Note: Descriptions are shown in the official language in which they were submitted.
CA 02351667 2001-05-16
XERION PHARMACEUTICALS GMBH X29166PCCA BO/HW
Method for modifying and identii=ying functional
sites in protein:>
The invention relates to a method for the
specific modification and identification of functional
sites in a target protein. A correlation can be made
between the specific amino acids) and their function
in the protein or their function as ligand binding
sites through the loss/gain of funcaion of the protein
and subsequent determination of the modified amino
acids (epitope mapping). The method is based on
combined use of chromophore-assisted laser inactivation
(CALI) and mass spectrometry or proi~ein sequencing. The
invention also relates to an apparatus for carrying out
this method.
The techiques of recombinant: DNA and of protein
preparation make deeper understanding of the relations
between protein structure and protean function easier.
Methods of random mutagenesis, specific genetic
selection and screening methods make it possible
rapidly to gain information at the protein level, but
have the disadvantage that particular functional
domains of the protein are very tolerant to amino acid
substitutions, that some mutagenesis reagents
preferentially attack particular DNA sequences, and
possible mutations are limited. The information
provided by these methods is thus limited.
The alternative of site-specific mutagenesis
methods provides a more direct and more specific way of
identifying amino acids which are important for the
biological function, such as, for example, the alanine
scanning mutagenesis method. In this method there is
systematic mutation of each amino acid to alanine, and
the function is determined. Each loss of function is
CA 02351667 2001-05-16
- 2 -
related to the specific amino acid. This technique can
be applied to proteins without i~he relevant three-
dimensional structure and without unknown functional
domains. In addition, this entails in each case
modification of only one amino acid, which is why
specific functions involving a plurality of amino acids
are not detected. In addition, the method becomes more
complicated as the molecular size _Lncreases. This is a
disadvantage for analysing the function of gene
products in connection with diseases because these are
often large proteins containing multiple structural
domains.
Furthermore, other recombinant DNA techniques
have been used for epitope mapping and for mapping
ligand binding sites, in which there is expression of
random antigen fragments with mutations introduced at
the DNA level (cf. J. Immunol. Meth., 141, 245-252,
1991, J. Immunol. Meth., 180, 53-61, 1995, Virology,
198, 346-349, 1994). Recently, the phage display of
random peptide libraries have been used for epitope
mapping (J. Immunol., 153, 724-729, 1994, PNAS, 93,
1997-2001, 1996).
Another variant of epitope mapping of proteins
is the rapid automatic synthesis of selected peptides
(J. Endocrinol., 145, 169-174, 1995) and the use of
combinatorial peptide libraries (FEBS Letters, 352,
167-170, 1994). Although these methods are reliable for
linear epitopes, they have been unsuccessful with
nonlinear or discontinuous epitopes. The variant of
using overlapping peptides was also unsatisfactory with
discontinuous epitopes. This entails screening
individual peptides or peptide mixures for binding to
the ligand by means of ELISA, w_Lth free and bound
peptides competing for the ligand (for example
antibody). However, this method is elaborate and time-
consuming.
CA 02351667 2001-05-16
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Another approach used a combination of protein
modification and mass spectrometry (Anal. Biochem.,
196, 120-125, 1991). This entails the ligand being
bound to the receptor, and the complex being modified
with acetic anhydride, resulting in acetylation of the
lysine residues. The proteolytic cleavage mixtures of
the two proteins are analysed by mass spectrometry and
compared with the corresponding fragments from the
untreated complex. The modified lysines can easily be
detected, these lysines not being protected in the
complex, and the unmodified lysines forming part of the
interaction between ligand and receptor. This technique
is thus confined to interactions involving lysine
residues. Another variant is based on differential
proteolysis (Protein Science, 4, 1088-1099, 1995), with
sites sensitive to proteolysis becoming protease-
resistant after complex formation.
There has also been a description of
identification by mass spectrometry of a proteolytic
cleavage of free peptide antigen comparing with the
pattern from the peptide antigen be>und to an antibody.
The identification took place by 252cJf plasma desorption
mass spectrometry (PNAS 87, 9848-9852, 1990). There has
furthermore been a description of. a combination of
immunoprecipitation and matrix-assisted laser
desorption mass spectrometry (MAhDI-MS) (PNAS, 93,
4020-4024, 1996). This entails an antigenic protein
being cleaved into smaller fragments and precipitated
with an. antibody of interest. The immunoprecipitated
peptides are identified by MALDI-MS, and the antibody-
binding region is determined. In this method there was
also separation of proteolytically cleaved peptides by
affinity capillary electrophoresis and identification
by electrospray mass spectrometry (ACE-MS) (Anal.
Chem., 69, 3008-3014, 1997). Injection of the peptide
mixture is followed by injection of the antibody.
Peptides which bind to the antibody are trapped and
CA 02351667 2001-05-16
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therefore do not migrate. The bound peptide is
investigated by the subtraction screening method in
order to determine the epitope residue on the peptide.
However, this technique is confined to linear epitopes
and cannot be applied to discontinuous epitopes.
The present invention is therefore based on the
object of overcoming the problems of the prior art
mentioned. The intention is to provide a method with
which any functional sites in any proteins can be
identified. It is intended prefer<~bly to be able to
identify sites involved in a ligand interaction, and
epitopes. In particular, the method according to the
invention should be applicable to nonlinear and
discontinuous epitopes and withoui= knowledge of the
three-dimensional structure of a ~>rotein. It is also
intended according to the invention for determination
of the protein function to be possible without
inactivating the molecule. It is additionally intended
that the method be simple to use, quickly carried out
and automatable. It is further intended to provide an
apparatus for carrying out the method according to the
invention simply.
This object is achieved by a method for
identifying one or more functional sites in a protein,
which is characterized in that
a) the target protein is contacted wi t-h a h; r,~l; r,n
partner linked to a laser-activatable marker (tag),
(BP-tag), to form a complex of target protein and BP-
tag,
b) the complex of target protein and BP-tag is
irradiated with laser light to generate free radicals
which selectively alter the bound target protein at the
binding site, and
c) the selectively altered region of the protein is
identified by a combination of protein cleavage and
mass spectrometry,
and by an apparatus for carrying out the method.
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It has been found, surprisingly, that
combination of the CALI technique with mass
spectrometry permits reliable and rapid identification
of functional sites on proteins. According to the
invention, a target protein is modified by CALI to
inactivate it. Subsequently, the modified region of the
protein is determined by mass spec, rometry. Tandem MS
and/or de novo sequencing are preferably used. This
makes it possible in a simple way to make a correlation
between the amino acid and its biological function.
This correlation between structure and function allows
information to be gained for example about the
correlation between a particular metabolic activity and
the corresponding site in the molecule, about proteins
with pathological alterations, cancer-promoting
proteins etc. It is thus also pross:ible specifically to
inactivate unwanted (for example pathological)
proteins.
The target protein is initially contacted with
a binding partner under condition. such that complex
formation takes place, but the proteins are not
denatured. Ideally, the conditions correspond to the
physiological conditions of the cellular environment of
the proteins. The binding partner i~~ linked to a laser-
activatable marker. The laser-activatable marker can be
any marker suitable for covalent or noncovalent linkage
to a binding partner. i. e. an am; n~ ar-; ("~ RPC'f7~PYlr"P at'1!'~
can be activated so that it is able to generate free
radicals. The marker is preferably activated with laser
light, but activation is also possible by peroxidases
(hydrogen peroxide/horseradish pc~roxidase system).
Examples of such markers are AMCA-;>, AMCA, BODIPY and
variants thereof, Cascade Blue, Cl-NERF, dansyl,
dialkylaminocoumarin, 4',5'-dichloro-2',7'-dimethoxy-
fluorescein, DM-NERF, eosin, eosin F3S, erythrosin,
hydroxycoumarin, Isosulfan Blue, lissamine rhodamine B,
malachite green, methoxycoumarin, naphthofluorescein,
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NBD, Oregon Green 488, 500, 5:14, PyMPO, pyrene,
Rhodamine 6G, Rhodamine Green, Rhodamine Red, Rhodol
Green, 2',4',5',7'-tetrabromosulphonefluorescein,
tetramethylrhodamine, Texas Red or X-rhodamine. The
marker is preferably malachite green isothiocyanate,
fluorescein isothiocyanate or 4',5'-bis(1,3,2-
dithioarsolan-2-yl)fluorescein. ThE: irradiation takes
place with laser light of a wavelength which is
absorbed by the particular chromophore.
The binding partner can be any binding partner
for the appropriate protein. It :is preferably scFv,
Fab, a diabody, an immunoglobulin-like molecule, a
peptide, RNA, DNA, PNA or a small organic molecule.
The binding partner is preferably derived from
a combinatorial library. This can be any combinatorial
library, for example protein libraz:y, peptide library,
cDNA library, mRNA library, library with organic
molecules, scFv library with immunoglobulin
superfamily, protein display library etc. The following
can be presented in the libraries: all types of
proteins, for example structural proteins, enzymes,
receptors, ligands, all types of peptides including
modifications, DNAs, RNAs, combinations of DNAs and
RNAs, hybrids of peptides and RNA or DNA, all types of
organic molecules, for example steroids, alkaloids,
natural substances, synthetic substances etc. The
presentation can take place in various ways, for
example as phage display system (for example
filamentous phages such as M13, fl, fd etc., lambda
phage display, viral display etc), presentation on
bacterial surfaces, ribosomes etc.
Using the CALI technique, target proteins are
directly and specifically inactivated (cf. PNAS, 85,
5454-5458, 1988; Trends in Cell B_Lology, 6, 442-445,
1996). CALI can be used to convert binding reagents
such as antibodies or other ligands into function-
blocking molecules. This entails a binding partner (BP)
CA 02351667 2001-05-16
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being linked for example to the dye malachite green
(MG). On irradiation with laser light of a wavelength
which is not significantly absorbed by the cellular
components, this dye generates free radicals. For the
determination, the BP linked to MG (=BP-MG) is
incubated with the protein sample of interest. The
region to be inactivated is selected and irradiated
with a laser beam at 620 nm. This light is absorbed by
MG to produce short-lived free radicals which
selectively inactivate the proteins bound to the BP-MG
in a radius of 15 A through irreversible chemical
modification. This system can be used for in vitro and
in vivo assays and for intra- and extracellular target
molecules.
The protein inactivated in this way is then
cleaved. It is possible to use for this purpose a
protease which cleaves specifically after a residue,
for example Lys-C, Glu-C, Asp-N. Examples thereof are
trypsin, chymotrypsin, papain etc. Chemical cleavage is
also possible, for example with. cyanogen bromide
(specific for Met), 3-bromo-3-methyl-2-(2-
nitrophenylmercapto)-3H-indole (BNPS-skatole; specific
for Trp), 2-nitro-5-thiocyanatobenz:oic acid (specific
for Cys) and Fe-EDTA.
The cleavage mixture is fractionated by
electrophoresis. It is then possible to evaluate the
modified amino acids by mass spectrometry and
comparison with the untreated target protein.
Very recent developments in mass spectrometry
have led to rapid identification of proteins (PNAS USA,
93, 14440-14445, 1996). As soon as the target protein
has been inactivated by CALI it is possible to identify
the modified amino acids of the inactivated protein
which are presumably responsible for the inactivation
by tandem mass spectrometry and, where appropriate, de
novo sequencing (Rapid Commun. Mass Spectrom., 11,
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1015-1024, 1997; Rapid Commun. N.fass Spectrom., 21,
1067-1075, 1997).
The identity of the proteins complexed
according to the invention with a binding partner is
established by a combination of protein cleavage and
mass spectrometry, where appropriate de novo
sequencing.
This entails a protein which has been treated
in this way and is provided with a marker initially
being separated from the target protein by an
electrophoresis or by a chromatography. The isolated
and modified protein is then cleaved either chemically
or proteolytically by the methods described above. This
can take place either in the gel (that is to say by
direct elution of the target protein from the gel after
the separation, followed by subsequent protein
separation) or in solution. Methods for cleavage in the
gel are known and described, for example, in Advanced
Methods in Biological Mass Spectrometry, EMBL-
Laboratory, Heidelberg, 1997 or in Shevchenko, A., et
al., Anal. Chem. 68:850-858, 1996.
The MALDI analysis is carried out in a manner
known per se.
It is necessary for the nanoelectrospray
analysis (nanoES} to extract the tryptic peptides from
the pieces of gel. To do this, they pieces of gel are
washed successively with ammonium bicarbonate,
acetonitrile, dilute formic acid and again with
acetonitrile. The supernatants are combined and dried
in a vacuum centrifuge. The sample is dissolved in 800
strength formic acid, rapidly diluted with water and
then desalted.
The analysis by mass spectrometry can be
carried out in various ways known per se, for example
using an ionization source such as electrospray
(Chapman, J.R., et al., Methods in Molecular biology,
61, JR Chapman editor, Humana Preas Inv. Totowa NJ,
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USA, 1996) including nanoelectrospray (VJilm. M. and
Mann, M., Anal. Chem. 68, 1-8, 1996) and matrix-
assisted laser desorption and ionization (MALDI)
(Siuzdak, G. Mass Spectrometry for Biotechnology,
Academic Press Inc. 1996) or using a combination of
mass analysers such as triple, quadrupole, time of
flight, magnetic sector, Fourier transformation ion
cyclotron resonance and quadrupole ion capture.
If the peptides in the c:Leavage mixture are
insufficient for unambiguous establishment of the
identity of the investigated site in the protein,
further sequence information can be obtained by further
fragmentation in the mass spectrometer such as, for
example, by decomposition downstream of the source in
MALDI-TOF, MS/MS (tandem mass spec;trometry), MS". the
proteins can additionally be identified by de novo
sequencing.
Since in CALI there is photochemical
modification of certain amino acids of the target
protein, the MS can be used in two stages to identify
which amino acids have been modified in what way. The
method can be carried out with low and high resolution.
At low resolution it is firstly possible to
determine by peptide mass mapping (Anal. Chem., 69,
4741-4750, 1997; Biochem. Soc. Transactions, 24, 893
896, 1996; Anal. Chem., 69, 1706-1714, 1997) which
segments of the protein have beers modified. In the
second stage, at higher resolution, tandem mass
spectrometry and/or de novo sequencing on selected
peptides can be used to determine the site of the
modifications. It is possible with a mass spectrometer
configuration able to resolve masses from 0 up to
0.03 dalton (Rapid Commun. Mass Spectrom., 11, 1015-
1024) (qQTOF and other mass analysers) to determine
which specific amino acids have been modified by CALI;
it is even possible to determine the type of
modification from the incremental mass gain or loss.
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The free radicals generated by CALI lead to a
modification of the oxidation-sensitive amino acid side
chains such as His, Met, Cys and Tr;p. There may also be
modification of other amino acid side chains. This
results in significant changes in rnass, because oxygen
has been added on. Comparison of the peptide fragments
after the cleavage between CALI-treated fragments and
untreated sample leads to an approximate localization
of the modified amino acids. These amino acids are
identified more accurately by de novo sequencing.
It is unnecessary to know the exact nature of
the modification. The method according to the invention
permits the modification to be recognized from the
difference between the treated and untreated samples.
The advantage of this method is that it can be used to
elucidate discontinuous epitopes even if the three-
dimensional structure of the protein. is unknown.
The modifications induced by CALI can also be
elucidated by using other methods such as parent ion
scans (J. Mass Spectrom, 32, 94-98, 1997), which
increases the speed of analysis. This detects only the
peptides altered by CALI.
If insufficient information is obtainable
because of limited peptide fragmentation or doubtful
assignment of mass, it is possible to carry out a de
novo mass sequencing (Rapid Commun. Mass Spectrom., 11,
1015-1024, 1997). In this method, the peptides are
labelled with 180. This is done by carrying out the
tryptic cleavage in the gel with a cleavage buffer
which contains 500 (vol/vol) H2180 purified by
microdistillation. A QqTOF mass spectrometer is
preferably used. Clear results are possible by reading
the doublet owing to the 1:1 16O/180 ratio. The mass
difference of the doublet is an indicator of the charge
state of the specific peptide. Using different charge
states it is possible to read off up to 15 amino acids
in a sequence for a given peptide. Comparison of the
CA 02351667 2001-05-16
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180-labelled spectra of the untreated and CALI-modified
proteins provides unambiguous information about the
modified amino acids.
Figure 1 shows the scheme for identifying
functional sites in proteins.
The invention also relates to an apparatus for
carrying out the method according to the invention.
This is shown in Figure 2. This apparatus is an
automated system of integrated independent units/parts
which can be used for identifying functional sites in
proteins. Part A relates to the preparation of the LBP-
tag. An automated LBP screening machine reveals
specific LBPs directed against specific target
molecules/ligands, while the chromophore synthesis
appratus produces chromophores of choice. The selected
LBPs and the synthesized chromophores are chemically
linked in an LBP-chromophore coupling apparatus,
resulting in the LBP-tag. This LBP-tag is transferred
into a loading apparatus which transfers the LBP-tab
into predetermined cavities coated with the target
molecule/ligand in the assay platform.
A transfer robot then moves the assay platform
into the laser system in order to initiate the second
part B. The samples are irradiated. with the laser at
the required wavelength in order to induce a
modification by free radicals. The irradiated samples
are then transferred by a sample transfer robot into an
LBT-tag/ligand separating apparatus in order to isolate
the ligand. The ligand is then cleaved in a protein
cleavage apparatus. The peptide fragments are then
analysed with a mass spectrometer in order to detect
changes in mass, or in order to carry out a direct
sequencing. Data from the mass spectra are then used
for the analysis in the data base, which finally leads
to identification of the amino acids or peptide
fragments. All parts of the apparatus are connected to
a central computer system for control and analysis.
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The invention is explained in detail by means
of the following examples.
Example 1
Epitope mapping of (3-galactosidase t>y CALI-MS
IgG antibodies against (3-galactosidase were
purchased from Sigma or Cappell and labelled with
malachite green isothiocyanate or fluorescein
isothiocyanate as described in PNAS 85, 5454-5458, 1988
or in PNAS, 95, 4293-4298, 1998. This entailed
malachite green isothiocyanate or fluorescein
isothiocyanate (from Molecular Probes, Eugene, OR)
being added from a stock solution (:?0 mg/ml or 2 mg/ml)
in DMSO stepwise until the concentration was 120 ug/ml
to the antibodies in a concentration of 600 ~g/ml in
500 ml of NaHC03 (pH 9.8). After incubation with
stirring at room temperature for one hour or incubation
in ice for four hours, the solution was desalted on a
column in 150 mM NaCl/50 mM Na3P04, pH 7.3 (for
malachite green it is necessary to centrifuge the
precipitate before changing the buffer) in order to
separate the labelled protein from free marker.
The laser arrangement and the irradiation with
the laser beam for carrying out CALL are essentially as
described in Methods Cell Biol., 44, 715-732, 1994 or
in PNAS, 95, 4293-4298, 1998. 20 ul of (3-galactosidase-
containing sample (10 ug/ml) and dye-labelled
antibodies against (3-galactosidase (200 ug/ml) were
placed in an ELISA 'plate (Nunc). The entire volume of
the well was irradiated with a laser beam with
different durations. The activity of these samples was
measured as described in Meth. Enzymol., 5, 212-219,
1962. After the CALI, each sample was placed on a 1D
PAGE gel in order to separate CALI-modified (3-
galactosidase from the antibody.
For the MS, both -the CALI-modified
(3-galactosidase and the untreated (3-galactosidase were
CA 02351667 2001-05-16
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cleaved in the gel with trypsin (Anal. Chem., 68, 850-
858, 1996; Protein Structure, A Practical Approach, 2na
edition, editor Creighton, T.E., Oxford University
Press: Oxford, UK, 1997, pp. 29-57). An aliquot of 0.3-
0.5 ul of cleaved protein was used for the MALDI
analysis, and the remainder was used for MS/MS and/or
de novo sequencing by electrospray MS.
To prepare samples for NfALDI for the low
resolution CALI-MS, 0.3 to 0.5 ~l of cleaved protein
was added as a drop to a 0.3 ul d.rop of 5-loo formic
acid on a vapor-deposited matrix/nitrocellulose surface
on the MALDI sample loading apparatus (cf. Anal. Chem.,
69, 4741-4750, 1997; Anal. Chem., E~6, 3281-3287, 1994:
J. Am. Soc. Mass Spectrom., 5, 955-958, 1994; Rapid
Commun. Mass Spectrom., 10, 1371-1378, 1996; Anal.
Chem., 68, 850-858, 1996; Org. Mass Spectrom., 27, 156-
158, 1992). The MALDI analyses of the untreated and
CALI-modified (3-galactosidase were carried out in
direct succession in order to ensure identical
instrumental conditions for the two samples. The MALDI
peptide mass mapping (Anal. Chem., ~~9, 4741-4750, 1997;
Biochem. Soc. Transactions, 24, 8'~3-896, 1996; Anal:
Chem., 69, 1706-1714, 1997) was previously carried out
on the untreated (3-galactosidase in order to identify
peaks in the MALDI mass spectrum corresponding to
~3-galactosidase part-sequences. Peaks not belonging to
(3-galactosidase are, for example, matrix and trypsin
autolysis peaks. It is additional:Ly possible on the
basis of a list of theoretical t.ryptic peptides to
establish the actual extent of the tryptic cleavage.
Analysis of the MALDI mass spectrum of the
untreated (3-galactosidase was followed by analysis of
the spectrum of the CALI-modified sample, and
differences in the spectra indicateo~ the regions of the
protein which were modified by CALI and are therefore
associated with a function.
CA 02351667 2001-05-16
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Tandem MS and de novo partial sequencing of
selected peptides based on the previously obtained
MALDI results were used for the high-resolution CALI-MS
to determine the actual amino acids) which has/have
been modified.
Based on the crystal structure of
(3-galactosidase, the amino acids responsible for
substrate binding and catalysis were identified in this
way. It emerged that the amino acids Met, His and Trp
are involved in the catalysis. The masses of these
amino acids result in peaks of increased mass, compared
with the unmodified amino acids, in the mass spectrum.
A de novo sequencing confirmed the presence of the
modified amino acids.
It is also possible to det=ermine the type of
modification through the incremental mass loss or gain.
As previously, the untreated and the CALI-modified
(3-galactosidase underwent successive MALDI analysis in
order to ensure identical instrumental conditions for
the two samples. The tandem MS or the MS/MS were
carried out with a nanoelectrospray source as
described, for example, in Nature, 379, 464-469, 1996
or in Biochem. Soc. Transactions, 24, 893-896, 1996.
The result obtained concerning the <~mino acids involved
in the catalysis was the same as previously.
The beta-galactosidase activity was followed as
a function of time after CALI. The activity was
measured as assay units and is shown in relation to the
activity of untreated beta-galactosidase.
Time (min) % active beta-galactosidase
+ CAL I - CAL I
0 100% 100°s
1 100% 100--°<s
3 75 0 100°>
5 22 0 100°>
10 5a 100°-<s
CA 02351667 2001-05-16
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30 <l0 1000
The ~i-galactosidase
inactivated
by CALI after
30 minutes was used for
further processing.
The tryptac
cleavage l ed to the fragments
lasted below
in the
sequence of
decreasing
mass as detected
by MALDI.
Pos.
refers to the number of the position of trypsin
cleavage in he stated ranges correspond
the enzyme. to
T
the number of the amino acid of the enzyme starting
from the N terminus. The underlined fragments showed
a
change in the molecular mass and correspond to those
modified after reatment. 'The amino acids His,
the CALI
t
Trp and Cy s in these
fragments
were modified,
as was
confirmed by resequencing,
and are consistent
with
structural investigationsfrom which it is evident that
the modified are responsible for the enzyme
amino acids
activity.
Pos from to mol. wt.
43 523 - 523 146.2
60 775 - 775 146.2
68 855 - 855 146.2
2 15 - 15. 174.2
57 756 - 756 174.2
37 448 - 449 289.3
65 811 - 812 332.4
35 441 - 443 374.4
26 335 - 337 402.4
75 940 - 943 435.5
69 856 - 858 438.5
28 354 - 357 444.5: His357 (new peptide
mass observed
= 460.5)
39 475 - 477 445.6
22 290 - 293 487.6
42 519 - 522 532.6
36 444 - 447 532.7
12 180 - 184 545.5
46 559 - 562 562.6
19 253 - 256 565.6
CA 02351667 2001-05-16
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21 284 - 289 637.6
62 783 - 787 665.7
39 - 44 704.7
206 - 211 709.9
5 33 427 - 432 715.7
67 849 - 854 735.9
58 757 - 761 749.8
13 185 - 191 801.1
23 294 - 300 812.9
10 61 775 - 782 840.0
7 54 - 60 860.9
45 553 - 558 870.0
73 911 - 918 896.0
17 232 - 238 899.9
15 30 382 - 389 962.0: Cys389 (new a tide
P P
mass observed = 978.0)
34 433 - 440 989.1
50 613 - 622 1064.2
10 159 - 167 1067.1
77 954 - 962 1083.2
64 802 - 810 1099.2
6 45 - 53 1100.2
4 28 - 38 1252.4
51 623 - 631 1265.4
76 944 - 953 1299.4
41 507 - 518 1341.5
24 301 - 311 1361.5
80 1015 - 1024 1367.6
11 168 - 179 1394.6
49 601 - 612 1400.6
71 883 - 895 1414.6
3 16 - 27 1428.5
63 788 - 801 1457.5
59 762 - 774 1496.7
78 963 - 974 1507.6
18 239 - 252 1547.8
1 1 - 14 1577.9
CA 02351667 2001-05-16
, - 17 -
52 632 - 646 1742.9
72 896 - 910 1757.9
27 338 - 353 1776.2
14 192 - 205 1787.9
47 563 - 578 1891.2: T:rp568 (new peptide
mass observed = 1907.2)
9 142 - 158 1949.2
3I 390 - 405 2005.2: His391 (new peptide
mass observed = 2021.2)
16 212 - 231 2265.5
32 406 - 426 2408.7: H_Ls418 (new peptide
mass observed = 2424.7)
25 312 - 334 2416.7
48 579 - 600 2447.4
70 859 - 882 2466.7
74 919 - 939 2500.8
54 679 - 700 2517.9
55 701 - 722 2522.8
38 450 - 474 2744.9
20 257 - 283 2848.1
29 358 - 381 2867 . I-iis359 ; His360 (new
3:
peptide 2883.3, 2899.3)
mass
observed
=
40 478 - 506 3071.3: Met502 (new peptide
mass observed = 3087.3, 3103.3)
44 524 - 552 3132.6
53 647 - 678 3424.9
56 723 - 755 3524.0
66 813 - 848 3776.2
79 975 - 1014 4325.7: Trp999 (new a tide
p p
mass observed = 4341.7)
8 61 - 141 9178.1
It was shown that free radicals lead to
oxidation. Thus, oxidation of the a~>ove amino acids led
to a mass gain of 16 da for each modified amino acid,
except for Met with a gain of up 32 da, depending on
the oxidation state. The modified amino acids are
CA 02351667 2001-05-16
' - 18 -
located near or in the active site o f the enzyme, which
explains the loss of function. This; is consistent with
independent studies which show the role of the amino
acid Met502 in catalysis (Arch Bioclzem Biophys 283:342-
350, 1990; J. Biol. Chem. 265:5512-5518, 1990).
Figures 3 and 4 depict part of the MALDI
spectrum of the tryptic beta-galactosidase peptides.
The peptide 478-506 with a molecular weight of 3071.3
is depicted. Met502 in this peptide is modified by CALI
so that masses are obtained at 3087.3 da (sulphoxide)
and/or 3103.3 da (sulphone). Only the alteration to the
sulphone is shown for the illustration. All the
expected peaks including trypsin autolysis peaks and
matrix peaks have been omitted.
The MALDI spectrum shows only the peaks which
represent beta-galactosidase peptides in addition to
the peptide 478-506. The masses from the MALDI-MS
spectra are monoisotopic molecular masses with the
addition of one hydrogen atom (MH+). The following
table indicates the average and monoisotopic peptide
ions MH+ in the spectrum:
Peptide position Average MH+ Monoisotopic MH+
257-283 2848.1 2847.42
358-381 2867.3 2866.38
478-506 3071.3 3070.43
478-506* 3103.3 3102.41
524-552 3132.6 3131.58
647-678 3424.9 3423.74
*CALI-modified peptide
The MS/MS or de novo sequencing
spectrum
shows
only the peaks of the peptide 478-506 (MH+ -
3070.43 da). MS/MS was performed the doubly charged
on
peak MH2+ at m/z 1 535.215 as parent ion. The
monoisotopic MH+ peak is observed
as in a MALDI
spectrum. The following table gives a list of peptides
CA 02351667 2001-05-16
- 19 -
from the tryptic peptide 478-506. Tryptic peptides
normally lead to a number of Y fragment ions which are
C-terminal ions resulting from the cleavage of the
amino acid bond between the carbonyl carbon and the
amide nitrogen. Leucine and isoleucine have identical
masses and cannot be distinguished. The Y ion on the
C-terminal side of proline gives a weak signal or may
be completely absent, but the following Y ion which
contains the proline itself gives a prominent signal.
Monoisotopic MH+ peaks in the de novo
sequencing of the tryptic peptide 478-506 from beta-
galactosidase (only partial list)
Y" ion Sequence of the MH+ (bf~fore MH+ (after
ion CALI) CALI)
1 R 175.12 175.12
2 AR 246.16 246.16
3 YAR 409.22 409.22
4 MYAR 540.26 572.25
5 PMYAR 637.31 669.30
6 CPMYAR 740.32 772.31
7 ICPMYAR 853.41 885.40
8 IICPMYAR 966.49 998.48
Example 2
Determination of the binding site of an RNA aptamer
The binding site of an RNA aptamer on the Ul-
snRNP-A protein was investigated using CALI and mass
spectrometery. RNA aptamers have a consensus sequence
for binding to the RNA hairpin structure complexed with
U1A. The 3D structure thereof has been determined.
Aptamers specific for the U1-snRNP-A protein
were labelled with fluorescein in a manner known per
se. Then 20 ul of sample containing 10 pg/ml protein
and a dye-labelled aptamer (200 ug/ml) were placed in
the well of an ELISA plate. A further 20 ul of sample
containing the UlA protein (10 ~g/ml) and an aptamer
CA 02351667 2001-05-16
- 20 -
(200 ug/ml) without dye was placed in another well.
Irradiation was carried out with laser light of a
wavelength of 488 nm.
Both the CALI-treated and t:he untreated sample
was mixed with SDS sample buffer. Electrophoresis was
carried out on a 12% SDS polyacrylamide gel. The gels
were stained and the bands corresponding to the UlA
proteins were cleaved with trypsin a.s described above.
A 0.3-0.5 ~tl aliquot of the cleaved protein was
used for the MALDI analysis, and thE~ remainder was kept
for the MS/MS analysis or the de novo sequencing with
nanoES/MS.
To prepare for the MALDI analysis, 0.3-0.5 ul
of cleaved protein was added as a di:op to a 0.3 ~zl drop
of 5-loo strength formic acid on a vapor-deposited
matrix/nitrocellulose surface on the MALDI target (cf.,
for example, Anal. Chem., 1997, Ei9, 4741-4750). The
MALDI analysis was then immediate:Ly carried out for
both samples. The spectra obtained in this way for the
treated and untreated samples were compared.
Differences in the spectra indicate the regions where
the protein has been modified by C:~LI and thus define
residues connected with the binding region. It is
additionally possible to use a list of theoretical
tryptic peptides for determining the actual extent of
the tryptic cleavage.
The result already obtained on the basis of the
X-ray structure concerning the binding of RNA ligands
was confirmed (Nature 1994, 372:432-438).
A high-resolution CALI-MS was then carried out.
This comprises a tandem MS and a partial de novo
sequencing. This makes it possible to determine the
amino acids which have been modified, and the nature
and extent of the modification. ThE: tandem MS (MS/MS)
was carried out as described in NATURE, 1996, 379, 464-
469 and Biochem. Soc. Transactions, 1996, 24, 893-896.
CA 02351667 2001-05-16
- 21 -
The tryptic cleavage of the N-terminal UlA
fragment led to the fragments l~~_sted below in the
sequence of decreasing mass as detected by MALDI. Pos.
refers to the number of the position of trypsin
cleavage in the enzyme. The stated :ranges correspond to
the number of the amino acid of the enzyme starting
from the N terminus. The underlined fragments showed a
change in the molecular mass and correspond to those
modified after the CALI treatment. The amino acids His
and Met in these fragments were modified, as was
confirmed by resequencing, and are consistent with
structural investigations from which it is evident that
the modified amino acids are responsible for the RNA
binding.
The amino acid sequence of N-terminal UlA
fragment is known to be as follows:
1
MAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQA
FVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK
98
Tryptic cleavage of the N-terminal U1A fragment
afforded the following fragments with the corresponding
molecular weights:
Pos from -to mol: wt.
3 23 - 23 146.2
5 28 - 28 146.2
3U 2 21 - 21 259.3
14 97 - 98 277.4
8 51 - 52 305.4: Met51 (new peptide mass observed
- 321.4, 337.4)
7 48 - 50 346.4
4 24 - 27 503.5
12 84 - 88 621.7
13 89 - 96 861.9
CA 02351667 2001-05-16
- 22 -
9 53 - 60 909.1
61 - 70 1047.1
11 71 - 83 1603.9: Met72;Met82 (new peptide mass
observed = 1619.9, 1635.9,
5 1651.9, 1667.9)
6 29 - 47 2170.5
1 1 - 20 2354.7: HislO (new peptide mass observed
~~~n w
10 It was shown that free radicals lead to the
oxidation. Thus, oxidation of the above amino acids led
to a mass gain of 16 da for each modified amino acid,
except for Met with a gain of up tc> 32 da depending on
the oxidation state. The modified amino acids are
located near the RNA binding site of the protein, which
is thus consistent with the structural studies which
show the binding site of the RNA.
Figures 5 and 6 depict part of the MALDI
spectrum of the tryptic peptides of the N-terminal U1A
fragment. The peptide 71-83 with a molecular weight of
1603.9 is depicted. Met72 and Met82 in this peptide are
modified by CALI to result in masses at 1667.9 da (two
sulphones). Only the alteration to the sulphone is
shown for the illustration. Other expected peaks
including trypsin autolysis peaks and matrix peaks have
been omitted.
The average and monoisotopic peptide ions MH+
in the spectrum are indicated in the table below:
Peptide position Average MH+ Monoisotopic MH+
89-96 862.96 862.96
53-60 910.10 909.52
61-70 1048.14 1047.54
71-83 1604.89 1603.74
71-83* 1668.89 1667.72
29-47 2171.55 2170.19
CA 02351667 2001-05-16
v - 23 -
*CALI-modified peptide
The MS/MS or de novo sequencing spectrum shows
only the peaks of the peptide 71-83 (MH+ = 1603.74 da).
MS/MS was performed on the doubly charged peak MH2+ at
m/z 801.87 as parent ion. The monoi.sotopic MH+ peak is
observed as in a MALDI spectrum. The following table
gives a list of peptides from the tryptic peptide 71-
83. Tryptic peptides normally lead to a number of ~Y
fragment ions which are C-terminal ions resulting from
the cleavage of the amino acid bond between the
carbonyl carbon and the amide nitrogen. Leucine and
isoleucine have identical masses and cannot be
distinguished. The Y ion on the C-terminal side of
proline gives a weak signal or may be completely
absent, but the following Y ion which contains the
proline itself gives a prominent signal.
Monoisotopic MH+ peaks in the de novo sequencing of the
Cryptic peptide 71-83 of the N-terminal U1A fragment
(only partial list)
Y" ion Sequence of the MH+ (before MH+ (after
ion CALI) CALI)
1 R 175.12 175.12
2 MR 306.1E~ 338.15
3 PMR 403.21. 435.20
4 KPMR 531.31 563.30
5 DKPMR 646.33 678.32
6 YDKPMR 809.40 841.39
7 FYDKPMR 956.47 988.46
8 PFYDKPMR 1053.52 1085.51
9 FPFYDKPMR 1200.59 1232.5$
10 GFPFYDKPMR 1257.61 1289.60
11 QGFPFYDKPMR 1385.67 1417.66
CA 02351667 2001-05-16
- 24 -
12 MQGFPFYDKPMR 1516.71 1580.69
13 SMQGFPFYDKPMR 1603.74 1667.72
