Note: Descriptions are shown in the official language in which they were submitted.
CA 02448534 2003-11-20
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PEPTIDE ANALYSIS USING
A SOLID SUPPORT
Technical field
The present invention relates to an improved method of identifying a
polypeptide,
wherein an acidic reagent is used to derivatize peptides before analysis
thereof using
mass spectrometry. The invention also relates to a kit, which comprises
reagents)
suitable for use in the present method.
t0 Back_r
The identification and sequencing of polypeptides has become of increased
impor-
tance with the rapid development of the field of proteomics, wherein the
expression
products of novel genes are examined as to their function and composition.
r 5 Matrix-assisted laser desorption ionization (MALDI) mass spectrometry is a
method
developed for peptide and polypeptide sequencing. (For a reference to the
principles
of MALDI mass spectrometry, see e.g. Spengler et al., "Peptide Sequencing by
Ma-
trix-assisted Laser-desorption Mass Spectrometry", Rapid Communications in
Mass
Spectrometry, Vol. 6, pp. 105-108 (1992).) MALDI mass spectrometry offers
several
2o advantages in the field of mass spectrometry. For example, it provides a
higher sensi-
tivity than the conventional electrospray triple quadrupole equipment. When
used in
combination with time-of flight (TOF) mass analyzers, MALDI mass spectrometry
is
also applicable to higher mass peptides than can be analyzed with triple
quadrupole
equipment. MALDI mass spectrometry is also useful for analyzing complex
mixtures
25 with minimal sample purification. Electrospray ionization, on the other
hand, is read-
ily interfaced to powerful separation techniques including liquid
chromatography
(LC) and various forms of capillary electrophoresis (CE). Highly automated
analyses
are possible when using LC and CE as the sample purification and introduction
de-
vices.
3a
However, current MALDI and, to a lesser extent, electrospray ionization mass
spec-
trometric methods fail to adequately offer predictable tandem mass
spectrometry
CA 02448534 2003-11-20
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fragmentation patterns. For example, multiple ion series (including a-ions, b-
ions, and
y-ions) are often observed, resulting in MALDI post-source decay spectra that
are too
complex for efficient interpretation and sequencing. Multiple ion series (b-
and y-
ions), plus internal fragments and both singly and multiply charged ions are
formed
from multiply charged precursor ions generated by electrospray ionization, and
the
resulting tandem mass spectra are often difficult to interpret de novo.
Accordingly,
problems with fragmentation have limited the ability to rapidly sequence
polypeptides
using mass spectrometry. As a result, mass spectrometry, and particularly
MALDI
mass spectrometry, has been of limited value in this area.
1 EI
Several research groups have attempted to improve the utility of mass
spectrometry in
the field of polypeptide sequencing through the use of chemical derivatization
tech-
niques. Such techniques have been utilized to promote and direct fragmentation
in the
MSMS spectra of peptides with the goal of increasing sensitivity and
decreasing the
15 complexity of the resulting spectra. Most of these methods provide cationic
deriva-
tives. For example, derivatization with a quaternary ammonium group, and
analysis
using the static SIMS ionization method has been suggested. However,
application of
such techniques using MALDI mass spectrometry and electrospray ionization with
low-energy collisional activation have not proven generally effective.
zo
More recently, for the determination of an amino acid sequence, Keough et al
(WO
00/43792, in the name of The Procter & Gamble Company) have suggested a de-
rivatization of the N-terminus of a polypeptide with one or more acidic
moieties hav-
ing pKa values of less than 2 before analysis by mass spectrometry of the
analyte,
25 such as with MALDI mass spectrometry. The acidic moiety is preferably a
sulfonic
acid or a disulfonic acid derivative. The derivatives promote a charge-site-
initiated
cleavage of backbone amide bonds and they enable the selective detection of
only a
single series of fragment ions comprising the y-ions. However, the reaction
according
to Keough et al is generally performed under non-aqueous conditions due to the
poor
3o water stability of the reagents utilized therein. Accordingly, for a
commercially useful
determination of amino acid sequences by mass spectrometry, there is still a
need for
improved methods that fulfill the requirements especially for automated
procedures.
CA 02448534 2003-11-20
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Summary of the Invention
One object of the present invention is to provide a method of identification
of a pep-
tide or polypeptide using a mass spectrometric technique, which due to its
robustness,
sensitivity and easily interpreted fragmentation spectra is more suitable for
automa-
tion than the prior art methods. This can be achieved by contacting acidic
derivatiza-
tion reagents with polypeptides immobilized to a solid support.
Thus, the present invention relates to a method of identifying a polypeptide,
which
method comprises the steps of:
1 t} (a) derivatization in an aqueous solution the N-terminus of the
polypeptide, or the N-
termini of one or more peptides of the polypeptide, with at least one acidic
reagent
comprising a sulfonyl or sulfonic acid moiety coupled to an activated acid
moiety
to provide one or more peptide derivatives, which reagent exhibits a half life
in
aqueous solution of not less than 10 minutes, preferably not less than about
20
~s minutes and most preferably not less than about 30 minutes at room
temperature;
(b) analyzing at least one such derivative using a mass spectrometric
technique to
provide a fragmentation pattern; and
(c) interpreting the fragmentation pattern obtained,
wherein the polypeptide is immobilized to a solid support at least during step
(a).
zo
The objects of the invention can more specifically be achieved as defined by
the ap
pended claims. Below, the present invention will be described in more detail
with ref
erence to specific embodiments and illustrative examples thereof.
2s Brief description of the drawings
Figure 1 shows the reflection spectrum of non-derivatized sample of horse
myoglobin
(15 fmol on MALDI target) as described in Example 2.
Figure 2 shows the reflection spectrum of derivatized sample (< l5fmol on the
MALDI target) described in relation to figure 1.
30 Figure 3 shows the PSD spectrum of m/z 1449.5, produced by derivatization
of a 1y-
sine-terminated peptide (mlz 1271 as shown in figure 2).
CA 02448534 2003-11-20
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Figure 4 shows how the protein above was identified in PepFrag, by submitting
the
masses (-42 Da from the reaction) of the seven y-ions obtained.
Figure 5 shows the fragmentation spectrum of an arginine-terminated peptide
(mlz
1742.8).
s Figure 6 shows the eight y-ions obtained were used for protein
identification in Pep-
Frag.
Figure 7 shows sulfonation of 500 femtomole of BSA tryptic peptides on solid
phase
as described in Example 3.
Figure 8 shows sulfonation of 4.5 picomole of BSA tryptic peptides in solution
as de-
1 o scribed in Example 3
Figure 9A -D show NMR-spectra as discussed in Example 12 below.
Figure 1 OA-B illustrate the stability of NHS-esters used according to the
invention.
More specifically, Fig 1 OA shows the stability of 3-sulfopropionic acid NHS-
ester in
D20 while Fig l OB shows the stability of 2-sulfobenzoic acid NHS-ester in
D20.
~s Figure 11A-C show MALDI PSD spectra and comparative reactivity data of
peptides
sulfonated as described in Example 17.
Figure 12 shows a reflectron spectrum, positive mode (showing average masses,
after
filtration, smoothing 5) of non-derivatized tryptic digest of 4VP-BSA obtained
with
the EttanTMMALDI-TOF.
Figure 13 shows a reflectron spectrum (showing average masses, after
filtration,
smoothing 5) of derivatized tryptic digest of 4VP-BSA (EttanTM MALDI-TOF).
Figure 14 shows the PSD spectrum (positive mode) showing a complete y-ion
series
of peptide (I) from the derivatized tryptic digest of 4VP-BSA (figure 13)
obtained
with the EttanTMMALDI-TOF.
?5 Figure 15 shows the PSD spectrum (positive mode) of peptide (II) from the
derivat-
ized tryptic digest of 4VP-BSA (figure 13).
Figure 16 shows the PSD spectrum (signals from 300 shots accumulated) of
peptide
(III) (figure 13), m/z1704, from the derivatized tryptic digest of 4VP-BSA.
CA 02448534 2003-11-20
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Figure 17 shows a ftrst example of a reflectron spectrum (positive mode, 100
shots
accumulated, showing average masses, after filtration, smoothing 5) of a non-
derivatized protein digest from a Coomassie-stained 2-D gel obtained with the
EttanTM MALDI-TOF.
Figure 18 shows a reflectron spectrum (positive mode, showing average masses,
after
filtration, smoothing 5) of the same 2-D sample as in figure 17 (remaining
95%), but
after N-terminal derivatization with NHS-ester.
Figure 19 shows a PSD spectrum (accumulated from 300 shots), of the
derivatized
peptide, m/z 1927.
~ 0 Figure 20 shows a second example of a reflectron spectrum (accumulated
from 100
shots, showing average masses, after filtration, smoothing 5) of a non-
derivatized
tryptic digest of a protein spot from a Coomassie-stained 2D gel, obtained
with Et-
tanTMMALDI -TOF.
Figure 21 shows a reflectron spectrum (positive mode showing average masses,
after
filtration, smoothing 5) of the same 2-D sample as in figure 19, but after
ZipTipTM
clean up and derivatization with NHS-ester in aqueous solution as described.
Figure 22 shows a PSD spectrum (signal from 300 shots accumulated) of the
derivat-
ized peptide, m/z 1705 (see figure 12).
Figure 23 shows sample-loaded ZipTipsT"" placed into a laboratory centrifuge
for sub-
let) sequent sulfonation in a multiplexed fashion.
Figure 24 illustrates sample washing in the centrifuge following the
sulfonation reac-
tion.
Figure 25 illustrates direct loading of the derivatized samples from the solid
supports
onto the MALDI sample stage.
Figure 26 shows the MALDI mass spectra obtained following sulfonation of
Fibrino-
peptide A on solid support. Duplicate samples were sulfonated at three
different pep-
tide levels (10, 1 and 0.1 pmoles).
Figure 27 shows the use of hydroxylamine hydrochloride for reversing unwanted
ester
side-products formed in the sulfonation reaction. The upper spectrum was
obtained
3C3 from ASHLGLAR sulfonated on solid support in the centrifuge. The lower
spectrum
was obtained from the same sulfonated peptide following treatment with
hydroxyla-
mine hydrochloride.
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Figure 28 demonstrates the sulfonation of a protein digest. The upper spectrum
was
obtained from the native protein digest. The lower spectrum was obtained
following
sulfonation of the digest.
Definitions
In the present specification, the term "identifying" is not necessarily
synonymous with
determining the complete sequence, since it also includes partial sequence
determina-
tion for identifying the polypeptide or characterizing it as similar to or
different from
a peptide derived from a known protein. Further, it also includes making a
tentative
identification based on the most probable of a small number of possibilities.
Further, the term "ionization" as used herein refers to the process of
creating or re-
taining on an analyte an electrical charge equal to plus or minus one or more
electron
units.
The term "aqueous environment" as used herein includes any water-based
solution,
t 5 suspension or any other form, which contains less than about 20% of
organic solvents.
As used herein, the term "electrospray ionization" refers to the process of
producing
ions from solution by electrostatically spraying the solution from a capillary
electrode
at high voltage with respect to a grounded counter electrode. The definition
is in-
tended to include both electrospray ionization and pneumatically assisted
electrospray
?(~ ionization, which is also referred to as ionspray. As used herein, the
term "electro-
spray ionization" applies to all liquid flow rates and is intended to include
microspray
and nanospray experiments. Moreover, the definition is intended to apply to
the
analyses of peptides directly infused into the ion source without separation,
and to the
analysis of peptides or peptide mixtures that are separated prior to
electrospray ioni-
25 zation. Suitable on-line separation methods include, but are not limited
to, HPLC,
capillary HPLC and capillary electrophoresis. Electrospray ionization
experiments
can be carried out with a variety of mass analyzers, including but not limited
to, triple
quadrupoles, ion traps, orthogonal-acceleration time-of flight analyzers and
Fourier
Transform Ion Cyclotron Resonance instruments.
30 As used herein, the term "polypeptide" refers to a molecule having two or
more
amino acid residues.
CA 02448534 2003-11-20
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As used herein, the term "wild-type" refers to a polypeptide produced by
unmutated
organisms.
As used herein, the term "variant" refers to a polypeptide having an amino
acid se-
quence that differs from that of the wild-type polypeptide.
The term "water stable" as used herein refers reagents having a half life in
aqueous
solution of not less than 10 minutes, preferably not less than about 20
minutes and
most preferably not less than about 30 minutes at room temperature.
The term "activated acid" refers to an acid derivative, preferably a
carboxylic acid de-
rivative, which is capable of forming amide bonds in an aqueous environment.
1 o The term "immobilized" as used herein to define how peptides and/or
polypeptides
axe adsorbed to a solid support means that peptide and/or polypeptide binding
is suffi-
ciently strong to last during the reaction. For example, when the support is
coated
with C18, a hydrophobic binding between the peptides and the support is strong
enough to retain peptides through the reaction and cleanup steps.
I$
As used herein, the following abbreviations axe used:
Tetrahydrofuran THF
N-hydroxysuccinamide NHS
Dichloromethane DCM
N,N - diisopropylethylamine DTEA
Trifluoroacetic acid TFA
Deuterated water D20
Hydrochloric acid HCl
Thionyl chloride SOC12
Ethyl acetate EtAc
Methanol MeOH
Room Temperature and Pressure RTP
Room Temperature RT
Milli-Q purified water MQ
O-(N-Succinimidyl)-N,N,N',N'- TSTLT
tetramethyluronium BF4
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Acetonitrile ACN
Deuterated chloroform CDC13
Thin layer chromatography TLC
Detailed description of the invention
A first aspect of the present invention is a method of identifying a
polypeptide, which
method comprises the steps of
(a) derivatization of the N-terminus of the polypeptide, or the N-termini of
one or
more peptides of the polypeptide, with a least ore acidic reagent comprising a
sul-
fonyl or sulfonic acid moiety coupled to an activated acid moiety to provide
one
or more peptide derivatives, which reagent exhibits a half life in aqueous
solution
of not less than 10 minutes, preferably not less than about 20 minutes and
most
preferably not less than about 30 minutes at RT;
(b) analyzing at least one such derivative using a mass spectrometric
technique to
provide a fragmentation pattern; and
(c) interpreting the fragmentation pattern obtained,
1 S wherein the peptide or polypeptide is immobilized to a solid support at
least during
step (a).
The solid support used according to the invention can be any suitable
substrate capa-
ble of immobilizing peptides or polypeptides under the conditions defined
herein.
zo Thus, in one embodiment, the above-mentioned solid support is comprised of
a silica-
based medium derivatized with C18. The solid support can e.g. be present on a
plastic
surface, such as the walls of microtiter wells, on a metal surface, such as a
MALDI-
slide, on the surface of a compact disc (Gyros AB, Uppsala, Sweden), or in
composite
structures, such as the commercially available ZipTipTM (Millipore
Corporation,
25 USA, see e.g. WO 9/37949). The high binding capacity of the present solid
support
results in a more efficient derivatization method. Also, the solid support is
a
convenient means to concentrate dilute peptide digests and to desalt e.g.
prior to
MALDI mapping, which greatly improves the signal/noise ratio. Other advantages
of
immobilizing polypeptides to a solid support is that it decreases reaction
times, it
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
reduces the number of sample manipulations required to guanidinate and/or
sulfonate
peptides and polypeptides and it increases the overall processing throughput.
The
spectra of protein digests that have been derivatized on solid supports often
show
increased numbers of tryptic peptides, improved protein sequence coverage and
higher database search scores. In fact, the present inventors also have been
able to
show an improvement in sensitivity as high as five times that obtained using
the
corresponding chemistry but performed in solution instead of on a solid
support.
Materials such as ZipTipTM have not been used before as supports for peptide
or
polypeptide derivatization prior to mass spectrometry-based sequencing, but
they
1 o been used simply to concentrate dilute solutions and to clean up the
solutions by
removing low-molecular weight contaminants such as alkali salts.
In an advantageous embodiment, the amount of ester side-products present after
step
(a) is reduced or eliminated by optionally adding a suitable chemical, such as
hydroxylamine, mercaptoethanol, dithiothreitol or acetic hydrazide, that
hydrolyzes
unwanted ester groups. The derivatized peptide or polypeptide is washed to
remove
excess reagent prior to analysis. In the present context, the term "acidic"
reagent
means a reagent that comprises one or more moieties having pica's of less than
2,
preferably less than 0 and more preferably less than -2 when coupled to a
peptide or
2() polypeptide.
The present method is useful for sequencing polypeptides, such as wild-type,
variant
and/or synthetic polypeptides. The method is especially useful for identifying
high
molecular weight polypeptides for use e.g. in the biological and
pharmaceutical field.
25 More specifically, the present method can be used to facilitate biological
studies re-
quiring rapid determination of peptide or polypeptide sequences; to identify
post-
translational modifications in proteins and to identify amino acid
modifications in
vaxiant proteins, such as those used in commercial laundry and cleansing
products; to
aid in the design of oligonucleotide probes for gene cloning; to rapidly
characterize
3o products formed in directed evolution studies; in combinatorial and peptide
library
identification; and in proteomics.
CA 02448534 2003-11-20
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Thus, in step (b), the present invention utilizes a mass spectrometric
technique for the
analysis of the derivative(s), which technique can include matrix-assisted
laser de-
sorption ionization (MALDI) mass spectrometry or electrospry ionization. These
ioni-
zation techniques can be carried out with a variety of mass analyzers,
including but
7 not limited to, triple quadrupoles, ion traps, reflector time-of flight
analysers, or-
thogonal-acceleration time-of flight analyzers and Fourier Transform Ion
Cyclotron
Resonance instruments. The spectra obtained are routinely interpreted de novo
in ac-
cordance with standard procedure. However, in the most preferred
embodiment,.in
step (b), MALDI mass spectrometry is used. MALDI mass spectrometers are com-
1t~ mercially available and described in the literature, see e.g. Kussmann M.
and Roep-
storff P., Spectroscopy 1998, 14: 1-27.
Thus, as mentioned above, in the prior art sulfonic groups have been added to
the N
termini of peptides to facilitate sequencing with MALDI mass spectrometry.
Reagents
r s suggested to this end include those exhibiting a low stability in water.
(In this context,
see e.g. T. Keough, R.S. Youngquist and M.P. Lacey, Proc.Natl. Acad. Sci.
USA., 96,
7131 (1999); T. Keough, M.P. Lacey, A.M. Fieno, R.A. Grant, Y. Sun, M.D. Bauer
and K.B. Begley, Elect~opho~esis, 66 2252 (1999); and T.Keough, M.P.Lacey and
. R.S.Youngquist, Rapid Commu~. Mass Spect~om. 14, 2348 (2000).) The present
in-
zt) vention relates to a method wherein such acidic reagents are used, which
method
contrary to what has been suggested before is performed on polypeptides
immobilized
to a solid support. In the most advantageous embodiment, the present invention
util-
izes an acidic reagent comprised of a sulfonyl or sulfonic acid moiety coupled
to an
ester moiety, such as an NHS-ester. Such reagents will be discussed in more
detail
25 below.
Thus, in one embodiment, the present invention provides an improved one-step
method wherein a water-stable reagent is used for the derivatization step
preceding
the actual mass spectrometry analyses. The advantages of working with a water-
3U soluble and water stable reagent and avoiding organic solvents are obvious
and in-
clude easier automation of the derivatization procedure because no dry down
steps
and solvent changes are required.
CA 02448534 2003-11-20
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The fact that the present invention utilizes tryptic polypeptides immobilized
to a solid
support will also contribute to an enhanced suitability for automation. In an
especially
advantageous embodiment, both step (a) and a preceding guanidination step are
per-
formed on a solid support. This embodiment is advantageously performed
simultane-
ously on a large number of samples, such as in the standard 96 well format in
order to
be easily adapted to,available automation systems, such as ProSpot~ (Amersham
Bi-
osciences AB, Uppsala, Sweden) or microfluidics sample preparation devices
like
compact disks (Gyros AB, Uppsala, Sweden). Such adaptation may include steps
such
as taking the solid support off pipettes, incubation etc. In this embodiment,
the gua-
nidination reaction and the sulfonation reaction are performed on the.peptide
or poly-
peptide contents of the same microtiter well, following immobilization on a
ZipTipTM
Accordingly, the samples need only be immobilized or bound once, which
simplifies
the procedure in total. Also, this embodiment has been shown to improve the
sensi-
1 S tivity as much as 5 times as compared to the corresponding method in
solution. As
regards further differences between using peptides in solution and
irmnobilized to a
solid support for the present purpose, see Example 4 below, where a comparison
of
the sulfonation step is presented.
zo Furthermore, the present invention also relates to a method of protecting
lysine resi-
dues by guanidination wherein the peptides and/or polypeptides are immobilized
to a
solid support.
In another embodiment, in order to reduce the duration of the sulfonation step
and to
25 provide an efficient derivatization procedure, the sulfonation reagent is
centrifuged
during step (a), which forces the liquids through the peptide or polypeptide-
loaded
ZipTipsT"", or any other solid phase used. This approach provides a
mechanically sim-
ple means to move chemical reagents over immobilized peptides or polypeptides.
The present inventors have unexpectedly shown that by using this embodiment, a
near
~t) quantitative derivatization can be performed, see Example 3 below.
11
CA 02448534 2003-11-20
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If the method according to this embodiment also includes a step for
guanidination (as
discussed in detail below), said reaction is conveniently performed during
tryptic elu-
tion from a 2D gel, see e.g. Hale et al (Anal. Biochem. 28, (2000), 110-117).
Gua-
nidination during peptide extraction from the gel can be done robotically, and
the
s tryptic peptides can subsequently be immobilized to a solid support and
sulfonated as
described above.
Accordingly, in an especially advantageous embodiment, the present method is a
computer-assisted method, wherein suitable software is utilized in step (c).
Thus, data
t 0 analysis of mass-to-charge ratios obtained by the mass spectrometry is
used for the
interpretation of the fragmentation pattern obtained. Several software
programs have
been developed to compare mass spectra of the peptides obtained e.g. from
MALDI-
TOF experiments with theoretical spectra from proteins. The subject has been
re-
viewed by Kussmann and Roepstorff (Kussmaml M. and Roepstorff P., Spectroscopy
r5 1998, 14: 1-27).
An advantage of the kind of reagents used in the present method resides in the
fact
that they are easily stored in a crystalline form. Thus, the stability during
storage and
accordingly the shelf life of the reagents is greatly improved. Consequently,
the pres-
20 ent invention utilizes reagents that make possible a less costly handling
and also sim-
plifies the practical use thereof in many routine procedures.
The acidic reagent used in the present method may have a pKa of less than
about 2,
preferably less than about 0 and most preferably less than about -2 when
coupled with
?5 a peptide or polypeptide. The skilled person in this field can measure pKa
values of
acidic moieties as covalently coupled to a polypeptide or peptide using
standard
methods well known in the art. For example, such methods may include titration
or an
electrochemical method. The activated acid moiety of the reagent can e.g. be
an N-
hydroxysuccinimide (NHS) ester, such as 3-sulfopropionic acid N-
30 hydroxysuccinimide ester or 2-sulfobenzoic acid N-hydroxysuccinimide ester.
1?
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
As the skilled in this field will realize, said reagents) can be used combined
with any
suitable buffer, as long as the buffer does not effectively compete with the
analyte for
the acidic reagent. In one embodiment, the buffer provides a pH within the
range of
about 8-12, such as 9-10 and in a specific embodiment about 9.4. One suitable
buffer
is 0.25 M NaHC03. Alternatively, they are simply used as dissolved in water,
in
which case the final solution pH will have to be adjusted, since the final
solution pH
must be basic for the reaction to occur. Furthermore, in the present method,
it is to be
understood that even though for practical reasons one single reagent is
normally used,
the invention also encompasses a method utilizing a mixture of two or more
such rea-
l o gents, each one of which being defined by comprising a sulfonyl or
sulfonic acid moi-
ety coupled to an NHS-ester moiety.
The preparation of the above mentioned exemplary reagents will be illustrated
below
in the experimental part of the present application. The activated acids used
in the
U 5 present method are prepared according to techniques well known to those
ordinarily
skilled in the art. The starting materials used in preparing the compounds of
the in-
vention are known, made by laiown methods, or are commercially available as a
starting material.
z0 It is recognized that the ordinarily skilled artisan in the art of organic
chemistry can
readily carry out standard manipulations of organic compounds without further
direc-
tion. Examples of such manipulations are discussed in standard texts such as
J. March,
Advanced Organic Chemistry, John Wiley & Sons, 1992.
?5 The ordinarily skilled artisan will readily appreciate that certain
reactions are best car-
ried out when other functionalities are masked or protected in the compound,
thus in-
creasing the yield of the reaction and/or avoiding any undesirable side
reactions. Of
ten, the ordinarily skilled artisan utilizes protecting groups to accomplish
such in-
creased yields or to avoid the undesired reactions. These reactions are found
in the
30 literature and are also well within the scope of the ordinarily skilled
artisan. Exam-
ples of many such manipulations can be found in, for example, T. Greene,
Protecting
Groups in Or anic Synthesis, John Wiley & Sons, 1981.
t3
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
The compounds used in the present method may be prepared using a variety of
proce-
dares known to those ordinarily skilled in the art. Non-limiting general
preparations
include the following.
The activated acids used according to the invention can be prepared by
activating the
acid in a compound of the general structure below followed by reaction to
generate a
water stable reagent of the invention.
O
1' Where:
HO S03H Y=a spacer which contains aliphatic and/or aromatic
fragments and may optionally include additional
sulfonic acids
Non-limiting examples of appropriate acids are e.g. 2-sulfoacetic acid, 3-
sulfopropionic acid, 3-sulfobenzoic acid 4-sulfobenzoic acid, 2-bromo-5-
sulfobenzoic
acid and 2-sulfobenzoic acid. For a general reference to sulfonyl groups
useful to this
t 5 end, see e.g. WO 00/43792.
Those skilled in the art will realize that in addition to the protonated acids
of these
compounds, the salts including, but not limited to sodium and potassium will
be use-
ful for the synthesis of compounds of the invention. Most of the activated
acids can
?o be easily prepaxed with common methods of the art (Recent reviews and books
for
peptide synthesis and preparation of activated esters: a) Alberico, F.;
Carpino, L.A.,
Coupling reagents and activation., Method. Enzynzol.,1997, 289, 104-126. b)
Bodan-
sky, M.; Principles of Peptide Synthesis, 2"a ed., Springer-Verlag: Berlin,
1993. c)
Humphrey, J.M., Chamberlin, A.R., Chemical Synthesis of Natural Product
Peptides:
25 Coupling Methods for the Incorporation of Noncoded Amino Acids into
Peptides.
Chem. Rev., 1997, 97, 2243-2266. d) Handbook of Reagents for Organic
Synthesis:
Activating Agents and Protecting Groups, Peaxson, A.J, and Roush, W.R., ed.,
John
Wiley & Sons, 1999). Reactive derivatives of this structure include, for
example, acti-
vated esters such as 1-hydroxybenzotriazole esters, mixed anhydrides of
organic or
14
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
inorganic acids such as hydrochloric acid and sulfonic acids, and symmetrical
anhy-
drides of the acids of this structure. These activated materials may be
directly useful
as water-stable reagents of the invention. However; highly reactive materials
such as
acid chlorides may not be water stable as defined herein but can be further
reacted
with reagents such as N-hydroxysuccinamide to generate active acids that are
water
stable reagents of the invention.
Of the numerous active esters found in the literature, N hydroxysuccinimide
derived
esters (Anderson, G.W.; Zimmerman, J. E.; Callahan, F.M.; J. Am. Chem. Soc.,
1964,
1t> 86, 1839, For a review see Klausner, Y.S.; Bodansky, M.S., Synthesis,
1972, 453),
ortho and para-nitrophenyl esters (Bodansky, M.; Funk, K.W., Fink, M.L.; J.
O~g.
Chem., 1973, 38, 3565, Bodansky, M.; Du Vigneaud, V.; J. Am. Chem. Soc., 1959,
81, 5688), 2,4,5-trichlorophenyl esters (Pless, J.; Boissonnas, R.A., Helv.
Chim. Acta;
1963, 46, 1609), pentachlorophenyl (Kovacs, J.; Kisfaludy, L., Ceprini, M.Q.,
J. Am.
1 S Chem. Soc.,1967, 89, 183) and pentafluorophenyl esters (Kisfaludy, L.,
Roberts, J.E.,
Johnson R.H., Mayers, G.L., Kovacs, J.; J.O~g. Chem., 1970, 35, 3563) are of
the
most practical interest. Other acid activating moieties include, thin esters
such as 2-
pyridylthio esters (Lloyd, K.; Young, G.T.; J. Chem.Soc. (C), 1971, 2890), cya-
nomethyl esters (Schwyzer, R.; Iselin, B.; Feurer; M., Helv. Chim. Acta; 1955,
38,
20 69), N acylimidazolides (Wieland, T.; Vogeler, K., Angew.Chem., 1961, 73,
435),
acyl azide (Curtius, T., Ber.dtsch.Chem.Ges., 1902, 35, 3226 Fujii, N.;
Yajima, H.,
J.Chem.Soc.Perki~ Ti~a~s 1,1981, 789) or benzotriazol derived intermediate
(Dormoy,
J.R.; Castro, B., Tet~ahedro~, 1981, 37, 3699) are as well considered.
?s The use of these activated esters can as well be combined with selected
acylation
catalysts such as for example 4-dimethylaminopyridine (Hoefle, G.; Steglich,
W.;
Vorbrueggen, H., Angew. Chem., Int. Ed. Engl., 1978, 17, 569. Scriven, E.F.V.,
Chem.Soc.Rev., 1983,12, 129). The exact molecular structure of the reagent is
not es-
sential, as long as said sulfonyl or sulfonic acid moiety and the activated
acid moiety
30 are present and provided that its water stable nature and chemical
reactivity with
amines are retained. Further routine experimentation can subsequently be
performed
1~
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
in order to identify e.g. an optimal pH for the reaction, or a specific
activated acid, for
which unwanted side reactions e.g. at hydroxyl groups are minimized.
The polypeptide, or peptides thereof, may be obtained by any means. For
example, if
necessary, the polypeptide of interest is isolated for analysis. Several
procedures may
be utilized for isolation including for example one-dimensional and two-
dimensional
electrophoresis. Alternatively, the polypeptides may have been synthesized
through
combinatorial chemistry methods well known in the art. In this instance, it is
most
preferable to synthesize a polypeptide having a basic or hydrophobic residue,
prefera-
1 t~ bly a basic (most preferably arginine or lysine), at or near the C-
terminus of the re-
sulting polypeptide.
Digestion may occur through any number of methods, including in-gel or on a
mem-
brane, preferably in-gel (see e.g. Shevchenko et al., "Mass Spectrometric
Sequencing
of Proteins from Silver-Stained Polyacrylamide Gels", Analytical Chemistry,
Vol. 68,
pp. 850-858 (1996)). Thus, in an advantageous embodiment, the present method
uses
in-gel digests: It is. possible to digest the polypeptide either enzymatically
or chemi-
cally, preferably enzymatically. It is most preferable to utilize a digestion
procedure
that yields a basic or hydrophobic residue, most preferably a basic, at or
near the C-
z0 terminus ~of the resulting peptides.
A polypeptide may be digested enzymatically e.g. using trypsin, endoproteinase
Lys
C, endoproteinase Arg C, or chymotrypsin. Trypsin, endoproteinase Lys C or
endo-
proteinase Arg C are preferred, since the resulting peptides of the
polypeptide will
?5 typically terminate at the C-terminus with an arginine or lysine residue
(basic resi-
due), with the exception of course of the C-terminus of the polypeptide. Other
en-
zymes can be used, especially if basic residues occur at or near the C-
terminus of the
resulting peptides. For example, chymotrypsin, which typically cleaves at
hydropho-
bic amino acid residues, may be used. Alternatively, chemical digestion can be
used,
3o such as by cyanogen bromide. (For a general reference to digestion methods,
see e.g.
US patent number 5 821 063.)
i~
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Thus, in a specific embodiment, the present method is used to identify a
polypeptide
or a protein, in which case a first step is included wherein said polypeptide
or protein
is digested, preferably enzymatically, to provide peptides. In a preferred
embodiment,
the enzyme is trypsin.
In an especially advantageous embodiment, the present method also includes a
step of
protecting specific residues before the derivatization step. For example, in a
case
where a polypeptide or protein is-digested by trypsin, Lys residues may be
protected
in order to avoid e.g. undesired sulfonation reactions. An example of such a
protection
t 0 procedure by guanidination will be described in detail below in the
experimental sec-
tion (see Example 8). Guanidination is advantageously used, since it is
capable of
selectively protecting Lys side chains without having any adverse effect on
peptide
recovery in subsequent steps such as mapping experiments. Furthermore, guanidi-
nated lysine residues in intact proteins are susceptible to trypsin digestion,
so lysine-
r S containing peptides can be used for a quantitative analysis. For example,
a set of
control proteins can be guanidinated with a reagent like O-methylisourea
hydrogen-
sulfate consisting of natural abundance isotopes. A treatment set of proteins
can be
guanidinated with the same reagent enriched in heavy isotopes e.g. O-
methylisourea
hydrogensulfate containing 13C and/or 15N. The protein mixtures can be
combined
z0 and separated prior to tryptic digestion. Interesting proteins are
identified with
MALDI mapping and sequencing, and they are quantitated by comparing abundance
ratios of isotopically labeled and unlabeled lysine-containing peptides.
The present method is preferably used with polypeptides from protein digests.
Poly-
25 peptides can be used which preferably includes less than about 50 amino
acid resi-
dues, more preferably less than about forty residues, even more preferably
less than
about thirty residues, still more preferably less than about twenty residues
and most
preferably less than about ten amino acid residues.
3o A second aspect of the present invention is the chemical compound 3-
sulfopropionic
acid N-hydroxysuccinimide ester as such, which is especially useful as a
reagent for
peptide derivatization on a solid support, as discussed above.
17
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
A third aspect of the present invention is the chemical compound 2-
sulfobenzoic acid
N-hydroxysuccinimide ester as such, which is also useful as a reagent for
peptide
derivatization on a solid support, as discussed above.
A fourth aspect of the invention is a kit for identifying a polypeptide, which
kit con-
tains an acidic reagent in a suitable container. The acidic reagent comprises
a sul-
fonyl or sulfonic acid moiety coupled to an activated acid moiety, and is
preferebly ~
present in the lcit in the solid state. In one embodiment, the reagent is pre-
weighed,
1 c> and in an alternative embodiment, it is present as a bulk reagent. Such
kit may also
contain a buffer providing a pH within the range of ~-11. For reasons of
stability, the
buffer solution will be added by the end-users just prior to use. A kit
according to the
invention can also comprise a model peptide. The kit can also be accompanied
by
written instructions, e.g. in the form of a booklet, as to the use thereof.
Thus, in one embodiment, the present kit contains the necessary devices and
means
for performing a method of identifying a peptide or polypeptide according to
the in
vention. A specific embodiment is a lcit which comprises one or more of the
novel
reagents according to the invention and further means necessary for use with
matrix-
2o assisted laser desorption ionization time of flight (MALDI-TOF) mass
spectrometry.
An alternative embodiment is a kit, which comprises one or more of the novel
rea-
gents according to the invention and further means necessary for use with
electrospray
ionization mass spectrometry (ESI-MS). In a specific embodiment, the present
lcit also
comprises hydroxylamine hydrochloride in a compartment separate from that of
the
reagent, which is useful to add to the reaction after finalized derivatization
in order to
reverse any unwanted ester side-products that have been formed by reaction
with in-
ternal amino acids having side-chain hydroxyl groups.
A fifth aspect of the present invention is the use of an acidic reagent
comprising a sul-
3U fonyl or sulfonic acid moiety coupled to an ester moiety, such as an N-
hydroxy-
succinimide (NHS) ester, e.g. a 3-sulfopropionic acid N-hydroxysuccinimide
ester or
a 2-sulfobenzoic acid N-hydroxysuccinimide ester, as a derivatization reagent
in a
vs
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
mass spectrometric technique wherein the peptides are immobilized to a solid
support
during derivatization. More specifically, the present invention relates to the
use of the
above- described reagent in a method according to the invention.
Detailed description of the drawings
Figure 1 shows the reflection spectrum of non-derivatized sample of horse
myoglobin
(15 fmol on MALDI target) as described in Example 2 below.
Figure 2 shows the reflection spectrum of derivatized sample (< l5fmol on the
1 o MALDI taxget) described in relation to figure 1. Due to the efficient
guanidination of
the lysines on solid support, and the improved response of guanidinated
peptides, the
signals for the lysine-terminated peptides were dramatically increased in the
reflection
spectrum of derivatized sample compared to the analysis of the non-derivatized
sam-
ple. Two derivatized peptides were used for PSD analysis (one lysine
terminated pep-
15 tide m/z~ 1449.5 and one arginine terminated peptide m/z, 1742.8).
Figure 3 shows the PSD spectrum of m/z 1449.5.
Figure 4 -shows how the protein above was identified in PepFrag, by submitting
the
2o masses of the seven observed y-ions (-42 Da mass increment resulting from
the gua-
nidination reaction).
Figure 5 shows the fragmentation spectrum of an arginine-terminated peptide
(m/z
1742.8).
Figure 6 shows the eight y-ions obtained were used for protein identification
in Pep-
Frag.
Figure 7 shows sulfonation of 500 femtomoles of BSA tryptic peptides on solid
phase
3o as described in Example 3.
19
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Figure 8 shows sulfonation of 4.5 picomoles of BSA tryptic peptides in
solution as
described in Example 3
Figure 9A -D show NMR-spectra as discussed in Example 4 below. More specifi-
cally, Fig 9A shows the spectrum of 3-sulfopropionic acid; Fig 9B shows the
13C
NMR spectrum of 3-sulfopropionic anhydride, Fig 9C shows an anhydride carbon
spectrum; and Fig 9D shows the spectrum of the NHS-ester from 3-sulfopropionic
anhydride.
Figures 1 OA-B illustrate the stability of NHS-esters according to the
invention. More
specifically, Fig 10A shows the stability of 3-sulfopropionic acid NHS-ester
in D20
while Fig 1 OB shows the stability of 2-sulfobenzoic acid NHS-ester in DZO.
The
analysis was conducted on a 270 MHz NMR-instrument from JEOL. NHS-ester were
put in a NMR-tube and diluted with D20 to 700.1. A single-pulse 1H-NMR was con-
z S ducted and the spectra analysed. The hydrolysis being measured by the
ratio of the
integration of the signal at 2,92 ppm for 3-sulfopropionic acid N-
hyrdoxysuccinimide,
3,01 ppm 2-sulfobenzoic acid N-hydroxysuccinimide and the signals of the
protons of
N-hydroxysucccinimide 2,76 ppm.
ztt Figure 1 lA-C show the MALDI PSD mass spectra produced from these
derivatives
and the comparative reactivities of peptides sulfonated as described in
Example 7.
More specifically, Fig 11A shows a comparison of the fragmentation patterns
pro-
duced from peptides containing 2-sulfobenzoic acetamides (upper) and 3-
sulfopropionamides (lower). 3-Sulfopropionamides are preferred because of less
loss
z5 of the derivative (which regenerates the starting peptide and is
uninformative) and
better yields of lower mass fragments, Fig 11B shows a comparison of the
reactivities
of propionyl sulfonate NHS ester (upper) and the 2-sulfobenoic acid NHS ester
(lower) with 1 nMole of a model peptide. The 3-sulfopropionic acid NHS ester
shows
better conversion of starting peptide to final product, and Fig 11C is as in
Fig 11B but
3U the reaction used 10 pmoles of FibA as the model peptide.
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Figure 12 shows a reflection spectrum, positive mode (showing average masses,
after
filtration, smoothing 5) of 250 fmols of a non-derivatized tryptic digest of
4VP-BSA
obtained with the EttanTMMALDI-TOF. (Peptides I-III were quantitatively
derivatized
after reaction with 3-sulfopropionic acid anhydride NHS-ester, see figure 13).
s
Figure 13 shows a reflection spectrum (showing average masses, after
filtration,
smoothing 5) of a derivatized tryptic digest of 4VP-BSA (Ettan MALDI-ToFTM).
The
peptides were derivatized with 3-sulfopropionic acid NHS ester using aqueous
condi-
tions as described. The peptides marked I-III were quantitatively derivatized
and used
1 o for PSD analyses.
Figure 14 shows a PSD spectrum (positive mode) showing a complete y-ion series
of
peptide (I) from the derivatized tryptic digest of 4VP-BSA (figure 13)
obtained with
the EttanTMMALDI-TOF. The ion gate was set on the mass of the derivatized
parent
r 5 ion, m/z1064, and the signals from 300 shots were accumulated.
Figure 15 shows a fragmentation spectrum (PSD, positive mode) of peptide (II)
from
the derivatized tryptic digest of 4VP-BSA (figure 13). The ion gate was here
set on
nz/z1616. Signals from 300 shots were accumulated. Gaps are marked with an X.
2c~
Figure 16 shows a PSD spectrum (signals from 300 shots accumulated) of peptide
(III) (figure 13), m/z 1704, from the derivatized tryptic digest of 4VP-BSA.
Gaps are
marked with an X. The peptide, MH+ m/z 1715, passed the ion gate together with
de-
rivatized peptide.
?5
21
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Figure 17 shows a first example of a reflection spectrum (positive mode, 100
shots
accumulated, showing average masses, after filtration, smoothing 5) of a non-
derivatized protein digest from a Coomassie-stained 2-D gel obtained with the
Ettan
MALDI-TOF. Five percent of the total eluted tryptic digest was used to obtain
his
spectrum. (The peak marked with a circle can be seen fully derivatized in
Figure 18.)
Figure 18 shows a reflection spectrum (positive mode, showing average masses,
after
filtration, smoothing 5) of the same 2-D sample as in figure 17 (remaining
95%), but
after N-terminal derivatization with NHS-ester. The sample was cleaned up on a
~,C18
ZipTipTM, and derivatized according the protocol. The peptide m/z 1791
(previous
figure) was quantitatively derivatized and is here observed with the extra
mass of the
label, m/z 1927.
Figure 19 shows a PSD spectrum (accumulated from 300 shots), of the
derivatized
peptide, m/z 1927. The masses of the fragments (y-ions) were used for
identification
in PepFrag. The protein was identified as actin.
Figure 20 shows a second example of a reflection spectrum (accumulated from
100
shots, showing average masses, after filtration, smoothing 5) of a non-
derivatized
2cj tryptic digest of a protein spot from a Coomassie-stained 2-D gel,
obtained with Et-
tanTMMALDI -TOF. Five percent of the sample was used in this analysis. The
marked
peptide was used for PSD analyses after derivatization (see figure 21).
Figure 21 shows a reflection spectrum (positive mode showing average masses,
after
?5 filtration, smoothing 5) of the same 2-D sample as in figure 19, but after
ZipTipTM
clean up and derivatization with NHS-ester in aqueous solution as described.
The
peptide m/z 1569.9 (figure 20) was quantitatively derivatized and is here
observed
with the extra mass of the label (+136) as m/z 1705.9.
3o Figure 22 shows a PSD spectrum (signal from 300 shots accumulated) of the
derivat-
ized peptide, m/z 1705 (see figure 20). The fragment masses (y-ions) were used
for
2?
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
protein identification in PepFrag. The protein was identified as E-coli
succinyl-CoA
synthetase.
Figure 23 shows sample-loaded ZipTipsT"" placed into a laboratory centrifuge
for sub-
sequent sulfonation in a multiplexed fashion.
Figure 24 illustrates sample washing in the centrifuge following the
sulfonation reac-
tion.
1 o Figure 25 illustrates direct loading of the derivatized samples from the
solid supports
onto the MALDI sample stage.
Figure 26 shows the MALDI mass spectra obtained following sulfonation of
Fibrino-
peptide A on solid support. Duplicate samples were sulfonated at three
different pep-
tide levels (10, 1 and 0.1 pmoles).
Figure 27 the use of hydroxylamine hydrochloride for reversing unwanted ester
side-
products formed in the sulfonation reaction. The upper spectrum was obtained
from
ASHLGLAR sulfonated on solid support in the centrifuge. The lower spectrum was
2c) obtained from the same sulfonated peptide following treatment with
hydroxylamine
hydrochloride.
Figure 28 demonstrates the sulfonation of a protein digest. The upper spectrum
was
obtained from the native protein digest. The lower spectrum was obtained
following
sulfonation of the digest.
EXPERIMENTAL PART
The present examples are intended for illustrative purposes only and should
not be
3o construed as limiting the invention as defined by the appended claims. All
references
given below and elsewhere in the present application are hereby included
herein by
reference.
23
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Example 1: Sulfonation on solid support, general scheme
Reagent: 3-sulfopropionic acid N-hydroxysuccinimide ester
Buffers and chemicals:
O- methylisourea hydrogen sulfate
0.25M NaHC03, pH~ 11.9
0.25M NaHC03, pH 9.4
50% hydroxylamine solution / 1 ~,1 of a 1 SM solution
1 o Acetonitrile (ACN)
Trifluoracetic acid (TFA) -
matrix for MALDI-TOF analyses of a-cyano-4-hydroxycinnamic acid
Buffers and solutions prepared from deionized 18.2 MS2 (DI) water
C1$ ZipTipTM (ZT) from Millipore (~.C18 ZipTips can alternatively be used)
1S
General procedure:
The sample can be dried down and reconstituted in 10 p.1 0.1 % TFA.
Alternatively,
the sample is dried down to about 20 uL, in which case the samples are made
acidic
before loading onto ZipTips.
2o Solid support in the form of C18 ZipTipTM (ZT) is activated with 50%
ACN;0.5%TFA
and the ZipTipT"~ is then equilibrated with 0.1 %TFA. A sample comprising
tryptic
peptides is loaded the sample on the ZipTipT"" (pipett 10 times slowly up and
down).
In a separate vessel, 2~,1 O-methylisourea hydrogensulfate solution (86 mg/ml
MQ
?5 H20) is mixed with 8~.10.25M NaHCO3, pH 11.9. The resulting mixture is
loaded on
the ZipTipT"" (pipett ~5 times up and down). The tip is removed with solution
on the
top and put in an eppendorf tube, the lid is closed and it is placed in a
heating block at
37°C for 2h.
3o The tip is then washed with 0.1%TFA (pipett ~5 times up and down).
2~.
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
The sulfonation reagent solution is made fresh just prior to incubation and
dissolved
in 0.25 M NaHC03, pH 9.4 (1 Omg/100~1).
Then, step (a) of the present method is performed by passing the sulfonation
reagent
solution through the ZipTipT"" by pipetting up and down 10 times. The solution
is left
on the tip for at least 3 minutes. If the reactions are being performed
manually using a
single-position micropipetter it may be convenient to take the tip, with
solution on the
top of the C1$ column, off of the micropipetter and set it aside. It is then
possible to
continue with the next sample, while waiting for completion of step (a).
~o
In order to reduce the amount of unwanted sulfonation of internal amino acids,
1 ~,l
15M hydroxylamine solution is added to the reagent solution. Mix and load to
the ZT
and pipett up and down 10 times. In the alternative embodiment, a small volume
of
the hydroxylamine solution is passed over the ZTs containing the sulfonated
peptides.
15 Thus, in this last mentioned embodiment, the hydroxylamine is never with
the original
reagent solution.
The ZT is the preferably washed with 0.1 % TFA and the sample is eluted in 10
~l
80% acetonitrile:0.5%TFA.
To analyze the derivatives) obtained, the sample is dried down and
reconstituted in
3 ~,1, 0.1 %TFA. A total drying in this step will allow a more exact analysis,
since it
compensates for differences in sample volumes by standardising the procedure,
which
is especially desired in automated procedures. The sample is mixed 1:1 with
saturated
~5 alpha-cyano-matrix solution in 50% ACN:0.5%. The sample is then loaded on
the
MALDI target and analyzed.
As mentioned above, in one embodiment, which is especially suited for low-
level
analytes, the samples are not dried down. The cleaned up products are then
eluted off
of the ZT directly onto the MALDI sample plate, for example using 2.5 uL of
50%
3o ACN:0.5% TFA containing the MALDI matrix. This way, sample handling losses
are reduced and preferably avoided altogether, so that all of the products can
be trans-
ferred to the MS.
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Example 2' Guanidination and sulfonation of a low level tryptic digest of
horse myo-
globin immobilized to solid support
Alkylation and try~sin di;~estion of the protein:
Horse myoglobin (Sigma) was dissolved in MQ water to a concentration of 1
~g/~.l
and 50.1 was mixed with 450 ~1 denaturation buffer (8 M UREA, 50 mM TRIS-HCl
pH 8.0, 50 mM DTT (all chemicals were plusoneTM)) and incubated for 1 hour at
37
1 t) °C, in order to denature the protein and disrupt any disulfide
bonds. The cysteine SH
groups were then chemically blocked by 2-Iodoacetamide (MERCK), by adding
5001 alkylation buffer (8 M UREA, 50 mM TRIS-HCl pH 8.0, 125 mM 2-
Iodoacetamide). The reaction was allowed to proceed for 1 hour at 37°C.
The sample
was thereafter purified on a NAP-10 column, equilibrated with 15 ml 10 mM
l5 NH4HCO3. The sample was applied (1000 ~.1) and eluted in 900 ~.1 10 mM
NH4HC03.
The protein was digested by adding 5 ~g of trypsin (Promega, VS 11A) to the
eluted
sample. The trypsin digestion reaction was left over night (approximately 14
hours) at
37 °C, and terminated by the addition of 5 ~,l of concentrated
triftzoroacetic acid
(TFA) (Pierce) to a final concentration of 0.5%. The digested sample was
diluted
2U stepwise in 0.1 % TFA to a final concentration of 15 fmol/~.1. The
resulting material
was stored at -20 °C.
Guanidination and sulfonation on solid support:
A C1$ ZipTipTM (Millipore) (ZT) was activated with 50% acetonitrile;0.5%TFA
(by
25 pipetting 2 times up and down). The ZT was thereafter equilibrated with 0.1
%TFA
(by pipetting 2 times up and down). Tryptic digest of horse myoglobin (150
fmol in
~.l 0.1 %TFA) was loaded to the ZT (pipett 10 times slowly up and down). A
stock
solution of O-methylisourea (84mg/ml MQ H20) was prepared. Two microliters of
the stock solution of O-methylisourea was mixed with 8,10.25 M, NaHC03 buffer,
30 pH 11.7 and the solution was loaded to the ZT. The ZT was left in a closed
eppendorf
tube in 37°C for 2 h, for the sample to react. The ZT was therefore
washed with 10 ~l
0.1% TFA (by pipetting 2 times up and down). NHS-ester of 3-sulfopropionic
acid
26
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
anhydride, was dissolved in 0.25 M NaHCO3 buffer, pH 9.4, to a final
concentration
of 100mg/ml. Ten ~,1 of the NHS-ester solution was loaded to the ZT. The
sample was
left to react for 3 minutes in RT. One microliter of 15M hydroxylamine
solution was
added to the NHS-ester reagent and loaded to the ZT (by pipetting 5 times up
and
down).
The tip was washed with 0.1% TFA and the sample eluted in 10 ~,l 80% acetoni-
trile:0.5%TFA. The sample was dried down under nitrogen and reconstituted in 3
~.1
50% acetonitrile. 0.3 ~.1 of the sample was loaded to the MALDI target, using
the Et-
1 o tan MALDI spotter and mixed with 0.3 ~l saturated a-cyano matrix solution.
The
sample was analyzed in reflectron and PSD modes using the Ettan MALDI ToF.
One-tenth of the 150 fmole tryptic digest of horse myoglobin, which was
guanidi-
nated and sulfonated following immobilization onto a ZipTipTM, was analyzed
using
the Ettan MALDI ToF. For comparison, Figure 1 shows the reflectron spectrum of
a
non-derivatized sample of horse myoglobin (15 fmol on MALDI target) and figure
2
the reflectron spectrum of derivatized sample (< 15 fmol on the MALDI target).
Due
to the efficient guanidination of the lysines on solid support the signals for
the lysine-
terminated peptides were dramatically increased in the reflectron spectrum of
de-
2c~ rivatized sample compared to the analysis of the non-derivatized sample.
Two de-
rivatized peptides were used for PSD analysis (one lysine terminated peptide
m/z,
1449.5 and one arginine terminated peptide m/z, 1742.8). Figure 3 shows the
PSD
spectrum of m/z 1449.5. The protein was identified in PepFrag, by submitting
the ob-
served y-ion masses (-42 Da mass increment from the guanidination reaction)
figure
2s 4. Figure 5 shows the fragmentation spectrum of an arginine-terminated
peptide (m/z
1742.8). The eight y-ions obtained were used for protein identification in
PepFrag
(figure 6).
The guanidination and sulfonation reaction times are reduced when the
reactions are
3o carried out with peptides or polypeptides immobilized to a solid support.
The overall
efficiency of the derivatization procedures is improved, and better
sensitivity results
because dilute analyte solutions can be concentrated prior to reaction and
because re-
27
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
duced sample losses occur as a result of reduced sample manipulation prior to
analy-
sis. The example shows protein identification by derivatization PSD analysis,
starting
with as little as 15 fmol of the protein.
s Example 3: alternative method to sulfonate peptides and poly~eptides
immobilized to
a solid support
Peptides and polypeptide mixtures in solution are concentrated to a final
volume be-
tween 10 to 50 ~,1. The pH of each solution is made acidic, and the pep-
tide/polypeptide solutions are loaded onto C18 ZipTipsT"". The sample-loaded
Zip-
TipsT"" are placed into the tops of drilled-out, closed microcentrifuge tubes,
which are
loaded into a laboratory centrifuge as shown in Figure 23. The sample-loaded
tips are
washed with 0.1 % TFA. This, is accomplished by adding 25 p,1 of 0.1 % TFA to
the
tops of each tip and spinning. The centrifugal force is sufficient to move the
solution
l 5 over the tip. The solution is collected into the bottom of the
microcentrifuge tube.
This wash step is repeated two more times. Samples are then sulfonated using
e.g.
propionylsulfonate-NHS ester. The sulfonation reagent is prepared at a
concentration
of 10 mg/100 ~,1 base (HZO:DIEA 19:1 v:v) just prior to use. The pH of the
reagent
solution is checked, and adjusted if necessary, to be sure that it is basic
prior to use.
?o The samples are sulfonated by loading 5 ~l of the sulfonation solution to
the top of
each sample-loaded tip. The samples are spun again to transport the
sulfonation rea-
gent over the tips. All samples in the centrifuge are sulfonated in parallel
using this
procedure. Optionally, the sample-loaded tips can be further treated with
hydroxyla-
mine hydrochloride to reverse any unwanted ester side-products that may have
been
25 formed during the sulfonation step. That reaction is carried out by loading
5 ~,1 of
fresh hydroxylamine hydrochloride solution (2M in H20:DIEA 19:1 v:v, pH
adjusted
to basic prior to use) to the top of each sample-loaded tip. The samples are
again spun
to transport that solution over the tips. The samples are then washed three
times with
25 ~,1 of 0.1% TFA, as shown in Figure 24. The derivatized samples are loaded
di-
3o rectly from the ZipTipsT"" onto a MALDI sample stage for analysis. The
samples axe
eluted onto the sample stage with a small volume (2.5 ~,1 of ACN:0.1% TFA (1:1
v:v)
28
WO 02/095419 PCT/US02/1624
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containing 10 mg/ml of a suitable MALDI matrix like a-cyano-4-hydroxycinnamic
acid or 2,5-dihydroxybenzoic acid, as shown in Figure 25.
The utility of this approach is illustrated with data presented in the next
few figures.
For example, Figure 26 shows the MALDI mass spectra obtained from varying quan-
tities of Fibrinopeptide A (ADSGEGDFLAEGGGVR) sulfonated according to the
method just discussed. The starting MH+ mass of Fib A is 1536.7 and the
desired
monosulfonate product weighs 1672.7 Da. The measured molecular masses are in
error about 0.5 Da because the mass scale was not accurately calibrated in
these ex-
l0 periments. The spectra indicate near quantitative sulfonation even at the
100-fmole
level. Note that the lower mass ions in the 10-pmole samples (lower two
traces) result
because too much sample was presented to the mass spectrometer in those two
analy-
ses. The ions having masses less than that of the sulfonation product mainly
result
from fragmentation processes that occurred within the ion source during
analysis.
is Figure 27 compares MALDI mass spectra of a small Arg-terminated peptide
(ASHLGLAR), which was sulfonated as just described. The top spectrum in the
fig-
ure was obtained following sulfonation. It shows signals for the desired
product at
about m/z 960, and a signal for an unwanted double sulfonation product at
about m/z
1096. The lower spectrum was obtained from the same sulfonated peptide after
ZO treatment with hydroxylamine hydrochloride as described above. Note that
the un-
wanted sulfonation product at about m/z 1096 has been greatly reduced in
relative
abundance. The spectra in Figure 28 demonstrate that protein digests can also
be effi-
ciently sulfonated using this method. The upper spectrum in the figure was
obtained
from the native tryptic digest, which was not sulfonated. The lower spectrum
was ob-
2,5 tamed from the protein digest that was sulfonated according to the present
method.
The peptide masses observed in the top spectrum shift upwards by 136 Da
following
sulfonation according to the present method. Near quantitative sulfonation of
the pro-
tein digest was observed in this experiment.
ao Example 4, comparative: Sulfonation in solution vs on solid support
29
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Sulfonation in solution
General method
The sample (BSA tryptic peptides) was dissolved in 5 ~l of water. 10 ~1 of 20%
DIEA
solution was added followed by 5 ~l of NHS ester solution. After 15 minutes,
hy-
s droxylamine was added to hydrolyse unwanted ester groups, which may have
been
formed during the sulfonation step. The pH of the resulting solution was made
acidic
(<4) by addition of 50% TFA. The reacted peptides were bound to reverse phase
chromatography (RPC) solid support (ZipTipTM, Millipore) and eluted using 80%
Acetonitrile and 0.5% TFA. The eluted sample was dried and reconstituted in 3
~,1 of
50% ACN, 0.5% TFA for further analysis on MALDI.
Sample: BSA tryptic peptides
Reaction vessel: 500 ~,l Eppendorff tube
Total volume: 20 ~1
~ 5 Water: 5 ~.1
Volume of base: 10 ~l of 20% DIEA (shake thoroughly before pipetting as it
is immiscible) or 2 ~1 neat DIEA
Volume of NHS ester: 5 q1 (10 mg/100~1)
Reaction time: 15 minutes or more
?0 Addition of hydroxylamine: 2 ~1
Neutralization: Add 3 ~l of 50% TFA to neutralize before cleaning up with
ZipTipTM.
Preparing ZipTipTM for binding peptides: Wet the C18 matrix with 50%
acetonitrile and then equilibrate with 0.1 % TFA.
?j Elution: 80% Acetonitrile and 0.5% TFA in another tube
For making matrix: 50% Acetonitrile, 0.5% TFA
Sulfonation on solid support
General method
3o Bind the sample (peptides having arginine or homoarginine as C-terminal) to
solid
support, preferably C18 on chemically resistant matrix. Here we have used
ZipTipTM
Clg 0.6 ~l supplied by Millipore). Leave in contact with reaction mixture (NHS
es-
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
ter+base) for a minimum of 3 minutes. Add hydroxylamine to the reaction
mixture, to
hydrolyze any unwanted ester side-products that may have been formed during
sul-
fonation, and aspirated it up and down five times. Wash the solid support with
0.1
TFA and elute it for further analysis.
Preparing ZipTip for Binding peptides: Wet the C18 matrix with 50%
acetonitrile
and then equilibrate with 0.1 % TFA
Sample: BSA tryptic peptides
Reaction vessel: 500 ~l eppendorf tubes
1c) Volume of NHS ester of propionic acid: 10 ~1 (10 mg/100~,1) dissolved in
0.25 M
Sodium bicarbonate.
Reaction time: minimum of 3 minutes
Addition of hydroxyl amine : 1 ~1
Elution: 80% Acetonitrile and 0.5% TFA in another tube
I S For making matrix: 50% Acetonitrile, 0.5% TFA
MALDI anal.
For this sulfonation reaction, the intensities of five arginine peptides (see
table below
z0 and figures 7 and 8) were studied and compared.
Table 1: Peptides studied
Sequence Peptide Native Sulfonated Tyrosine labeled
347-359 DAFLGSFLYEY 1567.8 1703.8 1839.8
SR
421-433 LGEYGFQNALI 1479.9 1615.9 1751.9
VR
360-371 RHPEYAVSVLL 1439.9 1575.9 1711.9
R
361-371 HPEYAVSVLLR 1283.7 1419.7 1555.7
31
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161-167 ~ YLYEIAR 927.49 1063.49 1199.4
Results
See discussion in relation to figures 7 and 8 above.
Comparison of results from reactions performed in solution and on solid phase
1. The reaction time in solid phase was about 3 minutes where as it was 15
minutes
for the solution.
t o 2. When sodium bicarbonate solution was used in solution phase, very high
signal to
noise ratio was observed on the spectra, whereas in solid phase there was no
effect
on the baseline.
3. A thorough mixing of solution is required when DIEA is used as base in
liquid
phase.
15 4. As seen from figure 7 and 8 that spectra of 500 fmole on solid phase and
4.5 pi-
comole of BSA peptides in solution had comparable sensitivity on MALDI.
Example 5: Preparation of 3-sulfopropionic acid N-hydroxysuccinimide ester
20 Materials
Chemicals for synthesis:
N-Hydroxysuccinimide (NHS), internal supply, Art-Nr 30070800
3-Mercaptopropionic acid from ALDRICH 99+%, CAS-107-96-0
Hydrogen peroxide (30%, aqueous solution)
35 Acetic acid (glacial) 100% from KEBO CAS-64-19-7
Potassium hydroxide from Merck , pellets
n-Heptane from Merck 99%
Thionyl chloride from ALDRICH 99+%, CAS-7719-09-7
n-Hexane from Merck 99%
3o Diisopropyl amine from ALDRICH 99%, CAS-7087-68-5
Dichloromethane from ALDRICH 99.8% anhydrous, CAS-75-09-2
32
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WO 02/095419 PCT/US02/16247
Argon gas-tube from Air Liquide
Ethyl acetate from KEBO, CAS-141-78-6
Methanol from KEBO, CAS-67-56-1
TLC Silica gel 60 F25ø on plastic sheets from Merck
Chemicals for anal
Chloroform-d from Cambridge Isotope Laboratories 99.8%, CAS-865-49-6
Deuteriwnoxide (D20) from Larodan Fine Chemicals CAS-7789-20-0
t o Methods
NMR-analysis:
The analysis was conducted on a 270 MHz NMR-instrument from JEOL.
10 mg of NHS-ester were put in a NMR-tube and diluted with CDCl3 to 7001. A
single-pulse 1H-NMR was conducted and the spectra analysed. The analysis was
conducted in the same way for 3-sulfopropionic anhydride. For the 3-
sulfopropionic
acid, D20 was used as a solvent instead of CDC13.
2o For the 3-sulfopropionic anhydride a decoupled 13C-NMR was carried out in
the same
way as with the 1H-NMR (see above).
Melting t~oint determination:
The melting point for the NHS-ester crystals was obtained on a BLTCHI Melting
Point
B-540 apparatus. A few crystals were put in a vial and heated until they
melted. The
temperature interval was from 160°C to 185°C and the temperature
gradient 1°C/min.
Stability test in water:
10 mg of NHS-ester were put in a NMR-tube and 7001 of DZO was added. A single-
:;0 pulse 1H-NMR was conducted and the spectrum analysed. The same sample was
stored at RT (20-25°C) and after 5 and 24 hours another 1H-NMR spectrum
was
collected.
33
CA 02448534 2003-11-20
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Stability test in air:
1 Omg of NHS-ester were put in a NMR Tube and analysed as above with
Chloroform-
D as solvent. About 100 mg of the NHS-ester were then put in a flaslc and kept
without lid in air and RT (20-25°C) for some days. The hydrolysis of
the ester was
followed with NMR.
Synthesis:
Synthesis of 3-Sulfopropionic acid
z c~
0
0
Acetic acid
SH OH + H/O\O/H 50 °C S03 OH
3-Mercaptopopanoic acid 3-Sulfopropionic acid
A 3-necked roundbottomed flask (SOOmI) was equipped with a thermometer,
dropping
1 s funnel and a degassing pipe. A gas-trap with two security-flasks (coupled
in series
after each other), the last containing 25% KOH-solution was fitted to the
pipe. During
the reaction a nitrogen-balloon kept an inert atmosphere through the system.
Acetic
acid (70m1) and hydrogen peroxide (70g, 30% aqueous solution, 620mmo1) were
put
in the flask and the solution was heated under stirring to 50°C on a
waterbath. 3-
2o Mercaptopropanoic acid (8,20m1, 94mmol) was added very carefully through
the
dropping funnel over a period of about 1 hour. An exothermic reaction started
at once
and the temperature rose to about 80°C. The solution was then cooled on
an
ethanol/COZ bath (-72°C) until the temperature was again 50°C,
this procedure was
repeated until all the 3-mercaptopropanoic acid had been added from the
dropping
25 funnel. The reaction was then left stirring at 50°C for two hours
and at RT over night.
The solvent was evaporated on a rotary evaporator (water-bath 40°C, 100
mbar) until
the volume had been reduced to about 30m1, the rest was then removed by
azeotropic
evaporation with 3x300m1 heptane. The resulting oil was dried in a desiccator
under
34
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
high vacuum over night. The crude product was a white precipitate in an oil.
The
yield was about 50%,.estimated from the NMR-spectrum, see Fig 1.
Synthesis of 3-sulfopropionic anh d
7
o\~ A o
O SOCIZ SAO
S03 OH Reflux, 3h
3-Sulfopropionic acid o
3-Sulfopropionic anhydride
The 3-sulfopropionic acid (20g of the crude product from the experiment above)
was
i o put in a 3-necked roundbottomed flask. A reflux-condenser and a septum
were fitted
to the flask. During magnetic stirring, SOCl2 (140m1) was carefully added
through the
septum over a period of 30 minutes. When all the SOCl2 had been added the
mixture
was refluxed for 3 hours. Everything had dissolved during reflux into a brown-
red
coloured solution. After cooling for about 5 minutes, hexane (140m1) was
added. A
15 white solid precipitated at once and a brown oil was formed at the bottom
of the flask.
The solution was then heated again until the white solid had dissolved and the
solution was decanted into another flask to get rid of the oil. The solution
was then
allowed to cool in RT for an hour and then put in a refrigerator over the
weekend for
crystallisation.
2t)
The precipitate was filtered under nitrogen atmosphere, washed with cold n-
hexane
(from the refrigerator) and dried in a desiccator under high vacuum over
night. All
equipment that was used for the filtration had been dried in an oven
beforehand and
cooled in a desiccator, since the anhydride is very sensitive to water.
Synthesis of NHS-ester from 3-sulfopropionic anhydride:
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
O
O OH
OH ~~~\ , O
O N O
S\ O \~~~ O O
p~
3-sulfopropionic anhydride NHS NHS-ester
All equipment that was used was dried in an oven (100°C) and put in a
desiccator
before the synthesis.
NHS (420mg, 3,68mmol) was weighed into a round-bottomed flask (100m1) equipped
with a septum and an argon balloon. DCM (20m1, anhydrous 99.5%) was added and
magnetic stirring began. DIEA (0.64m1, 3,68mmo1) and 3-sulfopropionic
anhydride
(O.SOg, 3,68mmo1) were added carefully during stirring. The reaction was left
stirring
1 o for three hours under an argon atmosphere. The solvent was evaporated (RT,
100mbar) and the product was dried in a vacuum oven over night (RT, 1 mbar).
The
resulting crystals were dissolved in the minimum amount of warm EtOAc/MeOH
(9:1 ). When everything had dissolved the solution was left to cool in RT for
about
three hours and then in the freezer over night. During the night white
crystals had
k 5 formed which were filtered on a glass filter (p3) and washed with cold
ethyl acetate
(5°C). Finally the crystals were dried under high vacuum in a
desiccator to get the
DIEA-salt of the NHS-ester as white crystals (42% yield).
Results & Discussion
20 S, nt
Synthesis of 3-Sulfopropionic acid:
The synthesis was quite simple and gave the crude 3-sulfopropionic acid as a
white
slurry. The tricky part was to lceep the reaction at 50°C, this was
done with alternating
ice-bath and oil-bath which perhaps is not the most effective way. The
temperature
25 during the reaction varied from 20°C up to 80°C. If a better
temperature control could
be maintained under the reaction maybe the yield would improve. No further
purification was done since it was not necessary for the next step (synthesis
of the
3 ti
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
anhydride) making the yield very hard to calculate. On the NMR-spectra you
could
see at least one bi-product and maybe some of the starting material (see NMR-
analysis) an estimation of the purity would be around 50%.
Synthesis of 3-sulfopropionic anhydride:
As expected the anhydride was very sensitive to water and~it was necessary to
dry all
equipment in an oven before use and to do the reaction and purification under
an
argon atmosphere. The reaction and recrystallisation was done in SOC12 which
is a
very toxic solvent. The product, 3-sulfopropionic anhydride, was collected as
light-
~ t) brown crystals. For a reliable calculation of the yield, it is essential
that the starting
material is pure.
Synthesis of NHS-ester from 3-sulfopropionic anh d
Once again the equipment was dried in an oven before the reaction which was
done
t s under an argon atmosphere. The reaction was quite simple and after two
hours of
stirring the solvent was evaporated to give the crude NHS-ester/DIEA-salt as a
white/yellow solid. The yield after purification was 42%. A longer reaction
time and
excess NHS and/or DIEA could possibly improve the yield. The yield is also
calculated on a 100% pure 3-sulfopropionic anhydride.
z0
Purification:
The crude NHS-ester/DIEA-salt was recrystallized. This was done in EtOAc/MEOH
(9:1) after first trying EtOAc/MeOH (7:3). The latter one gave no
crystallisation after
cooling.
In the synthesis of the anhydride (see above) a sort of recrystallisation was
done in
SOCl2. This however was in reality just a re-heating of the reaction mixture
and a
decantation to get rid of the oil in the bottom of the flask. A better purity
of the
anhydride will be achieved by a proper recrystallisation.
37
CA 02448534 2003-11-20
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Characterisation
Melting point determination:
The melting point of the crude NHS-ester/DIEA-salt was between 145-
155°C. After
recrystallisation however the melting point was determined to 176-
178°C. This higher
and much sharper melting point after purification indicates that the product
has indeed
become purer.
NMR-analysis:
The spectra obtained from NMR analysis is shown in Figure 1.
3-sulfopropionic acid:
Table 2: Interpretation of the 1H-NMR-spectra of 3-sulfopropionic acid CDC13
Proton numbershift (8 Interpretation Group
- ppm)
1,2 3.13 t, methylene 03S-CH2-CH2-
protons
COOH
3,4 2.75 t, methylene CHZ-CH2-COOH
protons
is
The spectra also contained some by-product and some starting material giving
some
peaks at 82.78, 82.85, 83.18 and at 83.52. This was expected when no
purification had
been done.
3-Sulfopropionic anh d
Table 3: Interpretation of the 1H-NMR-spectra of 3-sulfopropionic anhydride
CDCl3
Proton numbershift (8 Interpretation Group
ppm)
1,2,3,4 2.45-2.85 m, methylene -03S-CH2-CH2-
38
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
protons COO-
Table 4: Interpretation of the decoupled 13C-NMR -spectra of 3-sulfopropionic
acid
CDC 13
Carbon shift (8 Interpretation Group
number ppm)
1 47 Alkyl carbon 03S-CH2-CH2-
COOH
2 31 Alkyl carbon 03S -CHZ-CH2-
COOH
3 174 Carbonyl carbon03S -CH2-CH2-
COOH
Both spectra were compared and confirmed with reference spectra.
NHS-ester from 3-propionic anh d
Table 5: Interpretation of the 1H-NMR-spectra in CDC13
Proton number Shift (8 Interpretation Group
ppm)
1,2 3.20 m, methylene 03S-CH2-CH2-COO-
~
protons
3,4 3.08 m, methylene 03S-CH2-CH2-COO-
protons
5,6,7,8 2.80 s, methylene -CO-CH2-CH2-CO-
protons
DIEA(2 protons)3.67 m, methine protons(CH3)2CH-N(C2H5)-
CH(CH3)2
DIEA(2 protons)3.20 m, methylene -N-CH2-CH3
protons
39
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WO 02/095419 PCT/US02/16247
DIEA(IS protons) ~ 1.40 ~ dd, methyl protons ~ ((CH3)2-CH)2N-CH2-CH3
Typical inpurities in the crude product are NHS and DIEA. NHS gives a peak at
82.68(s) and DIEA gives peaks at almost the same ppm as seen above in the
table.
This makes the DIEA impurity harder to spot than NHS but it can be estimated
by
looking at the integral of the peaks. If there are any solvent left the MeOH
gives a
peak at b 3.49(s), EtOAc at 82.05(s), 51.26(t) and at 84.12(q) and finally DCM
at
85.30(s).
~o
Example 6: alternative preparation of 3-sulfopropionic acid
N-h d~ysuccinimide ester
Preparation of 3-sulfo~ropionic acid
A 1L 3-neck flask was fitted with mechanical stirrer, thermometer and N2
inlet, an
addition funnel, and a heating mantle and set up in an efficient fume hood.
Acetic
acid, 165.4 ml, was added to the vessel as was 165.4 ml of 30% H202, 1.46
mole. This
mixture was stirred and heated to 50 deg. C. At 50 deg. C. dropwise addition
of 3-
2c) mercaptopropionic acid, 50 gm 0.471 mole, was begun after the mantle was
removed.
The reaction is exothermic requiring external cooling. Temperature was
maintained
at 50-55 deg. C. with a dry ice/acetone bath. When the addition was complete
(re-
quired about 5 minutes) the reaction remained exothermic for about 30 minutes
then
the temperature started to drop. When the exothermic activity had ceased, the
mantle
was replaced and used to maintain the temperature at 50 deg. C. for 2 more
hours.
Periodic testing of the solution using starch iodide paper indicated the
continued pres-
ence of peroxide. After 2 hours the clear, colorless solution was allowed to
cool and
was transferred to a flask for flash evaporation. The rotary evaporator bath
was set to
50 deg. C. and used a vacuum source of about 5-6 mm Hg. This step was
necessary to
remove as much acetic acid as possible so as not to interfere with the
subsequent ex-
traction with ethyl acetate. When no more acetic acid/water/H202 could be
collected
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
at this temperature and vacuum (about 1-1.5 hr), the sample was removed and
weighed about 100-120 gm. This is greater than the 72 gm theoretical weight of
the
product and represents water that is very difficult to remove using our
evaporative
techniques. Freeze drying did not work to remove additional water as the
material
will not stay frozen even at -20 deg. C. Possibly greatly diluting the
material would
allow the sample to remain frozen but adding the extra water represents an
undesir-
able step. The concentrated solution was dissolved in 500 ml of water and
extracted 3
times with 300 ml each time of ethyl acetate. The ethyl acetate extracts
tested posi-
tive for HZOZ decreasing in intensity with each subsequent extraction. The
water layer
t 0 was concentrated to about 100 gm one final time. The product was a viscous
oily
product that contained a white precipitate. 1H NMR analysis in D20 with a
trace of
acetonitrile (2.06 ppm) added to serve as an internal standard revealed
singlets at 3.23
ppm and 2.78 ppm. Note: these peaks can shift depending on concentration. Mi-
nor impurities were observed at 3.58, 2.9, and 2.23 ppm. A 13C NMR on the same
~ 5 sample revealed peaks at 174.8, 45.5, and 28.4 ppm.
Preparation of ~3-sulfopropionic anh d
The entire sample obtained in the reaction described above 0100 gm) was
treated
20 with 652.4 gm, 5.48 mole, of thionyl chloride again using an efficient fume
hood.
The thionyl chloride was added incrementally since reaction with the residual
water
can be vigorous. No violent fuming was observed although HCl and S02 are
evolved
which were directed to the rear of the fume hood using tygon tubing attached
to the
top of the condenser using an adapter. When addition was complete, the mixture
was
25 stirred magnetically at reflux for 12 hours. While cooling yet still
stirring the (3-
sulfopropionic anhydride precipitated. The flaslc was stoppered and placed in
the
freezer for 2 hours to maximize the amount of precipitate. The solid anhydride
was
then collected by filtration in a glove bag under NZ and the filter cake
rinsed twice
with 50 ml portions of petroleum ether. The use of the glove bag (a dry box
would
30 work as well) is very important since the anhydride is extremely water
sensitive re-
acting to give the starting 3-sulfopropionic acid. The solid anhydride was
transferred
to a stoppered flask inside the glove bag, then removed to a vacuum desicator
where it
41
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
was unstoppered and subject to a 1 mm vacuum over P205. The dried anhydride
weighed 39 gm, a yield of 61%. 1H NMR analysis in CDC13 revealed singlets at
3.8
ppm and 3.45 ppm. A 13C NMR on the same sample revealed peaks at 161.9, 48,
and
32 ppm. M.p. was 74.6 deg. C. Lit. 76-77 deg. C.
Reproducibility_
This entire sequence (both reactions) was repeated using the same scale and
tech-
t c} niques. Nearly identical results were observed. The crude material
weighed 84 gm.
Note: close observation of the mixture following addition of the thionyl
chloride re-
vealed that as the water was consumed in the reaction with excess thionyl
chloride in
30-45 minutes, a beautiful white solid precipitated that is believed to be the
anhydrous
3-sulfopropionic acid. As the stirring at reflux was continued for another
hour, this all
~ s dissolved and reacted as observed earlier. The final weight of the second
sample of ~3-
sulfopropionic anhydride was 40.7 gm. A yield of 63.5%. %. 1H NMR analysis in
CDCl3 revealed singlets at 3.8 ppm and 3.45 ppm. A 13C NMR on the same sample
revealed peaks at 161.9, 48, and 32 ppm.
zo
N-H~roxysuccinimide ester of 3-sulfopropionic acid, diisopropylethylamine salt
A 500 ml 3-neck flask was prepared with magnetic stirring bar, thermometer and
N2
inlet, and addition funnel. 3.9 gm, 0.0338 mole, of N-hydroxysuccinimide was
placed
?s into the flask at room temperature. 100 ml of CH2Cl2 was added and the
mixture
stirred as 4.37 gm, 5.9 ml, 0.0338 mole, of diisopropylethylamine were added.
Note:
the N-hydroxysuccinimide dissolved upon addition of the diisopropylethylamine.
4.6
gm, 0.0338 mole, of (3-sulfopropionic anhydride was dissolved in 80 ml of
CH2C12
and added to the stirred solution using the addition funnel. The reaction
mixture dark-
30 ened as the addition progressed. When addition was complete, the mixture
was stirred
for 3 additional hours at room temperature then transferred to a single neck
flask and
the solvent removed on the rotary evaporator yielding a light brown solid
residue. The
~.?
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
residue was dissolved in 50 ml of CH2C12 and stirred for 1 hour at room
temperature
with 2 gm of activated charcoal followed by filtration through glass fiber
filter paper
and a bed of celite. The celite was rinsed once with 25 ml of CH2C12, The
CHZC12 was
removed on the rotary evaporator. The solid residue was dissolved in 20 ml of
50
deg. C. methanol. This solution was poured into 180 ml of ethyl acetate and
the solu-
tion placed in the freezer overnight. The next morning a tan solid had
precipitated
that was collected by filtration. The solid was rinsed on the filter paper
with about 50
ml of cold (freezer temperature) ethyl acetate. This f ltration was performed
in a NZ
filled glove bag although the ester may be expected to have far less water
sensitivity
1 Ca than the starting anhydride, if any. The dried sample weighed 7.3 gm and
represents a
yield of 86%. An 1H NMR in CDC13 revealed: 9.175 (1H-bs), 3.6 ppm (2H-m), 3.1
ppm (4H-s), 3.0 ppm (2H-m), and 1.35 ppm (15H-m). A 13C NMR on the same sam-
ple revealed peaks at 173.3, 168.8, 167.4, 53.9, 45.7, 42.2, 27.4, 25.3, 18.3,
17.1, and
11.9 ppm. The sample had a m.p. of 175-176 deg. C. Lit. 176-178 deg. C.
IS
Note: Care should be taken to use a minimum amount of the methanol/ethyl
acetate
solvent for the recrystallization step. Too much may result in little or no
precipitation
of product.
Example 7: Preparation of 2-sulfobenzoic acid N-hydroxysuccinimide ester
The N-hydroxysuccinimide (NHS) ester of 2-sulfo benzoic cyclic anhydride was
prepared as DIPEA salt according to scheme 3 and as explained below:
?5
0
O O O I ;N-oH, DIPEA O ~N O O OH
S=O ~o ~O~ N-O S; O
\N-O S ~ O Dowex (H * ) _ ~ O
O - O
3 6
43
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
All equipment was dried in an oven and transferred in an exiccator filled with
argon
prior to use. The reaction was carried out under an argon atmosphere. NHS and
2-
sulfo benzoic acid cyclic anhydride were dried under vacuum prior to use.
Methylene chloride (1.9 ml) and DIEA (1.019 ml, 5.85 mmol) were added to a
round
bottle flask containing NHS (673.2, 5.85 mmol). A solution of 2-sulfo benzoic
acid
cyclic anhydride (1.077 g, 5.85 mmol) in methylene chloride (19 ml) was then
added
in portions (7x) to the reaction mixture, which was then left at room
temperature for 2
h 20 min. The reaction mixture was split in two parts, which were evaporated
to give
a light yellow highly viscous residue (1. 1.11 g and 2. 1.24 g, respectively).
to
Fraction 1 was dissolved in MQ (11.098 ml, 100 mg/ml), filtered and used 3X1
ml in
reversed phase preparative HPLC; Column: Supelcosil LC-18, 10 cm X 21.2 mm,
2~.;
Flow: 10 ml/min, Method: 0-10 min. isocratic 5% acetonitrile containing 0.1 %
TFA
B in water, 2 min. sample injection, 10-15 min. Gradient 5-12 % B in water.
The
fractions were evaporated and freeze dried to give a white solid/transparent
viscous
oil (totally 237.7 mg) of not purified product in DIEA salt form, NHS, DIEA
and side
product. A previous more successful attempt using reversed phase preparative
HPLC
with the same column and system but another method: 0-6 min. isocratic 5
acetonitrile containing 0.1 % TFA B in water, 2 min. sample injection, 6-18
min.
Gradient 5-25% B in water, resulted in the product as a DIEA salt with
approximately
5% NHS left and some traces from side-product in the aromatic area.
Hl NMR (D20) b:8.0-8.1 (dd, 1H) 7.9-8.0 (dd, 1H) 7.7-7.8 (m, 2H) 3.6-3.8 (m,
2H)
3.1-3.2 (m, 2H) 3.0 (s, 4H) 1.2-1.3 (m, IS H) and 2.7 (s, 0.2 H, NHS peak).
Acetone (2.5 ml cold, 0°C, ice-water bath) was added to fraction 2
dropwise to give a
white precipitation after 20 min. in room temperature and 25 min. in
4°C. The
precipitate was filtered and washed carefully in acetone (24 ml cold, OOC, ice-
water
bath) to give the product as a DIEA salt (612.7 mg, 46.3 %).
H1 NMR (D20) 8:8.0-8.1 (dd, 1H) 7.9-8.0 (dd, 1H) 7.7-7.8 (m, 2H) 3.6-3.8 (m,
2H)
3.1-3.3 (m, 2H) 3.0 (s, 4H) 1.2-1.3 (m, 15 H).
44
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WO 02/095419 PCT/US02/16247
Example 8: Synthesis of another type of NHS-ester
Br O O N Br O
O
i I ~ i I
-" o-N
-I- ~N'-~ w
_ ~z O 2:1 Dioxane:water
O~ O OH BF4 O~' I~ OH O
O
2-bromo-5-sulfobenzoic acid is dissolved in 1 mL dioxane and 0.5 mL water. The
diisopropylethylamine, 2 eq., is added. To this well stirred solution is added
the O-
(N-Succinimidyl)-N,N,N',N'-tetramethyluronium BF4 (TSTU), 1.2 eq., as a solid.
The
reaction is stirred for 30 minutes then concentrated by rotary evaporation
followed by
to drying under high vac. A silica gel column is prepared with 2%
water:acetonitrile as
the mobile phase. The sample is loaded in 2% water:acetonitrile. The column is
started with 2% water:acetonitrile and polarity is progressively increased to
5%
water:acetonitrile and finally 80 mL 10% water:acetonitrile. The fractions
containing
product are identified by TLC in 10% water acetonitrile and confirmed by
negative
ion MS. This material has approximately 1 equivalent of DIEA by NMR.
Example 9: Sulfonation of peptides
Model peptides and Cryptic digests of various proteins were dissolved in about
20 pL
of base which was prepaxed by mixing deionized water with
diisopropylethylamine
2U (DIEA) in the ratio of 19:1 v:v. Peptide mixtures from in-gel digests were
concentrated to a final volume of about 20 p.L and 1 p,L of DIEA was added to
make
the solution basic. 5 ~.L of sulfonic acid active ester reagent at 100 mg/mL
is added
and the solution vortexed. The pH of each reaction is checked to ensure that
it is still
basic and adjusted if necessary. The reaction is allowed to proceed for 30
min. at RT.
2~ The samples are acidified with 5 p.L of 1 N HCl and cleaned up directly
using C1$
mini-columns (p,ClB ZipTipT~, Millipore, Bedford MA). The sulfonated peptides
4~
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
were eluted from the columns in 4-20 ~,L of acetonitrile:H20 (1:1 v:v)
containing
0.1 % TFA.
Example 10: Protection of Lys side chains by ~uanidination and subsequent
sulfonation of the trYptic peptides
Model peptides and tryptic digests of various proteins were dissolved in about
20 ~,L
of base which was prepared by mixing deionized water with
diisopropylethylamine
(DIEA) in the ratio of 19:1 v:v. Peptide mixtures from in-gel digests were
1.o concentrated to a final volume of about 20 ~L and 1 ~L of DIEA was added
to make
the solution basic. Two-~,L of aqueous 0.5 M O-methylisourea hydrogensulfate
was
added and the solutions were vortexed. The pH of each solution was checked,
and
adjusted if necessary, to insure that they were still basic after addition of
the reagent.
The reactions were then allowed to proceed at room temperature (RT) for
varying
a.5 lengths of time (a few hours to two days). Typically, the room temperature
reactions
were allowed to proceed overnight. In the morning, 5 ~.L of sulfonic acid
active ester
reagent at 100 mg/mL is added and the solution vortexed. The pH of each
reaction is
checked to ensure that it is still basic and adjusted if necessary. The
reaction is
allowed to proceed for 30 min. at RT. The samples are acidified with 5 ~.L of
1 N
~o HCl and cleaned up directly using C18 mini-columns (~,C18 ZipTipTM,
Millipore,
Bedford MA). The guanidinated-sulfonated peptides were eluted from the columns
in
4-20 ~.L of acetonitrile:H20 (1:1 v:v) containing 0.1% TFA.
Example 11: Experimental description of the instrument used (Fig 3~
Derivatized peptides were analyzed on an Applied Biosystems (Framingham, MA
01701) Voyager DE-STR time-of flight mass spectrometer equipped with a NZ
laser
(337 nm, 3 nsec pulse width, 20 Hz repetition rate). All mass spectra were
acquired in
the reflectron mode with delayed extraction. External mass calibration was
performed
with low-mass peptide standards, and mass measurement accuracy was typically ~
0.2
Da. PSD fragment ion spectra were obtained after isolation of the appropriate
derivatized precursor ions using timed ion selection. Fragment ions were
refocused
46
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
onto the final detector by stepping the voltage applied to the reflectron in
the
following ratios: 1.0000 (precursor ion segment), 0.9126, 0.6049, 0.4125,
0.2738,
0.1975 and 0.1273 (fragment ion segments). The individual segments were
stitched
together using software developed by Applied Biosystems. All precursor ion
segments were acquired at low laser power (variable attenuator =1800) for <
256
laser pulses to avoid detector saturation. The laser power was increased
(variable
attenuator = 2100) for the remaining segments of the PSD acquisitions. The PSD
data
were acquired at a digitization rate of 20 MHz; therefore, all fragment ions
were
measured as chemically averaged and not monoisotopic masses. Mass calibration
was
I c} done externally with peptide standards. Metastable ion decompositions
were
measured in all PSD experiments.
The PSD tandem mass spectra were searched in two ways against the NCBI non-
redundant protein sequence database (most recent update at the time of the
present
is filing was 3/2/2001). First, uninterpreted PSD spectra were searched with
the MS-
Tag program from the Protein Prospector suite of search tools developed at
UCSF
(see P.R. Baker and I~.R. Clauser, http://prospector.ucsf.edu). Search inputs
included
the measured precursor and fragment ion masses. The measured fragment ion
masses
of guanidinated peptides were decreased by 42 Da, the mass of the added
guanidinium
2o group, before searching against either database. The conservative error
tolerances
typically used were ~ 0.6 Da for the monoisotopic precursor ion and ~ 2.0 Da
for the
chemically averaged fragment ions. Only y-type fragment ions were allowed
possibilities. Other types of fragment ions like a, b, (b + H20), (b-NH3) and
internal
cleavages were not considered because they are not prominent in the PSD
spectra
?5 following sulfonation. Alternatively, the PSD data were manually
interpreted. The
derived sequence tags were searched using the MS-Edman program from the
Protein
Prospector software package. MS-Edman does not require the precursor or
fragment
ion masses as inputs. It only uses the measured sequence tags. The program
considers
all combinations of ambiguous residues, like (K, Q and E) or (I, L, N and D),
which
3U have similar masses.
47
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
Example 12: Database description
The sequences of the polypeptide, and peptides thereof, may also be
efficiently and
accurately determined using software which accepts mass spectral fragmentation
data,
either uninterpreted y-ion series masses or sequence tags derived from the y-
ion
masses, as inputs for sequence database searches. Such search software
commonly
utilized by the skilled artisan include, but are not limited to, "Protein
Prospector"
(commercially available from the University of California at San Francisco or
http://prospector.ucsf.edu) and "Peptide Search" (commercially available from
the
1 o European Molecular Biology Laboratory at Heidelberg, Germany or
http://www.mann.embl-heidelberg.de).
The fragmentation pattern produced by this invention can be searched against a
number of sequence databases including, but not limited to, the NCBI non-
redundant
r5 database (ncbi.nlm.nih.gov/blast/db.nr.z), SWISPROT
(ncbi.nlm.gov/repository/SWISS-PROT/sprot33.dat.z), EMBL
(FTP://ftp.ebi.ac.uk/pub/databases/peptidesearch/), OWL
(ncbi.nlm.nih.gov/repository/owI/FASTA.z),dbEST
(ncbi.nlm.nih.gov/repository/dbEST/dbEST.weekly.fasta.mmddyy.z) and Geneba,uc
z0 (ncbi.nlm.nih.gov/genebank/genpept.fsa.z). The entire sequence of the
polypeptide of
interest can often be retrieved from the sequence database by searching the
fragmentation data produced from one or more of the relevant peptide
derivatives
formed using the methods of this invention.
?5 Of course, when using database searching.techniques, it is most efficient
to limit the
searches by specifying that only y-ions or (y-NH3) ions are allowed fragments
because y- and (y-NH3) ions are the most prominent species observed in the
fragmentation patterns wherein the present methods are utilized. Other
fragment ion
types like a-, b-, (b+H20), (b-H20), (b-NH3) and internal cleavage ions can be
3o disallowed because they are not prominent in the spectra of the peptides
derivatized
using the methods of the present invention. The derivatives formed with the
present
invention provide simple fragmentation patterns that often yield greater
database
48
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
search specificity than can be obtained from the spectra of the same peptides
without
derivatization.
Example 13: dPSD of NHS-ester derivatized peptides
dPSD of NHS-ester derivatized tryptic digest of a model protein:
4-vinyl-pyridine alcylated bovine serum albumin (4VP-BSA) (Sigma) was used as
model protein for dPSD using NHS-esters.
Acplation with vin~p, rid The lyophilised protein (2.4 mg) was dissolved in
800
~1 of a buffer solution consisting of 8M urea, SOmM Tris-HCl pH 8.0 and SOmM
DTT
and incubated at 30°C for 30 min. 10.14-vinyl pyridine was added (to
prevent forma-
tion of disulfide bonds) and the sample was incubated for another 1h at
30°C. The
t s sample was desalted using a NAP-10 column (Amersham Pharmacia Biotech),
equili-
brated with 100mM NH4HC02, pH8.8 and eluted in 1.2 ml.
The sample was digested with trypsin (Promega), lp,g trypsin/100~.g protein,
for 6h at
30°C and the reaction was stopped by the addition of TFA to a final
concentration of
1 %. The digest was diluted in 50% AcN:0.5% TFA to a final concentration of
z0 ~ 100ng/~.l (l.5pmo1/~.l).
N-terminal derivatization with NHS-ester of 3-Sulfopropionic acid anh, d~
Tryptic
digest of 4VP-BSA (3pmole) were dried on a speed vac and reconstituted in 101
of
deionized H2O:diisopropylethylamine (19:1, v:v). The NHS-ester was dissolved
in
25 deionized H20 (lOmg NHS-ester/100~,1 H20) and 5~,1 were added to each
sample. The
reaction mixture was vortexed and left for 15 minutes at room temperature to
react.
The samples were .acidified by adding 1 p1 10% TFA and purified through p,C 18
Zip-
TipTM (Millipore) according the instructions of the manufacturer. The sample
was
eluted directly on the MALDI-target with a saturated solution of alpha-cyano-4-
3o hydroxycinnamic acid in 50% AcN:O.l%TFA and analyzed in reflectron positive
mode and PSD mode positive mode using the EttanT"~ MALDI-ToF.
49
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
dPSD of NHS-ester derivatized tryptic digests of proteins from E-coli
Preparation of low speed supernatant of Esche~ichia coli- Escherichia coli (E-
coli),
(40 ~g stain B, ATCC 11303) was put in 20 m1 reducing buffer containing 8M
urea/4
chaps, 2% 3-10 pharmalyt; 65 mM DTT. The cells were disrupted by sonication (7
s x 20s with cooling on ice in between). The lysate was centrifuged at 10.000
x g for 40
min at 8°C. The Iow speed supernatant (LSS) was stored in -20°C
until used.
Separation by 2-dimensional (2D) electrophoresis- LSS of E-coli (lmg) was
diluted in
IPG rehydration buffer (8M urea/2% CHAPS/ 2% IPG buffer 4-7/ 10 mM DTT) and
rt3 rehydrated into the IPG strips (24cm, pH 3-lONL, Amersham Pharmacia
Biotech)
overnight. 2D-electophoresis was performed following the instructions of the
manu-
facture. After separation by 2D-electrophoresis, the gels were fixed in 40%
ethanol
(EtOH), 10% acetic acid (HAc) for 1h, stained with, 0.1% Commassie brilliant
blue in
40% EtOH, 10% HAc, for 30 min and destained in 20% EtOH, 5 % HAc overnight.
l.5
Trypsin di ,estion: Spots of proteins (1.4mm in diameter) of medium Glow
pmole) to
low intensity (high fmole) were picked and transferred to a microtiter plate
using the
EttanT"" spot picker (Amersham Pharmacia Biotech). The proteins were destained
with
100u1, 50% methanol, SOmM ammonium bicarbonate IAMBIC), 3x30minutes, dried
zt~ in a TuboVap for 15 minutes and digested with 5 u1 trypsin for 60 minutes
at 37°C
(40ng/ u1 20mM AMBIC, Promega) using the EttanT"" TA Digester (Amersham Phar-
macia Biotech). The peptides were extracted using 35u150% acetonitrile, 0.5%
TFA
2x,20 minutes. The extracts were dried at room temperature overnight.
25 N-terminal derivatization: The samples were reconstituted in 20.1 deionized
H20. One
~.I (20%) of each sample was mixed 1:1 with alpha cyano matrix solution and
ana-
lysed in reflectrone positive mode using the EttanT"" MALDI-ToF. To the
remaining
19,1 of each sample, 1 ~l DIEA and S~,I sulfopropionic NHS-ester solution, 10
mg/100~,1 were added. The samples were thoroughly mixed by pipeting and left
to
~0 react for 15 minutes at room temperature. TFA (1~.1, 10%) was added to each
sample
and purified through ~.C18 ZipTipTM (Millipore). The samples were eluted
directly on
the MALDI-target with a saturated solution of alpha-cyano-4-hydroxycinnamic
acid
SO
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
in 50% AcN:O.l%TFA and analyzed in reflector positive mode and PSD positive
mode using the EttanT"" MALDI-ToF.
Automated dPSD using NHS-esters
The current chemistry is well suited for automation. Using EttanTM digester
and Et-
tanTM spotter the sample handling and reaction mixtures can be automatically
proc-
essed. Experimentally, the model peptides or peptide mixtures placed in
individual
wells of a microtiterplate are reconstituted in 100 u1 water (quality of 18
MS2 or bet-
i.() ter). At this point the liquid handler can split the sample into two
reactions. One, con-
taining Sul, for direct analysis in the MS, and the other for chemical
modification. The
material designated for chemical modification is dried at room temperature for
one
hour. The handler (e.g. a Gilson 215 multiprobe) then reconstitutes the dried
material
by addition of 10 u1 of the reactive derivatisation reagent in a buffer
containing DIEA
a s (Diisopropylethylamine). The reactants are mixed by repeated aspiration.
The chemi-
cal modification step is allowed to proceed for approximately 15 minutes at
room
temperature. The samples are finally worked up in the same fashion as
previously de-
scribed, and analysed in the MS.
?o
Results
Quantitative N-terminal derivatization of tryptic peptides of 4VP-BSA was
obtained
with NHS-ester of 3-sulfopropionic acid anhydride in aqueous solution. Figure
4 and
show the reflectron spectra of non-derivatized and derivatized 4VP-BSA respec-
2~ tively. The peptides I-III were used for dPSD analyses, (figure 6-8). The
fragmenta-
tion spectra showed exclusively y-ions. The fragmentation data from each of
the three
peptides could be used for unambiguous identification against the NCBInr
protein se-
quence database (PepFrag, www.proteometric.com).
a0 Two gel plugs, containing proteins of E-coli from a commassie stained 2D-
gel were
identified with dPSD using NHS-ester. The proteins were digested with trypsin,
ex-
tracted from the gel plug and derivatized as described. Figure 9 and 10 show
the re-
51
CA 02448534 2003-11-20
WO 02/095419 PCT/US02/16247
flectrone spectra of non-derivatized and derivatized sample from one of the
gel plugs.
The peptide marked with a circle was quantitatively derivatized and used for
PSD
analysis (figure 11 ). The masses of the fragment ions (y-ions) were used for
protein
identification in PepFrag. The suggested candidate from PepFrag agreed with
the can-
didate obtained by searching the tryptic map in ProFound (proteometrics.com).
Re-
flectron spectra of non-derivatized and NHS-ester derivatized sample from the
second
gel plug are shown in figure 12 and 13. The peptide, m/z 1569 was
quantitatively de-
rivatized (m/z 1705) and used for PSD analyses (figure 14). The y-ions
obtained were
used for protein identification in PepFrag, showing the same candidate as
obtained
1 c3 with peptide masses in ProFound.
It is apparent that many modifications and variations of the invention as
hereinabove
set forth may be made without departing from the spirit and scope thereof. The
spe-
cific embodiments described are given by way of example only, and the
invention is
t 5 limited only by the terms of the appended claims.
52
