JP2006506647A - Method for analyzing amino acids, peptides and proteins - Google Patents

Abstract

The present invention provides methods, reagents and kits for the identification, differential quantification of amino acids, peptides and proteins, and for the analysis of post-translational modifications and crosslinking states. The method includes the step of derivatizing a mixture of amino acids, peptides or proteins to form one or more amino acids, peptides or proteins containing a fixed charge ion at a site other than the C-terminus or N-terminus, wherein the fixed charge is included. Introducing a mixture of amino acids, peptides or proteins containing derivatized amino acids, peptides or proteins into a mass spectrometer, amino acids derivatized to contain the fixed charge, amino acids containing peptides or proteins, peptides or proteins Passing the mixture through a first mass resolving analyzer to select a protein or peptide precursor ion having a first mass-to-charge ratio, dissociating the precursor ion having the first mass-to-charge ratio and fixing charge Is a characteristic of fragmentation that occurs in the vicinity of Step to produce product ions having 2 mass to charge ratio, and comprising the step ,, for detecting the product ions having a second mass-to-charge ratio. The product ion having the second mass-to-charge ratio is a product ion formed by neutral desorption of fixed charge from the precursor ion, or a product formed by charge desorption of fixed charge from the precursor ion It may be an ion.

Description

  The present invention relates to a method for analyzing amino acids, peptides or proteins.

  With the completion of the first draft of the human genome sequence, researchers are now facing and challenging to understand the function of the gene. However, the biological function of a gene cannot be judged from a simple survey of its DNA sequence. Thus, a comprehensive analysis of the proteins expressed by the genome will bridge between the gene and its biological function. The term proteomics refers to (i) the identification and characterization of all proteins synthesized by a particular cell type or tissue at any given time, and the overall level of protein expression observed between two different cell states. Quantification of changes (collectively known as expression proteomics) and (ii) identification of components of functionally active protein complexes and characterization of intrinsic protein-protein interactions involved in intercellular protein trafficking and signaling pathways Synonymous with decision (all known as cell mapping proteomics). Taken together, these methods allow for comprehensive investigations at the protein level of complex cellular changes that occur following transformation of cells from one state to another [Blackstock, W. et al. P. And Weir, M .; P. Trends Biotechnol. 1999, 17, 121-127. Pandey, A .; And Mann, M .; Nature 2000, 405, 837-846].

  Recent advances in mass spectrometry (MS), along with advances in advanced bioinformatics tools for database retrieval of MS-derived data, have become a major factor in realizing proteomics [Mann, M. et al. Hendrickson, R .; C. And Pandey, A .; Annu. Rev. Biochem. 2001, 70, 437-473]. In particular, the rapidity, specificity, and sensitivity of these mass spectrometry has become particularly attractive for use in methods that require rapid protein identification and characterization. Conventional MS methods for proteomics usually involve separation of protein mixtures by one-dimensional or two-dimensional electrophoresis (2DE), then excising protein spots or gel slices and subjecting them to in situ proteolysis using trypsin. The peptide is then extracted and subjected to mass spectroscopic analysis. The mass of these peptides is a property of the protein and provides a “mass fingerprint” that can be used in database searches to identify proteins for the peptide [Henzel, W. et al. J. et al. Billec T. M.M. , Stults J .; T.A. , Wong S. C. , Grimley C.M. And Watanabe C.I. Proc. Natl. Acad. Sci. USA. 1993, 90, 5011-5015]. A more comprehensive method, particularly a more comprehensive method for the identification and quantification of individual components present in a complex protein mixture, is to subject each proteolytic peptide to tandem mass spectrometry. Subsequent identification of each peptide is accomplished by database search of partially derivatized amino acids “sequence tags” [Mann, M. et al. And Wilm, M .; Anal. Chem. 1994, 66, 4390-4399], database analysis of uninterpreted product ion spectra [Eng, J. et al. K. McCorack, A.M. L. And Yates, J .; R. J. et al. Am. Soc. Mass Spectrom. 1994, 5, 976-989], or “de-novo” sequence analysis [Hunt, D. et al. F. Yates, J .; R. , Shabanowitz J. et al. , Winston S. And Hauer, C .; R. Proc. Natl. Acad. Sci. USA 1986, 83, 6233-6237].

  However, this general analysis method is complicated by several limitations.

  First, 2D-gels can only separate about 1500-2000 proteins, and cells typically have more than 8000 proteins expressed. Thus, only the most abundant proteins are observed, and the major part of the 2-DE separation gel spot is one or more by the same protein in a form that has undergone co-electrophoresis and / or different modification (or processing) Of protein. In addition, several classes of proteins, particularly hydrophobic proteins, low abundance proteins, and proteins with extreme pI and molecular weight, do not appear very well in 2D-gel based separations [Gygi, S. et al. P. Corthals, G .; L. , Zhang, Y .; Rochon, Y .; Aebersold, R .; Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 9390-9395]. Also, 2-DE is labor intensive, slow, requires technology, and often has low reproducibility, so it cannot be easily adapted to high throughput or automation.

  Second, identification of a unique peptide from a protein digest by MS / MS is necessary for a clear identification of that protein, provided that the peptide is unique to a single protein. In fact, when analyzing either the same peptide ion (eg, in a continuous scan between chromatographic separations of a complex peptide mixture) or different peptides of the same protein, A very long time is spent between analyses. Thus, peptides that are present in high relative amounts are generally preferred for samples, and information regarding the identification of proteins present in complex mixtures is generally not available for small amounts of peptides. Moreover, up to 1/4 of the MS / MS spectrum obtained in the process of peptide sequence analysis based on peptide MS cannot be analyzed (that is, the product ion spectrum is too complex, the amount is too small, or the interpretation thereof) ) [Simpson, R. et al. J. et al. , Connelly, L .; M.M. Eddes, J .; S. Pereira, J .; J. et al. Moritz, R .; L. And Reid, G .; E. Electrophoresis. 2000, 21, 1707-1732].

By using dynamic exclusion during MS / MS data acquisition to partially overcome these limitations, a greater number of individual peptide ions are selected throughout the LC / MS / MS experimental process [Davis, M . T. T. et al. Spahr, C .; S. McGinley, M .; D. Robinson, J .; H. Bures, E .; J. et al. Beierle, J .; Mort, J .; Yu, W .; Luethy, R .; Patterson, S .; D. Proteomics 2001, 1, 108-117]. Multidimensional chromatography [Washburn, M .; P. , Wolters, D .; And Yates, J .; R. Nat. Biotechnol. 2001, 19, 242-247], or a method of selectively concentrating only peptides containing specific amino acids in solution by affinity selection [Spahr, C. et al. S. Susin, S .; A. Bures, E .; J. et al. Robinson, J .; H. Davis, M .; T. T. et al. McGinley, M .; D. , Kroemer, G .; And Scott D. et al. Patterson, S.M. D. Electrophoresis. 2000, 21, 1635-1650], or differential chromatography [Gevaert, K. et al. , Van Damme, J. et al. Goethals, M .; , Thomas, G .; R. Hoorelbeke, B .; , Demol, H .; Martens, L .; , Puype, M .; , Staes, A .; And Vandekerckhove, J. et al. Mol. Cell. Proteomics. Several methods for simplifying peptide mixtures prior to mass spectroscopic analysis are also described according to any of US 2002, 1,896-903]. By combining dynamic exclusion and the multidimensional chromatographic methods outlined above, these methods can further increase the number of proteins that can be identified by mass spectrometry. However, identification of these low abundance in a complex mixture (the expression of individual proteins may be in the range of cells per 10-10 ⁶ copies) proteins, important information of the structure and position of the post-translational modification Is still an important attempt.

  A basic aspect of proteomics research is to determine protein expression levels (ie, relative quantification of protein levels) between two different states of a biological system, such as found between normal and diseased cells or tissues. It is. There is a significant discrepancy between the mRNA expression level (transcriptomics) and the corresponding protein (proteomics), so array-based gene expression monitoring methods or other gene expression methods alone to measure mRNA abundance It is clear that it is insufficient to analyze the protein complement of cells [Gygi, S .; P. Rochon, Y .; , Franza, B .; R. And Aebersold, R .; Mol. Cell. Biol. 1999, 19, 1720-1730].

  Qualitative and quantitative analysis of protein expression profile changes as an effect of cell cycle regulation, pathology or drug exposure has traditionally been achieved by the high dimensionality obtained by orthogonal isoelectric molecular weight separation mode, and It has been performed by two-dimensional electrophoresis through image analysis [Pattern & Bechem Curr Opinion Chem Biol 2002, 6, 63-69. Zhou, G .; Li, H .; , DeCamp, D.M. Chen, S .; , Shu, H .; , Gong, Y .; , Flaig, M .; Gillespie, J .; W. Hu, N .; Taylor, P .; R. Emmert-Buck, M .; R. Liotta, L .; A. Petricoin, E .; F. And Zhao, Y .; Mol Cell Proteomics 2002, 1, 117-123]. However, these 2DE gel-based quantification methods are subject to the same limitations as described above for 2DE gel-based protein identification, so that the quantitative analytes are relatively abundant and highly pure spots on the gel. Or proteins that can be visualized appropriately by the staining method used [Gygi, S .; P. Corthals, G .; L. , Zhang, Y .; Rochon, Y .; Aebersold, R .; Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 9390-9395]. Some of these drawbacks include the SYPRO ruby fluorescent protein staining method [Bergren, K. et al. , Chernokalskaya, E .; Steinberg, T .; H. Kemper, C .; Lopez, M .; F. , Diwu, Z .; Haugland, R .; P. And Patton, W .; F. Electrophoresis 2000, 21, 2509-2521] and 2-D differential gel electrophoresis (DAGE) [Unlu, M. et al. Morgan, M .; E. And Minden, J .; S. Have been overcome by using more sensitive detection methods such as Electrophoresis, 1997, 18, 2071-2077]. However, these methods rely essentially on the use of appropriate software packages for spot detection, gel matching, and spot quantification [Raman, B. et al. , Cheung, A .; And Martin, M .; R. Electrophoresis, 2002, 23, 2194-2202], the situation where overlapping protein spots exist cannot be explained.

  Two general methods have been developed to quantify differential protein expression levels between two different cell / tissue states based on MS utilizing isotope labeling.

First, Smith's group [Pasa-Tolic, L; Jensen, P .; K. Anderson, G .; A. Lipton, M .; S. Peden, K .; K. Martinovic, S; Tolic, N; Bruce, J .; E. Smith, R .; D. J. et al. Am. Chem. Soc. 1999, 121, 7949-7950. Venstra, T .; D. Martinovic, S .; , Anderson, G. A. Pasa-Tolic, L .; And Smith, R .; D. J. et al. Am. Soc. Mass Spectrom. 2000, 11, 78-82. Conrads, T .; P. Et al. Anal. Chem. 2001, 73, 2132-2139; Smith, R .; D. Anderson, G .; A. Lipton, M .; S. Pasa-Tolic, L .; Shen, Y .; Conrads, T .; P. Venstra, T .; D. Udseth, H .; R. Proteomics 2002, 2, 513-523], Chait's group [Oda, Y. et al. Huang, K .; Cross, F .; R. Cowburn, D .; Chait, B .; T. T. et al. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 6591-6596], and others [Chen, X. et al. Smith, L .; M.M. Bradbury, E .; M.M. . Anal. Chem. 2001, 72, 1134-1143; Ong, Shao-En. , Bragoev, B .; , Krachmarova, I .; Bach-Kristensen, D .; Stein, H .; , Pandey, A .; And Mann, M .; Mol. Cell. Proteomics. 2002, 1, 376-386; Jiang, H .; And English, A .; M.M. J. et al. Proteome. Res. 2002, 1, 345-350; Zhu, H .; Hunter, T .; C. Pan, S .; Yau, P .; M.M. Bradbury, E .; M.M. And Chen, X. et al. Anal. Chem. 2002.74, 1687-1694], all of which are amino acids depleted (eg ¹³ C-, ¹⁵ N-, and ² H- depleted) or enriched ( ¹⁵ Metabolism into cellular protein populations either by homogenous labeling with N- or by incorporation of selected amino acids containing heavy isotopes (eg ¹³ C, ¹⁵ N, ² H) Represents the results with dynamic uptake. After isolating the protein from the cell substrate, the sample is mixed with a sample that has incorporated the natural isotope, then the individual proteins are separated by electrophoresis or chromatography, digested, and the peptide is analyzed by MS. Measure the mass. Site-specific labeling allows for efficient identification of peptides rich in amino acid mass “tags” by comparison with those in unlabeled form. In addition, changes in protein expression levels between two samples can be quantified by comparing the relative amount of peptide from an isotopically enriched sample with the relative amount from a sample prepared using a natural abundance of isotopes. sell. However, this in vivo labeling method is limited to systems where cells can be cultured under conditions suitable for incorporation of isotope labels.

  The second method involves in vitro chemical derivatization with isotopically enriched labels after isolation of the protein from the cellular substrate. One method that has received great attention to date is the isotope-coded affinity tag (ICAT) method developed by Abersold and colleagues [Gygi, S .; P. Rist, B; Gerber, S .; A. Turecek, F; Gelb, M .; H. Aebersold, R .; Nat. Biotechnol. 1999, 17, 994-999. Smolka, M; Zhou, H; Aebersold, R .; Mol. Cell. Proteomics. 2002, 1, 19-29. Shio, Y; Donooe, S; Yi, E .; C. Goodlett, D .; R. Aebersold, R; Eisenman, R .; N. EMBO 2002, 21, 5088-5096. Han, D .; K. Eng, J .; Zhou, H .; And Aebersold, R .; Nature Biotechnol. 2001, 19, 946-951. Griffin, T .; J. et al. Han, D .; K. M.M. , Gygi, S .; P. Rist, B .; Lee, H .; Aebersold, R .; And Parker, K .; C. J. et al. Am. Soc. Mass. Spectrom. 2001, 12, 1238-1246. Gygi, S .; P. Rist, B .; And Aebersold, R .; Current Opinions in Biotechnol. 2000, 11, 396-401]. Proteins obtained from two different cell / tissue states are reduced and S-alkylated using either natural abundance (light) or isotope enriched (heavy) ICAT reagents, respectively. Each of these contains a biotin moiety for subsequent affinity selection of a cysteine-containing peptide by streptavidin affinity purification, resulting in simplification of the mixture prior to MS analysis. Similar to that described above for metabolically labeled samples, for each of these methods, a proteolytic derivatized peptide comprising a light isotope “normal” sample and a heavy isotope “disease state” sample (or structure). The abundance ratio compared (with those containing the target label) is an indicator of changes in protein expression levels between the two samples, thereby allowing their differential quantification.

  A number of similar methods for selective peptide identification and differential quantification have been described previously [Spahr, C. et al. S. Susin, S .; A. Bures, E .; J. et al. Robinson, J .; H. Davis, M .; T.A. McGinley, M .; D. , Kroemer, G .; And Patterson, S .; D. Electrophoresis. 2000, 21, 1635-1650. Adamczyk, M .; And Gebler, J. et al. C. Wu, J .; Rapid. Commun. Mass Spectrom. 1999, 13, 1813-1817. Sechi, S .; Anal. Chem. 1998, 70, 5750-5158. Sechi, Salvatore. Rapid Commun. Mass Spectrom. 2002, 16, 1416-1424. Gehanne, S .; , Cecconi, D .; , Carboni, L .; Giorgio Righetti, P .; , Domenici, E .; And Handam, M .; Rapid Commun. Mass Spectrom. 2002, 16, 1692-1698. Wang, S .; Regnier, F .; E. J. et al. Chrom. A 2001, 924 (1-2), 345-357. Amini, A; Chakraborty, A; Regnier, Fred E .; . J. et al. Chrom. B. 2002, 772, 35-44. Wang, S .; , Zhang X. And Regnier, F .; E. J. et al. Chrom. A. 2002, 949, 153-162. Miragorskaya, O .; A. Kozmin, Y .; P. Titov, M .; I. Korner, R .; , Sonsen, C.I. P. And Roepstorff, P .; Rapid Commun. Mass Spectrom. 2000, 14, 1226-1232. Stewart, I .; I. Thomson, T; Figeys, D .; Rapid Commun. Mass Spectrom. 2001, 15, 2456-2465. Yao, X .; Freas, A; Ramirez, J .; Demirev, P .; A. Fenselau, C .; Anal. Chem. 2001, 73, 2836-2842. Peters, E .; C. Horn, E .; C. Tully, D .; C. And Block, A .; Rapid Commun. Mass Spectrom. 2001, 15, 2387-2392. Muenchbach, M .; , Quadroni, M .; Miotto, G .; And James, P .; Anal. Chem. 2000, 72, 4047-4057; Chakraborty, A; Regnier, F .; E. J. et al. Chrom. A 2002, 949, 173-184. Geng, M .; Ji, J .; Regnier, F .; E. . J. et al. Chrom. A 2000, 870, 295-313. Ji, J .; Chakraborty, A .; Geng, M .; Zhang, X .; Amini, A .; Bina, M .; Regnier, F .; J. et al. Chrom. B 2000, 745, 197-210. Goodlett, D .; R. Keller, A .; Watts, J .; D. Newt, R .; Yi, E .; C. Purvine, S .; Eng, J .; , Von Haller, P .; Aebersold, R .; And Kolker, E .; Rapid Commun. Mass Spectrom. 2001, 15, 1214-1221. Liu, P; Regnier, F .; E. J. et al. Proteome Res. 2002, 1, 443-450]. More recently, two groups have described covalent solid phase cysteine capture methods for mixture simplification [Zhou, H. et al. Ranish, J .; A. Watts, J .; D. Aebersold, R .; Nature Biotechnol. 2002, 20, 512-515. Qiu, Y; Sousa, E .; A. Hewick, R .; M.M. Wang, J .; H. Anal. Chem. 2002, 74, 4969-4979]. Several non-isotopic labeling methods using structure labeling [Cagney, G. et al. , Emili, A .; Nature Biotechnol. 2002, 20, 163-170. Beardsley, R .; L. , And Reilly, J. et al. P. J. et al. Proteome. Res. 2003, 2, 15-21], as well as methods for performing differential quantification by comparing the abundance of MS ions separated by chromatography between a sample of interest and a control [Bondarenko, P. V. , Cheleus, D .; And Shaler, T .; A. Anal. Chem. 2002, 74, 4741-4749. Cheleus, D .; Bondarenko. P. V. J. et al. Proteome Res. 2002, 1, 317-323]. A number of reviews compare the relative advantages and limitations of these several methods [Mosley, M., et al. A. Trends in Biotechnol. 2001, 19, S10-S16. Patton, W .; F. , Schuleenberg, B .; And Steinberg, T .; H. Curr. Opin. Biotechnol. 2002, 13, 321-328. Turecek, F .; J. et al. Mass Spectrom. 2002, 37, 1-14. Regnier, F .; E. Riggs, L .; Zhang, R .; Xiong, L .; Liu, P .; Chakraborty, A .; Seeley, E .; Sima, C .; Thompson, R .; A. J. et al. Mass Spectrom. 2002, 37, 133-145].

  The two major post-translational modifications (PTM's) of proteins are phosphorylation and glycosylation. Of these, reversible phosphorylation of proteins is positioned as the most important PTM that occurs in cells. Usually, two methods are used to enrich for phosphorylated proteins / phosphopeptides prior to their detection and subsequent detailed characterization. (I) Immunoprecipitation method; [Gronberg, M .; , Kristiansen, T .; Z. , Tensvalle, A .; , Andersen, J .; S. Ohara, O .; Mann, M .; Jensen, O .; N. And Pandey, A .; Mol. Cell. Proteomics. 2002, 1, 517-527] and (ii) immobilized metal ion affinity chromatography (IMAC); [Posewitz, M .; C. And Tempst, P .; Anal. Chem. 1999, 71, 2883-2892, Ficarro, Scott B. et al. McCleland, Mark L .; Stukenberg, P .; Todd; Burke, Daniel J. et al. Ross, Mark M .; Shabanowitz, Jeffrey; Hunt, Donald F .; White, Forest M .; Nat Biotechnol. 2002, 20, 301-305].

Proteins separated by 2D gels or 1D SDS-PAGE can be obtained by autoradiography or conserved phosphoimaging using ³² P labeling in vivo or in vitro [Ji H, Baldwin GS, Burgess AW, Moritz RL, Ward LD and Simpson. RJ. J Biol Chem 1993, 268, 13396-13405. Boyle W .; J. et al. , Geer Van der P. And Hunter T. Methods Enzymol. 1991, 201, 110-149. Yan J .; X. , Packer N. H. , Gory A. A. And Williams K.K. L. J. et al. Chromatogr. 1998, 808, 23-41], or detection by Western blotting using antibodies to detect phosphorylated proteins. Historically, sequence analysis of ³² P-labeled peptides is performed by Edman degradation [Wetttenhall, R .; E. Aebersold, R .; H. , And Hood, L .; E. Methods Enzymol 1991, 201, 186-99]. However, this method usually requires prior knowledge of the protein sequence in order to correlate the loss of radioactivity, which is an indicator of the phosphorylation site, with the amino acid sequence of the peptide, and the phosphorylation at a given site. When oxidation is stoichiometrically low [Katze, M .; G. Kwieczezewski, B .; , Goodlett, D .; R. Blakely, C .; M.M. , Nedderman, P .; Tan, SL, Abersold, R .; Virology. 2000, 278, 501-513], or when there are multiple different phosphorylated forms of the same protein [Storm, S .; M.M. , And Kawaja, X. Z. Brain. Res. Mol. Brain. Res. 1999, 71, 50-60], which can be disturbed.

More recently, mass spectrometry has been shown to be particularly useful for the analysis of protein phosphorylation [Neubauer, G. et al. And Mann, M .; Anal Chem 1999, 71, 235-242]. However, mass spectrometric analysis of phosphorylated peptides has shown that ionization efficiency is low in the cation MS analysis mode [Liao P. et al. C. Leykam J .; , Andrews P.M. C. , Cage D. A. And Allison J. et al. Anal. Biochem. 1994, 219, 9-20] and the difficulties encountered in rapidly changing ionization polarity to allow both identification (negative mode) and characterization (positive mode) during a single experimental step. [Janek K. Wenschuh H .; , Bienert M. et al. , And Krause E. Rapid Commun. Mass Spectrom. 2001, 15, 1593-1599. Ma Y. , Lu Y. Mo W .; , And Neubert T. A. . Rapid Commun. Mass Spectrom. 2001, 15, 1693-1700], which is more complex than the analysis for unmodified peptides. In addition, phosphoserine- and phosphothreonine-containing peptides are prone to detachment of phosphoric acid (H ₃ PO ₄ ) during ESI and MALDI ionization and during low energy CID.

There are several ways to avoid the difficulties associated with suppressing ionization of phosphorylated peptides in the positive ion mode. To reduce excess amounts of unmodified peptide that suppress ionization, the sample can be enriched for phosphorylated peptides as described above. However, despite attempts made to overcome the loss of signal in the analysis of phosphorylated peptides, there are still limitations due to phosphate side chain instability when attempting to identify phosphorylation sites by tandem mass spectrometry To do. A simple solution is to perform analysis of phosphorylated peptides by parallel analysis of samples treated with and without alkaline phosphatase [Larsen, M., et al. R. Sorensen, G .; L. , Fey, S .; J. et al. Larsen, P .; M.M. And Roepstorff, P .; Proteomics, 2001, 1, 223-238]. In addition, the identification of phosphorylated peptides is followed by a precursor ion scan mode that monitors the characteristic phosphate-specific product ions at m / z 79 (PO ₃ ⁻ ) and m / z 89 (H ₂ PO ₄ ⁻ ). By collision-induced dissociation (CID) of phosphorylated peptides in negative ion mode or by monitoring the desorption of H ₃ PO ₄ (98 Da) or HPO ₃ (80 Da) in cation neutral desorption scan mode [Carr S. A. , Huddleston M .; J. et al. And Anna R.R. S. Anal. Biochem. 1996, 239, 180-192. Schlosser A .; , Pipkorn R. Bossemeyer D. And Lehmann W. et al. D. Anal. Chem. 2001, 73, 170-176].

Product ion scan mode MS / MS for intact phosphopeptides may contain information about the specific position of the amino acid residue in which the b- and y-type fragments formed by cleavage of the peptide backbone are phosphorylated As such, the exact position of the phosphate within the peptide may be used to characterize it. However, as noted above, this method is often hampered by long-term dephosphorylation in the gas phase and removal of phosphoric acid (H ₃ PO ₄ , 98 Da) from phosphoserine and phosphothreonine, thereby It is difficult to accurately grasp the modification position. However, the phosphorylated tyrosine side chain is relatively stable under MS and MS / MS conditions because it has a relatively high stability due to aryl phosphate modification, thus the position of the phosphorylated tyrosine residue is 243 Da. It can be easily determined by the difference in mass between two consecutive fragment ions. In fact, the phosphotyrosine “reporter” immonium ion, which is characteristic at 216.043 Da, has been used in positive ion mode MS / MS precursor ion experiments for the selective identification of peptides containing phosphotyrosine. [Steen H. Kuster B. , Fernandez M .; , Pandey A. , And Mann M.M. 2001. Anal. Chem. 73: 1440-1448].

  A common method to overcome the instability of phosphoserine and phosphothreonine side chains in MS / MS is to replace the phosphate group with a more stable, less acidic side chain functional group. This can be achieved by β-removal of phosphoserine and phosphothreonine residues under strongly alkaline conditions such that dehydroalanine or dehydroaminobutyric acid groups are obtained, respectively. Subsequent Michael addition of the nucleophile allows a simple method to derivatize serine or threonine residues that have been previously phosphorylated prior to mass spectrometric analysis [Weckwerth, W. et al. , Willmitzer, L .; And Fiehn, O .; Rapid Commun. Mass Spectrom. 2000, 14, 1677-1681. Molloy, M .; P. And Andrews, P .; C. Anal. Chem. 2001, 73, 5387-5394. Li, W .; Boykins, R .; A. Bucklund, P .; S. , Wang, G .; And Chen, HC. Anal. Chem. 2002 74, 5701-5710. Steen, H .; And Mann, M .; J. et al. Am. Soc. Mass Spectrom. 2002, 13, 996-1003]. This method can also be used for incorporation of affinity “tags”, which allows derivatized peptides to be enriched prior to their analysis [Adamczzy, M. et al. Gebler, J .; C. And Wu, J .; Rapid Commun Mass Spectrom. 2001, 15, 1481-1488. Oda, Y .; Nagasu, T .; And Chait, B .; T.A. Nature Biotechnol. 2001, 19, 379-382] and can be used to quantify the degree of phosphorylation observed between two different sets of samples when combined with isotope labeling [Goshe M. et al. B. Conrads, T .; P. Panisko, E .; A. , Angel, N .; H. Venstra, T .; D. And Smith, R .; D. Anal Chem. 2001, 73, 2578-2586. Goshe, M .; B. Venstra, T .; D. Panisko, E .; A. Conrads, T .; P. , Angel, N .; H. And Smith, R .; D. Anal. Chem, 2002, 74, 607-616. Adamczyk, M; Gebler, J .; C. And Wu, J .; Rapid Commun. Mass Spectrom. 2002, 16, 999-1001].

  Note that β-removal has a similar effect on both O-phospho linkages and O-glycoside linkages. Therefore, the β-removal method cannot discriminate O-linked post-translational modifications of serine and threonine residues.

  Other chemical methods for phosphoproteome analysis (also applicable to phosphotyrosine-containing peptides and peptides containing phosphoserine and phosphothreonine residues) have been described recently [Zhou, H. et al. Watts, J .; D. And Aebersold, R .; Nature Biotechnol 2001, 19, 375-378]. For a recent review of quantitative phosphoproteome analysis see [Mann, M. et al. , Ong, S-E. Gronberg, M .; Stein, H .; Jensen, O .; N. And Pandey, A .; Trends Biotechnol. 2002, 20, 261-268].

  Measurement of protein-protein interactions is an important aspect in integrated biological research aimed at understanding the complex pathways involved in cell signaling and protein trafficking. Already, most of the known protein-protein interactions in yeast [Bader, G. et al. D. And Hogue, C.I. W. V. Nature Biotechnol. 2002, 20, 991-997] have been identified by a genome-scale yeast two-hybrid assay [Uetz, P. et al. Et al. Nature 2000, 403, 623-627. Ito, T .; , Chiba, T .; Ozawa, R .; Yoshida, M .; , Hattori, M .; And Sakaki, Y. et al. Proc. Natl. Acad. Sci. USA, 2001, 98, 4569-4574. Legrain, P .; Nature. Biotechnol. 2002, 20, 128-129], and (i) coprecipitation with recombinant proteins tagged with affinity tags [Gavin, AC. Nature 2002, 415, 141-147. Ho, Y .; Et al. Nature. 2002, 415, 180-183], (ii) “pull-down” techniques using antibodies to one of the constituent proteins, (iii) protein-affinity-interaction (eg, recombinant glutathione S-transferase (GST) — Direct affinity capture methods such as fusion proteins and glutathione-affinity chromatography), or (iv) isolation of intact multiple protein complexes (eg, nuclear pore complex, ribosome complex, and spliceosome) Has been identified. These latter methods, in combination with mass spectrometry for protein identification, constitute a major method for cell mapping proteomics, which is a function of all protein-protein interactions within a given cell ( The purpose is to explain (spatial and temporal interactions) [Blackstock, W. et al. P. And Weir, M .; P. Trends Biotechnol. 1999, 17, 121-127]. However, the interactions detected by these physical methods can include a number of non-specific interactions that have no biological significance [Bader, G. et al. D. And Hogue, C.I. W. V. Nature Biotechnol. 2002, 20, 991-997].

  Mass spectrometry combined with cross-linking [Rappsilber, J. et al. Siniossoglou, S .; , Hurt, E .; C. , And Mann, M .; Anal. Chem. 2000, 72, 267-275], or mass spectrometry combined with hydrogen / deuterium exchange [Yamada, N .; , Suzuki, E .; , And Hirayama, K .; Rapid Commun. Mass Spectrom. 2002, 16, 293-299] can be used for rapid low-resolution evaluation of the three-dimensional structure of proteins and protein complexes. Cross-linking usually involves chemical cross-linking of isolated protein complexes [Uy, R .; And Wald, F .; 1977, In: “Protein Cross-linking” (Friedman, M. Ed.), Plenum, New York. Fancy, D .; A. Current Opin. Chem. Biol. 2000, 4, 28-33] or photochemical cross-linking [Chowdhry, V .; And Westheimer, F .; H. , Annu. Rev. Biochem. 1979, 48, 293-325. Fancy, D .; A. And Kodadek, T .; Proc. Natl. Acad. Sci. USA 1999, 96, 6020-6024], which then includes proteolytic digestion and MS and / or MS / MS analysis of the resulting peptide mixture to bring adjacent regions of the examined protein close together . However, the high number of peptide species that occurs following digestion of cross-linked proteins often makes it difficult to quickly and unambiguously identify cross-linked peptides. Many different groups have developed methods to partially overcome this limitation. The method involves prior reduction with a cross-linking agent capable of cleaving thiols [Bennett, K. et al. L. Kussmann, M .; Bjork, P .; , Godzwon, M .; Mikkelsen, M .; Sorensen, P .; And Roepstorff, P .; Prorein Sci. 2000, 9, 1503-1518], use of isotope labeling techniques [Chen, X. et al. Chen, Y .; H. , And Anderson, V .; E. Anal. Biochem 1999, 273, 192-203. Pearson, K .; M.M. Panell, L .; K. And Fales, H .; M.M. Rapid Commun. Mass Spectrom. 2002, 16, 149-159. Muller, D .; R. Schindler, P .; Towbin, H .; , Wirth, U.S. , Voshol, H .; , Hoving, S .; , And Steinmetz, M .; O. Anal. Chem. 2001, 73, 1927-1934. Back, J .; W. , Notenboom, V .; , De Koning, L. J. et al. , Muijsers, A .; O. , Sixma, T .; K. , De Koster, C.I. G. , And de Jong, L. Anal. Chem. 2002, 74, 4417-4422. Taberner, T .; Hall, N .; E. , O'Hair, R. A. J. et al. And Simpson, R .; J. et al. J. et al. Biol. Chem. 2002, 277, 46487-46492], or incorporation of unstable MS / MS “tags” into the crosslinker itself [Back, J. et al. W. , Hartog, A .; F. Dekker, H .; L. , Muijsers, A .; O. , De Koning, L. J. et al. And de Jong, L .; J. et al. Am. Soc. Mass Spectrom. 2001, 12, 222-227], etc.

  It is important to recognize that all of the above methods for selective identification and differential quantification based on the incorporation of the differential isotope “signature” depend on general methods for peptide identification. That is, the isotope signature of the peptide ion of interest is detected by mass spectrometry of its intact precursor ion. Therefore, the limitations of this method occur when: (I) When the abundance of ions of interest is low (ie, close to or below the level of chemical noise present in the spectrum) [Kruchinsky, A .; N. And Chait, B .; T.A. J. et al. Am. Soc. Mass Spectrom. 2002, 13, 129-134], (ii) when the mass of a less abundant peptide with a different label overlaps with the mass of other abundant peptides in the mixture, (iii) with a different label When separation of “heavy” and “light” peptides occurs in the chromatographic fraction of the peptide mixture [Zhang, R .; Sima, C .; S. Wang, S .; Regnier, F .; E. Anal. Chem. 2001, 73, 5142-5149, Zhang, R .; , Sima, C .; S. Thompson, R .; A. Xiong, L .; Regnier, F .; E. Anal. Chem. 2002, 4, 3661-3669. Zhang, R .; And Regnier, F .; E. J. et al. Proteome Res. 2002, 1, 139-147], or (iv) when the mass spectrometer does not have sufficient resolution to adequately separate the two labeled components so that they cannot be detected .

  Tandem mass spectrometry (MS / MS) dissociation method [McLuckey, S .; A. And Goeringer, D .; E. J. et al. Mass Spectrom. 1997, 32, 461-474] (by which the mass of the target precursor ion is selected and subjected to fragmentation via collision-induced dissociation (CID)), and then the mass of the resulting product ion is At least some of the limitations described above can be addressed by using structural information to derive structural information about the amino acid sequence or analyzed to indicate the presence and location of post-translational modifications. Reduced chemical noise associated with the formation of product ions at different m / Z values than those for the selected precursor ions would result in successful detection and detection of low abundance ions that would otherwise not be apparent in the mass spectrum. Can be analyzed. In fact, the precursor ion MS / MS scan mode [Schwartz, J. et al. C. Wade, A .; P. Enke, C .; G. Cooks, R .; G. Anal. Chem. 1990, 62, 1809-1818. Schlosser A .; , Pipkorn R. Bossemeyer D. , And Lehmann W. D. 2001. Anal. Chem. 73: 170-176. McClellan, J .; E. Quarmby, S .; T.A. Yost, R .; A. Anal. Chem. 2002, 74, 5799-5806], or the neutral loss MS / MS scan mode [Schwartz, J. et al. C. Wade, A .; P. Enke, C .; G. Cooks, R .; G. Anal. Chem. 1990, 62, 1809-1818. Wilm M; Neubauer G; Mann M .; Anal. Chem. 1996, 68, 527-33. McClellan, J .; E. Quarmby, S .; T.A. Yost, R .; A. Anal. Chem. The operation of tandem mass spectrometry in 2002, 74, 5799-5806] allows the mass spectrometer to detect low mass product ions for diagnosis, or product ions that are corrected by being given mass from precursor ions. Although set, each has been shown to improve the sensitivity of conventional MS-based detection methods by 1-2 orders of magnitude [Wilm M; Neubauer G; Mann M. et al. Anal. Chem. 1996, 68, 527-533]. By using these precursor ions or neutral desorption scan modes, the presence of peptides containing specific structural features is shown through the formation of diagnostic “signature ions” formed during CID MS / MS be able to. This allows the scan mode to provide greater specificity compared to MS-based methods and overcome the problem of low abundance peptides overlapping with other more abundant peptides present in the mixture.

However, one of the limitations of MS / MS-based methods is that fragmentation that gives rise to product ions of interest or neutral desorption is usually only one of many dissociation channels, whereby the spectrum is “diluted”. Can limit sensitivity and complicate subsequent spectral interpretation. Furthermore, no MS / MS method for quantification of differential protein expression is described.

  We have fixed charge derivatization of selected amino acids, peptides or proteins with specific structural features (eg, side chains of selected amino acids, or those side chains including post-translational modifications and crosslinkers) A novel method has been developed for protein identification, differential quantification, and analysis of post-translational modification states and crosslinking, comprising The method, in a preferred embodiment, is capable of solving all the MS and MS / MS limitations described above, and is a key requirement for various existing methods based on MS, selectivity and An improvement in sensitivity is expected.

  Derivatization methods for mass spectrometry are common and have been reviewed previously [Knapp, D. et al. R. Methods Enzymology 1990, 193, 314-329. Anderegg, R .; J. et al. Mass Spectrom. Rev. 1988, 7, 395-424. Roth, K .; D. W. , Huang, ZH. , Sadagopan N, and Watson J. et al. T.A. Mass Spectrom. Rev. 1998, 17, 255-274. Sadagopan, N .; And Watson J. et al. T.A. J. et al. Am. Soc. Mass. Spectrom. 2001, 12, 399-409. Jones, M .; B. Jeffrey, W .; A. Hansen, H .; F. , Pappin, D.M. J. et al. C. Rapid Commun. Mass Spectrom. 1994, 8, 737-42. Spengler, B .; Luetzenkirchen, F .; Metzger, S .; , Chaurand, P .; Kaufmann, R .; Jeffery, W .; Bartlet-Jones, M .; And Pappin, D .; J. et al. C. Int. J Mass Spectrom. Ion Proc. 1997, 169/170, 127-140; , Lacey, M .; P. , And Youngquist, R .; S. Rapid. Commum. Mass Spectrom. 2000, 14, 2348].

  Fixed charge derivatization of peptides is used continuously with tandem mass spectrometry and is used for sequence analysis applications, but all previous work produces a specific series of basic backbone cleavages derived from sequence ions To concentrate on fragmentation (ie maximizing sequence cleavage). And it was limited to derivatization of N-terminal and C-terminal and lysine and arginine side chains.

On the other hand, the present invention is based on a method of fixed charge derivatization and is designed to allow dissociation towards a single predictable fragmentation pathway, producing a single product, resulting in precursor ions or MS / MS with a method of scanning neutral desorption allows it to be selectively identified from complex mixtures, which can then be used with further MS / MS, or multi-stage MS / MS (MS ⁿ ) Or subjected to further structural investigation by examining the “exact mass tag” of the precursor ion or product ion to allow its characterization [Conrads, T .; P. , Anderson, G. A. Venstra, T .; D. Pasa-Tolic, L .; And Smith, R .; D. Anal. Chem. 2000, 72, 3349-3354. Goodlet, D .; R. Bruce, J .; E. , Anderson, G. A. Rist, B .; Pasa-Tolic, L .; , Fiehn, O .; Smith, R .; D. And Aebersold, R .; Anal. Chem. 2000, 72, 1112-1118. Strittmatter, E .; F. Ferguson, P .; L. Tang, K .; And Smith, R .; D. J. et al. Am. Soc. Mass Spectrom. 2003, 14, 980-991].

The general principle behind the use of fixed charge peptide ion derivatization methods to perform CID MS / MS fragmentation is based on the selective identification and differential quantification of side chain fixed charge sulfonium ion derivatives of peptides containing methionine. Specified in the description (fixed charge sulfonium ion derivatives of the amino acids methionine and cysteine fragment only through neutral elimination of the side chain CH ₃ SR (R is a substituted alkyl group). [Reid, G. E., Simpson, R. J. and O'Hair, R. A. J. J. Am. Soc. Mass Spectrom. 2000, 11, 1047-1060 .; O'Hair R.A.J. and Reid, G.E.Eur.Mass Spectrom. 5-334], has been adopted in the past for the purpose of combining the gas phase of potential product ions contained in the side chain fragmentation reaction of amino acids independently). Similar methods for selective analysis of peptides containing other amino acids, methods containing post-translationally modified amino acids, and methods for characterizing cross-linked peptides are also described.

The present invention is an amino acid, peptide or protein analysis method comprising:
(1) derivatizing a mixture of amino acids, peptides or proteins to form one or more amino acids, peptides or proteins derivatized to contain fixed charge ions at sites other than the C-terminus or N-terminus;
(2) introducing a mixture of one or more amino acids, peptides or proteins derivatized to contain fixed charge ions at sites other than the C-terminal or N-terminal, a peptide or protein mixture into a mass spectrometer;
(3) A mixture of amino acids, peptides or proteins containing one or more amino acids, peptides or proteins derivatized to contain fixed charge ions at sites other than the C-terminal or N-terminal is passed through the first mass resolving analyzer. Selecting a protein or peptide precursor ion having a first mass to charge ratio;
(4) dissociating the precursor ion having the first mass-to-charge ratio to generate a product ion having a second mass-to-charge ratio, which is a characteristic of fragmentation occurring in a site near the fixed charge; and (5) An analysis method comprising: detecting a product ion having a second mass-to-charge ratio.

  A product ion having a characteristic second mass-to-charge ratio is a charged amino acid, peptide or protein, a product ion formed by neutral elimination of a fixed charge from a precursor ion, or a charge of a fixed charge from a precursor ion Any of the product product ions formed by a charged loss.

  Preferably, the method of the present invention further comprises the step of (6) determining the identity of the derivatized peptide or protein.

  Step (6) is performed by the first iteration steps (1), (2), (3) and (4), and then (i) from a precursor that will have the same charge state as the precursor has. Product ion complementary to the charged product that is generated by neutral desorption or by (ii) charge desorption from the precursor ion and corresponds to a protein or peptide containing a product ion having a lower charge state than the precursor Is carried out by subjecting a product ion having a characteristic second mass-to-charge ratio formed by a further dissociation step, the purpose of which is to examine the amino acid sequence of the peptide and subsequently identify the protein of origin Forming a series of product ions having a mass to charge ratio in a predetermined range.

  Alternatively, step (6) has a mass accuracy of about 1-5 ppm with respect to the product ions detected in step (5) or their complementary product ions (ie to obtain an “accurate product ion mass tag”). This is done by using a high resolution mass spectrometer. This step is performed in conjunction with a database search and can be a subsequent tool for identification of peptides found to contain fixed charge derivatives. In the past, when an “exact product ion mass tag” for a precursor ion has been obtained, and when the presence of a particular amino acid (eg, the presence of a cysteine residue) is known, The specificity is improved, and it is possible to clearly identify the protein from which the peptide is derived from only this information.

  Amino acids, peptides or proteins may be dissociated by any suitable dissociation method, including but not limited to (i) collision with inert gas (collision-induced dissociation (CID) or collision activation) (Known as dissociation (CAD)), (ii) collision with the surface (known as surface-induced dissociation or SID), (iii) photodissociation by interaction with photoparticles (eg via a laser), (iv) Thermal / Blackbody Infrared Radiation Dissociation (BIRD), and (v) Electron-induced dissociation (EID) for monovalent cations, electron capture dissociation (ECD) for multivalent cations or combinations thereof by interaction with an electron beam including.

  The analysis method of the present invention can be used for identification of amino acids, peptides or proteins, differential quantification, analysis of post-translational modification states, analysis of crosslinking states and protein interactions.

  The present invention provides a method for analyzing an amino acid, peptide or protein containing a fixed charge at a site other than the C-terminal or N-terminal.

  The fixed charge derivative is included in the side chain of the selected amino acid residue or in the side chain of the selected amino acid residue included in the protein or peptide. Preferably, the selected amino acid residue is a “rare” amino acid as detailed below. Fixed charge derivatives can be included in the side chain of post-translationally modified amino acid residues, as described in detail below. Fixed charge derivatives can be included in the cross-linking between two proteins or peptides, as detailed below.

  The selected amino acid residue may contain an S atom in its side chain. Preferred amino acid residues are methionine, cysteine, homocysteine or selenocysteine. The selected amino acid residue can also be tryptophan or tyrosine. The side chain may contain an S-alkyl group. Preferred amino acid residues are methionine, S-alkylcysteine, S-alkylhomocysteine, S-alkyltryptophan or S-alkyltyrosine. Derivatization of the side chain of the amino acid residue selected to introduce charge can be achieved using various methods known to those skilled in the art.

  The fixed charge is an O-linked post-translationally modified amino acid residue in the peptide or protein (eg, a dehydroalanine residue formed by β-removal from an O-linked post-translationally modified serine amino acid residue, or O- On the dehydroamino-2-butyric acid residue formed by β-removal from the attached post-translationally modified threonine amino acid residue. Derivatization of previously post-translationally modified amino acid residues to introduce charge can be accomplished using various methods known to those skilled in the art.

  A fixed charge may also be included in the cross-linking reagent or in the cross-link between two amino acids, peptides or proteins. Derivatization of cross-linking reagents or cross-links between two amino acids, peptides or proteins to introduce charge can be achieved using various methods known to those skilled in the art.

  Non-limiting examples of fixed charges include sulfonium ions, quaternary alkyl ammonium ions, or quaternary alkyl phosphonium ions.

  Analysis of amino acid, peptide or protein ions can be performed by tandem mass spectrometry. The tandem mass spectrometer can be equipped with an electrospray ionization (ESI) interface or a matrix-assisted laser desorption ionization (MALDI) interface for converting protein or peptide ions from the liquid phase to the gas phase. Mass spectrometers applicable to tandem mass spectrometry fall into two basic categories: tandem in-space mass analyzers and tandem in time mass spectrometers. Combinations of these types can also be used. A tandem in-space mass spectrometer has a separate mass spectrometer for each stage of mass spectrometry; examples include sector-type mass analyzers (typically dual-converging sector-type quality and sector-type instruments and four Hybrid type combining a quadrupole device), a time-of-flight mass spectrometer and a triple quadrupole mass analyzer, and a hybrid mass spectrometer combining a time-of-flight device and a quadrupole device It is. A tandem-in-time mass spectrometer has only one mass spectrometer, and each stage of mass spectrometry occurs in the same place, but they are separated in time through a sequence of processing steps. Tandem-in-time mass spectrometers include two-dimensional and three-dimensional quadrupole ion trap mass spectrometers and Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers.

  The method of the invention in certain embodiments includes one or more protein extraction steps, protein separation steps, cysteine disulfide reduction and alkylation steps, and / or digestion steps.

The method of derivatization of the fixed charge in the method of the present invention involves the incorporation of an appropriate isotope label (eg ¹³ C, ¹⁵ N, and ² H) into the fixed charge, or differential protein expression based on structural labeling. It can be applied to quantification. The method of fixed charge derivatization in the method of the present invention can also be applied to the identification and quantification of post-translational modification states of proteins, which are stable derivatives in mass spectrometry, O-phosphorylation and O-glycosylation Incorporation of serine or fixed charge derivatives via the β-removal / Michael addition method to form O-phosphorylated and O-glycosylated threonine is described in, for example, Meyer, H. et al. Hoffman-Posorske, E .; Korte, H .; And Heilmeyer, L .; J. et al. FEBS. Lett. 1986, 204, 61-66. , The disclosure of which is incorporated herein by reference. The method of derivatization of the fixed charge in the method of the present invention can also be applied to identify and characterize protein-protein interactions through incorporation of the fixed charge derivative into an appropriate cross-linking reagent.

  As already mentioned above, tandem mass spectrometry of fixed charge derivatized peptides causes limited production of product ions during dissociation, which is characteristic of fragmentation at fixed charge sites during dissociation, thereby complicating complex Enables specific identification of peptides alone, including derivatization, without the need to pre-separate or concentrate the mixture prior to analysis.

  The methods of the present invention are described in certain embodiments, with reference to selective identification and differential quantification of peptides present in different proportions, and by incorporating isotopically labeled fixed charge derivatives. Available in “light” (including only natural isotopes) and “heavy” (including isotopes or structural labels in substituents), and tandem mass spectrometry in neutral elimination mode for peptide level selection Identification and differential quantification are performed. Application of general methods of selective identification of protein-protein interactions by post-translational modification states as well as cross-linking analysis is also disclosed.

  In a preferred embodiment of the present invention, the above method is carried out using an amino acid, peptide or protein derivatized with a substituted acetophenone of the formula: or a salt or solvate thereof.

In the formula, X is preferably halogen, sulfonic acid ester, perchloric acid ester or chlorosulfonic acid ester. In certain embodiments, R ₁ -R ₅ are H and R ₁ ′ -R ₆ ′ are ¹² C. In another embodiment, the substituted acetophenone is an isotope-labeled substituted acetophenone or a salt thereof or a solvate thereof, and preferably 1 or more, more preferably 2 or more, and even more preferably among R _{1 to} R _5. 3 or more is ² H, R ₁ ′ to R ₆ ′ is ¹² C, or R _{1 to} R ₅ is H, and any one or more of R ₁ ′ to R ₆ ′, preferably 2 or more More preferably, 3 or more is ¹³ C.

In certain embodiments, any one or more of R _{1 to} R ₅ is a functional group containing an atom other than hydrogen or carbon.

Preferably, the substituted acetophenone is water soluble. Specific water soluble molecules include R ₁ is SO ₂ H, R _{2 to} R ₅ are H, R ₁ ′ to R ₆ ′ are ¹² C, or R ₁ is H and R ₂ is SO ₂ H R _{3 to} R ₅ are H and R ₁ ′ to R ₆ ′ are ¹² C, or R _1-2 is H, R ₃ is SO ₂ H, R _{4 to} R ₅ are H, R ₁ ′ to R ₆ ′ is ¹² C, R ₁ is SO ₃ H, R _{2 to} R ₅ are H, R ₁ ′ to R ₆ ′ are ¹² C, or R ₁ is H , R ₂ is SO ₃ H, R _{3 to} R ₅ are H, R ₁ ′ to R ₆ ′ are ¹² C, or R _1-2 is H, R ₃ is SO ₃ H, R Including those molecules described above wherein _{4 to} R ₅ are H and R ₁ ′ to R ₆ ′ are ¹² C.

In the isotopically labeled water-soluble substituted acetophenone molecule, R ₁ is SO ₂ H, any one or more of R _{2 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² Or R ₁ is SO ₂ H, R _{2 to} R ₅ are H, and any one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C, or R ₂ is SO _{2 2} H, any one or more of R ₁ and R _{3 to} R ₅ , preferably 3 or more is ² H, R ₁ ′ to R ₆ ′ is ¹² C, or R ₂ is SO ₂ H, R ₁ and R _{3 to} R ₅ are H and any one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C, or R ₃ is SO ₂ H, and R _{1 to} R ₂ and R ₄ to R ₅ in any one or more, preferably 3 or more ² _H, or R 1 'to R _6' is ¹² C, in _{R 3} is SO _{2 H, R} 1 ~ ₂ and _R 4 to R ₅ is _H, R 1 'to R _6' to any one or more, or preferably 3 or more ¹³ C, in _{which R 1} is SO _{3 H,} either R 2 to R ₅ 1 or more, preferably 3 or more is ² H, R ₁ ′ to R ₆ ′ is ¹² C, or R ₁ is SO ₃ H, R _{2 to} R ₅ are H, and R ₁ ′ to R ₆ ′, Preferably 3 or more is ¹³ C, R ₂ is SO ₃ H, R ₁ and any _one of R _{3 to} R ₅ , preferably 3 or more is ² H, R ₁ ′ to R ₆ ′ is ¹² C, R ₂ is SO ₃ H, R ₁ and R _{3 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more or There is a ¹³ C, in _{R 3} is SO _{3 H,} R 1 to R ₂ and _R 4 to R ₅ or one or more, preferably 3 or more ^{_{2 H, R 1 '~R 6}} ' Or a ¹² C, or _{R 3} in SO _{3 H,} in R 1 to R ₂ and _R 4 to R ₅ is _H, R 1 '~R _6' any one or more, preferably 3 or more ¹³ C Including those described above.

  It will be appreciated that other combinations and other amounts of the listed substitutions are possible in these molecules, and the present invention encompasses such combinations.

  According to another aspect of the present invention, the substituted acetophenone (or salt thereof, or solvate thereof) is represented by the following formula:

In the formula, X is a sulfonic acid ester, a perchloric acid ester or a chlorosulfonic acid ester. Preferred substituted acetophenones are those in which R _{1 to} R ₅ are H and R ₁ ′ to R ₆ ′ are ¹² C.

The present invention also includes substituted acetophenones that are isotopically labeled substituted acetophenones or salts or solvates thereof. Certain preferred substituted isotope-labeled acetophenones are any one or more of R _{1 to} R ₅ , preferably 3 or more is ² H and R ₁ ′ to R ₆ ′ is ¹² C, or R _{1 to} R _5. Is H and any one or more, preferably 3 or more, of R ₁ ′ to R ₅ ′ is ¹³ C.

  The present invention further includes a water-soluble substituted acetophenone of the following formula or a salt thereof or a solvate thereof.

  In certain embodiments, X is a halogen, sulfonate ester, perchlorate ester or chlorosulfonate ester. In preferred embodiments, X is Br or I.

In other embodiments, the water-soluble substituted acetophenone is R ₁ is SO ₂ H, R _{2 to} R ₅ are H, R ₁ ′ to R ₆ ′ are ¹² C, or R ₁ is H, R ₂ is SO ₂ H, R _{3 to} R ₅ are H, R ₁ ′ to R ₆ ′ is ¹² C, or R _1-2 is H, R ₃ is SO ₂ H, R _{4 to} R ₅ is H and R ₁ ′ to R ₆ ′ is ¹² C, or R ₁ is SO ₃ H, R _{2 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C, R ₁ is H, R ₂ is SO ₃ H, R _{3 to} R ₅ are H, R ₁ ′ to R ₆ ′ are ¹² C, or R _1-2 is H and R ₃ is SO ₃ H, R _{4 to} R ₅ are H and R ₁ ′ to R ₆ ′ are ¹² C.

The water-soluble substituted acetophenone can also be in the isotopically labeled form described herein, preferably X is a halogen, sulfonate ester, perchlorate ester or chlorosulfonate ester . In particular, the isotope-labeled water-soluble substituted acetophenone has R ₁ of SO ₂ H, one or more of R _{2 to} R ₅ , preferably 3 or more of ² H, and R ₁ ′ to R ₆ ′ of ¹² Or R ₁ is SO ₂ H, R _{2 to} R ₅ are H, and any one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C, or R ₂ is SO _{2 2} H, any one or more of R ₁ and R _{3 to} R ₅ , preferably 3 or more is ² H, R ₁ ′ to R ₆ ′ is ¹² C, or R ₂ is SO ₂ H, R ₁ and R _{3 to} R ₅ are H and any one or more of R ₁ ′ to R ₅ ′, preferably 3 or more is ¹³ C, or R ₃ is SO ₂ H, and R _{1 to} R ₂ and R ₄ to R ₅ in any one or more, preferably 3 or more ² _H, or R 1 'to R _6' is ¹² C, in _{R 3} is SO _{2 H, R} 1 ~ ₂ and _R 4 to R ₅ is _H, R 1 'to R _6' to any one or more, or preferably 3 or more ¹³ C, in _{which R 1} is SO _{3 H,} either R 2 to R ₅ 1 or more, preferably 3 or more is ² H, R ₁ ′ to R ₆ ′ is ¹² C, or R ₁ is SO ₃ H, R _{2 to} R ₅ are H, and R ₁ ′ to R ₆ ′, Preferably 3 or more is ¹³ C, R ₂ is SO ₃ H, R ₁ and any _one of R _{3 to} R ₅ , preferably 3 or more is ² H, R ₁ ′ to R ₆ ′ is ¹² C, R ₂ is SO ₃ H, R ₁ and R _{3 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more Is ¹³ C, or R ₃ is SO ₃ H, one or more of R _{1 to} R ₂ and R _{4 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C. Or R ₃ is SO ₃ H, R _{1 to} R ₂ and R _{4 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C. Is.

  It will be appreciated that other combinations and other amounts of the listed substitutions are possible in these molecules, and the present invention encompasses such combinations.

  In another aspect, the present invention includes a reagent kit for the analysis of amino acids, peptides or proteins by mass spectrometry. The kit includes one or more containers containing a substituted acetophenone as described herein.

  A particular reagent kit for the analysis of amino acids, peptides or proteins by mass spectrometry of the present invention comprises a container containing a substituted acetophenone of the formula: or a salt or solvate thereof.

In which X is a halide, preferably X is Br or I. Certain preferred substituted acetophenones in the above kit have R _{1 to} R ₅ as H and R ₁ ′ to R ₆ ′ as ¹² C. The preferred substituted acetophenone in other kits is an isotope-labeled substituted acetophenone or a salt thereof or a solvate thereof, preferably any one or more of R _{1 to} R ₅ , preferably 3 or more is ² H. , R ₁ ′ to R ₆ ′ are ¹² C, or R _{1 to} R ₅ are H and any one or more of R ₁ ′ to R ₆ ′, preferably 3 or more are ¹³ C. . Water-soluble derivatives of the substituted acetophenones listed above can also be included in the kit. Instructions for use of the substituted acetophenone in the analysis of amino acids, peptides or proteins by mass spectrometry can also be included.

  The kit can further include one or more containers containing a cysteine disulfide reducing agent, a cysteine alkylating reagent, a protease or chemical cleavage agent, and / or a solvent. The cysteine disulfide reducing agent preferably comprises dithiothreitol (DTT), β-mercaptoethanol, tris-carboxyethylphosphine (TCEP), and / or tributylphosphine (TBP). The cysteine alkylating reagent preferably comprises an alkyl halide (eg iodoacetic acid, iodoacetamide), vinyl pyridine and / or acrylamide. The protease or chemical cleavage agent is preferably trypsin, endoproteinase Lys-C, endoproteinase Asp-N, endoproteinase Glu-C, pepsin, papain, thermolysin, cyanogen bromide, hydroxylamine hydrochloride, 2- [2 ′ -Nitrophenylsulfenyl] -3-methyl-3'-bromoindole (BNPS-skatole), iodosobenzoic acid, pentafluoropropionic acid, and / or dilute hydrochloric acid. The solvent preferably comprises urea, guanidine hydrochloride, acetonitrile, methanol and / or water.

  The invention also includes amino acids that are derivatized to include side chain fixed charge sulfonium ions, quaternary alkyl ammonium ions, or quaternary alkyl phosphonium ions, or peptides that include such amino acids. Preferably, the amino acid is derivatized with a substituted acetophenone as described herein. In a particularly preferred embodiment, the amino acid is derivatized with a substituted acetophenone of the formula: or a salt or solvate thereof.

In which X is a halide, preferably X is Br or I. Certain preferred substituted acetophenones used to derivatize amino acids or peptides are R _{1 to} R ₅ are H and R ₁ ′ to R ₆ ′ are ¹² C. Other preferred substituted acetophenones used to derivatize amino acids or peptides are isotopically labeled substituted acetophenones or salts or solvates thereof, preferably one or more of R _{1 to} R ₅ , preferably 3 or more is ² H and R ₁ ′ to R ₆ ′ is ¹² C, or R _{1 to} R ₅ is H and any one or more of R ₁ ′ to R ₆ ′, preferably 3 or more ¹³ C. The water-soluble derivatives of the substituted acetophenones listed above can be used to derivatize amino acids or peptides. In a preferred embodiment, the amino acid derivative is isotopically labeled.

  In another aspect, the present invention provides a method for providing an internal standard in mass spectrometry, which comprises adding a predetermined amount of the above-described amino acid or peptide derivatized with a fixed charge to a sample.

  The present invention will now be described with reference to particular embodiments, but the method of the invention is not limited to these particular embodiments.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood in the technical field to which this invention belongs.

  “Fixed charge” as used herein, derivatization to the addition of any component, to a specific heteroatom contained in a protein or peptide, or (eg, in solution or in the gas phase) The specific heteroatom contained within the reagent contains any localized charge.

  “Fixed charge derivatization” as used herein means the introduction of a fixed charge as defined above. For example, by introducing a neutral reagent and then forming a fixed charge at a specific site within the protein or peptide, or by introducing a reagent containing a fixed charge at a specific site within the protein or peptide, A fixed charge can be introduced.

  The fixed charge derivative thus formed preferably has a structure that allows exclusive formation of product ions at the time of desorption characteristic of the fixed charge derivative.

  “Protein” as used herein means any protein including, but not limited to, peptides, enzymes, glycoproteins, hormones, receptors, antigens, antibodies, growth factors, and the like. To do. Proteins may be endogenous or produced from other proteins by chemical or proteolytic cleavage. A preferred protein is a protein consisting of at least 15 to 20 amino acid residues. The term also includes cross-linked proteins.

  “Peptide” as used herein includes any substance consisting of two or more amino acids, and according to the number of amino acids linked by amide bonds, di-, tri-, oligo and poly Includes peptides and the like. Peptides may be endogenous or produced by chemical or proteolytic cleavage from other peptides or proteins. A preferred peptide is a peptide consisting of 15 to 20 amino acid residues. The term also includes cross-linked peptides.

  When the amino acid is an α-amino acid, either the L-optical isomer or the D-optical isomer can be used. The L-isomer is usually preferred. For a general review see [Spatola, A. et al. F. , In Chemistry and Biochemistry of amino acids, peptides and proteins. 1983, B.E. Weinstein, Hen, Marc Dekker, New York, p. 267].

  “Alkyl” as used herein means a branched or unbranched, saturated or unsaturated monovalent hydrocarbon. Suitable alkyl groups include, for example, structures containing one or more methylene, methine and / or mesine groups. The branched chain structure has a branch motif similar to i-propyl, t-butyl, i-butyl, 2-ethylpropyl and the like. As used herein, the term includes “substituted alkyl” and “cyclic alkyl”.

“Substituted alkyl” refers to one or more substituents such as alkyl, aryl, acyl, halogen (ie alkyl halogen, eg CF ₃ ), hydroxy, amino, amide, alkoxy, alkylamino, acylamino, thioamide, acyloxy, The above alkyls including aryloxy, aryloxyalkyl, ether, ester, disulfide, mercapto, thia, aza, oxo, saturated and unsaturated cyclic hydrocarbons, heterocyclic rings and the like are meant. These groups may be bonded to any carbon atom or substituent of the alkyl moiety. Furthermore, these groups may overhang from the alkyl chain or may be incorporated. In order to pre-enrich in a solid phase to which a fixed charge derivative is covalently bound, the substituted alkyl group is covalently bound to an insoluble bead or polymer and chemically bonded between the insoluble bead or polymer and the alkyl group. Or photochemical cleavage sites can be included.

  The term “aryl” is used herein to mean an aromatic substituent, which may be a single aromatic ring or covalently linked together. It may be a condensed or multi-aromatic ring bonded to a common group such as a methylene or ethylene moiety. The common linking group may also be carbonyl in the case of acetophenone. Aromatic rings can include, for example, phenyl, naphthyl, biphenyl, diphenylmethyl, and benzophenone, among others. The term “aryl” includes “arylalkyl” and “substituted aryl”.

“Substituted aryl” means one or more functional groups, for example, the above aryls including lower alkyl, acyl, halogen, alkylhalo (eg, CF ₃ ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, phenoxy, mercapto As well as saturated and unsaturated cyclic hydrocarbons fused to an aromatic ring linked by a covalent bond or linked to a common group such as a methylene or ethylene moiety. The common linking group can also be carbonyl in the case of cyclohexyl phenyl ketone. The term “substituted aryl” includes “substituted arylalkyl”.

  The term “arylalkyl” means herein a subset of “aryl”, characterized in that an aryl group is attached to another group by an alkyl group as defined herein. Use it as something to do.

  “Substituted arylalkyl” is defined as a subset of the “substituted aryl” characterized in that a substituted aryl group is attached to another group by an alkyl group as defined herein.

  The term “acyl” is used to represent a ketone substituent — (O) R where R is alkyl or substituted alkyl, aryl or substituted aryl as defined herein.

Abbreviation CID Collision-induced dissociation ESI Electrospray ionization MALDI Matrix-assisted laser desorption ionization MS Mass spectrometry MS / MS Tandem mass spectrometry MS ⁿ Multistage tandem mass spectrometry (n> 2).

Materials and methods
Material model synthetic peptides (GAILMGGAILA (SEQ ID NO: 1), GAILMGAILK (SEQ ID NO: 2), GAILMGAILR (SEQ ID NO: 3), GAILAGAILA (SEQ ID NO: 4), GAILAGAILK (SEQ ID NO: 5) and GAILAGAILR (SEQ ID NO: 6) are Auspep (Melbourne, Vic, Australia) and used directly without further purification. Iodomethane, iodoethane, iodobenzene, iodomethylbenzene, d ₅ -acetophenone and bromoacetophenone were purchased from Aldrich (Castle Hill, NSW, Australia). Polymer supported pyridyl bromide perbromide, iodoacetic acid and iodoacetamide were obtained from Sigma (St. Louis, MI, USA). Methanol and acetonitrile (Chromar grade) were purchased from Mallinkrodt (Paris, KY, USA). Formic acid and acetic acid were obtained from the BDH laboratory (Pool, England). All solutions were prepared using deionized water purified by a tandem Milli-Q / Milli-RO system (Millipore, Bedford, MA, USA).

d ₅ - Synthesis d ₅ of bromoacetophenone - bromoacetophenone Frechet et al method [Frechet, J. M.M. J. et al. Farall, M .; J. et al. And Nuyens, L .; J. et al. J. et al. Macromol. Sci. -Chem. 1977, A11, 507-514]. Briefly, 1.25 mL of d ₅ -acetophenone is added to 30 mL methanol containing 4.8 g polymer supported pyridyl bromide perbromide (about ₃ meq Br ₃ ⁻ / resin 1 g) and then stirred at room temperature for 4 hours. It was made to react. The α-bromoketone obtained was obtained in pure form by filtering the reaction mixture and evaporating the solvent and then recrystallized before use.

Side Chain Fixed Charge Derivatization of Methionine-Containing Peptide Side chain fixed charge sulfonium ion derivative of methionine-containing peptide is 10 μL of 1M solution alkylating agent and 100 μg of peptide containing 30% CH ₃ CN containing 20% acetic acid aqueous solution 100 μL. It was prepared by adding to The reaction was allowed to proceed for 16 hours at room temperature, after which the sample was diluted and then introduced into the mass spectrometer without further purification.

Mass spectrometry mass spectrometry is performed using (i) quadrupole ion trap type (Finigan-MAT model LCQ-DECA, San Jose, CA), (ii) quadrupole time-of-flight type (Micromass model Q-TOF2, Manchester, UK). Or (ii) with a triple quadrupole (Finigan model TSQ, San Jose, CA) mass spectrometer. All devices are equipped with an electrospray ionization interface.

A quadrupole ion trap mass spectrometry sample (10 pmol / μL, 50: 50: 1 H ₂ 0: CH ₃ CN: in acetic acid) was introduced into the mass spectrometer at a flow rate of 2 μL / min. The ESI conditions were optimized so that the intensity of the target ions was maximized. Standard conditions were a spray voltage of −5 kV, a nitrogen sheath gas of 30 psi, and a heated capillary temperature of 150 ° C. MS / MS and MS ³ experiments were performed on selected ion masses using standard isolation and excitation procedures. The total spectrum acquired is an average of 10 scans.

After injecting a sample (same concentration as the solution composition for ion trap experiments) at a quadrupole time-of-flight mass spectrometry flow rate of 1 μL / min, the spectrum is obtained using the V-optics mode of the time-of-flight mass spectrometer. I got it. The electrospray interface conditions were optimized to maximize the intensity of the target ions. Standard conditions are: spray voltage −4.5 kV; nitrogen source gas 1 psi; cone gas 100 (arbitrary unit); source temperature 50 ° C .; desolvation temperature 150 ° C .; cone voltage 50 V (for monovalent ions), 30 V (divalent) (In the case of ions) and 20 V (in the case of trivalent ions). An energy-resolved CID MS / MS experiment by using argon as the inert collision gas at a pressure of 10 psi to automatically measure the charge state of the product ions for the isolated isotope envelope precursor ion group Was needed. The collision energy increases from 4V to 58V when increasing by 1V with respect to monovalent ions, from 4V to 20V when increasing by 1V with respect to bivalent and trivalent ions, and from 20V to 46V when increasing by 2V. Increased to. The spectrum acquired for each impact energy value is an average of 10 scans.

Triple quadrupole mass spectrometry sample (1 pmol / μL, 50: 50: 1 H ₂ 0: CH ₃ CN: in acetic acid) introduced into mass spectrometer with handmade nanoelectrospray ionization source at a flow rate of 200 nL / min did. The spray voltage was maintained at -1.8 kV. The temperature of the heated capillary was 150 ° C. Argon collision gas pressure was maintained at 1.5 mtorr. The mass spectrometer was operated under unit separation conditions. Neutral desorption mode MS / MS scans _{(d 0} - and _{d 5} - 83 and 85.5Da for divalent ions containing, _{d 0} - and _{d 5} - to trivalent ions containing 53.3 and 57 Da neutral desorption) were performed at 18V and 13V collision energies, respectively. Product ion CID MS / MS spectra of peptide ions selectively identified by neutral desorption scans were then acquired at 18 V and 31 V and 13 V and 18 V for divalent and trivalent charged ions, respectively. All spectra shown are an average of 20 scans.

  Capillary RP-HPLC was developed at a flow rate of 3.6 μL / min using the following (i): Brownlee RP-300, 7 μm dimethyloctyl silica, and using a linear 60 minute gradient of solvent B 0-100%. Column (200 μm ID x 150 mm OD quartz glass) (in this case, solvent A is a 0.1 M acetic acid aqueous solution and solvent B is a 0.1 M acetic acid aqueous solution containing 60% acetonitrile), or (Ii): Column (75 μm ID x 150 mm OD quartz glass) packed with Vydac C4 and developed at a flow rate of 200 nL / min using a linearity 60 min gradient of solvent B 0-100% ( In this case, solvent A is a 0.1 M acetic acid aqueous solution, and solvent B is a 0.1 M acetic acid aqueous solution containing 60% acetonitrile. ) Was performed using.

Results and examination
Identification of Proteins by CID MS / MS of Peptides Containing Fixed Charge Derivatives To show a general method for selective identification and quantification of peptides by selective CID MS / MS separation, in the present invention, methionine Side chain fixed charge derivatization of peptides containing The basis for utilizing fixed charge derivatization of methionine-containing peptides is that the method for selective identification and quantification of differential protein expression is specific for the detection of peptides containing rare amino acids, thereby enabling analysis. Based on the idea that the number of peptides required should be limited and that the protein should be comprehensively covered in the sequence database. Release 40.0 of the SWISS-PROT database includes a total of 101,602 protein entries. The expression rate of methionine in this database is 2.37%. In contrast, the expression rate of cysteine, the target amino acid in the well-known method ICAT and related methods for selective mass spectral detection and quantification, is 1.63%. Of the individual database entries, 85.33% contain cysteine versus 96.9% contain methionine. Only 1.65% of entries do not contain either methionine or cysteine, whereas 98.35% coverage is achieved when targeting proteins containing both amino acids. . Thus, the method performed for the identification of proteins containing methionine (or both methionine and cysteine) covers significantly more of the proteins shown in the database than the method targeting only cysteine. There is a need.

  Conceived in the present invention for the selective identification and subsequent differential quantification of methionine-containing peptides and S-alkylcysteines with fixed charge sulfonium ion derivatives (ie, derivatives in which the methionine or S-alkylcysteine side chain is alkylated). The main method and tandem mass spectrometry are outlined below. Similar methods for selective identification and differential quantification of cysteine-containing peptides via sulfonium or quaternary alkyl fixed charge derivatives, and for selective identification and differential quantification of peptides containing tryptophan (expression rate 1.21%) A similar method is also described.

Fixed charge sulfonium ion derivatization of methionine containing peptides Identification of methionine containing peptides via the CID of sulfonium ion fixed charge derivatives is attractive for several reasons. Methionine is a rare amino acid (see above). Many chemical and biological applications of sulfonium ions have been reported so far, and have been a precedent for developing reagents that form these ions in applications involving specific peptide derivatives [Stirling , M.M. J. et al. And Patai, S .; The Chemistry of the Sulfonium Group, Wiley: New York, 1981. Lundblad. Techniques in Protein Modification, Chapter 8 “The Modification of Methyline” CRC Press. Florida, 1995. Liu T .; -H. “The role of Sulfur in Proteins” In: The Proteins. Volume III, Neuroth, Hill and Boeder. Hen, Academic NY, 1977, Chapter 3, pp 239-402. Gundlach, H .; G. Moore, S, and Stein, W .; H. J. et al. Biol. Chem. , 1959, 234, 1761-1764. Lawson, W .; B. Gross, E .; , Foltz, C.I. M.M. And Wickkop, B .; J. et al. Am. Chem. Soc. , 1961, 83, 1509-1510. Lawson, W .; B. Gross, E .; , Foltz, C.I. M.M. And Wickkop, B .; J. et al. Am. Chem. Soc. 1962, 84, 1715-1718. Gross E .; Methods Enzymology 1967, 11, 238-255. Degen, J .; And Kyte, J. et al. Anal. Biochem. 1978, 89, 529-539. Kyte, J .; , Degen, J .; And Harkins, R .; N. Methods Enzymology 1983, 91, 367-377]. Alkylation of methionine residues in proteins and peptides is specific at low pH, regardless of the alkylating agent used [Lundblad. Techniques in Protein Modification, Chapter 8 “The Modification of Methyline” CRC Press. Florida, 1995. Liu T .; -H. “The role of Sulfur in Proteins” In: The Proteins. Volume III, Neuroth, Hill and Boeder. Ed., Academic NY, 1977, Chapter 3, pp 239-402]. The reason for this selectivity is that the rate of alkylation of the thioether sulfur atom in the methionine side chain is actually pH-dependent (even as low as pH 1), whereas all other nucleophilic functional groups (eg cysteine Lysine and histidine residues) decrease at low pH (due to deprotonation). To date, sulfonium ions have (a) been tagged to methionine sites of peptides and proteins [Lundblad. Techniques in Protein Modification, Chapter 8 “The Modification of Methyline” CRC Press. Florida, 1995. Liu T .; -H. “The role of Sulfur in Proteins” In: The Proteins. Volume III, Neuroth, Hill and Boeder. Hen, Academic NY, 1977, Chapter 3, pp 239-402. Gundlach, H .; G. Moore, S, and Stein, W .; H. J. et al. Biol. Chem. , 1959, 234, 1761-1764. Rogers, G .; A. Saltiel, N, and Boyer, P .; D. J. et al. Biol. Chem. 1976, 251, 5711-5717. Toennies, G .; And Kolb, J .; J. et al. J. et al. Am. Chem. Soc. , 1945, 67, 849-851. Lavine, T .; F. Floyd, N .; F, and Cammaroti, M .; S. J. et al. Am. Chem. Soc. , 1945, 67, 849-851. Lawson, W .; B. And Schramm, H .; J. et al. J. et al. Am. Chem. Soc. 1962, 84, 2017-2018], (b) to induce cleavage of peptide bonds adjacent to methionine residues via fragmentation of sulfonium ions in the liquid phase [Lawson, W. et al. B. Gross, E .; , Foltz, C.I. M.M. And Wickkop, B .; J. et al. Am. Chem. Soc. , 1961, 83, 1509-1510. Lawson, W .; B. Gross, E .; , Foltz, C.I. M.M. And Wickkop, B .; J. et al. Am. Chem. Soc. 1962, 84, 1715-1718. Jendrek, J .; P. Barker, R .; H. And Altschul, A .; M.M. Biochim Biophys Acta, 1967, 136, 409-411. Gross E .; Methods Enzymology 1967, 11, 238-255]. Other alkylating agents have also been used for similar effects [Tang, J. et al. R. And Hartley, B .; S. Biochem. J. et al. 1967, 102, 593. Tang, J .; R. And Hartley, B .; S. Biochem. J. et al. 1970, 118, 611], the most famous and widely studied system is the cyanogen bromide reaction [Gross E., et al. Methods Enzymology 1967, 11, 238-255]. Purification of peptides [Tang, J. et al. R. And Hartley, B .; S. Biochem. J. et al. 1967, 102, 593. Degen, J .; And Kyte, J. et al. Anal. Biochem. 1978, 89, 529-539. Kyte, J .; , Degen, J .; And Harkins, R .; N. Methods Enzymology 1983, 91, 367-377. Weinberger, S .; R. Viner, R .; I. And Ho, P .; Electrophoresis. 2002, 23, 3182-3192] and conversion of methionine residues to homocysteine residues [Chassasaing, G; Lavielle, S; Marquet, A. et al. J. et al. Org. Chem. 1983, 48, 1757-60], an autolytic protein cleavage reaction [Taylor, KL. Pohl, J; Kinkade, J .; M.M. , J .; Biol. Chem. Note that related reactions have been observed in peptide synthesis, such as [1992, 267, 25282-25288] [Gairi, M .; Lloyd-Williams, P .; Albericio, F .; And Girart, E .; Tetrahedron Lett. 1994, 35, 175-178]. Preliminary experiments involving sulfonium ion derivatives of methionine were performed with substituted alkyl halides R ₁ X, where X is a halogen and R is a substituted alkyl group [O'Hair, R .; A. J. et al. And Reid, G .; E. Eur. Mass Spectrom. 1999, 5, 325-334]. The reagents used in these preliminary studies react specifically with methionine side chains in liquids under acidic pH conditions and are stable side chain sulfonium ion derivatives (—CH (CH ₂ ) ₂ S (CH ₃ ) R ₁ ⁺ ) (Step (i) of Scheme 1). Under these conditions, the reagent does not react with the free thiol of the cysteine-containing peptide. However, the reagent can be used for derivatization of cysteine-containing peptides after the first S-alkylation of the cysteine thiol side chain under basic pH conditions to give the derivative (—CHCH ₂ SR ₂ ) (Scheme). Stage 2 (i)). Next, these S-alkylcysteine-containing peptides are further alkylated under the same conditions as used for alkylation of methionine to produce stable sulfonium ion derivatives of cysteine (—CHCH ₂ SR ₁ R ₂ ⁺ ) Is formed [Foti, S .; Saletti, R .; And Marletta, D .; Org. Mass Spectrom. 1991, 26,903-907. Lapko, V .; N. Smith, D .; L. And Smith, J. et al. B Mass Spectrom. 2000, 35, 572-575] (where the substituent R ₂ used for the initial cysteine alkylation is one that enhances the stability of the sulfonium ion (ie, the charge is stable)) (stage of Scheme 2 (Ii)). Importantly, conditions can be chosen such that the initial S-alkylcysteine derivative is not stable to sulfonium ion formation, so that in the case where the cysteine has been previously reduced and S-alkylated, The specific derivatization of methionine-containing peptides is possible for efficient proteolysis during preparation. Neutral Desorption Mode Scan Mode From a characteristic methionine using neutral desorption product ion detection software for post data acquisition after CID tandem mass spectrometry (MS / MS) scan or normal CID MS / MS Through the elimination of CH ₃ SR _{1 and} the elimination of R ₁ SR ₂ from cysteine, the respective product ions [M + nH—CH ₃ SR ₁ ] ^{(n + 1) +} and [M + nH—R ₁ SR ₂ ] ⁽ With the formation of ^{n + 1) +} , methionine-containing peptides and cysteine-containing peptides can be selectively identified. Thus, a methionine-containing peptide and a cysteine-containing peptide can be produced by the use of an R ₂ group that does not correspond to —CH ₃ (ie, a side chain alkyl group of methionine), or at the same time, cysteine when the R ₂ group corresponds to —CH _3. Can be identified individually by first alkylation.

In order to make this derivative method successful, it is necessary to study the chemical reaction in the liquid phase and gas phase of the resulting sulfonium ion. A series of model peptides (ie, tandem masses) designed to resemble the fragmentation behavior of these liquid and gas phase chemistry and fixed charge sulfonium ion derivatives of methionine-containing peptides similar to those obtained by trypsinization. The most commonly used peptides for protein identification by analysis were investigated. The amino acid sequence of the model peptide (GAILX ₁ GAILX ₂ (SEQ ID NO: 7), where X ₁ is alanine or methionine and X ₂ is either alanine, lysine or arginine) is the charge in the observed fragmentation behavior The conditions were selected so as to enable a comprehensive investigation on the effects of amino acid composition and amino acid composition.

The liquid phase reaction for forming the sulfonium ion needs to be easily performed using a readily available reagent. Many potentially useful reagents are commercially available. Other potentially useful reagents can be synthesized by simple methods described in the literature. Importantly, whether or not a suitable isotope label can be incorporated into the selected derivative is an important consideration when considering the realization of quantification following differential protein expression using the same method. Accordingly, isotopically labeled derivatives of these reagents are either commercially available or need to be easily synthesized. For example, in the present invention, the brominating agent poly (4-vinylpyridinium tribromide) was used to easily prepare isotope labeled d ₅ -bromoacetophenone from d ₅ -acetophenone in a one-step process (Materials and Methods section). reference). The reaction must proceed rapidly and to completion, and the resulting sulfonium ions must be chemically and thermally stable in solution [Stirling, M., et al. J. et al. And Patai, S .; The Chemistry of the Sulfonium Group, Wiley: New York, 1981. Stirling C .; J. et al. M.M. Sulfonium Salts in Organic Chemistry of Sulfur Oae, S. Ed. Plenum Press NY 1977, Chapter 9, pp 473-525. Capozzi, G, and Moderna, G .; Stud. Org. Chem. 1985, 19, 246-298]. A series of potential alkylating agents for the formation of methionine side chain fixed charge sulfonium ions, iodomethane, iodoethane, iodobenzene, iodomethylbenzene, iodoacetic acid, iodoacetamide and bromoacetophenone using the model peptide GAILGGAILK (SEQ ID NO: 2) Was evaluated. The peptides were reacted with a 100-fold molar excess of each alkylating agent for 4, 8, 16 and 32 hours and then analyzed by mass spectrometry to determine the extent of the reaction. After 16 hours, only the acetophenonesulfonium ion derivative reacted until completion of the reaction [March J. et al. “Advanced Organic Chemistry”, 4th edition, Wiley, New York, 1992, pp 343. Halvorsen, S .; J. et al. Chem. Soc. Chem Comm, 1978, 327. Yoh, L .; Tetrahedron Lett, 1988, 29, 4431]. The ethyl derivative was found to have the lowest reactivity and stability in solution (only 1% reaction after 16 hours). All other reagents were located between these extremes in terms of reactivity. The acetophenone derivatized sulfonium ion of the peptide was found to be stable in solution for several weeks without significant degradation. Free SH groups of cysteine residues have been shown to have an effect on the reverse reaction of alkylation of methionine residues [Scheitjter, A. et al. And Aviram, I. et al. FEBS Lett 1972, 21, 293-296], and the transfer of a methyl group from a sulfonium ion salt to a free sulfhydryl receptor is a well-known possible side reaction [Toennies, G. et al. And Kolb, J .; J. et al. J. et al. Am. Chem. Soc. , 1945, 67, 1141-1144. Naider, F .; And Bohak, Z .; Biochemistry 1972, 11, 3208-3211. Cantoni, G .; L. Comp. Biochem. 1960, 1, 181], a routine step involving S-alkylation of cysteine prior to proteolysis is useful and may be a necessary reaction.

Gas Phase Conditions for Sulfonium Ion Fragmentation All tested sulfonium ions (including those that are less reactive in solution) were found to be stable in the gas phase, so they can be used for MS / MS experiments. The mass could be selected and manipulated. Fragmentation through neutral elimination of CH ₃ SR ₁ was found to yield unique fingerprints for all ions (see Scheme 1). The desorption of CH ₃ SR ₁ from the fixed charge derivative is a small model system [Reid, G. E. , Simpson, R .; J. et al. And O'Hair, R .; A. J. et al. J. et al. Am. Soc. Mass Spectrom. 2000, 11, 1047-1060. O'Hair, R .; A. J. et al. And Reid, G .; E. Eur. Mass Spectrom. 1999, 5, 325-334], peptides [Foti, S .; Saletti, R .; And Mar1etta, D .; Org. Mass Spectrom. 199, 26, 903-907] as well as proteins [Lapko, V .; N. Smith, D .; L. And Smith, J. et al. B. Mass Spectrom. 2000, 35, 572-575]. Note that these gas phase reactions are similar to the liquid phase reactions described above. In order to obtain stable alkyl cations or alkenes, side reactions such as charge transfer via intramolecular proton transfer, alkyl group transfer, and bond breakage should be avoided [O'Hair, R .; A. J. et al. Freitas, M .; A. Gronert, S .; Schmidt, J .; A. R. And Williams, T .; D. J. et al. Org. Chem. 1995, 60, 1990-1998. Mudd, S .; H. Biochem, 1966, 5, 1653. Deakyne, C .; A. Et al. Mol. Struct. 1999, 485/486, 33-41. Markham, G .; D. And Bock, C.I. W. J Phys Chem, 1993, 97, 5562-5569. Katritzky, A .; R. Int J. et al. Mass Spectrom, 1997, 165/166, 577-583. Buckley, N .; J Org. Chem. 1996, 61, 2753-62. Mestdagh, H .; Org Mass Spectrom. 1986, 21, 321-327. Mestdagh, H .; Org Mass Spectrom. 1988, 23, 246-251]. Proton transfer (Scheme 3, Route 1) can give sulfur ylides. Although the acidity of (CH ₃ ) ₃ S ⁺ ion (or the proton affinity of the conjugate base (CH ₃ ) ₂ S (CH ₂ )) is unknown, a base such as a hydroxide ion (HO-) or an alkyllithium reagent can be used. Can be used to deprotonate sulfonium ions to form sulfur ylides in solution [Trost, B. et al. M.M. And Melvin, L .; S. “Sulfur Ylids: Emerging Synthetic Intermediates”. Academic Press: NY 1975]. Due to the high energy transition state (colinear geometry), intramolecular S _N 2 occurs rarely, so the alkyl transition (Scheme 3, pathway 2) does not immediately become a problem [O'Hair, R .; A. J. et al. Freitas, M .; A. Gronert, S .; Schmidt, J .; A. R. And Williams, T .; D. J. et al. Org. Chem. 1995, 60, 1990-1998]. Several biological sulfur-based liquid phase methyl cation affinities (MCAs) have been studied [Mudd, W. et al. A. , Klee P. et al. D. Biochem, 1966, 5, 1653]. Unfortunately, gas phase MCAs are experimental and theoretical estimates of dimethyl sulfide [Deakyne, C .; A. Knud, D .; M.M. Meot-Ner, M .; Breneman, C .; M.M. Liebman, J .; F. J. et al. Mol. Struct. 1999, 485/486, 33-41], as well as theoretical estimates of some other sulfides [Markham, G. et al. D. And Bock, C.I. W. J Phys Chem, 1993, 97, 5562-5569]. The binding strength of other cleaved alkyl groups appears to be unknown both in the concentrated phase and in the gas phase. However, there are several studies on the fragmentation reaction of sulfonium ions analyzed by mass spectrometry [Katritzky, AR Shipkova, PA Watson, CH Eyler, JR. And Kevil DN. Int J Mass Spectrom, 1997, 165/166, 577-583. Buckley, N; Maltby, D; Burlingame, AL. Openheimer, NJ. J. et al. Org. Chem. 1996, 61, 2753-62, (d) Mestdagh, H; Morin, N; Rolando, C .; Org. Mass Spectrom. 1986, 21, 321-327. Mestdagh, H; Morin, N; Rolando, C .; Org. Mass Spectrom. 1988, 23, 246-51]. Scheme 3 (Route 3) is very difficult to form the least stable carbocation (CH ₃ ⁺ ). The problems with Scheme 3 (routes 4 and 5) are to select R ₁ groups that do not produce stable ions, respectively (for example, avoid benzyl groups, etc.) and to select R ₁ groups that do not form alkenes. (E.g. avoiding ethyl or substituted ethyl groups and higher homologues). Differences between fragmentation reactions at the sulfonium ion of methionine and S-alkyl cysteine residues (ie, elimination of CH ₃ SR ₁ from methionine as opposed to elimination of R ₁ SR ₂ from cysteine (Scheme 2) (Scheme 1)) is clear. This can be achieved by using an alkyl group R ₂ that is not a methyl group (ie, R ₂ ≠ CH ₃ ).

General Structure of Sulfonium Ion Reagents Suitable for the Work Described herein Among the reagents investigated so far in the present invention, α-carbonyl-containing alkylating agents, iodoacetic acid, iodoacetamide and bromoacetophenone are preferred. The liquid phase and gas phase reaction characteristics are shown. Of these, acetophenone facilitates incorporation of a suitable isotope label (eg, via the synthesis of d ₅ -bromoacetophenone or ¹³ C ₆ -bromoacetophenone) and allows subsequent quantification, It is a reagent used after this. However, any reagent having the general structure XR, where X is any suitable leaving group consisting of, for example, halide, sulfonate ester, perchlorate ester or chlorosulfonate ester [March J. " Advanced Organic Chemistry ", 4th edition, Wiley, New York, 1992, pp 352-357], and R is any substrate with a carbon adjacent to the leaving group, such as an alkyl, aryl, or α-carbonyl group It may be important to be able to incorporate isotopic or structural labels into it, so that it can be used for differential quantification, with the exceptions suggested above.

Fragmentation behavior test of methionine fixed charge sulfonium ion derivatives
GILDGAILX (SEQ ID NO: 8), a peptide containing sulfonium ion methionine unmodified and derivatized in the acetophenone side chain in a CID MS / MS and MS ³ test in a quadrupole ion trap mass spectrometer (X is alanine, lysine or Trivalently charged lysine and arginine containing ions in a quadrupole ion trap mass analysis of a peptide containing arginine and a sulfonium ion methionine derivatized with an acetophenone side chain. Product ion spectra obtained by CID MS / MS of charged ions are shown in FIGS. All monovalent and divalent charged forms of unmodified peptides that are ionized by the addition of only protons are first ionized from dissociation according to the peptide backbone, predominating the b- and y-type ions.

In contrast, peptide ion derivatives containing acetophenone side chain sulfonium methionine each have a single fixed charge derived from the sulfonium ion, with 0, 1, 2 protons, monovalent, divalent or Creates an equilibrium for trivalently charged ions. The overwhelmingly dominant fragmentation pathway for these ions in each case following the CID MS / MS of the isolated precursor ion is the α-CH ₂ − at the site of fixed charge on the methionine side chain. corresponds to the selective cleavage of the S bond, S- methyl acetophenone _{(CH 3 SCH 2 COC 6 H} 5) - resulting in elimination of the neutral side chains (CH 3 S (AP) to as). Therefore, it is important to note that the product ions formed by neutral elimination from the side chains have the same charge state (number of ionized protons) that the precursor ions have. The example of a trivalent charged ion is the only partial exception, and in FIGS. 5A and 8A, protonated S-methylacetophenone (CH ₃ SCH ₂ COC ₆ H ₅ + H ⁺ ) ((— CH ₃ S (AP) + elimination of several charged side chains of ^{H +} to as) it is (corresponding protonated S- methylacetophenone _(CH 3 S (AP) + ^{H +} and forth) with an ion) is observed The elimination of the charged side chain from the trivalently charged precursor ion occurs by proton transfer resulting from neutral elimination following the dissociation of the α-CH ₂ —S covalent bond of the precursor ion. It is expected that the resulting ionic molecule complex will occur before dissociating into individual products, and the reason is expected to be due to the repulsion of niobium.

The potential mechanism by two possible pathways for the neutral elimination of CH ₃ SCH ₂ COC ₆ H ₅ from a fixed charge derivative is shown in Scheme 4.

  Route 1 in Scheme 4 involves direct proton transfer by intramolecular nucleophiles (Nu) such as arginine or lysine basic side chains, N-terminal amino groups, or basic skeleton amide carbonyl groups. With separation, it produces 3-amino-1-butenoic acid (vinyl glycine), a novel amino acid derivative having a mass of 83 Da. Alternatively, fragmentation is caused by the nucleophile attack mechanism, whereby the adjacent amide carbonyl attacks the side chain, causing neutral elimination while producing a cyclic product (the N-terminus of the modified methionine residue). On the side, shown as route 6 in Scheme 4 as a 6-membered ring product formed by nucleophilic attack by amide carbonyl). This mechanism is the main process in which the nucleophilic attack process is involved in the fragmentation of protonated peptide ions [O'Hair, R .; A. J. et al. J. et al. Mass Spectrom. 2000, 35, 1377-1381. Schlosser, A.M. And Lehmann, W .; D. J. et al. Mass Spectrom. 2000, 35, 1382-1390], which is consistent with the increasing recognition. The intramolecular movement of acidic protons present in either the precursor ion (path 1) or the product ion (path 2A or 2B) formed following cleavage of the methionine side chain is the “mobilization of this proton. Which allows it to participate in subsequent fragmentation reactions (following the mobile proton model built to theoretically explain fragmentation of protonated peptide ions) [Wysocki, V., et al. H. Tsaprailis, G .; Smith, L .; L. And Breci, L .; A. J. et al. Mass Spectrom. 2000, 35, 1399-1406]. If this reaction process occurs via Route 2 in Scheme 4, the amide bond incorporated into the cyclic product will not be applicable to subsequent cleavage. However, opening of the cyclic product via intramolecular transfer (path 2A) yields an acyclic amino acid derivative, thereby causing subsequent fragmentation of the product ion, and amino acids at the N-terminal and C-terminal sides. Causes new formation of the derivative.

Under the assumption that the sulfonium ion derivatized peptide fragments almost exclusively through CH ₃ SCH ₂ COC ₆ H ₅ neutral elimination, resulting in a charged product ion, neutral desorption for each peptide ion. The isolated product ions are selected by mass and then further tandem mass spectrometry (MS ³ ) to provide additional structural information for obtaining evidence for a preferred fragmentation pathway and also for its identification by sequence analysis. Provided to the stage. The spectra obtained after CID MS ³ for each of the neutral desorption product ions discussed above are shown in the insets of FIGS. 1B-4B, the insets of FIG. 5A, the insets of FIGS. 6B and 7B, and Shown in the inset of FIG. 8A. [M + nH—CH ₃ SCH ₂ COC ₆ H ₅ ] ^{(n + 1) +} product generated by neutral elimination from a fixed charge derivative, similar to the case of monovalent and divalent protonated unmodified peptide ions The ionic MS ³ results in the formation of a product that initially results from dissociation according to the peptide backbone, predominating the b- and y-type ions. MS / MS product ion spectrum of the unmodified peptide, and _{[M + nH-CH 3 SCH} 2 COC 6 H 5] (n + 1) + direct comparison MS ³ spectrum of product ions reveals points several interesting.

(I) In most cases, cleavage of the amide bond on either side chain of the modified vinylglycine residue formed by the elimination of the sulfonium ion side chain is observed following MS ³ , indicating that the fragment This suggests that the species to be converted comprises an acyclic vinylglycine residue generated by either ring opening of the cyclic product formed by Route 1 of Scheme 4 or Route 2 of Scheme 4.

(Ii) [M + nH—CH ₃ SCH ₂ C ₆ H ₅ ] ^{(n + 1) +} product for MS ^{3 of} monovalently charged methionine side chain fixed charge peptide ion (inset of FIGS. 1B, 3B and 6B ⁾ More “series” ion fragmentation was observed following MS ³ of the ion and compared to the results obtained by MS / MS of the unmodified methionine-containing peptide, water and ammonia desorption and a-type product Less cleavage of “non-series” ions, such as ion production. This finding can be easily explained by a mobile proton model for peptide fragmentation [Wysocki, V. et al. H. Tsaprailis, G .; Smith, L .; L. And Breci, L .; A. J. et al. Mass Spectrom. 2000, 35, 1399-1406]. In the case of monovalently charged sulfonium ion peptide derivatives, the acidic protons generated in the elimination of the side chain are shown by the direct proton transfer mechanism for cleavage in Route 4 of Scheme 4 or by Route 2A of Scheme 4. Ring opening of the cyclic product allows rapid movement onto the peptide backbone, allowing subsequent fragmentation to occur according to the backbone. On the other hand, in the case of monovalent protonated ions generated directly by electrospray, the proton to be ionized is first located on a basic side chain such as arginine or lysine, causing fragmentation of the next basic skeleton. To do so, a great deal of energy must first be supplied to overcome the barrier to proton transfer. As such, other processes, including the elimination of small molecules such as water and ammonia from precursor and product ions, and the generation of a-type product ions, therefore compete well with basic skeletal cleavage. Product produced by MS ³ from methionine side chain fixed charge [M + nH—CH ₃ SCH ₂ COC ₆ H ₅ ] ^{(n + 1) +} MS / MS product ion containing divalent protonated lysine and arginine for other peptides The ions (insets in FIGS. 4B and 7B) were similar to those obtained from MS / MS of unmodified divalent protonated peptide ions. However, the MS ³ of the divalent protonated product ion generated from the dissociation of the acetophenone-modified GAILGGAILA (SEQ ID NO: 1) peptide (inset in FIG. 2B) is the unmodified peptide with the b ₉ and b ₉ ²⁺ ions predominating. Produces less product than the results obtained by MS / MS. This means that in this case, the first proton present in the precursor and the acidic proton produced by side chain elimination from the divalently charged sulfonium ion derivative are present on the amino terminus, And suggests that it is not as “mobile” as in the case of a peptide that is divalently protonated at a position where another proton is present on the basic skeleton; this is again formed from a fixed charge derivative. Consistent with the first cyclic product. The methionine side chain fixed charge [M + nH—CH ₃ SCH ₂ COC ₆ H ₅ ] ^{(n + 1) +} MS ³ product ion (inset of FIG. 5A and FIG. 8A) containing trivalent protonated lysine and arginine is the main It was found that y ₈ ²⁺ product ions were produced in the reaction, and other y-type ions were also produced in small amounts.

_{(Iii) [M + nH-} CH 3 S (AP)] (n + 1) + collision energy required to MS ³ of product ions, relative to the unmodified peptide [M ^{+ nH] n} + of the precursor ion MS / MS for each charge state Consistently higher than what is needed. This suggests that fragmentation of the precursor ions caused by neutral elimination has a higher activation barrier compared to the precursors produced directly by electrospray ionization and the subsequent dissociation. Previously requiring ring opening again suggests the initial generation of a cyclic product ion from the fixed charge derivative in pathway 2 of Scheme 4.

Energy-resolved CID MS / MS and MS ^{3 in a} quadrupole time-of-flight mass spectrometer
Unmodified methionine-containing peptides as well as those containing an alanine residue at a position corresponding to methionine (Alanine has the closest structure as a common acid for vinylglycine residues formed by neutral elimination from fixed charge derivatives. Therefore, relative to the observed fragmentation process from fixed charge derivatives compared to methionine for direct comparison of the relative energy required for fragmentation) In order to investigate dynamic energetics and to obtain evidence regarding the structure of the resulting product ions, the fragmentation reaction of the sulfonium ion-containing peptides described above was performed using a quadrupole time-of-flight (Q-TOF) mass spectrometer. Was investigated by energy decomposition CID.

FIG. 9A shows a decrease curve and an appearance curve of energy-resolved CID for a divalent sulfonium ion derivative of GAILGGAILK (SEQ ID NO: 2). Precursor [M (AP) + H] ²⁺ depletion curve, first neutral desorption product ion (−CH ₃ S (AP)) appearance / decrease curve, and individual product ions and total product ion content ( The appearance curve of the sum of product ions) is shown. It appears that in principle there is no overlap between the decrease of [M (AP) + H] ²⁺ ions and the appearance of individual product ions, which means that these ions directly from the first precursor It suggests that it is not formed. Rather, these ions are generated from the initial neutral elimination of the product ion (—CH ₃ S (AP)). A collision energy (12 eV) sufficient to obtain a 70-90% reduction in precursor ion content, and a 70-90% reduction in initial neutral desorption (—CH ₃ S (AP)) product ion content. 9B and C show the spectra of typical product ions obtained under conditions of sufficient impact energy (22 eV), respectively. The product ion spectra observed under these “low” and “high” energy quadrupole CID conditions are in principle the ones observed in the quadrupole ion trap mass spectrometry MS / MS and MS ³ experiments, respectively. Were identical. This suggests that there is no improvement in the fragmentation process due to the time frame (on the millisecond vs. microsecond scale for ion trap and quadrupole experiments, respectively) and the ion activation mode. In fact, all peptides tested under these “low” and “high” energy quadrupole CID conditions are very similar products compared to the corresponding ion traps derived from MS / MS and MS ³ spectra. Ion spectrum was shown (data not shown). That is, fragmentation of directed fixed charge derivatives enables the use of low energy CID conditions for the formation of initial neutral desorption product ions, and then “high” energy CID to obtain further structural information. A “pseudo MS ³ ” spectrum is obtained under conditions. For comparison, an appearance curve with respect to the sum of product ions generated from a divalent protonated unmodified methionine and an alanine-containing peptide is shown in FIG.

  Table 1 shows the total collision energy required to achieve 50% product ion formation from each of the protonated unmodified peptides and from the first neutral desorption product ion from the fixed charge derivative. Shown in These data can be used to measure the stability of the sulfonium ion derivative neutral elimination product ion to the unmodified form. This data suggests that the collision energy required for the dissociation of the first neutral elimination product ion derived from the sulfonium ion derivative is in principle higher than for alanine and unmodified methionine-containing peptides and continues It further supports the formation of the first cyclic product ion required to overcome the barrier to ring opening prior to dissociation (Scheme 4, pathway 2A).

  Collision energy required to achieve 50% reduction in unmodified precursor ion content or initial neutral desorption product ion content from a fixed charge sulfonium ion derivative in a Q-TOF mass spectrometer.

  As an alternative to the additional CID MS / MS step being performed to identify these peptides known to contain fixed charge derivatives, high resolution mass accuracy combined with database searching ( Note that there may be the use of accurate mass tags).

  Conventionally, when an “accurate mass tag” close to 1 ppm has been obtained and the presence of a specific amino acid is known (for example, the presence of a cysteine residue), the characteristics of the database search algorithm are improved. From the information alone, a clear identification of the protein from which the peptide is derived has been achieved [Conrads, T .; P. , Anderson, G. A. Venstra, T .; D. Pasa-Tolic, L .; And Smith, R .; D. Anal. Chem. 2000, 72, 3349-3354. Goodlet, D .; R. Bruce, J .; E. , Anderson, G. A. Rist, B .; Pasa-Tolic, L .; , Fiehn, O .; Smith, R .; D. And Aebersold, R .; Anal. Chem. 2000, 72, 1112-1118. Strittmatter, E .; F. Ferguson, P .; L. Tang, K .; And Smith, R .; D. J. et al. Am. Soc. Mass Spectrom. 2003, 14, 980-991].

Identification of methionine-containing peptides by neutral desorption scan mode CID MS / MS of sulfonium ion fixed charge derivatives using a triple quadrupole mass spectrometer Using neutral desorption scan mode CID MS / MS, To verify the usefulness of fixed charge derivatives to selectively identify peptides containing specific amino acid residues in complex mixtures, reduced, S-carboxyamidomethylated and derivatized with bromoacetophenone 10 pmol of the digested bovine serum albumin trypsin was subjected to capillary RP-HPLC-MS (FIG. 11). And in a series of experiments, these peptides giving neutral detachment of m / z 83 (ie, detachment of CH ₃ SCH ₂ COC ₆ H ₅ from a divalent charged precursor ion) Identified in desorption scan mode CID MS / MS. The resulting total ion flow trace for this experiment is shown in FIG. 12A. Four main peaks are observed, and mass spectra obtained from two of them are shown in FIGS. 12B and 12C. The mass of these peaks was found to correspond to the four methionine-containing trypsin digested peptides of bovine serum albumin. Detected in a neutral desorption scan mode experiment by comparing the data in FIGS. 11B and C (mass spectra in two regions of the chromatogram of the total ion stream obtained from the MS experiment are shown by arrows) and FIGS. A relatively small amount of ions (m / z 799.4 in FIG. 12B is only the eighth most abundant ion in FIG. 11C, and m / z 759.8 in FIG. 12C is 3 in FIG. 11C. It can be observed that it is only the second most abundant ion). Thus, these data demonstrate the utility of fixed charge derivatives to selectively identify peptides containing specific amino acid residues using neutral charge scan mode CID MS / MS of fixed charge derivatives ( The four methionine-containing tryptic peptides of bovine serum albumin are selectively identified from a total of about 100 peptide groups present in this digest).

Method for protein identification by neutral desorption scan mode CID MS / MS of peptides containing other amino acid fixed charge derivatives
Modification of tryptophan by electrophilic aromatic substitution to obtain tryptophan and cysteine fixed charge sulfonium ion derivatives can be achieved by first reacting a tryptophan-containing peptide with a halogenated sulfen (R ₁ SX), an indole A derivative having a thioether functional group at the 2-position of the side chain is obtained (Scheme 5A) [Scoffone, E .; , Fontana, A .; And Rochhi, R .; Biochemistry. 1968, 7, 971] and then sulfonium ion derivatives of tryptophan are formed under conditions similar to those described above for methionine and S-alkylcysteine sulfonium ion derivatives. It should be noted that halogenated sulfenes also react with cysteine-containing peptides to yield asymmetric disulfides (Scheme 5B), however, the specificity of this reaction for tryptophan is due to the initial S-alkylation of cysteine. Or by reduction of asymmetric disulfides prior to sulfonium ion formation. Liquid phase and gas phase conditions for sulfonium ion production from 2-indole S-alkyltryptophan modified residues are similar to those described above for methionine and S-alkylcysteine sulfonium ion production. The 2-indole S-alkyl derivative of tryptophan is generated under the same conditions as described above for methionine and S-alkylcysteine, both residues being modified in this way. However, a method which has been described above with respect to methionine and S- alkylcysteine sulfonium ions, through the use of -CH ₃ groups R ₁ group that does not correspond to (e.g., methionine side chain alkyl group), or at the same time, the R ₁ group Through initial alkylation of tryptophan at the site corresponding to the —CH ₃ group, methionine and tryptophan containing peptides are individually identified. Similarly, 2-indole S-alkyltryptophan and S-alkylcysteine-containing peptides are introduced via the introduction of R ₁ groups into S-alkyltryptophan derivatives that are not compatible as R ₁ groups used for the initial alkylation of cysteine. Or simultaneously, can be individually identified via the first alkylation of tryptophan using the same R ₁ group used for the alkylation of the first cysteine. Note that under appropriate conditions, a similar electrophilic aromatic substitution reaction can be used to introduce an S-alkyl group into the side chain of a tyrosine residue, followed by the formation of a sulfonium ion derivative.

General Structure of Fixed Charged Ion Reagent Suitable for the Work Described herein Any reagent having the general structure R ₁ SX ₁ (X ₁ is any suitable leaving group such as halide, sulfonate ester, Chlorate ester or chlorosulfonate ester [March J. “Advanced Organic Chemistry”, 4th edition, Wiley, New York, 1992, pp 352-357], and R ₁ is optional with carbon adjacent to the leaving group Substrates, eg, alkyl, aryl, or α-carbonyl groups, with the exceptions suggested above, that can incorporate isotopic or structural labels so that they can be used for differential quantification) , and any reagent (X ₂ is any suitable leaving group with the general structure R ₂ X _2, for example a halide, Sulfonic acid ester, perchlorate ester or chlorosulfonic acid ester [March J. "Advanced Organic Chemistry" , 4th Edition, Wiley, New York, 1992, pp352-357] are, and _{R 2} is a leaving group (e.g. Any substrate with a carbon adjacent to an alkyl, aryl, or α-carbonyl group), with an isotopic or structural label attached to it for use in differential quantification, with the exceptions suggested above Can be captured).

The sulfonium ions of tryptophan and cysteine-containing peptides can also be formed directly by an initial reaction with a halogenated sulfene (X ₂ RSX ₁ ) containing the appropriate leaving group X ₂ , resulting in tryptophan (Scheme Cyclic sulfonium derivatives of 6A) and cysteine (Scheme 6B) are obtained.

General Structure of Fixed Charged Ion Reagents Suitable for the Work Described herein Any reagent having the general structure X ₂ RSX ₁ (X ₁ is any suitable leaving group such as halide, sulfonate ester, peroxide Chlorate ester or chlorosulfonate ester [March J. “Advanced Organic Chemistry”, 4th edition, Wiley, New York, 1992, pp 352-357], and R is a radical (eg, alkyl, aryl, or α- Any substrate with a carbon adjacent to the carbonyl group), into which an isotopic or structural label can be incorporated so that it can be used for differential quantification with the exceptions suggested above, and X ₂ any suitable leaving group, such as halides, sulfonic acid esters, perchloric acid ester or A Rorosuruhon ester [March J. "Advanced Organic Chemistry", 4th Edition, Wiley, New York, 1992, pp352-357]).

Cysteine cyclic sulfonium ions are specifically detected from cysteine-containing peptides by initial alkylation of the reagent (X ₂ RX ₁ ) (Scheme 7) or by initial disulfide exchange of the reagent (X ₂ RSSX ₁ ) (Scheme 7B). Each of which contains a suitable leaving group X ₂ , resulting in a cyclic sulfonium ion derivative.

General Structure of Fixed Charged Ion Reagent Suitable for the Work Described herein General Structure (i) Any reagent having X ₂ RX _l (X _l is any suitable leaving group such as halide, sulfonic acid Ester, perchlorate or chlorosulfonate [March J. “Advanced Organic Chemistry”, 4th edition, Wiley, New York, 1992, pp 352-357], and R is a leaving group (eg, alkyl, aryl Or any substrate with a carbon adjacent to the α-carbonyl group), incorporating an isotopic or structural label therein for use in differential quantification, with the exceptions suggested above. can be further X ₂ is any suitable leaving group, for example, halogenation under neutral pH conditions or basic , Sulfonate, perchlorate or chlorosulfonate, or protonated amines or alcohols under acidic pH conditions [March J. “Advanced Organic Chemistry”, 4th edition, Wiley, New York, 1992. , Pp 352-357]), or (ii) any reagent having X ₂ RSSX ₁ (X ₁ is, for example, nitrobenzoic acid and R is any substrate with a carbon adjacent to the leaving group, such as alkyl, aryl Or an α-carbonyl group, with the exception suggested above, that can incorporate an isotopic or structural label so that it can be used for differential quantification, and X ₂ can be any Suitable leaving groups such as halides, sulfonate esters, perchlorate esters or chlorides Consisting sulfonate ester [March J. "Advanced Organic Chemistry", 4th Edition, Wiley, New York, 1992, pp352-357]).

Methods of protein identification by precursor ion scan mode CID MS / MS for peptides containing other amino acid fixed charge derivatives In contrast to the fragmentation reactions discussed above, where neutral detachment is observed (in these derivatives Note that the site of fixed charge is immediately adjacent to the site of bond cleavage), and the peptide derivatized on the side chain where the fixed charge is away from the site of bond cleavage site is This results in desorption, which can be selectively detected using precursor ion scan mode MS / MS experiments (Scheme 8). For example, fixed charge sulfonium, quaternary alkylammonium or quaternary alkylphosphonium ion reagents provide side chain charge elimination via cis 1,2 elimination, and complementary neutral peptide species (if the precursor's initial charge Produces a predominantly small molecule product with the generation of a charged peptide ionic species (if the state was 1) or 1 less charged state than the precursor (if the initial charge state of the precursor was greater than 1) . These characteristic small molecule product ions can be selectively detected using parent ion scan mode MS / MS experiments. This side chain cleavage can be further promoted by oxidation of thioethers [Steen, H. et al. And Mann, M .; J. et al. Am. Soc. Mass Spectrom. 2002, 13, 996-1003]. It is noted that fragmentation of cysteine-containing peptides alkylated with reagents with basic functionality (eg iodoacetamide, acrylamide and vinylpyridine) is observed before any fragmentation at the side chain occurs. [Moritz, R. L. Eddes, J. et al. S. Reid, G .; E. And Simpson, R .; J. et al. Electrophoresis. 1995, 17, 907-917].

The liquid and gas phase requirements for quaternary alkylammonium or quaternary alkylphosphonium ion formation and fragmentation are similar to those for sulfonium ion formation and fragmentation discussed above. Before the carboxypeptidase C-terminal peptide sequence analysis becomes possible, a fixed charge derivative of cysteine (2-bromoethyl for charge introduction into the side chain thiol of the cysteine residue) trimethylammonium bromide (BrCH ₂ CH ₂ (CH ₃ Note that ₃ ) NBr)) has been used with mass spectrometry for reaction product detection [Bonetto, V. et al. Bergman, AC. Jonvall, H .; And Sillard, R .; Anal. Chem. 1997, 69, 1315-1319].

Any reagent having the general structure R ⁺ X (X is any suitable leaving group such as halide, sulfonate ester, perchlorate ester or chlorosulfonate [March J. “Advanced Organic Chemistry”, 4th Plate, Wiley, New York, 1992, pp 352-357], and R ⁺ is any substrate with carbon adjacent to the leaving group (eg, alkyl, aryl, or α-carbonyl group), and terminal sulfonium, 4 With a quaternary alkylammonium or quaternary alkylphosphonium ion, an isotopic or structural label can be incorporated therein so that it can be used for differential quantification).

Other mass spectrometer and ion source studies Recognizing that the dissociation of a fixed charge derivative with neutral side-chain desorption results in a peptide product ion having the same charge state (and number of ionized protons) as the precursor ion That is important. Therefore, these derivatives are ESI (multiple charged ions are usually observed) and MALDI (major single single) when trying to achieve peptide sequence identification by “high” energy MS / MS or MS ³ Charged ions are observed). On the other hand, dissociation of the fixed charge derivative with side chain charge desorption (ie, production of low molecular product ions) results in a peptide product having a charge one less than the value of the precursor ion. Thus, if the identification of this peptide sequence is achieved by further dissociation, this method is only applicable when using the ESI method, where in addition to the side chain cleaved small molecule product ion, the peptide Charged product ions containing will be produced.

Differential quantification of proteins by CID MS / MS of peptides containing fixed charge derivatives
Neutral Desorption Scan Mode CID MS / MS in Triple Quadrupole Mass Spectrometer
In the general method of differential quantification of protein expression using the fixed charge derivatization method described herein, sulfonium or tertiary containing natural (light) isotopes prior to introducing the sample into the mass spectrometer. A first sample is derivatized with an alkyl fixed charge reagent and then mixed with a second sample derivatized with a similar reagent containing an isotopically enriched (heavy) isotope. And using either neutral desorption or parent ion scan mode MS / MS, neutral desorption or parent ion scan mode MS of peptide ions containing light isotopes compared to those containing heavy isotope tags The presence ratio of product ions formed by / MS can be examined. The ratio indicates the difference in protein abundance level between the two samples. This product ion representing the peptide product is then automatically selected for further structural investigation by “high” energy MS / MS or MS ³ . The methods described herein can also be readily combined with “global” labeling (ie, N- or C-terminal labeling) methods, thereby separating the isotope label from the fixed charge site. Introduced at the site, a common neutral elimination can be used to identify derivatives containing light and heavy isotopes.

Nominal 1.0 pmol / μL of GAILMGAILR (SEQ ID NO: 3) peptide derivatized with either d ₀ -or d ₅ -acetophenone and introduced into a triple quadrupole mass spectrometer by electrospray ionization: 1 The ESI mass spectrum of the 0.0 pmol / μL mixture is shown in FIG. 13A. From this mass spectrum, a ratio of 1.04: 1 is obtained relative to the divalent (m / z 567.2 and 569.7) charged peptides, and trivalent (m / z 378.4). And 380.1) a ratio of 1.06: 1 to the charged state is obtained. The 10 times magnified region of the spectrum around m / z 700-800 indicates the level of chemical noise associated with the mass spectrum. In comparison, a neutral desorption mode MS / MS spectrum of the same mixture (83 Da and 85.5 Da neutral desorption for each of the divalent charged precursors and 55.50 for each of the trivalent charged precursors. Neutral desorption of 3 Da and 57 Da) is shown in FIG. 13B. Again, the m / z 700-800 region of each spectrum was magnified 10 times to display the chemical noise level. The ratios observed for the increase in MS / MS neutral desorption scan mode ions in FIG. 13B are essentially the same as those observed for the increase in MS ions in FIG. 13A (divalent and trivalent peptides). The chemical noise is reduced in magnitude by at least two orders of magnitude compared to that observed in the mass spectrum in FIG. 13A. In fact, with the exception of electrical noise, characterized by a single data point noise “spike”, the neutral desorption MS / MS spectrum does not show any nonspecific chemical noise on the baseline. It was.

FIG. 14 shows the mass spectrum of a 1.0 pmol / μL: 0.1 pmol / μL mixture of the d ₀ -and d ₅ -containing methionine side chain fixed charge acetophenone (AP) sulfonium ion derivative of GAILGGAILR (SEQ ID NO: 3). The divalent and trivalent charged ions were found to be 10.12: 1 and 9.72: 1, respectively. The ion ratios observed by the neutral desorption scan mode MS / MS experiment (FIG. 15) were 9.45: 1 and 10.83: 1. Again, the chemical noise level in the MS / MS neutral desorption scan mode experiment was reduced in magnitude by at least two orders of magnitude compared to that observed in the mass spectrum. For all neutral desorption scan mode MS / MS experiments (0.1: 1, 0.2: 1.0, 0.5: 1.0, 1.0: 1.0, 1.0: 0. 5, 1.0: 0.2 and 1.0: 0.1 ratio), and all the errors were within 10% of the expected values.

For neutral elimination product ions formed from sulfonium ion derivative peptide precursors, a common product is formed from both “heavy” and “light” ions, so either product is a “high” energy CID. Note that alternatively, it is selected for further structural analysis by MS ³ .

  One of the additional advantages of the MS / MS method described herein, including performing these identifications by augmenting a single MS peak, is to make both of the ion pairs detectable. The presence of certain ions (ie light and heavy ions). Also, in these examples, the mass of neutral desorption from the parent ion indicates its origin (ie, from a “light” or “heavy” sample).

To verify the usefulness of the isotope-labeled fixed charge derivatization technique in combination with the use of neutral desorption scan mode MS / MS for differential quantification of peptides containing specific amino acid residues, d ₀ − And 1 pmol of GAILGGAILR (SEQ ID NO: 3) peptide derivatized with d ₅ -acetophenone were subjected to capillary RP-HPLC-MS and neutral desorption scan mode CID MS / MS (FIGS. 16A and 16C, respectively). As a result, the masses obtained by MS (1.06: 1 and 1.04: 1 ratio for divalent and trivalent charged ions) and neutral desorption MS / MS scans (divalent and trivalent) The ratios of 1.02: 1 and 0.99: 1 for charged ions) are shown in FIGS. 16B and 16C. _D charged bivalent ₀ - and _{d 5} - and _{d 5} - - that for a labeled precursor, neutral desorption of 83Da and 85.5Da, and _{d 0} charged trivalent for labeling precursor, the 55.3Da and 57Da All neutral desorption scans were summed. The d ₀ -and d ₅ -forms of fixed charge derivatives were observed with a resolution within 10%, suggesting the absence of strong deuterium isotopes in chromatographic separations for these fixed charge derivatives. This is consistent with recent reports on factors controlling the effect of deuterium isotopes in chromatography [Zhang, R .; Sima, C .; S. Wang, S .; Regnier, F .; E. Anal. Chem. 2001, 73, 5142-5149. Zhang, R .; , Sima, C .; S. Thompson, R .; A. Xiong, L .; Regnier, F .; E. Anal. Chem. 2002, 74, 3662-3669. Zhang, R .; And Regnier, F .; E. J. et al. Proteome Res. 2002, 1, 139-147].

O-linked serine and threonine, which are stable derivatives for O-linked post-translational modified protein identification and differential quantitative mass spectrometry by CID MS / MS or ion-ion reaction MS / MS of peptides containing fixed charge derivatives When combined with β-removal / Michael addition chemistry to form a peptide containing a linked post-translationally modified amino acid, the fixed charge derivatization approach is the state of O-linked post-translational modification in the protein. Can be extended to identification and quantification. Following β-removal of post-translational modification of O-linked serine or threonine, a one or two-step Michael addition reaction step can be used for fixed charge introduction. Scheme 9 illustrates a one-step process for directly introducing a fixed charge derivative (eg, a quaternary ammonium or phosphonium ion derivative). Alternatively, in a two-step process, neutral side chain derivatives can be introduced (eg, alkyl thiols) and subsequently derivatized to form fixed charge sulfonium ions. Thus, the sulfonium, quaternary alkylammonium or quaternary alkylphosphonium ion chemistry described above can be used for selective detection of peptides containing modified amino acid residues. A similar method has been described by Steen and Mann for the introduction of dimethylamine-containing sulfenic acid derivatives into β-removed phospho-serine and phospho-threonine residues, whereby protonated low mass fragments are , Detected at m / z 122.06 by precursor ion scan mode MS / MS at low energy CID [Steen, H. et al. And Mann, M .; J. et al. Am. Soc. Mass Spectrom. 2002, 13, 996-1003]. However, this ion is not always observed as the main product. This is because the production of this ion is competitive with the fragmentation process of the basic skeleton. Thus, there could be a mixture of product ions without modified side chains and product ions with modified side chains, which at the same time resulted in a complex product ion spectrum, which is expected Interpretation in the absence of knowledge of peptide sequences is potentially difficult. However, the fixed charge derivatization method described herein results in limited elimination of the modified side chain, which improves the detection of the peptide to a higher level of sensitivity and the subsequent structure. The next dissociation step for analysis can be made better controllable. When combined with the different isotope labeling methods outlined above, the fixed charge derivatization methods of the present invention may also allow differential quantification of O-linked post-translational modification states.

Characterization of protein-protein interactions by CID MS / MS or ion-ion reaction MS / MS of cross-linked peptides containing fixed charge derivatives The fixed charge derivatization approach extends to the characterization of protein-protein interactions It can be incorporated into a suitable cross-linking reagent prior to the cross-linking reaction or derivatized the cross-link contained between two proteins or peptides after the cross-linking reaction (in CID MS / MS, Fragmentation via specific neutral elimination from the crosslinks or elimination of charged entities). Incorporation of unstable MS / MS “tags” on the crosslinker was used by Back et al. To identify cross-linked peptides via detection of the characteristic low mass product ions generated at low energy CID. [Back, J. et al. W. , Hartog, A .; F. Dekker, H .; L. , Muijsers, A .; O. , De Koning, L. J. et al. And de Jong, L .; J. et al. Am. Soc. Mass Spectrom. 2001, 12, 222-227]. On the other hand, it was seen that the product ions generated in this way were only product ions with a relatively small increase. Conversely, in a similar method employed to perform peptide identification and quantification using the fixed charge derivatives outlined above, fragmentation of the cross-linked peptide towards limited cleavage of the fixed charge is performed. In order to obtain characteristic neutral desorption or low mass ions (Scheme 10), the method of the present invention can be employed. The product ions containing cross-linked peptides are then automatically selected for further structural investigation by higher energy MS / MS or MS ³ . Isotopic labels can also be incorporated into the cross-links when a quantitative comparison of cross-links between two different samples is required.

  If desired, any of the above samples containing a fixed charge derivative may be pre-concentrated prior to mass spectrometry by known chromatographic means [Tang, J. et al. R. And Hartley, B .; S. Biochem. J. et al. 1967, 102, 593. Degen, J .; And Kyte, J. et al. Anal. Biochem. 1978, 89, 529-539. Kyte, J .; , Degen, J .; And Harkins, R .; N. Methods Enzymology 1983, 91, 367-377. Gevaert, K .; , Van Damrne, J .; Goethals, M .; , Thomas, G .; R. Hoorelbeke, B .; , Demol, H .; Martens, L .; , Puype, M .; , Staes, A .; And Vandekerckhove, J .; Mol. Cell. Proteomics. 2002, 1, 896-903].

  In another method, the fixed charge derivative can be selectively pre-concentrated by a solid phase capture method using a fixed charge reagent covalently linked to beads or an insoluble polymer [Weinberger, S .; R. Viner, R .; I. And Ho, P .; Electrophoresis. 2002, 23, 3182-3192. Zhou, H .; Ranish, J .; A. Watts, J .; D. Aebersold, R .; Nature Biotechnol. 2002, 20, 512-515. Qiu, Y; Sousa, E .; A. Hewick, R .; M.M. Wang, J .; H. Anal. Chem. 2002, 74, 4969-4979. Qian, WJ, Goshe, M .; B. , Camp D. G. Yu, LR, Tang, K .; And Smith. R. D. Anal. Chem. 2003, 75, 5441-5450]. Chemical or photolytic cleavage is then used to release the fixed charge derivative of the peptide, thereby reducing the complexity of the mixture sample to be analyzed prior to introduction into the mass spectrometer (sulfonium ion). The fixed charge derivative is shown in Scheme 11).

Preferred Embodiments of Methionine Fixed Charge Derivatization Reagent The two structural formulas shown below have been used to date in the studies described herein. These are bromoacetophenone and it is isotopically encoded and labeled with deuterium (d ₅ ).

Some preferred isotopic encoding reagents of this type are labeled with ¹³ C ₆ and the structure is shown below. For the purpose of minimizing any chromatographic separation of isotope labeled against unlabeled reagents, ¹³ C labeling is preferred over deuterium labeling. Those skilled in the art will expect that this reagent will have properties similar to those of unlabeled reagents.

Further preferred reagents are the water-soluble forms of the aforementioned reagents. This added property provides the convenience of allowing a more efficient sample preparation procedure flow. Examples of the water-soluble form of this reagent and its ¹³ C ₆ labeled form are shown below. Note that the addition of the sulfinic acid or sulfonic acid functionality is accomplished in the ortho-, meta-, or para-position relative to the acetyl group. The method for synthesis is selected to allow synthesis at any one of these positions; the para substitution position is shown in the figure below.

  Non-limiting methods for the synthesis of these derivatives include (i) reacting benzene with 2,4-dinitrophenylsulfenyl chloride to give phenyl 2,4-dinitrophenyl sulfide, (ii) Step of reacting with acetic anhydride to obtain p-acetylbenzene 2,4-dinitrophenylsulfide (iii) Oxidation to obtain p-acetylbenzene 2,4-dinitrophenylsulfone (iv) Cleavage with methanolic alkali through the step of obtaining p-acetylbenzene 2,4-dinitrophenylsulfinic acid, then (v) further oxidation and subsequent bromination step to obtain sulfonic acid, or (v) direct bromination step. And methods known to those skilled in the art can be used [Buess, C .; M.M. And Kharasch, N .; J. et al. Am. Chem. Soc. 1950, 72, 3529-3532. Kharasch, N .; And Swidler, R .; J. et al. Org. Chem. 1954, 19, 1704-1707. Vogels Textbook of Practical Organic Chemistry. 4th edition 1978. p773-778. Szmant, H .; H. And Irwin, D .; A. J. et al. Am. Chem. Soc. 1956, 78, 4386-4389].

  In another method, following steps (i) and (ii), the derivative is (iii) cleaved by thiocleavage to give a p-acetylthiobenzene derivative (iv) oxidized to sulfinic acid or sulfonic acid, It can then be (v) brominated.

  The bromine atom is shown as the leaving group in the above structure (ie, the X moiety of RX), and iodine is also preferred. Other usable leaving groups include: halides such as chlorine atoms, and sulfonate esters, perchlorate esters, chlorinated sulfonate esters, or protonated amines or alcohols [March J. et al. “Advanced Organic Chemistry”, 4th edition, Wiley, New York, 1992, pp 352-357].

  In addition to the free acids shown above, these compounds can be prepared in the form of their salts (eg, sodium salts or hydrochlorides) and can be prepared as solvates.

  The present invention also includes a reagent kit for analysis of amino acids, peptides or proteins by mass spectrometry. The reagent kit comprises one or more of the above-described compounds and a protein disulfide reducing agent (eg, dithiothreitol (DTT), β-mercaptoethanol, tris-carboxyethylphosphine (TCEP), tributylphosphine (TBP)), cysteine Alkylating reagents (eg alkyl halides (eg iodoacetic acid, iodoacetamide) and vinylpyridine or acrylamide), proteases or chemical cleaving agents (eg trypsin, Lys-C, Asp-N, pepsin, thermolysin, bromide) Other reagents such as cyan, dilute acid). The reagent also includes a solvent. Typical solvents used include urea, guanidine hydrochloride, acetonitrile, methanol, water.

  These various reagents can be used to perform the above-described method steps in liquid phase or in solid phase (eg, by immobilizing this reagent on a column).

  Amino acids or peptides labeled using the methods of the invention can be used as internal standards in addition to amino acid, peptide and protein analysis. For example, somewhat similar to some recent reports using known isotopically encoded amounts of peptide derivatives that are injected into a sample as an internal standard to quantify the absolute amount of the selected peptide of interest. The derivatized peptides described herein can be used [Gygi, Steven P. et al. Peng, Junmin. PCT Int. Appl. (2003) WO 2003078962; Gerber, Scott A. et al. Rush, John; Steman, Olaf; Kirschner, Marc W .; Gygi, Steven P .; Proc. Natl. Acad. Sci. U. S. A. (2003), 100, 6940-6945]. It should be noted that these reports do not incorporate the use of fixed charge derivatives that are amenable to tandem mass spectrometry detection as described herein.

  It will be appreciated by those skilled in the art that many changes and / or improvements can be made to the invention as illustrated in the examples without departing from the spirit or scope of the invention as broadly described. The embodiments of the present invention are therefore to be regarded as illustrative in nature and not as restrictive.

  The disclosures of the references cited in this specification are hereby incorporated by reference.

  Throughout this example, the word “comprising” is understood to mean the inclusion of a specified element, number, step or group of elements, a group of numbers or a group of steps, but any other element It does not exclude numbers, stages, groups of elements, groups of numbers or groups of stages.

  Any discussion, acts, materials, devices, articles or the like in this specification that are included in this example are set forth only to provide an explanation of the invention. Any or all of these matters form part of the prior art existing prior to the priority date of each claim of this application, or are common general knowledge in the field related to the present invention. Should not be accepted as self-identifying.

Quadrupole ion trap tandem mass spectrometry of monovalent GAILGGAILA (SEQ ID NO: 1) and its methionine side chain fixed charge derivative. (A) CID MS / MS of [M + H] ⁺ ions. (B) Methionine side chain fixed charge acetophenone (AP) sulfonium [M (AP)] ⁺ CID MS / MS of ion. (Inset) CID MS ³ of the product ion [M-CH ₃ S (AP)] ⁺ in FIG. 1B. Quadrupole ion trap tandem mass spectrometry analysis of divalent GAILGGAILA (SEQ ID NO: 1) and its methionine side chain fixed charge derivative. (A) CID MS / MS of [M + 2H] ²⁺ ions. (B) CID MS / MS of methionine side chain fixed charge acetophenone (AP) sulfonium [M (AP) + H] ²⁺ ion. (Inset) CID MS ³ of product ion [M-CH ₃ S (AP)] ²⁺ in FIG. 2B. Quadrupole ion trap tandem mass spectrometry analysis of monovalent GAILGGAILK (SEQ ID NO: 2) and its methionine side chain fixed charge derivative. (A) CID MS / MS of [M + H] ⁺ ions. (B) Methionine side chain fixed charge acetophenone (AP) sulfonium [M (AP)] ⁺ CID MS / MS of ion. (Inset) CID MS ³ of product ion [M-CH ₃ S (AP)] ⁺ in FIG. 3B. Quadrupole ion trap tandem mass spectrometry analysis of divalent GAILGGAILK (SEQ ID NO: 2) and its methionine side chain fixed charge derivative. (A) CID MS / MS of [M + 2H] ²⁺ ions. (B) CID MS / MS of methionine side chain fixed charge acetophenone (AP) sulfonium [M (AP) + H] ²⁺ ion. (Inset) CID MS ³ of the product ion [M-CH ₃ S (AP)] ²⁺ in FIG. 4B. Quadrupole ion trap tandem mass spectrometry analysis of the trivalent acetophenone (AP) sulfonium derivative of GAILGGAILK (SEQ ID NO: 2). (A) CID MS / MS of [M (AP) + 2H] ³⁺ ion. (Inset) CID MS ^{3 of} product ion [M-CH ₃ S (AP)] ³⁺ . Quadrupole ion trap tandem mass spectrometry of monovalent GAILGGAILR (SEQ ID NO: 3) and its methionine side chain fixed charge derivative. (A) CID MS / MS of [M + H] ⁺ ions. (B) Methionine side chain fixed charge acetophenone (AP) sulfonium [M (AP)] ⁺ CID MS / MS of ion. (Inset) CID MS ³ of product ion [M-CH ₃ S (AP)] ⁺ in FIG. 6B. Quadrupole ion trap tandem mass spectrometry of the divalent GAILGGAILR (SEQ ID NO: 3) and its methionine side chain fixed charge derivative. (A) CID MS / MS of [M + 2H] ²⁺ ions. (B) CID MS / MS of methionine side chain fixed charge acetophenone (AP) sulfonium [M (AP) + H] ²⁺ ion. (Inset) CID MS ³ of the product ion [M-CH ₃ S (AP)] ²⁺ in FIG. 7B. Quadrupole ion trap tandem mass spectrometry analysis of the trivalent acetophenone (AP) sulfonium derivative of GAILGGAILR (SEQ ID NO: 3). (A) CID MS / MS of [M (AP) + 2H] ³⁺ ion. (Inset) CID MS ^{3 of} product ion [M-CH ₃ S (AP)] ³⁺ . (A) Energy-resolved CID MS / MS of divalent methionine side chain fixed charge acetophenone (AP) sulfonium [M (AP) + H] ²⁺ ions of GAILGGAILK (SEQ ID NO: 2). (B) CID MS / MS product ion spectrum of [M (AP) + H] ²⁺ ions obtained with 12V collision energy (laboratory frame). (C) CID MS / MS product ion spectrum of [M (AP) + H] ²⁺ ions obtained with 22V collision energy (laboratory frame). Energy-resolved CID MS / MS of divalent [M + 2H] ²⁺ ions of GAILAGALK (SEQ ID NO: 5) and GAILGGAILK (SEQ ID NO: 2). (A) Reduction of bovine serum albumin and S-carboxamidomethylated trypsin digest was then derivatized with bromoacetophenone. D. Total ion flow trace by capillary RP-HPLC MS analysis. Individual mass spectra in two regions of the total ion flow chromatogram are indicated by arrows and are represented in FIGS. 11B and 11C. (A) Reduction of bovine serum albumin and S-carboxamidomethylated trypsin digest was then derivatized with bromoacetophenone. D. Capillary RP-HPLC-total ion flow trace by neutral desorption scan mode CID MS / MS analysis. The peptides are IETMR (SEQ ID NO: 9), ETYGDMACDCEK (SEQ ID NO: 10), TVMENFVAFVDK (SEQ ID NO: 11), and MPCTEDYLSLILNR (SEQ ID NO: 12). Individual neutral desorption scan mode mass spectra in two regions of the total ion flow chromatogram are indicated by arrows and are shown in FIGS. 12B and 12C. (A) Mass spectrum and (B) Neutral of 1.0: 1.0 pmol / μL mixture of d ₀ and d ₅ containing methionine side chain fixed charge acetophenone (AP) sulfonium ion derivative of GAILGGAILR (SEQ ID NO: 3) Desorption scan mode CID MS / MS spectrum (83.0 Da and 85.5 Da neutral desorption for divalent d ₀ − and d ₅ − containing [M (AP) + H] ²⁺ ions, and [M (AP) + 2H] Neutral elimination of 55.3 Da and 57.0 Da for trivalent d ₀ -and d _5- containing ³⁺ ions). (A) Mass spectrum of a 1.0: 0.1 pmol / μL mixture of d ₀ and d ₅ containing methionine side chain fixed charge acetophenone sulfonium ion derivative of GAILGGAILR (SEQ ID NO: 3). Panels (B) and (C) show enlarged m / z range regions for trivalent and divalent precursors, respectively. (A) Summed neutral desorption scan mode CID MS of 1.0: 0.1 pmol / μL mixture of d ₀ and d ₅ containing methionine side chain fixed charge acetophenone sulfonium ion derivative of GAILGGAILR (SEQ ID NO: 3) / MS spectrum. Panels (B) and (C) show the individual neutral desorption mass spectra for the trivalent and divalent precursors, respectively. (A) 75 μm of a 1.0: 1.0 pmol mixture of d ₀ and d ₅ containing a methionine side chain fixed charge acetophenone (AP) sulfonium ion derivative of GAILGGAILR (SEQ ID NO: 3). D. Total ion flow trace by capillary RP-HPLC MS analysis. (B) Mass spectrum summed from the area shown in panel (A). (C) 75 μm of 1.0: 1.0 pmol mixture of d ₀ and d ₅ containing the methionine side chain fixed charge acetophenone (AP) sulfonium ion derivative of GAILGGAILR (SEQ ID NO: 3). D. Capillary RP-HPLC-total ion flow trace by neutral desorption scan mode CID MS / MS analysis. (D) Mass spectrum summed from the area shown in panel (C).

Claims (89)

A method for analyzing amino acids, peptides or proteins comprising:
(1) derivatizing a mixture of amino acids, peptides or proteins to form one or more amino acids, peptides or proteins derivatized to contain fixed charge ions at sites other than the C-terminus or N-terminus;
(2) introducing a mixture of one or more amino acids, peptides or proteins derivatized to contain fixed charge ions at sites other than the C-terminal or N-terminal, a peptide or protein mixture into a mass spectrometer;
(3) A mixture of amino acids, peptides or proteins containing one or more amino acids, peptides or proteins derivatized to contain fixed charge ions at sites other than the C-terminal or N-terminal is passed through the first mass resolving analyzer. Selecting a protein or peptide precursor ion having a first mass to charge ratio;
(4) dissociating the precursor ion having the first mass-to-charge ratio to generate a product ion having a second mass-to-charge ratio, which is a characteristic of fragmentation occurring in a site near the fixed charge; and (5) A method comprising detecting product ions having a second mass to charge ratio. The method of claim 1, wherein the product ions having a second mass to charge ratio are product ions formed by neutral desorption of fixed charges from precursor ions. The method of claim 1, wherein the product ions having a second mass to charge ratio are product ions formed by charge desorption of a fixed charge from the precursor ions. (6) The method of claim 1, further comprising determining the identity of the derivatized peptide or protein. Determining the identity of the derivatized peptide or protein includes the first iteration steps (1), (2), (3) and (4), followed by a second to determine the amino acid sequence of the peptide or protein. 5. The method of claim 4, wherein the method is performed by dissociating product ions having a mass to charge ratio to form a series of product ions having a predetermined range of mass to charge ratios. 6. The method of claim 5, wherein product ions having a second mass to charge ratio are formed by neutral desorption from the precursor ions. 6. The method of claim 5, wherein product ions having a second mass to charge ratio are formed by charge desorption of precursor ions. The method according to claim 4, wherein the step of determining the identification of the derivatized peptide or protein is carried out by use of a high resolution mass spectrometer. 9. The method of claim 8, wherein use of a high resolution mass spectrometer provides a mass accuracy of about 1-5 ppm for the product ion detected in step (5) or its complementary product ion. 5. The method of claim 4, wherein determining the identity of the derivatized peptide or protein comprises a database search to identify peptides that are known to contain a fixed charge derivative. The step of dissociation includes (i) collision with an inert gas (collision-induced dissociation (CID)) or collision-activated dissociation (CAD)), (ii) collision with a surface (surface-induced dissociation (SID)), (iii ) Photodissociation by interaction with photoparticles using an appropriate laser, (iv) thermal / blackbody infrared radiation dissociation (BIRD), and (v) monovalent cations by interaction with electron beams The method of claim 1, comprising a method selected from the group consisting of electron induced dissociation (EID), electron capture dissociation (ECD) for multivalent cations, or a combination thereof. The method according to any one of claims 1 to 11, wherein the method is used for identification of amino acids, peptides or proteins. The method according to any one of claims 1 to 11, wherein the method is used for quantification of amino acids, peptides or proteins. 12. The method according to any one of claims 1 to 11, wherein the method is used for differential quantification of amino acids, peptides or proteins based on incorporation of a suitable isotope or structural label into a fixed charge. Isotope label is a 1 or more ^{13 C, 15 N,} and ² H, The method of claim 14, wherein. The method according to any one of claims 1 to 11, wherein the method is used for analysis of a post-translational modification state of an amino acid, peptide or protein. Β-to form an O-phosphorylated and O-glycosylated serine or O-phosphorylated and O-glycosylated threonine, which are stable derivatives in mass spectrometry, for the analysis of post-translational modification states of amino acids, peptides or proteins 17. The method of claim 16, comprising incorporation of a fixed charge derivative via a removal / Michael addition method. The method according to any one of claims 1 to 11, wherein the method is used for analysis of a crosslinking state of an amino acid, a peptide or a protein. The method according to any one of claims 1 to 11, wherein the method is used for analysis of protein interaction. 20. The method according to any one of claims 1 to 19, wherein the fixed charge derivative is included in a side chain of a selected amino acid residue or a side chain of a selected amino acid residue included in a protein or peptide. . 21. The method of claim 20, wherein the selected amino acid residue is that of a rare amino acid. 21. The method of claim 20, wherein the selected amino acid residue comprises an S atom in its side chain. The method according to claim 22, wherein the amino acid residue is methionine, cysteine, homocysteine or selenocysteine. 24. The method of claim 23, wherein the amino acid residue is methionine and the method is performed according to any of Schemes 1, 3, and 4. 24. The method of claim 23, wherein the amino acid residue is cysteine and the method is performed according to any of Schemes 2, 5, 6, 7, and 8. 21. The method of claim 20, wherein the selected amino acid residue is tryptophan or tyrosine. 27. The method of claim 26, wherein the amino acid residue is tyrosine or tryptophan and the method is performed according to either scheme 5 or 6. 21. The method of claim 20, wherein the side chain comprises an S-alkyl group. 29. The method of claim 28, wherein the amino acid residue is methionine, S-alkylcysteine, S-alkylhomocysteine, S-alkyltryptophan or S-alkyltyrosine. 20. A method according to any one of claims 1 to 19, wherein the fixed charge derivative is included in the side chain of a post-translationally modified amino acid residue. 32. The method of claim 30, wherein the fixed charge derivative is included in an O-linked post-translationally modified amino acid residue. 31. The method of claim 30, wherein the O-linked post-translationally modified amino acid residue is a dehydroalanine residue formed by β-removal from an O-linked post-translationally modified serine amino acid residue (Scheme 9). 31. The method of claim 30, wherein the O-linked post-translationally modified amino acid residue is a dehydroamino-2-butyric acid residue formed by β-removal from an O-linked post-translationally modified threonine amino acid residue. 20. A method according to any one of claims 1 to 19, wherein the fixed charge derivative is comprised in a cross-linking between two amino acids, peptides or proteins (Scheme 10). 20. A method according to any one of claims 1 to 19, wherein a fixed charge derivative is included in the crosslinking reagent. 36. The fixed charge derivative is selectively pre-enriched by a solid phase capture method using a fixed charge reagent covalently bound to a bead or an insoluble polymer (Scheme 11), any one of claims 1 to 35. The method described. The method according to any one of claims 1 to 36, wherein the fixed charge ion is a sulfonium ion, a quaternary alkylammonium ion, or a quaternary alkylphosphonium ion. 38. A method according to any one of claims 1 to 37, wherein the analysis of amino acid, peptide or protein ions is performed by tandem mass spectrometry. 39. The method of claim 38, wherein the tandem mass spectrometer comprises an electrospray ionization (ESI) interface or a matrix-assisted laser desorption ionization (MALDI) interface for converting protein or peptide ions to the gas phase. 39. The method of claim 38, wherein the tandem mass spectrometer is a tandem in-space mass spectrometer, a tandem in time mass spectrometer, or a combination thereof. Tandem in-space mass spectrometer is a sector mass spectrometer, time-of-flight mass spectrometer, triple quadrupole mass spectrometer, or a hybrid mass that combines time-of-flight and quadrupole instruments 41. The method of claim 40, wherein the method is an analytical device. 42. The method according to claim 41, wherein the sector mass spectrometer is a double-focusing sector mass spectrometer or a hybrid mass spectrometer combining a sector apparatus and a quadrupole apparatus. The tandem-in-time mass spectrometer is a two-dimensional quadrupole ion trap mass spectrometer, a three-dimensional quadrupole ion trap mass spectrometer, or a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. 39. The method according to Item 38. 44. The method of any one of claims 1 to 43, further comprising one or more protein extraction steps, protein separation steps, cysteine disulfide reduction and alkylation steps, and / or digestion steps. 45. A method according to any one of claims 1 to 44, wherein the amino acid, peptide or protein is derivatized with a substituted acetophenone of the formula: or a salt or solvate thereof.

46. The method of claim 45, wherein X is a halogen, sulfonate ester, perchlorate ester or chlorosulfonate ester. A R ₁ to R ₅ is _H, R 1 '~R _6' is ¹² C, 46. The method of claim 45, wherein. 46. The method according to claim 45, wherein the substituted acetophenone is an isotopically labeled substituted acetophenone or a salt thereof or a solvate thereof. 49. The method of claim 48, wherein X is a halogen, sulfonate ester, perchlorate ester or chlorosulfonate ester. R ₁ to R ₅ or one or more, preferably 3 or more ^{_{2 H, R 1 '~R 6}} ' is ¹² C, The method of claim 49. A R ₁ to R ₅ is _H, R 1 '~R _6' any one or more, preferably 3 or more is ¹³ C, The method of claim 49. Any one or more of R ₁ to R ₅ is a functional group containing an atom other than hydrogen or carbon, any one method according to claim 45 or claim 51. 53. The method of any one of claims 45 to 52, wherein the substituted acetophenone is water soluble. R ₁ is SO ₂ H, R _{2 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R ₁ is H, R ₂ is SO ₂ H, R _{3 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R _1-2 is H, R ₃ is SO ₂ H, R _{4 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R ₁ is SO ₃ H, R _{2 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R ₁ is H, R ₂ is SO ₃ H, R _{3 to} R ₅ are H, R ₁ ′ to R ₆ ′ are ¹² C, or R _1-2 is H and R ₃ is SO in _{3 H,} with R 4 to R ₅ is _H, 54. the method of claim 53, wherein R 1 '~R _6' is ¹² C. R ₁ is SO ₂ H, any one or more of R _{2 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₁ is SO ₂ H, R _{2 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₂ is SO ₂ H, one or more of R ₁ and R _{3 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₂ is SO ₂ H, R ₁ and R _{3 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₃ is SO ₂ H, one or more of R _{1 to} R ₂ and R _{4 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₃ is SO ₂ H, R _{1 to} R ₂ and R _{4 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₁ is SO ₃ H, any one or more of R _{2 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₁ is SO ₃ H, R _{2 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₂ is SO ₃ H, and one or more of R ₁ and R _{3 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₂ is SO ₃ H, R ₁ and R _{3 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₃ is SO ₃ H and any one or more of R _{1 to} R ₂ and R _{4 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C, or R ₃ is SO ₃ H, R _{1 to} R ₂ and R _{4 to} R ₅ are H, and any one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C;
54. The method of claim 53. A substituted acetophenone of the following formula or a salt thereof or a solvate thereof.

In the formula, X is a sulfonic acid ester, a perchloric acid ester or a chlorosulfonic acid ester. R ₁ to R ₅ is _H, R 1 '~R _6' is ¹² C, substituted acetophenones of claim 56. 57. The substituted acetophenone according to claim 56, wherein the substituted acetophenone is an isotopically labeled substituted acetophenone or a salt thereof or a solvate thereof. 59. A substituted acetophenone according to claim 58, wherein any one or more of R _{1 to} R ₅ , preferably 3 or more is ² H and R ₁ ′ to R ₆ ′ is ¹² C. A R ₁ to R ₅ is _H, R 1 '~R _6' any one or more, preferably 3 or more ¹³ C, substituted acetophenones of claim 58. A water-soluble substituted acetophenone of the following formula or a salt thereof or a solvate thereof:

62. The water soluble substituted acetophenone of claim 61, wherein X is halogen, sulfonate ester, perchlorate ester or chlorosulfonate ester. 64. The water soluble substituted acetophenone of claim 62, wherein X is Br or I. R ₁ is SO ₂ H, R _{2 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R ₁ is H, R ₂ is SO ₂ H, R _{3 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R _1-2 is H, R ₃ is SO ₂ H, R _{4 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R ₁ is SO ₃ H, R _{2 to} R ₅ are H, and R ₁ ′ to R ₆ ′ are ¹² C,
R ₁ is H, R ₂ is SO ₃ H, R _{3 to} R ₅ are H, R ₁ ′ to R ₆ ′ are ¹² C, or R _1-2 is H and R ₃ is SO in _{3 H,} with R 4 to R ₅ is _H, R 1 '~R _6' is ¹² C, a water-soluble substituted acetophenone derivatives of claim 63. 62. The water soluble substituted acetophenone of claim 61 in isotopically labeled form. 65. The isotopically labeled water-soluble substituted acetophenone of claim 64, wherein X is halogen, sulfonate ester, perchlorate ester or chlorosulfonate ester. R ₁ is SO ₂ H, any one or more of R _{2 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₁ is SO ₂ H, R _{2 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₂ is SO ₂ H, one or more of R ₁ and R _{3 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₂ is SO ₂ H, R ₁ and R _{3 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₃ is SO ₂ H, one or more of R _{1 to} R ₂ and R _{4 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₃ is SO ₂ H, R _{1 to} R ₂ and R _{4 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₁ is SO ₃ H, any one or more of R _{2 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₁ is SO ₃ H, R _{2 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₂ is SO ₃ H, and one or more of R ₁ and R _{3 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C,
R ₂ is SO ₃ H, R ₁ and R _{3 to} R ₅ are H, and one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C,
R ₃ is SO ₃ H and any one or more of R _{1 to} R ₂ and R _{4 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C, or R ₃ is SO ₃ H, R _{1 to} R ₂ and R _{4 to} R ₅ are H, and any one or more of R ₁ ′ to R ₆ ′, preferably 3 or more is ¹³ C;
The isotopically labeled water-soluble substituted acetophenone of claim 66. 68. A reagent kit for analysis of amino acids, peptides, or proteins by a mass spectrometer comprising a container containing the substituted acetophenone according to any one of claims 56 to 67. A reagent kit for analysis of amino acids, peptides or proteins by mass spectrometry, comprising a container containing a substituted acetophenone of the following formula or a salt thereof or a solvate thereof.

In the formula, X is a halide. 70. The reagent kit according to claim 69, wherein X is Br or I. The reagent kit according to claim 69 or 70, wherein R _{1 to} R ₅ are H, and R ₁ 'to R ₆ ' are ¹² C. 70. The reagent kit according to claim 69, wherein the substituted acetophenone is an isotope-labeled substituted acetophenone, a salt thereof, or a solvate thereof. The reagent kit according to claim 72, wherein any one or more of R _{1 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C. The reagent kit according to claim 72, wherein R _{1 to} R ₅ are H, and any one or more of R ₁ 'to R ₆ ', preferably 3 or more, is ¹³ C. 75. The reagent kit of any one of claims 68 to 74, further comprising one or more containers comprising a cysteine disulfide reducing agent, a cysteine alkylating reagent, a protease or chemical cleavage agent, and a solvent. The reagent kit according to claim 75, wherein the cysteine disulfide reducing agent is dithiothreitol (DTT), β-mercaptoethanol, tris-carboxyethylphosphine (TCEP), and / or tributylphosphine (TBP). The reagent kit according to claim 75, wherein the cysteine alkylating reagent is an alkyl halide (eg, iodoacetic acid, iodoacetamide), vinylpyridine, or acrylamide. Protease or chemical cleaving agent is trypsin, endoproteinase Lys-C, endoproteinase Asp-N, endoproteinase Glu-C, pepsin, papain, thermolysin, cyanogen bromide, hydroxylamine hydrochloride, 2- [2'-nitro 76. The reagent kit of claim 75, which is phenylsulfenyl] -3-methyl-3'-bromoindole (BNPS-skatole), iodosobenzoic acid, pentafluoropropionic acid, and / or dilute hydrochloric acid. The reagent kit according to claim 75, wherein the solvent is urea, guanidine hydrochloride, acetonitrile, methanol and / or water. Amino acids that are derivatized to contain side chain fixed charge sulfonium ions, quaternary alkyl ammonium ions, or quaternary alkyl phosphonium ions, or peptides containing such amino acids. 81. The amino acid or peptide of claim 80, wherein the amino acid is derivatized with the substituted acetophenone of any one of claims 55-66. 81. The amino acid or peptide of claim 80, wherein the amino acid is derivatized with a substituted acetophenone of the following formula or a salt or solvate thereof.

In the formula, X is a halide. 83. The amino acid or peptide of claim 82, wherein X is Br or I. R ₁ to R ₅ is _H, R 1 '~R _6' is ¹² C, according to claim 82 or 83 amino acids or peptides described. The amino acid or peptide according to claim 82, wherein the substituted acetophenone is an isotopically labeled substituted acetophenone, a salt thereof, or a solvate thereof. 86. The amino acid or peptide according to claim 85, wherein any one or more of R _{1 to} R ₅ , preferably 3 or more is ² H, and R ₁ ′ to R ₆ ′ is ¹² C. The amino acid or peptide according to claim 85, wherein R _{1 to} R ₅ are H, and any one or more, preferably 3 or more, of R ₁ 'to R ₆ ' is ¹³ C. 88. The amino acid or peptide according to any one of claims 80 to 87, wherein the amino acid derivative is isotopically labeled. 90. A method for providing an internal standard in mass spectrometry, comprising adding a predetermined amount of the amino acid or peptide of any one of claims 80 to 88 derivatized with a fixed charge to a sample.

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