WO2005012247A1

WO2005012247A1 - Compounds and methods for the rapid quantitative analysis of proteins and polypeptides

Info

Publication number: WO2005012247A1
Application number: PCT/CA2004/001427
Authority: WO
Inventors: Alexei Pshezhetsky; Ilya Nifant'ev
Original assignee: Hôpital Sainte-Justine
Priority date: 2003-07-30
Filing date: 2004-07-30
Publication date: 2005-02-10

Abstract

A polypeptide reactive reagent having the formula PRG-Z wherein 'PRG' is a polypeptide reactive group which optionally binds to a capture reagent, and wherein 'Z' is an aryl, substituted aryl, alkyl, substituted alkyl, lower alkyl or substituted lower alkyl group in which one or more atoms can be differentially labeled with one or more stable isotopes 'X', wherein 'X' is selected from the group consisting of H and D, is described.

Description

TITLE OF THE INVENTION

COMPOUNDS AND METHODS FOR THE RAPID QUANTITATIVE ANALYSIS OF PROTEINS AND POLYPEPTIDES

FIELD OF THE INVENTION

[0001] The present invention relates generally to compounds and methods for the rapid quantitative analysis of proteins and polypeptides. More specifically, the present invention relates to compounds and methods for the quantitative analysis of proteins in a proteome or in a complex mixture using stable isotope labeling and analysis by mass spectroscopy.

BACKGROUND OF THE INVENTION

[0002] Proteins are among the most abundant of organic molecules, often encompassing as much as 50 percent or more of a living organisms dry weight. Proteins play crucial roles in virtually all biological processes. Their significance and the remarkable scope of their activity are exemplified in the following functions: (a) Enzymatic catalysis; (b) Transport and storage; (c) Coordinated motion; (d) Mechanical support; (e) Immune protection; (f) Generation and transmission of nerve impulses; and (g) Control of growth and differentiation. Many proteins are present only in very minute quantities within living organisms, yet are nevertheless critical to the life of the organism. For example loss of Factor VIII in humans leads to hemophilia.

[0003] The sequencing of the human genome provided the first step towards the complete understanding of the countless biological processes taking place within the human body. In order to fully exploit the potential of the sequenced human genome in drug development, an experimental approach aimed at determining the function of the various proteins making up the human genome needs to be applied.

[0004] The proteome is known as the total protein complement expressed by a genome. The proteome is modulated by genetics, disease, pharmaceutical intervention and metabolic state. The comparison of two proteomic sets allows for the identification of "key" proteins involved in human biological processes, providing for the discovery or confirmation of new pharmaceutical targets as well as for the identification of pharmacologically important metabolic pathways. The analysis of the whole proteome (proteomics) has emerged as a new experimental approach primarily because of the numerous advances in mass spectroscopy (MS), extending the capability of this technology to the analysis of complex protein mixtures.

[0005] Although MS is unrivaled in its ability to characterize proteins, it is a purely qualitative method and requires coupling to other analytical methods for the quantitative profiling of the proteome. The presently used quantification methods are primarily based on 2-dimensional electrophoresis (2DE), and dye- or isotope-labeling of proteins.

[0006] The most common implementation of proteome analysis is based on the separation of complex protein samples, most commonly by two- dimensional gel electrophoresis (2DE), and the subsequent identification of the separated protein species by mass-spectroscopy (MS) using bioinformatics. This approach has been revolutionized by the development of powerful mass spectrometric techniques and the development of computer algorithms which correlate protein and peptide mass spectral data with available sequence databases, and thus rapidly and conclusively identify proteins. This technology has reached a level of sensitivity (femtomols) which permits the identification of essentially any protein which is detectable by conventional protein staining methods including silver staining. However, 2-dimensional electrophoresis followed by mass-spectrometry (2DE-MS) suffers from significant limitations. The sequential manner in which samples are being processed in 2DE (the protein spots are individually processed and identified) limits the sample throughput. Moreover, since 2DE is labor intensive and demands a high level of technical skill, the potential for full automation is severely limited. Moreover, a substantial fraction of the protein spots contains more than one protein and/or differentially modified or processed forms of a protein, further complicating the quantification. Additionally, a limited sample capacity, in addition to a limited detection sensitivity, makes the detection of low abundance proteins such as regulatory proteins very difficult if not impossible without prior enrichment. For example, in total yeast cell lysates separated by 2DE and marked by silver staining, proteins present in amounts of less than 1000 copies per cell could not be detected (Gygi, S. P., Corthals, G. L., Zhang, Y., Rochon, Y., and Aebersold, R.; (2000), Proc. Natl. Acad. Sci. U.S.A. 97, 9390-9395). Since proteins expressed at low abundance may represent a significant portion of a given proteome, it becomes readily apparent that 2DE does not provide a true representation of all the expressed proteins. Furthermore, the separation using 2DE of trans-membrane proteins, as well as proteins having high or low isoelectric points (pi), or with high apparent molecular weights (Mr), remains challenging.

[0007] As an alternative to the 2DE-MS approach to proteome analysis, the direct analysis by tandem mass spectrometry of peptide mixtures generated by digestion of complex protein mixtures has been proposed (Dongr'e, A.R.; Eng, J.K.; and Yates, J.R.: Emerging tandem-mass-spectrometry techniques for the rapid identification of proteins. Trends Biotechnol. 15:418-425). While this method considerably accelerates protein identification, the quantities of the analyzed proteins cannot be easily determined.

[0008] The development of methods and instrumentation for automated, data-dependent electrospray ionization (ESI) tandem mass spectrometry (MS) in conjunction with microcapillary liquid chromatography (μLC) and database searching has significantly increased the sensitivity and speed of identification of gel-separated proteins. μLC-MS-MS has been used successfully for the large-scale identification of individual proteins directly from mixtures without gel electrophorectic separation (Washburn, M.P., Walters, D., Yates, J.R.: Large scale analysis of the yeast proteome by multi-dimensional protein identification technology. Nat. Biotechnol. 2001, Mar 19(3):242-247; Opiteck, G.J. et al.: A strategy for rapid, high-confidence on-line LC/LC/MS of proteins. Anal. Chem. 69:1518-1524). However, low abundance proteins in complex samples are difficult to analyze by the μLC-MS-MS method without their prior enrichment.

[0009] A broadly applicable approach for protein analysis using an

Isotope-Coded Affinity Tag (ICAT) has been recently reported (Gygi, S.P.; Rist, B.; Gerber, S.A.; Turecek, F.; Gelb, M.H.; and Aebersold, R.: Rapid quantitative analysis of proteins or protein function in complex mixtures. Nat. Biotechnol. 17, 994-999 (1999); Aebersold, R. θf al.: WO 00/11208). The ICAT approach represents an alternative to 2DE, and embodies a combination of selective protein labeling using stable isotope-containing affinity reagents and multidimensional liquid chromatography in conjunction with automated data-dependent tandem mass spectrometry and sequence database searching. This method has proven to be effective and has been shown to overcome many of the main limitations of the 2DE-MS approach. The ICAT approach allows for the rapid quantification and identification of the components of complex protein mixtures with a high degree of automation and without the need for separation of the protein mixtures prior to analysis. The ICAT approach involves the labeling of intact proteins with reagents comprising a Cys-reactive group and a biotin tag for the affinity purification of the labeled peptides following enzymatic digestion (e.g. with trypsin). However, the ICAT approach has important shortcomings since cystein (Cys) is one of the least abundant amino acids. Furthermore, the mass tags add a number of steps to ICAT synthesis, making the reagent slow and expensive to prepare, prohibitively so in large quantities. Moreover, the ICAT approach involves the addition of a relatively large chemical group to cystein-containing peptides, which group is very labile under collision-induced dissociation conditions rendering the analysis of the corresponding MS data considerably more difficult.

[0010] Solid supports, chemically modified with either heavy or light isotopic labels, have been proposed to label digested peptides containing cystein residues (Zhou, H., Ranish, J. A., Watts, J. D., and Aebersold, R. Nature Biotechnology, 20, 512-515, 2002; Aebersold et al.: US 2004/0110186). The digested peptides are released from the support, together with a mass tag, following photolytic cleavage. This method requires the use of two different types of solid support, one comprising a heavy isotopic mass tag (e.g. deuterium), and one comprising a light isotopic mass tag (e.g. hydrogen), for the analysis of cystein-containing peptides from two samples. However, this approach poses a technically challenging problem since it requires the synthesis of two solid supports, chemically modified with either heavy or light isotopic labels. Moreover, any variation in the properties of the solid supports, occurring in the process of their preparation or storage, will result in uneven binding of the peptides, which in turn results in significant quantification errors. This is particularly true for iodoacetamide-activated supports which are unstable and which rapidly decompose in the course of storage.

[0011] International application WO 02/48717A2 discloses Acid-Labile

Isotope-Coded Extractants (ALICE) for the quantitative mass spectrometric analysis of protein mixtures. The extractants comprise a thiol-reactive group for the capture of cystein-containing peptides from peptide mixtures. However, this approach requires the use of two types of acid-labile extractants, chemically modified with either heavy or light isotopic mass tags, in order to enable the direct quantification of peptides/proteins through mass spectrometric analysis. Furthermore, as was the case with ICAT, the ALICE approach is limited to the quantification of peptides containing cystein residues.

[0012] There thus remains a need for a method and reagents that overcome the limitations inherent in traditional techniques employed in proteome analysis. More specifically, there remains a need in the art for a method capable of identifying and quantifying proteins and peptides in a complex mixture or in a proteome, including those present in only small quantities. Furthermore, there remains a need for analytical reagents for use in such a method, that can be synthesized quickly and inexpensively from commercially available materials.

[0013] The present invention seeks to meet these and other needs.

[0014] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0015] The present invention relates to a method for identifying and quantifying proteins and peptides in complex mixtures or in a proteome, including those present in only small quantities. More specifically, the present invention relates to a method for the identification and quantitative analysis of proteins and peptides in complex mixtures or in a proteome using stable isotope labeling and analysis by mass spectroscopy. Yet more specifically, the present invention relates to a method using labeled polypeptide reactive reagents capable of reacting with proteins and peptides and which function as mass tags.

[0016] The present invention relates to a polypeptide reactive reagent having the formula "PRG-Z" wherein "PRG" is a polypeptide reactive group which optionally binds to a capture reagent, and wherein "Z" is an aryl, substituted aryl, alkyl, substituted alkyl, lower alkyl or substituted lower alkyl group in which one or more atoms can be differentially labeled with one or more stable isotopes "X", wherein "X" is selected from the group consisting of H and D.

[0017] In a preferred embodiment, the present invention relates to a polypeptide reactive reagent having the formula "PRG-Z" wherein "PRG" is of the general formula:

wherein "Y" is an amino acid reactive moiety that selectively reacts with certain protein functional groups selected from the group consisting of an -NH₂ moiety, an arginine moiety, a lactone moiety and a cystein moiety; "n" is an integer selected from the group consisting of 1 , 2 and 3; "X" is either hydrogen (H) or deuterium (D), and wherein "Z" is an aryl, substituted aryl, alkyl, substituted alkyl, lower alkyl or substituted lower alkyl group in which one or more atoms can be differentially labeled with one or more stable isotopes.

[0018] In a further preferred embodiment, the present invention relates to a polypeptide reactive reagent selected from the group consisting of

"X" is either hydrogen (H) or deuterium (D).

[0019] The present invention also relates to a method for identifying and quantifying one or more proteins in a sample comprising the steps of: (a) reacting the proteins with proteolytic enzymes providing peptide digestion products; (b) reacting a first portion of the peptide digestion products with at least one of the polypeptide reactive reagents as shown above wherein "X" is D; (c) reacting a second portion of the peptide digestion products with at least one of the polypeptide reactive reagents as shown above wherein "X" is

(d) mixing the products of steps (b) and (c); and (e) identifying and quantifying the peptide digestion products by mass spectroscopy. [0020] Furthermore, the present invention relates to a method for identifying and quantifying one or more proteins in a sample comprising the steps of: (a) reacting a first portion of the sample with at least one of the polypeptide reactive reagents as shown above wherein "X" is D, to provide labeled proteins; (b) reacting a second portion of the sample with at least one of the polypeptide reactive reagents as shown above wherein "X" is H to provide labeled proteins; (c) mixing the products of steps (b) and (c) to provide a solution of labeled proteins; (d) reacting the solution of labeled proteins with proteolytic enzymes providing peptide digestion products; and (e) identifying and quantifying the peptide digestion products by mass spectroscopy.

[0021] Finally, the present invention relates to polypeptides (protein digestion products) labeled with polypeptide reactive reagents capable of serving as internal standards thus facilitating the quantitative determination by mass spectroscopy of the relative amounts of the proteins in samples.

[0022] Further scope and applicability will become apparent from the detailed description given hereinafter. It should be understood however, that this detailed description, while indicating preferred embodiments of the invention, is given by. way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 illustrates elution profiles of Cys-dO (blue) and Cys-d5

(red) labeled peptides obtained by reverse-phase chromatography. The peptides were separated by reverse phase HPLC on a Zorbax 300SB-C18 column (Agilent) with a gradient of 3-90% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. The elution profiles of the peptide modified with labels containing heavy (D) and light isotopes (H) coincides, indicating the absence of isotopic effects.

[0024] Figure 2 illustrates MS (top) and MS/MS spectra of the tryptic

BSA peptide modified with a cysteine-specific label. The MS-Scan at 30.9 min from a BSA digest mixture labeled with Cys-dO and Cys-d5 in a 1 :2 ratio, shows that the intensities of the spectra are proportional to the abundance of the peptides (Top). The MS/MS spectra of the EYEATLEE CCAK peptide labeled on two adjustment cystein residues shows an effective fragmentation (bottom).

[0025] Figure 3 illustrates examples of Cys-containing peptides identified in a dO-SIMPL-BSA / d5-SIMPL-BSA 1:1 mixture. 14 peptide pairs modified with d5 and dO-SIMPL labels were detected with an abundance ratio between 0.7 and 1.2 (mean ratio was 0.97). Samples were analyzed by an LC-

MS/MS system consisting of a nanoflow liquid chromatography system and an ion trap 1100 series LC MSD mass spectrometer (NanoFlow Proteomics Solution, Agilent Technologies, Santa Clara, CA). Peptides were separated by reverse phase HPLC on a Zorbax 300SB-C18 column (Agilent) with a gradient of 3-90% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. The column eluent was sprayed directly into the mass spectrometer. The spectra were searched against the NCBI NR database (NCBI, Bethesda, MD) using a Spectrum Mill software (Agilent). [0026] Figure 4 illustrates the selectivity of the binding and the release of peptides modified with SIMPL, using dimetoxybenzaldehyde polymer beads as the capture reagent. The tryptic digest of a dO-SIMPL-BSA / d5-SIMPL-BSA 1:1 mixture was incubated with 2,6-dimetoxybenzaldehyde polymer beads for one hour at 37 °C. The beads were collected by centrifugation and subsequently washed with DMF (50%; 1ml), NaCI (1 ml; 1M), and twice with DMF (20%; 1 ml). The bound peptides were separated from the capture reagent by suspending it in hydrazine (100 ml; 1 mM in 20% DMF). The beads flow-through (supernatant 1) and cleaved material (supernatant 2) were analyzed by LC-MS/MS as described above. Cys-containing peptides are selectively bound to the polymer beads.

[0027] Figure 5 illustrates the correlation between the expected ratios of BSA concentration and those measured using the cystein-specific labels.

[0028] Figure 6 illustrates examples of MS/MS spectra of BSA tryptic peptides modified and non-modified with a label for the terminal NH₂-group showing that labeling improves collision induced fragmentation.

[0029] Figure 7 illustrates peptides identified in a BSA tryptic digest, modified with the label specific the terminal NH₂-group showing that the majority (90%) of peptides are labeled exclusively on N-terminus.

[0030] Figure 8 illustrates the quantification of a BSA 1 :1 mixture. All together 15 peptide pairs modified with d5 and dO-SIMPL labels were detected with an abundance ratio between 0.8 and 1.4. The mean ratio was 1.04 with a standard deviation of 0.16. DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0031] The present invention relates to novel polypeptide reactive reagents for the isotopic labeling of proteins and peptide digestion products. The present invention also relates to a novel method for the identification and quantification of proteins and peptides in complex mixtures or in a proteome, including those present in only small quantities, using stable isotope labeling and analysis by mass spectroscopy. The method uses labeled polypeptide reactive reagents capable of reacting with proteins and peptides and which function as mass tags.

[0032] Using general covalent capture-and release chemistries, labeled molecules of a complex sample can be attached to a solid support and subsequently released. By incorporating the ability to attach and subsequently release a molecule, the method of the present invention can be advantageously used to isolate or purify labeled molecules. Furthermore, the method of the present invention is advantageous in that it can be used to selectively isolate and label molecules from a sample, allowing quantitative analysis of complex mixtures, preferably by methods such as mass spectroscopy.

[0033] The present invention relates to a method that, in one particular embodiment, utilizes a solid phase based approach to capture labeled polypeptides (protein digest products) or other molecules of interest covalently via a cleavable linker that allows enzymatic or chemical recovery of the captured molecule.

[0034] The method of the present invention is particularly useful for the identification and quantitative analysis of molecules contained in biological samples, in particular the analysis of proteins for quantitative proteomics. The present invention also relates to reagents that are useful for labeling molecules. The method of the present invention is particularly useful for transferring labels or tags to molecules suitable for mass spectrometry (MS) analysis.

[0035] As used herein, the term "amino acid" is intended to mean both naturally occurring and non-naturally occurring amino acids as well as amino acid analogs. Naturally occurring amino acids include the 20 (L)-amino acids utilized during protein biosynthesis as well as others such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine, for example. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine and the like. Amino acid analogs include modified forms of naturally and non-naturally occurring amino acids. Such modifications can include, for example, substitution or replacement of chemical groups and moieties on the amino acid or derivitization of the amino acid.

[0036] As used herein, a "functional group" is any chemical group that has desirable functional properties. A desirable functional property is any property that imparts a desirable chemical characteristic to a molecule. A functional group can include a group that changes the physicochemical properties of a molecule, for example, changing the mass, charge, hydrophobicity, and the like. A particularly useful functional group is a label or tag, for example, fluorophores, chromophores, spin labels, isotope distribution tags, and the like.

[0037] As used herein, the term "label" is intended to mean any moiety that can be attached to a molecule that results in a change in mass of that molecule. The label can be bound to the molecule either covalently or non- covalently, although generally the label will be covalently bound. A particularly useful label is a mass label useful for analysis of a sample by MS. The change in mass of the molecule due to the incorporation of a mass label should be within the sensitivity range of the instrument selected for mass determination. In addition, one skilled in the art will know or can determine the appropriate mass of a label for molecules of different sizes and different compositions. Moreover, when using heavy and light mass labels, for example, for differential labeling of molecules, a mass difference as small as between about 1-3 mass units can be used or as large as greater than about 10 mass units. Mass labels suitable for differentially labeling two samples are chemically identical but differ in mass.

[0038] As used herein, a "tag" refers to a label that is detectable. The tag imparts a characteristic to a molecule such that it can be detected by any of a variety of analytical methods, including MS, chromatography, fluorography, spectrophotometry, immunological techniques, and the like. A tag can be, for example, an isotope, fluor, chromagen, ferromagnetic substance, luminescent tag, or an epitope tag recognized by an antibody or antibody fragment. A particularly useful tag is a mass tag, which is a mass label suitable for detection and analysis of a molecule by MS. Exemplary mass tags include, for example, a stable isotope tag, an isotope distribution tag, a charged amino acid, differentially isotopically labeled tags, and the like. A tag can also be an element having a characteristic isotope distribution, for example, chlorine, bromine, or any elements having distinguishable isotopic distribution. Additionally, a tag can have a bond that breaks in a collision cell or ion source of a mass spectrometer under appropriate conditions and produces a reporter ion.

[0039] As used herein, a "cleavable functional group" is a chemical group that can be cleaved by a variety of methods, including input of energy, a chemical, an enzyme, and the like. For use in methods of the present invention, the cleavable functional group is generally specific, that is, one which can be specifically cleaved without altering or damaging the molecule being cleaved or which relatively uniformly alters the molecule in a reproducible manner. For example, the cleavable functional group can be a chemical cleavable group. If desired, a chemical cleavage reaction can be carried out under relatively mild conditions in which the chemical cleavable group is essentially the only chemical bond cleaved. A chemical cleavable group can also be a group cleavable by a chemical such as CNBr, which can cleave a methionine residue. CNBr can be particularly useful for releasing a molecule if a chemical cleavable group such as methionine has been added to the molecule, particularly in a polypeptide that does not have a methionine residue. Suitable chemical cleavable groups are well known to those skilled in the art (see, for example Wilson and Czarnik, eds., Combinatorial Chemistry: Synthesis and Application, John Wiley & Sons, New York (1997); Sewald and Jakubke, Peptides: Chemistry and Biology, Wiley-VCH, Weinheim (2002); Merrifield, J. Am. Chem. Soc. 85:2149 (1964); Bodanszky, M., Principles of Peptide Synthesis (Springer-Veriag, 1984)). The cleavable functional group can also be an enzymatic cleavable group. For example, a protease can be used to cleave a cleavable functional group having a suitable recognition sequence for the protease. Particularly useful proteases are the endopeptidases such as trypsin and the like. The protease can be selected based on the incorporation of a particular cleavable recognition sequence as a functional group. Other considerations for selecting a protease include the presence or absence of a recognition sequence in the molecule being captured and released.

[0040] As used herein, the term "isotopic label" or "isotopic tag" refers to a chemical group that can be generated in two distinct isotopic forms, for example, heavy and light isotopic versions of the constituent elements making up the chemical group. Such constituent elements include, for example, hydrogen, carbon, nitrogen, oxygen, and sulfur. Particularly useful isotopic labels or tags are those that allow convenient analysis by MS.

[0041] As used herein, the term "reactive group" is intended to mean any of a variety of chemical groups having useful chemical properties suitable for reacting and covalently binding to a molecule such as a polypeptide and the like. For example, a reactive group can react with carboxyl groups found in Asp or Glu, or the reactive group can react with other amino acids such as His, Tyr, Arg, and Met. A reactive group can also react with amines such as Lys as well as with oxygen or sulfur using chemistry well known in the art.

[0042] As used herein, the term "polypeptide" refers to a peptide or polypeptide of two or more amino acids. A polypeptide includes small polypeptides having a few or several amino acids as well as large polypeptides having several hundred or more amino acids. Non-limiting examples of a polypeptide include proteins, peptide digestion products and enzymes. Usually, the covalent bond between the amino acid residues is an amide bond. However, the amino acids can be joined together by various other means known to those skilled in the peptide and chemical arts.

[0043] As used herein, the term "protein function" is intended to mean the determination of peptide or polypeptide sequences, the identification of amino acid modifications in variant proteins such as those used in, for example, drug discovery, physiological function, and quantity.

[0044] As used herein, the term "aryl" is intended to mean phenyl, 1- naphthyl, and 2-naphthyl. The term "substituted aryl" as used herein, is intended to mean phenyl, 1 -naphthyl and 2-naphthyl having a substituent selected from the group consisting of phenyl, lower alkyl, lower alkoxy, lower alkylthio, halo, as well as mono-, di- and tri-substituted phenyl, 1 -naphthyl, and 2-naphthyl comprising substituents selected from the group consisting of lower alkyl, lower alkoxy, lower alkylthio and halo.

[0045] The term "alkyl" as used herein, is intended to mean straight or branched chain radicals having up to seven carbon atoms. The term "lower alkyl" as used herein, is understood as being straight or branched radicals having up to four carbon atoms and is a preferred sub-grouping for the term "alkyl". [0046] The terms "lower alkoxy" and "lower alkylthio" as used herein, are intended to mean such lower alkyl groups as defined above attached to an oxygen or sulfur atom.

[0047] As used herein, the term "capture reagent" is intended to mean a reagent capable of reacting with the labeled proteins, protein or peptide digestion products and peptides. Non-limiting examples of capture reagents comprise solid supports composed of a homopolymer or a heteropolymer containing polystyrene, polyethylene, polyacrylamide, polyacrylein, or the like. Solid supports, e.g. resins, matrices, beads, glass beads or the like are available from a variety of commercial sources. Further non-limiting examples of capture reagents include functionalized solid supports such as those functionalized with hydrazine or with benzaldehyde and its derivatives. Yet further non-limiting examples of capture reagents include substituted and unsubstituted benzaldehyde as well as 2-halo-2-imidazoline salts.

[0048] In a first embodiment of the present invention, the proteins in a first portion of a mixture or proteome are reduced and denatured, followed by treatment with proteolytic enzymes, non-limiting examples of which are trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid endopeptidase or glutamic acid endopeptidase, generating peptide digestion products comprising several to several tens of amino acid residues. The digested peptides are then chemically modified using labeled polypeptide reactive reagents comprising heavy isotopes (D) and capable of reacting with functional groups such as the thiol group

(SH) found on cystein (Cys) residues, the amino group (NH₂); the carboxylate group (COO^"); and the lactone group, formed following the cleavage of the S-C bond of methionine (Met) using CNBr. The procedure is repeated with a second portion of the mixture or proteome using labeled polypeptide reactive reagents comprising light isotopes (H). The peptides modified with the reagents comprising heavy and light isotopes are then mixed. [0049] Alternatively, the proteins in a first portion of a mixture or proteome are reduced and denatured, followed by treatment with proteolytic enzymes, non-limiting examples of which are trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid endopeptidase or glutamic acid endopeptidase, generating peptide digestion products comprising several to several tens of amino acid residues. The digested peptides are then chemically modified using labeled polypeptide reactive reagents comprising light isotopes (H) and capable of reacting with functional groups such as the thiol group (SH) found on cystein (Cys) residues, the amino group (NH₂); the carboxylate group (COO-); and the lactone group, formed following the cleavage of the S-C bond of methionine (Met) using CNBr. The procedure is repeated with a second portion of the mixture or proteome using labeled polypeptide reactive reagents comprising heavy isotopes (D). The peptides modified with the reagents comprising heavy and light isotopes are then mixed.

[0050] If the Arg-specific, the lactone specific or the Cys-specific label, as shown below, (polypeptide reactive reagents) is used, the peptide digestion products may be captured on a solid matrix. This is particularly useful in the selective identification of cysteine and arginine residues, since these amino acids have relatively low natural abundances; the lactone group resulting from the cleavage of the S-C bond of methionine using CNBr represents a further selective target aiding in the identification of a methionine residue. After rigorous washing, eliminating all non-specifically bound products, the labeled peptides digestion products are released from the matrix by enzymatic or chemical oxidative cleavage. The protein digestion products are then analyzed by either 2DHPLC- MS-MS or LC-MS-MS tandem mass spectroscopy. The former technique is best suited for the analysis of complex protein mixtures, whereas the latter technique is best suited for the analysis of mixtures containing a few to several proteins.

Cys-Specific Label Lactone-Specific Label

[0051] It is important to note that peptide digestion products labeled with the -NH₂ specific labels, as shown below, may also be captured on a solid matrix. However, since all of the amino acids comprise an NH₂ group, the use of - NH₂ specific labels is non-selective and thus useful in quantifying total protein content of a given sample.

NH₂-Specific Label Terminal NH₂-Specific Label

[0052] In a second embodiment of the present invention, the proteins in a mixture or proteome are first labeled with mass tags (labeled polypeptide reactive reagents), analyzed by SDS-PAGE (Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis), digested and finally analyzed by LC-MS-MS or 2DHPLC-MS-MS tandem mass spectroscopy. More specifically, the proteins in a mixture or proteome are treated under reducing conditions with a denaturing buffer containing either sodium dodecyl sulfate, urea, guanidinium chloride or any other protein denaturing agent. They are then labeled with polypeptide reactive reagents, i.e. reagents interacting with thiol groups, amino groups or lactone groups such as those illustrated below, wherein "X" is either hydrogen (H) or deuterium (D). The isotopically labeled mixtures are then resolved using one- dimensional SDS-PAGE electrophoresis. The gel portions comprising protein bands are excised and subjected to in gel-digest with proteolytic enzymes, non- limiting examples of which include trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid peptidase and glutamic acid endopeptidase. The resulting peptide digestion products are extracted and analyzed by LC-MS-MS or by 2DHPLC-MS-MS tandem mass spectroscopy.

Cys-Specific Label Lactone-Specific Label

NH₂-Specific Label

Terminal NH₂-Specific Label Terminal NH₂-Specific Label

[0053] In a third embodiment of the present invention, the_. proteins in a mixture or proteome are first labeled with mass tags (labeled polypeptide reactive reagents), analyzed by 2DE-GE (2-Dimensional Gel Electrophoresis), digested, and finally analyzed by LC-MS-MS or 2DHPLC-MS-MS tandem mass spectroscopy. More specifically, the proteins in a mixture or proteome are treated under reducing conditions with a denaturing buffer containing either sodium dodecyl sulfate, urea, guanidinium chloride or any other protein denaturing agent. They are then labeled with polypeptide reactive reagents, i.e. reagents interacting with thiol groups, amino groups or lactone groups such as those illustrated above wherein "X" is either hydrogen (H) or deuterium (D). The isotopically labeled mixtures are then resolved using two-dimensional gel electrophoresis. The gel portions comprising protein spots are excised and subjected to in gel-digest with proteolytic enzymes, non-limiting examples of which include trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid peptidase and glutamic acid endopeptidase. The resulting peptide digestion products are extracted and analyzed by LC-MS-MS or by 2DHPLC-MS-MS tandem mass spectroscopy.

EXAMPLE 1 Synthesis and application of a label specific for the NH₂-group

[0054] The synthesis of an amino-specific labeled polypeptide reactive reagent, as well as its application for the quantitative analysis of a peptide mixture is illustrated below, wherein "X" is either hydrogen (H) or deuterium (D).

EXAMPLE 2 Synthesis and application of a label specific for Arginine (Arg)

[0055] The synthesis of an arginine-specific labeled polypeptide reactive reagent, as well as its application for the quantitative analysis of a peptide mixture is illustrated below, wherein "X" is either hydrogen (H) or deuterium (D).

EXAMPLE 3 Synthesis and application of a label specific for the -SH group

[0056] The synthesis of a thiol-specific labeled polypeptide reactive reagent, as well as its application for the quantitative analysis of a peptide mixture is illustrated below, wherein "X" is either hydrogen (H) or deuterium (D).

.0 ^{x x} Peptide — SH

Cys-Specific Label

Enzymatic or Chemical Oxidative Cleavage

EXAMPLE 4 Synthesis and application of a label specific for the lactone group

[0057] The CNBr-cleavage of methionine-containing proteins results in peptides comprising a lactone functionality. The synthesis of a lactone-specific labeled polypeptide reactive reagent, as well as its application for the quantitative analysis of a peptide mixture is illustrated below, wherein "X" is either hydrogen (H) or deuterium (D).

Lactone-Specific Label

EXAMPLE 5 Further application of a label specific for the -SH group

[0058] The application of a thiol-specific labeled polypeptide reactive reagent for the quantitative analysis of a peptide mixture is illustrated below, wherein "X" is either hydrogen (H) or deuterium (D).

Peptide-SH

O 2005/012247

27

EXAMPLE 6

Synthesis and application of a label specific for the terminal NH₂-group

[0059] The synthesis of an amino-specific labeled polypeptide reactive reagent specific for the terminal amino group, as well as its application for the quantitative analysis of a peptide mixture is illustrated below, wherein "X" is either hydrogen (H) or deuterium (D).

Peptide-NH₂

EXAMPLE 7 Synthesis of N-phenylmaleimide (d0/d5), a label specific for the -SH group

[0060] Maleic anhydride (2.0 g; 20 mmol) was dissolved in 30 ml of diethyl ether. Aniline (1.86 g; 20 mmol) or aniline-d5 (1.96 g; 20 mmol) in 5 ml of diethyl ether was then added. The reaction mixture was allowed to stir for 1 hour after which the solvent was eliminated. [¹H-NMR (DMSO-d₆) 6.24 (d, 1H), 6.42 (d, 1H), 7.35 (m, 3H*), 7.46 (m, 2H*); * -dO only].

[0061] To the compound obtained as described above (3.82 g (dO) or

3.91 g (d5); 20 mmol) was then added acetic anhydride (9.5 ml; 0.1 mol) and sodium acetate (0.92 g; 10 mmol). The reaction mixture was allowed to stir for 2 hours at 90 °C, and was then quenched with a saturated potassium carbonate solution (10 ml). The aqueous layer was extracted several times with dichloromethane (50 ml) and the combined organic layers evaporated. The so- obtained residue was recrystallized from benzene to yield 2.6 g (dO) or 2.8 g (d5) of the desired compound. [¹H-NMR (CDCI₃) 6.87 (s, 2H), 7.37 (m, 3H*), 7.49 (m, 2H*); * -dO only].

EXAMPLE 8 Synthesis of pentafluorophenyl-4-anilino-4-oxobutanoate (d0/d5/d9), a label specific for the -NH₂ group

[0062] To a solution of succinic anhydride (5.0 g (dO) or 5.2 g (d4); 50.0 mmol) in diethyl ether (50 ml) was added dropwise aniline (4.65 g (dO) or 4.9 g (d5); 50.0 mmol) in diethyl ether (5 ml). The reaction mixture was allowed to stir for 2 hours after which the so-obtained white precipitate was filtered and washed with diethyl ether. Removal of the solvent yielded about 8.0 g of desired product. [¹H-NMR DMSO-d₆) 2.83 (t, 2H*), 3.22 (t, 2H*), 7.05 (m, 1H**), 7.28 (m, 2H**), 7.60 (m, 2H**), 10.10 (s,1H); * -d0/d5 only; ^** -dO only].

[0063] The compound obtained as described above (8.0 mmol) was dissolved in 1,4-dioxane (30 ml), followed by the addition of pentafluorophenol (1.45 g; 9.0 mmol) and DCC (1.33 ml; 10.0 mmol). The reaction mixture was allowed to stir for 12 hours at room temperature after which the so-obtained precipitate was removed and washed with cold dioxane. Removal of the solvent and crystallization of the residue from chloroform yielded approximately 2.2 g of desired compound. [¹H-NMR DMSO-d₆) 2.81 (t, 2H*), 3.07 (t, 2H*), 7.03 (m, 1H**), 7.29 (m, 2H**), 7.60 (m, 2H**); * -d0/d5 only; ** -dO only]. EXAMPLE 9 Synthesis of 2-chloro-2-imidazoline sulfate

-NN⁺⁺HH^{Η H!S}°⁴ -CI ~NH

[0064] Chlorine was passed through a cooled aqueous mixture (0 °C; 7 ml) of imidazolidine-2-thione (4.13 g; 4.0 mmol) over a period of 2 hours. The reaction mixture was allowed to stir for an additional 30 minutes at 45 °C, after which the solvent was evaporated. The so-obtained crude residue was washed on a glass filter with acetone (30 ml) and then with diethyl ether (30 ml). The washed residue was then dissolved in concentrated sulfuric acid (10 ml). Following the removal of hydrogen chloride, acetone (60 ml) was added and the mixture cooled on ice. The so-obtained white precipitate was filtered, washed with acetone, dried and recrystallized from methanol to yield the desired compound (6.2 g). [¹H-NMR (DMSO-de) 3.33 (s), 3.47 (m), 3.78(m). 3.93 (s)].

EXAMPLE 10 Quantitative analysis of a protein mixture

[0065] Sample preparation Two samples of BSA (5 mg each) were dissolved in 1 ml of 50 mM ammonium carbonate buffer (pH 8.5) containing urea (6 mol). DTT was then added to a final concentration of 5 mM, and the solution heated for 1 hour at 37 °C.

[0066] Labeling The pH of the protein solution was reduced to 5.0 using concentrated acetic acid. The protein solution (1 μl) was then mixed with a DMF solution (19 μl) comprising the label N-phenylmaleimide (dO) or N- phenylmaleimide (d5) in DMF (final concentration 15 mM). The labeling reaction mixture was allowed to proceed for 1 hour at 37 °C after which the samples were combined and hydrazine hydrate was added to a final concentration of 0.1 M. The combined sample was twice diluted with Laemmli buffer, and 10 μl of sample (comprising 5 μg and 2.5 μg of BSA respectively) were loaded per SDS-PAGE lane. One-dimensional gel electrophoresis followed by in-gel digestion with trypsin (Promega, sequencing grade ratio 1 :10) and peptide extraction was then performed (Shevchenko, A., Wilm, M., Vorm, O., and Mann, M.; (1996), Anal. Chem. 68, 850-858).

[0067] Binding to the capture reagent Capture reagent (5 mg)

(Sigma; 2,6-dimetoxybenzaldehyde polymer-bound, 1mM/g) was added to the peptide extract (100 μl) and the mixture agitated for one hour at 37 °C. The bound product was separated from any unbound peptides (supernatant 1) by centrifugation and subsequently washed with DMF (50%; 1ml), NaCI (1 ml; 1M), and twice with DMF (20%; 1 ml).

[0068] Cleavage The bound product was separated from the capture reagent by suspending it in hydrazine (100 μl; 1 mM in 20% DMF). The reaction mixture was vortexed at 37 °C for 30 minutes. The capture reagent was then separated from the supernatant (supernatant 2) by centrifugation.

[0069] Modification I p-Nitrobenzaldehyde was added to supernatant

1 to a final concentration of 3 mM. The so-obtained mixture was then analyzed by LC-MS/MS tandem mass spectroscopy. The set-up consisted of a nanoflow liquid chromatography system and an ion trap 1100 series LC/MSD mass spectrometer (NanoFlow Proteomics Solution, Agilent Technologies, Santa Clara, CA). The peptides were separated by reverse-phase HPLC on a Zorbax 300SB-C18 column (Agilent) using a gradient of 3-90% acetonitrile in 0.1% formic acid at a flow rate of 300 nl/min. The column eluent was sprayed directly into the mass spectrometer and the obtained spectra analyzed against the NCBI NR database (NCBI, Bethesda, MD) using Spectrum Mill software (Agilent). Analysis of the elution profiles of the peptides modified with labels containing heavy and light isotopes (Zorbax 300SB-C18 column) showed that they coincide, indicating the absence of an isotopic effect during reverse-phase chromatography. The peptide sequences were identified based on their collision-induced fragmentation (MS/MS) spectra. Their relative amounts in the two samples was determined by comparing the signal intensities of the peptides labeled with the heavy (D) and light (H) reagents. 29 unique peptides (cystein-containing peptides only) were identified. The obtained ratio between d5 and dO-modified peptides was 0.97±0.33 (expected ratio 1.0). Comparative LC-MS/MS analysis of supernatant 1 and supernatant 2 showed that the Cys-containing peptides were selectively captured by the polymer beads (solid support). Further experiments performed with several other BSA samples of known concentration, resulted in a good correlation between the expected and measured concentration ratios.

[0070] Modification II 3,5-Bis-trifluromethylbenzaldehyde was added to supernatant 1 to a final concentration of 3 mM. The so-obtained mixture was then analyzed by LC-MS/MS tandem mass spectroscopy as described above. This chemical modification leads to significant increases of the retention times on reverse-phase chromatography allowing for the separation of modified and non- modified peptides. 15 unique peptides (cystein-containing peptides only) were identified. The obtained ratio between d5 and dO-modified peptides was 1.1 ±0.3 (expected ratio 1.0).

[0071] Modification III 2-Chloro-2-imidazoline sulfate was added to supernatant 1 to a final concentration of 10 mM. The so-obtained mixture was then analyzed by MALDI-Q-TOF mass spectroscopy. This chemical modification leads to significant increases in the sensitivity of peptide detection by MALDI mass spectroscopy. 15 unique peptides (cystein-containing peptides only) were identified. The obtained ratio between d5 and dO-modified peptides was 1.2±0.24 (expected ratio 1.0). EXAMPLE 11 Quantitative analysis of a protein mixture

[0072] A BSA sample (2 mg) was dissolved in 200 μl of 50 mM Tris-

HCI buffer (pH 8.5) containing urea (6 mol). DTT was then added to a final concentration of 5 mM, and the solution heated for 1 hour at 37 °C. lodoacetamide was subsequently added to a final concentration of 10mM. The samples were incubated at room temperature in the dark over a period of 1 hour and then desalted on a Fast Desalting FPLC column (Pharmacia) equilibrated with ammonium carbonate buffer (50 mM; pH 7.8). Digestion in solution with trypsin (Promega, sequencing grade ratio 1 :100) was then performed (Shevchenko, A., Wilm, M., Vorm, O., and Mann, M.; (1996), Anal. Chem. 68, 850-858). The digested sample (2.5 μl) was then mixed with acetonitrile (17.5 μl), pyridine-HCI buffer (2.5 μl, 4.0 M; pH = 5.0) and the label pentafluorophenyl-4-anilino-4- oxobutanoate (dO) in DMF (3.75 μl of a 1M solution in DMF; final concentration in the reaction mixture was 150 mM). The sample was incubated for 3 hours at 25 °C and then analyzed using LC/MS/MS tandem mass spectroscopy as previously described. This chemical modification results in an improved collision-induced fragmentation of the labeled peptides, resulting in them being more readily identified from MS/MS spectra. 47 unique peptides were identified; coverage was 70% [i.e. 70% of the protein sequence was identified]. 92% of the peptides had their terminal NH₂-groups modified with the label. In a second experiment, BSA samples of 6 known concentrations were labeled with pentafluorophenyl-4-anilino- 4-oxobutanoate (dO) and pentafluorophenyl-4-anilino-4-oxobutanoate (d5), mixed and analyzed by LC/MS/MS as described above. The obtained ratio between d5 and dO-modified peptides was 0.5±0.01 (expected ratio 0.5); 1.1±0.16 (expected ratio 1.0); and 4.0±0.01 (expected ratio 4.0).

[0073] Although the present invention has been described hereinabove by way of embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide reactive reagent having the formula: PRG-Z wherein "PRG" is a polypeptide reactive group which optionally binds to a capture reagent, and wherein "Z" is an aryl, substituted aryl, alkyl, substituted alkyl, lower alkyl or substituted lower alkyl group in which one or more atoms can be differentially labeled with one or more stable isotopes "X", wherein "X" is selected from the group consisting of H and D.

2. The polypeptide reactive reagent as defined in claim 1 , having the formula:

wherein Z is as previously defined and wherein:

"Y" is an amino acid reactive moiety that selectively reacts with certain protein functional groups selected from the group consisting of an -NH₂ moiety, an arginine moiety, a lactone moiety and a cystein moiety; and

"n" is an integer selected from the group consisting of 1 , 2 and 3; and

3. The polypeptide reactive reagent as defined in claim

2, having the formula:

wherein "X" is either hydrogen (H) or deuterium (D) and wherein said reagent selectively reacts with an -NH₂ moiety.

4. The polypeptide reactive reagent as defined in claim 3, wherein the -NH₂ moiety is embodied in a protein, peptide, or peptide digestion product.

5. The polypeptide reactive reagent as defined in claim 4, wherein the -NH₂ moiety includes an -NH₂ moiety and a terminal -NH₂ moiety.

6. The polypeptide reactive reagent as defined in claim 2, having the formula:

wherein "X" is either hydrogen (H) or deuterium (D), and wherein said reagent selectively reacts with the arginine moiety.

7. The polypeptide reactive reagent as defined in claim 6, wherein the arginine moiety is embodied in a protein, peptide, or peptide digestion product.

8. The polypeptide reactive reagent as defined in claim 2, having the formula:

wherein "X" is either hydrogen (H) or deuterium (D), and wherein said reagent selectively reacts with the lactone moiety.

9. The polypeptide reactive reagent as defined in claim 8, wherein the lactone moiety is embodied in a protein, peptide, or peptide digestion product.

10. The polypeptide reactive reagent as defined in claim 9, wherein the lactone moiety is generated following cleavage of the S-C bond of methionine using CNBr.

11. The polypeptide reactive reagent as defined in claim 2, having the formula:

wherein "X" is either hydrogen (H) or deuterium (D), and wherein said reagent selectively reacts with a terminal -NH₂ moiety.

.

12. The polypeptide reactive reagent as defined in claim

11 , wherein the terminal amino moiety is embodied in a protein, peptide, or peptide digestion product.

13. The polypeptide reactive reagent as defined in claim

1 , having the formula:

wherein Z is as previously defined.

14. The polypeptide reactive reagent as defined in claim

13, having the formula:

wherein "X" is either hydrogen (H) or deuterium (D) and wherein said reagent selectively reacts with the cystein moiety.

15. The polypeptide reactive reagent as defined in claim

14, wherein the cysteine moiety is embodied in a protein, peptide, or peptide digestion product.

16. The polypeptide reactive reagent as defined in claim 1 , having the formula:

wherein Z is as previously defined and wherein "X" is either hydrogen (H) or deuterium (D).

17. The polypeptide reactive reagent as defined in claim 16, having the formula:

wherein "X" is either hydrogen (H) or deuterium (D) and wherein said reagent selectively reacts with a terminal -NH₂ moiety.

18. The polypeptide reactive reagent as defined in claim

17, wherein the terminal amino moiety is embodied in a protein, peptide, or peptide digestion product.

19. A protein reactive reagent selected from the group consisting of

wherein "X" is either hydrogen (H) or deuterium (D).

20. A method for identifying and quantifying one or more proteins in a sample comprising the steps of: a. reacting the proteins with proteolytic enzymes providing peptide digestion products; b. reacting a first portion of the peptide digestion products with at least one of the polypeptide reactive reagents as defined in claim 1 , wherein "X" is D; c. reacting a second portion of the peptide digestion products with at least one of the polypeptide reactive reagents as defined in claim 1 , wherein "X" is H; d. mixing the products of steps (b) and (c); and e. identifying and quantifying the peptide digestion products by mass spectroscopy.

21. The method as defined in claim 20, wherein the polypeptide reactive reagents are selected from the group consisting of:

wherein "X" is either hydrogen (H) or deuterium (D).

22. The method as defined in claim 21 , further comprising the steps of: (f) reacting the peptide digestion products of step (d) with a capture reagent to provide captured peptide digestion products; and (g) releasing the captured peptide digestion products.

23. The method as defined in claim 22, wherein the proteolytic enzymes are selected from the group consisting of trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid endopeptidase and glutamic acid endopeptidase.

24. The method as defined in claim 22, wherein the capture reagent is a solid support.

25. The method as defined in claim 24, wherein the solid support is a solid support functionalized with hydrazine.

26. The method as defined in claim 24, wherein the solid support is a solid support functionalized with benzaldehyde or a benzaldehyde derivative.

27. The method as defined in claim 26, wherein the benzaldehyde derivative is p-nitrobenzaldehyde, or bis- trifluoromethylbenzaldehyde.

28. The method as defined in claim 22, wherein the releasing comprises enzymatic or chemical oxidative cleavage.

29. The method as defined in claim 22, wherein the capture reagent is a 2-halo-2-imidazoline salt.

30. The method as defined in claim 29, wherein the capture reagent is 2-chloro-2-imidazoline sulfate.

31. The method as defined in claim 20, wherein the polypeptide reactive reagents are selected from the group consisting of:

wherein "X" is either hydrogen (H) or deuterium (D).

32. The method as defined in claim 29, wherein the proteolytic enzymes are selected from the group consisting of trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid endopeptidase and glutamic acid endopeptidase.

33. A method for identifying and quantifying one or more proteins in a sample comprising the steps of: a. reacting a first portion of the sample with at least one of the polypeptide reactive reagents as defined in claim 1 wherein "X" is D, to provide labeled proteins; b. reacting a second portion of the sample with at least one of the polypeptide reactive reagents as defined in claim 1 wherein "X" is H, to provide labeled proteins; c. mixing the products of steps (b) and (c) to provide a solution of labeled proteins; d. reacting the solution of labeled proteins with proteolytic enzymes providing peptide digestion products; and e. identifying and quantifying the peptide digestion products by mass spectroscopy.

34. The method as defined in claim 33, wherein the polypeptide reactive reagents are selected from the group consisting of:

wherein "X" is either hydrogen (H) or deuterium (D).

35. The method as defined in claim 34, further comprising the steps of: f. reacting the peptide digestion products of step (d) with a capture reagent to provide captured peptide digestion products; and g. releasing the captured peptide digestion products.

36. The method as defined in claim 35, wherein the proteolytic enzymes are selected from the group consisting of trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid endopeptidase and glutamic acid endopeptidase.

37. The method as defined in claim 35, wherein the capture reagent is a solid support.

38. The method as defined in claim 37, wherein the solid support is a solid support functionalized with hydrazine.

39. The method as defined in claim 38, wherein the solid support is a solid support functionalized with benzaldehyde or a benzaldehyde derivative.

40. The method as defined in claim 39, wherein the benzaldehyde derivative is p-nitrobenzaldehyde, or bis- trifluoromethylbenzaldehyde.

41. The method as defined in claim 35, wherein the releasing comprises enzymatic or chemical oxidative cleavage.

42. The method as defined in claim 35, wherein the capture reagent is a 2-halo-2-imidazoline salt.

43. The method as defined in claim 42, wherein the capture reagent is 2-chloro-2-imidazoline sulfate.

44. The method as defined in claim 33, wherein the polypeptide reactive reagents are selected from the group consisting of:

wherein "X" is either hydrogen (H) or deuterium (D).

45. The method as defined in claim 44, wherein the proteolytic enzymes are selected from the group consisting of trypsin, lysine endopeptidase, arginine endopeptidase, aspartic acid endopeptidase and glutamic acid endopeptidase.

46. Use of the polypeptide reactive reagent as defined in claim 1 for identifying and quantifying one or more proteins in a sample.

47. The use as defined in claim 46, wherein the polypeptide reagent is selected from the group consisting of :

wherein "X" is either hydrogen (H) or deuterium (D).