25 56
L2 3<_ nHe.< 4 and nH~NAc = 2 13 20 <1
L3+ i + 1< nHe.< i +2 and nH~NAc = i> 3 1 18 1
Gb nH, = 4 and nH~NAc = 1 11 4 16
0 other types 10 24 <1 a)
F fucosylated, ndHeX > 1 28 27 1
T non-reducing terminal HexNAc, 37 21 26
nHeu :~ nHeuNAc + 1
SA1 monosialylated, nNeõ5Ac = 1 86
SA2 disialylated, nNeOAc = 2 14
SP sul hated or phosphorylated, +80 Da <1
Examples of 0-linked glycan classification Neutral Sialylated
glycans glycans
Class Definition
01 nH, = 1 and nH~NAc = 1 <1 43 72
02 nH, = 2 and nH~NAc = 2 53 35 26
03+ nH, = i and nH~NAc = i> 3 13 13 2
0 other types 34 a) 9 <1
F fucosylated, ndHeX > 1 24 10 5
T non-reducing terminal HexNAc, 12 <1 <1
nHeu :~ nHeuNAc + 1
SA1 monosialylated, nNeõ5Ac = 1 39 55
SA2 disialylated, nNe,,sAc = 2 52 44
SP sulphated or phosphorylated, +80 Da 14 2
a) not included in present quantitative analysis.
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