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Profiling the Global Tyrosine Phosphorylation State

2003; Elsevier BV; Volume: 2; Issue: 4 Linguagem: Inglês

10.1074/mcp.r300002-mcp200

1535-9484

Kazuya Machida, Bruce J. Mayer, Peter Nollau,

Advanced Proteomics Techniques and Applications

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome. Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome. Over the past two decades, it has become clear that tyrosine phosphorylation plays a pivotal role in a variety of important signaling pathways in multicellular organisms. In the typical vertebrate cell, phosphotyrosine (pTyr) 1The abbreviations used are: pTyrphosphotyrosinePTKprotein tyrosine kinasePTPprotein tyrosine phosphataseMSmass spectrometrySH2Src homology 2pSerphosphoserinepThrphosphothreonine2DEtwo-dimensional gel electrophoresis1Done dimensionalIPimmunoprecipitationGSTglutathione S-transferaseIMACimmobilized metal affinity chromatographyICATisotope-coded affinity tagsMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightPhIATphosphoprotein isotope-coded affinity tagsphospho-Abphosphorylation site-specific antibodyPTB domainphosphotyrosine binding domainGSH-HRPglutathione-horseradish peroxidase conjugatePDGFplatelet-derived growth factorPI3-kinasephosphatidylinositol 3-kinasePLCγphospholipase CγGAPGTPase-activating proteinRAretinoic acidMMmultiple myelomaGISTgastrointestinal stromal tumorPBMCperipheral blood mononucleated cellsAMLacute myeloid leukemiaSELDI-TOFsurface enhanced laser desorption/ionization time-of-flightPCRpolymerase chain reaction. represents only a tiny fraction of total protein phosphorylation (∼0.05%). The presence of a constitutively activated protein tyrosine kinase (PTK) oncogene product might increase this percentage 10-fold (to ∼0.5% of the total), and this relatively minor change is sufficient to induce malignant transformation (1Hunter T. Protein modification: phosphorylation on tyrosine residues.Curr. Opin. Cell Biol. 1989; 1: 1168-1181Google Scholar). Because unregulated PTK signaling causes a breakdown in the normal regulation of processes such as cell proliferation and motility, leading directly to human diseases including cancer (2Kolibaba K.S. Druker B.J. Protein tyrosine kinases and cancer.Biochim. Biophys. Acta. 1997; 1333: F217-F248Google Scholar), tyrosine kinase signaling pathways are now a major focus of biomedical research. phosphotyrosine protein tyrosine kinase protein tyrosine phosphatase mass spectrometry Src homology 2 phosphoserine phosphothreonine two-dimensional gel electrophoresis one dimensional immunoprecipitation glutathione S-transferase immobilized metal affinity chromatography isotope-coded affinity tags matrix-assisted laser desorption ionization time-of-flight phosphoprotein isotope-coded affinity tags phosphorylation site-specific antibody phosphotyrosine binding domain glutathione-horseradish peroxidase conjugate platelet-derived growth factor phosphatidylinositol 3-kinase phospholipase Cγ GTPase-activating protein retinoic acid multiple myeloma gastrointestinal stromal tumor peripheral blood mononucleated cells acute myeloid leukemia surface enhanced laser desorption/ionization time-of-flight polymerase chain reaction Given the importance of tyrosine phosphorylation, a major challenge is to develop the means to rationally control and manipulate the cellular tyrosine phosphorylation state. The potential benefits are clearly illustrated by the remarkable success of the small molecule drug Imatinib (Gleevec, STI571; Novartis, Basel, Switzerland) in treating chronic myelogenous leukemia and other malignancies (3Druker B.J. Perspectives on the development of a molecularly targeted agent.Cancer Cell. 2002; 1: 31-36Google Scholar). Chronic myelogenous leukemia is caused by a chromosome rearrangement leading to the expression of a constitutively active PTK, the BCR-Abl fusion protein, which is strongly inhibited by Imatinib. A number of other agents that target PTKs are also in various stages of development; for example Trastuzumab (Herceptin; Genentech, South San Francisco, CA), an inhibitor of HER2/Neu/Erb2 receptor-type tyrosine kinase, has shown some success in combination with other anticancer agents in treating advanced HER2-overexpressing breast cancers (4Montemurro F. Choa G. Faggiuolo R. Sperti E. Capaldi A. Donadio M. Minischetti M. Salomone A. Vietti-Ramus G. Alabiso O. Aglietta M. Safety and activity of docetaxel and trastuzumab in HER2 overexpressing metastatic breast cancer: a pilot phase II study.Am. J. Clin. Oncol. 2003; 26: 95-97Google Scholar). These success stories have clearly validated the usefulness of specific PTK inhibitors for treating human disease and also provide a persuasive justification for the identification of downstream effectors of PTK signaling, which may be expected to include novel therapeutic targets. How should we best explore the downstream target molecules in PTK or protein tyrosine phosphatase (PTP) signaling pathways? Recent technical advances, including the availability of the complete human genome sequence, have set the stage for comprehensive or global analyses of PTK/PTP signaling. There are two broad approaches to this goal. One is the comprehensive identification of all PTKs/PTPs existing in the human genome, followed by elucidation of their function and regulation in the cell. Manning et al. have termed the full complement of protein kinases the "kinome," and they currently estimate there are 90 individual PTKs in the human genome (5Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Google Scholar). Also taking into consideration the number of PTPs in the genome, the spatiotemporal regulation PTK/PTP activity is sure to be extremely complex. Therefore, the comprehensive profiling of these enzymatic activities in vivo would be an enormous challenge at the moment, although activity-based probe technology has begun to emerge as a promising tool for such studies (6Adam G.C. Sorensen E.J. Cravatt B.F. Chemical strategies for functional proteomics.Mol. Cell. Proteomics. 2002; 1: 828-835Google Scholar, 7Lo L.C. Pang T.L. Kuo C.H. Chiang Y.L. Wang H.Y. Lin J.J. Design and synthesis of class-selective activity probes for protein tyrosine phosphatases.J. Proteome Res. 2002; 1: 35-40Google Scholar). A second direction is the comprehensive identification of all tyrosine-phosphorylated proteins in the cell, the tyrosine phosphoproteome. This may be a more realistic goal, in part due to the availability of tools such as pTyr-specific antibodies (anti-pTyr) that can be used to detect or enrich for tyrosine-phosphorylated proteins. Historically, major tyrosine-phosphorylated proteins such as FAK, paxillin, p130CAS, etc. have been identified as prominent substrates in cells transformed by PTK oncogenes (8–10), implying biological relevance to transformation parameters such as cell growth, morphological alteration, or adhesion/motility, and indeed their biological importance has been validated in many cases (11–13). In contrast, the comprehensive detection of phosphoproteins must be unbiased. Furthermore, low-abundance proteins (or those phosphorylated at relatively low stoichiometry) are likely to play critical roles in vivo. Therefore, in order to explore new molecular targets in PTK/PTP signaling, reliable technologies that are both highly sensitive and selective are clearly needed. To address this challenging problem, we have taken advantage of Src homology 2 (SH2) domains to develop a strategy for profiling the global tyrosine phosphorylation state (14Nollau P. Mayer B.J. Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13531-13536Google Scholar). The SH2 domain is a small modular protein domain that binds specifically to tyrosine-phosphorylated peptide ligands; it is the most prevalent type of tyrosine phosphorylation binding motif in the cell, found in a large number of different proteins in metazoan organisms (15Bradshaw J.M. Waksman G. Molecular recognition by SH2 domains.Adv. Protein Chem. 2002; 61: 161-210Google Scholar, 16Pawson T. Gish G.D. Nash P. SH2 domains, interaction modules and cellular wiring.Trends Cell Biol. 2001; 11: 504-511Google Scholar). Because these domains play a critical role in normal signaling by mediating the formation of protein-protein complexes in response to changes in tyrosine phosphorylation, the SH2 binding pattern is likely to reflect functionally relevant aspects of the PTK signaling state. In the first part of this article, advantages and disadvantages of current mass spectrometry (MS)-based approaches for analysis of the phosphoproteome are discussed. In the next section, the SH2 domain assay (SH2 profiling) will be described, and finally prospects for the development of high-throughput SH2 profiling formats will be discussed. The detection, identification, and quantitation of phosphoproteins, and mapping of their phosphorylated sites, are the fundamental aims of phosphoproteomics. Practically, phosphoproteomic approaches can be evaluated by how many phosphoproteins, especially low-abundance proteins, are identified from complex samples such as whole-cell protein extracts. Current MS-based phosphoproteomic approaches are outlined in Table I. While the downstream MS analysis obviously plays a key role in the output of each phosphoproteomic approach, sample preparation/purification approaches will be considered first, followed by a very brief overview of MS technologies. Extensive reviews and protocols for current MS-based phosphoproteomic methods are available and should be consulted for further details (17Adam G.C. Sorensen E.J. Cravatt B.F. Chemical strategies for functional proteomics.Mol. Cell. Proteomics. 2002; 1: 781-790Scopus (169) Google Scholar, 18Conrads T.P. Issaq H.J. Veenstra T.D. New tools for quantitative phosphoproteome analysis.Biochem. Biophys. Res. Commun. 2002; 290: 885-890Google Scholar, 19Mann M. Ong S.E. Gronborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.Trends Biotechnol. 2002; 20: 261-268Google Scholar, 20Pandey A. Andersen J.S. Mann M. Use of mass spectrometry to study signaling pathways.Sci. STKE. 2000; 2000: L1Google Scholar).Table IMS-based phosphoproteomic approachesCategoryNo.FeatureAdvantagespTyrRef.EnrichmentQuantitationSite IDSensitivitySelectivityThroughputConventional12D gel21, 22 32p232P-2D gelX23–25332P-IP-2D gelX26Immunoaffinity4pTyr-IP-1D gelXXX27–30, 575pTyr-IP-2D gelXX31–336pSer/Thr-IP-1D gelX407IP/pulldown-1D gelXXXXX34–368pTyr-2D WesternXX22, 37–399pSer-2D Western37102D WesternX41112D far-WesternXX67Metal affinity12IMACXXXXX42–46131D/2D gel-IMACXXX4814pTyr-IP-IMACXXXX39, 60153H labeling-IMACXXXXX39Chemical/isotopic16BiotinylationXXX49 modification17Sulfhydryl trapXXXX5018PhIATXXXXX51, 5219d5-EtSH labelingXXX5320In vivo label-1D gelXX34, 5421NIT-1D gelXX55 Open table in a new tab Two-dimensional gel electrophoresis (2DE) is a standard tool for proteomics. However, given the low abundance of phosphoproteins, particularly those containing phosphotyrosine, this is not an efficient approach unless phosphopeptides can be enriched or specifically labeled. In a few cases, however, 2DE was successfully used in combination with functional information for analysis of phosphoproteins (21, 22). For example, Lewis et al. identified novel proteins involved in mitogen-activated protein kinase pathways by 2DE combined with MS based on the kinetics of change in abundance of spots in response to specific kinase activators (21Lewis T.S. Hunt J.B. Aveline L.D. Jonscher K.R. Louie D.F. Yeh J.M. Nahreini T.S. Resing K.A. Ahn N.G. Identification of novel MAP kinase pathway signaling targets by functional proteomics and mass spectrometry.Mol. Cell. 2000; 6: 1343-1354Google Scholar). 32P labeling is frequently used for phosphoproteomics as a highly selective and sensitive means of detecting phosphopeptides (23Immler D. Gremm D. Kirsch D. Spengler B. Presek P. Meyer H.E. Identification of phosphorylated proteins from thrombin-activated human platelets isolated by two-dimensional gel electrophoresis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS).Electrophoresis. 1998; 19: 1015-1023Google Scholar, 24Larsen M.R. Sorensen G.L. Fey S.J. Larsen P.M. Roepstorff P. Phospho-proteomics: evaluation of the use of enzymatic dephosphorylation and differential mass spectrometric peptide mass mapping for site specific phosphorylation assignment in proteins separated by gel electrophoresis.Proteomics. 2001; 1: 223-238Google Scholar, 25Yoshimura Y. Shinkawa T. Taoka M. Kobayashi K. Isobe T. Yamauchi T. Identification of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II in the postsynaptic density by protein sequencing and mass spectrometry.Biochem. Biophys. Res. Commun. 2002; 290: 948-954Google Scholar). Direct visualization and quantitation of phosphoprotein spots on 2D gels are possible with 32P labeling. Using high-resolution narrow-range 2DE, it is feasible to detect and quantitate differentially phosphorylated forms of a protein, which exhibit similar molecular mass but different isoelectric points (23Immler D. Gremm D. Kirsch D. Spengler B. Presek P. Meyer H.E. Identification of phosphorylated proteins from thrombin-activated human platelets isolated by two-dimensional gel electrophoresis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS).Electrophoresis. 1998; 19: 1015-1023Google Scholar). Moreover, 32P labeling coupled to immunoprecipitation (IP) allows phosphoproteomic analysis of particular protein complexes or organelles such as the 26S proteasome (26Mason G.G. Murray R.Z. Pappin D. Rivett A.J. Phosphorylation of ATPase subunits of the 26S proteasome.FEBS Lett. 1998; 430: 269-274Google Scholar). However, the advantages of 32P are diminishing with the emergence of MS technologies that enable the direct detection of phosphoproteins as well as identification of the phosphorylation site. In addition, inconvenience and safety issues regarding the handling of radioactive materials are an unavoidable drawback of 32P labeling and preclude its use for human tissue samples. IP with anti-pTyr is a powerful tool to enrich for low-abundance tyrosine-phosphorylated proteins, thereby improving sensitivity of detection in subsequent MS analysis. Anti-pTyr IP coupled to either one-dimensional (1D) gel electrophoresis (20, 27–30) or 2DE (31–33) are now the most prevalent formats for tyrosine phosphoproteomic analysis, allowing unambiguous identification of tyrosine-phosphorylated proteins. Gel staining can be a critical factor for the sensitivity of this approach; Maguire et al. used anti-pTyr IP and 2DE stained with SYPRO-Ruby, which is more sensitive than conventional silver staining, ultimately detecting 67 spots by 2DE and identifying 10 proteins including FAK and Syk (32). As an alternative to anti-pTyr, phosphoprotein binding motifs such as SH2 domains or 14-3-3 proteins can also be used to purify phosphoproteins from complex mixtures (34–36). Using a glutathione S-transferase (GST)-Grb2 SH2 domain fusion, Blagoev et al. identified 228 proteins from epidermal growth factor-stimulated cells, of which 28 were specifically induced upon epidermal growth factor stimulation and 2 were unknown (34Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat. Biotechnol. 2003; 21: 315-318Google Scholar). It should be noted that not all detected proteins are phosphorylated when IP or pull-down methods are used for enrichment, because unmodified proteins can bind to and coprecipitate with phosphoproteins. Combining anti-pTyr immunoblotting with 2DE provides considerable detection sensitivity for tyrosine phosphoproteins. Phosphoproteins can be identified by MS analysis of gel spots excised from a reference gel, which correspond to spots detected by immunoblotting (22Iwafune Y. Kawasaki H. Hirano H. Electrophoretic analysis of phosphorylation of the yeast 20S proteasome.Electrophoresis. 2002; 23: 329-338Google Scholar, 37Soskic V. Gorlach M. Poznanovic S. Boehmer F.D. Godovac-Zimmermann J. Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor.Biochemistry. 1999; 38: 1757-1764Google Scholar, 38Marcus K. Immler D. Sternberger J. Meyer H.E. Identification of platelet proteins separated by two-dimensional gel electrophoresis and analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry and detection of tyrosine-phosphorylated proteins.Electrophoresis. 2000; 21: 2622-2636Google Scholar, 39Ficarro S. Chertihin O. Westbrook V.A. White F. Jayes F. Kalab P. Marto J.A. Shabanowitz J. Herr J.C. Hunt D. Visconti P.E. Phosphoproteome analysis of human sperm. Evidence of tyrosine phosphorylation of AKAP 3 and valosin containing protein/P97 during capacitation.J. Biol. Chem. 2003; 278: 11579-11589Google Scholar). Although this approach can be sensitive for protein identification, in many cases the amount of protein in a spot is not sufficient for identification of the specific phosphorylation site. Because antibodies for phosphoserine (pSer) and phosphothreonine (pThr) are generally not thought to have sufficient specificity or affinity for IP, enrichment of serine- or threonine-phosphorylated proteins by IP with those antibodies has not been widely used. Recently, however, Gronborg et al. demonstrated that an anti-pSer/pThr PKA substrate antibody was capable of enriching pSer/pThr-containing proteins, leading to the identification of a novel signaling molecule (40Gronborg M. Kristiansen T.Z. Stensballe A. Andersen J.S. Ohara O. Mann M. Jensen O.N. Pandey A. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho- specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate.Mol. Cell. Proteomics. 2002; 1: 517-527Google Scholar). In addition, two-dimensional Western blot analysis with an AKT kinase substrate antibody was also used for sensitive detection of phosphosubstrates (41Jenkins L.W. Peters G.W. Dixon C.E. Zhang X. Clark R.S. Skinner J.C. Marion D.W. Adelson P.D. Kochanek P.M. Conventional and functional proteomics using large format two-dimensional gel electrophoresis 24 hours after controlled cortical impact in postnatal day 17 rats.J Neurotrauma. 2002; 19: 715-740Google Scholar). Therefore, improved pSer/pThr antibodies have the potential to play a larger role in the future. Gel-based "off-line" approaches have inherently low throughput, because electrophoresis, staining, and spot-picking are relatively slow compared with MS analysis. Immobilized metal affinity chromatography (IMAC) is a chromatographic technique for phosphopeptide enrichment based on the affinity of Fe(III) or Ga(III) for the negatively charged phosphate group and can be used in an on-line high-throughput format; accordingly, it has been widely used for phosphoproteomic studies (42–46). Although IMAC purification is not absolute due to the binding of acidic peptides (19, 47), Ficarro et al. have reduced this background binding by methylester modification of carboxyl groups prior to IMAC, thereby improving detectability of phosphopeptides in subsequent MS (45Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Google Scholar). They could detect more than a thousand phosphopeptides and identified 383 phosphorylation sites in 216 peptides starting with 500 μg of yeast protein. This is outstanding sensitivity and throughput compared with other published reports. Stensballe et al. reported that custom-made nanoscale Fe(III)-IMAC columns, in combination with 2DE, increased the likelihood of identification of phosphorylation sites (48Stensballe A. Andersen S. Jensen O.N. Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis.Proteomics. 2001; 1: 207-222Google Scholar). Of course this off-line format is incompatible with an automated high-throughput system. Tagging phosphopeptides by specific chemical modification is attractive because it is amenable to large-scale analysis. Methods in which the phosphate moiety is chemically modified, e.g. by biotinylation, allow enrichment of phosphopeptides by affinity chromatography and the subsequent unambiguous identification of the phosphorylated site (Table I, nos. 16–18) (49Oda Y. Nagasu T. Chait B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome.Nat. Biotechnol. 2001; 19: 379-382Google Scholar, 50Zhou H. Watts J.D. Aebersold R. A systematic approach to the analysis of protein phosphorylation.Nat. Biotechnol. 2001; 19: 375-378Google Scholar, 51Goshe M.B. Conrads T.P. Panisko E.A. Angell N.H. Veenstra T.D. Smith R.D. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.Anal. Chem. 2001; 73: 2578-2586Google Scholar, 52Goshe M.B. Veenstra T.D. Panisko E.A. Conrads T.P. Angell N.H. Smith R.D. Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins.Anal. Chem. 2002; 74: 607-616Google Scholar). On the other hand, methods in which peptides are differentially labeled with stable isotopes such as 12C/13C or 14N/15N allow accurate determination of the abundance of specific phosphopeptides in one sample relative to another by measuring relative MS signal intensity (Table I, nos. 15, 18–21) (34Blagoev B. Kratchmarova I. Ong S.E. Nielsen M. Foster L.J. Mann M. A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling.Nat. Biotechnol. 2003; 21: 315-318Google Scholar, 39Ficarro S. Chertihin O. Westbrook V.A. White F. Jayes F. Kalab P. Marto J.A. Shabanowitz J. Herr J.C. Hunt D. Visconti P.E. Phosphoproteome analysis of human sperm. Evidence of tyrosine phosphorylation of AKAP 3 and valosin containing protein/P97 during capacitation.J. Biol. Chem. 2003; 278: 11579-11589Google Scholar, 51Goshe M.B. Conrads T.P. Panisko E.A. Angell N.H. Veenstra T.D. Smith R.D. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.Anal. Chem. 2001; 73: 2578-2586Google Scholar, 53Weckwerth W. Willmitzer L. Fiehn O. Comparative quantification and identification of phosphoproteins using stable isotope labeling and liquid chromatography/mass spectrometry.Rapid Commun. Mass Spectrom. 2000; 14: 1677-1681Google Scholar, 54Oda Y. Huang K. Cross F.R. Cowburn D. Chait B.T. Accurate quantitation of protein expression and site-specific phosphorylation.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6591-6596Google Scholar, 55Zhang X. Jin Q.K. Carr S.A. Annan R.S. N-Terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry.Rapid Commun. Mass Spectrom. 2002; 16: 2325-2332Google Scholar). The goal of the isotope-coded affinity tag (ICAT) method is to quantitate relative protein amounts in two samples without separation by 2DE. All cysteine residues in one sample are modified with a biotinylated "heavy" isotope tag, and those of a second sample with a similar "light" isotope tag; the two samples are then combined and the relative intensity of corresponding heavy and light peptides is determined by MS (56Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Google Scholar). Phosphoprotein isotope-coded affinity tags (PhIATs) is conceptually similar to ICAT, but pSer and pThr residues are tagged instead of cysteine residues. This permits the simultaneous enrichment, quantitation, and identification of phosphopeptides via a biotinylated isotope tag (51Goshe M.B. Conrads T.P. Panisko E.A. Angell N.H. Veenstra T.D. Smith R.D. Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.Anal. Chem. 2001; 73: 2578-2586Google Scholar). While chemical modification methods based on the β-elimination reaction, including PhIAT, cannot modify tyrosine residues (Table I, nos. 16, 18, and 19), a different approach based on a carbodiimide condensation reaction can be applied to pTyr (Table I, no. 17) (50Zhou H. Watts J.D. Aebersold R. A systematic approach to the analysis of protein phosphorylation.Nat. Biotechnol. 2001; 19: 375-378Google Scholar). It has been pointed out that current chemical modification approaches can only detect relatively abundant phosphoproteins (19Mann M. Ong S.E. Gronborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.Trends Biotechnol. 2002; 20: 261-268Google Scholar). Methods aimed at enriching phosphorylated peptides prior to modification have been somewhat disappointing, with increased sensitivity compromised by losses due to multiple additional purification steps. However, a recent report demonstrated the identification of low-abundance phosphoproteins by the PhIAT system (52). There is no doubt that current MS technologies have enormous advantages over traditional Edman sequencing, both in sensitivity and throughput. For instance, Yoshimura et al. reported the identification of a total of 28 32P-labeled spots (potential substrates of Ca2+/calmodulin-dependent protein kinase II) by nanoflow liquid chromatography-tandem mass spectrometry (MS/MS) analysis, of which 12 were previously not detectable by Edman sequencing (25Yoshimura Y. Shinkawa T. Taoka M. Kobayashi K. Isobe T. Yamauchi T. Identification of protein substrates of Ca(2+)/calmodulin-dependent protein kinase II in the postsynaptic density by protein sequencing and mass spectrometry.Biochem. Biophys. Res. C