Proteomics Analysis of Protein Kinases by Target Class-selective Prefractionation and Tandem Mass Spectrometry

Abstract

Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis. Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis. The basic principle of reversible phosphorylation of proteins represents a fundamental regulatory mechanism of central importance to signal transmission in higher organisms. In human cells, most protein phosphorylation events are catalyzed by the more than 500 members of the protein kinase superfamily of enzymes, also known as the human kinome (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome..Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6259) Google Scholar). Therefore, protein kinases take the center stage in the complex signaling networks regulating a wide range of biological processes such as cell proliferation, differentiation, and survival (2Blume-Jensen P. Hunter T. Oncogenic kinase signalling..Nature. 2001; 411: 355-365Crossref PubMed Scopus (3144) Google Scholar, 3Chang L. Karin M. Mammalian MAP kinase signalling cascades..Nature. 2001; 410: 37-40Crossref PubMed Scopus (4399) Google Scholar). Many members of the protein kinase superfamily are positively or negatively regulated by either interacting proteins or through specific phosphorylation events mediated by other protein kinases. For example, in the case of the central cell cycle regulator cyclin-dependent kinase 2 (CDK2), 1The abbreviations used are: CDK, cyclin-dependent kinase; DMF, dimethylformamide; EDC, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride; bisX, bisindolylmaleimide X; purB, purvalanol B; AMP-PNP, adenosine 5′-(β,γ-imino)triphosphate; GSK3, glycogen synthase kinase 3; RIPK2, receptor-interacting protein kinase 2; PAK4, p21-activated kinase 4; CaMK, calcium/calmodulin-dependent protein kinase; MARK, microtubule-associated protein-regulating kinase; ERK, extracellular signal-regulated kinase. both phosphorylation in the kinase domain activation loop on Thr-160 by CDK activating kinase and binding of cyclin A or cyclin E are required for the full activation of CDK2 activity, whereas phosphorylation at Thr-14 and Tyr-15 by the kinases Wee1 or Myt1 or binding of the specific protein inhibitors p21CIP1 or p27KIP1 result in inhibition of catalytic activity (4Harper J.W. Adams P.D. Cyclin-dependent kinases..Chem. Rev. 2001; 101: 2511-2526Crossref PubMed Scopus (199) Google Scholar). Upon transition to the active state, various protein kinases further undergo autophosphorylation reactions on specific serine, threonine, or tyrosine residues, which can stabilize their active conformations or lead to the generation of additional docking sites for phosphorylation-dependent protein-protein interactions (5Pawson T. Scott J.D. Signaling through scaffold, anchoring, and adaptor proteins..Science. 1997; 278: 2075-2580Crossref PubMed Scopus (1900) Google Scholar, 6Nolen B. Taylor S. Ghosh G. Regulation of protein kinases; controlling activity through activation segment conformation..Mol. Cell. 2004; 15: 661-675Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar). Although only a fraction of all human protein kinases has been studied in mechanistic detail, extrapolation of available data suggests that autophosphorylation reactions and/or phosphorylation events by upstream kinases can provide a readout for the cellular activities of almost all members of the human protein kinase superfamily. However, because protein kinases are usually of much lower abundance than their cellular substrate proteins, previous global phosphoproteome studies have resulted in the identification of rather small numbers of kinase-derived phosphopeptides (7Ballif B.A. Villen J. Beausoleil S.A. Schwartz D. Gygi S.P. Phosphoproteomic analysis of the developing mouse brain..Mol. Cell. Proteomics. 2004; 3: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 8Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins..Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1237) Google Scholar, 9Cantin G.T. Venable J.D. Cociorva D. Yates III, J.R. Quantitative phosphoproteomic analysis of the tumor necrosis factor pathway..J. Proteome Res. 2006; 5: 127-134Crossref PubMed Scopus (92) Google Scholar, 10Trinidad J.C. Specht C.G. Thalhammer A. Schoepfer R. Burlingame A.L. Comprehensive identification of phosphorylation sites in postsynaptic density preparations..Mol. Cell. Proteomics. 2006; 5: 914-922Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Even the largest dataset of 2002 phosphorylation sites reported in a study by Gygi and co-workers (8Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins..Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12130-12135Crossref PubMed Scopus (1237) Google Scholar) included only 61 from protein kinases. Consequently it appears that selective prefractionation strategies are required for the in-depth analysis of protein kinases and their cellular phosphorylation sites with currently available MS techniques. To date, the only biochemical techniques permitting the efficient isolation of certain subfractions of the expressed kinome use immobilized low molecular weight kinase inhibitors as selective capture molecules for the affinity enrichment of their cellular target proteins (11Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. Cotten M. Daub H. An efficient proteomics method to identify the cellular targets of protein kinase inhibitors..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15434-15439Crossref PubMed Scopus (324) Google Scholar, 12Brehmer D. Greff Z. Godl K. Blencke S. Kurtenbach A. Weber M. Müller S. Klebl B. Cotten M. Kéri G. Wissing J. Daub H. Cellular targets of gefitinib..Cancer Res. 2005; 65: 379-382PubMed Google Scholar, 13Daub H. Characterisation of kinase-selective inhibitors by chemical proteomics..Biochim. Biophys. Acta. 2005; 1754: 183-190Crossref PubMed Scopus (39) Google Scholar). Because up to 35 different protein kinases could be identified by the combination of kinase inhibitor affinity chromatography and MS analysis, this type of chemical proteomics strategy might be useful for comprehensive approaches to cellular kinase biochemistry in case the coverage of protein kinases can be substantially increased (14Wissing J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Here we report the development of a chromatographic method using four different kinase-selective affinity resins with distinct target specificities. This fractionation procedure efficiently isolates a large variety of protein kinases from total cell extracts and permitted the detection of up to 113 protein kinases cell by analysis. The technique further with phosphoproteomics and more than 200 phosphorylation sites from protein kinases could be in this in with and to to the to a density of 2 by and in at 2 at and in the of A and for to and of the cell in in or with and and to on to cell and in at a of For inhibitor and to and and the at in the with with and the as described D. Greff Z. Godl K. Blencke S. Kurtenbach A. Weber M. Müller S. Klebl B. Cotten M. Kéri G. Wissing J. Daub H. Cellular targets of gefitinib..Cancer Res. 2005; 65: 379-382PubMed Google Scholar, D. Godl K. B. Wissing J. Daub H. identification of cellular targets by protein kinase inhibitors..Mol. Cell. Proteomics. 2004; 5: Full Text Full Text PDF Scopus Google Scholar). For of purvalanol from in to of and by the of of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide in DMF, The on a at in the with of DMF, a by of DMF, in DMF, and of the as described (14Wissing J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). affinity resins in at in the of with of inhibitor inhibitor 2 for at and with of the cell extracts and by for at for at and through a For affinity chromatography on an system, four chromatography with of different kinase inhibitor resins to and The cell from up to or of or through four inhibitor in which the immobilized bisindolylmaleimide and purvalanol inhibitors as affinity of the the with of A and to low with of and the and with either and or 2 and of AMP-PNP, a of the and all of in with of to complex the in the the of which included in the for the the inhibitors as used for to the specific all to and to a with from the total cell and by and protein detection either by or by with the mouse synthase kinase protein kinase 2 and and kinase from Signaling in a and to protein to the method of and D. A method for the of protein in in the of and PubMed Scopus Google Scholar). to the of protein a of fraction by of as the technique to G. M. L. L. B. P. L. a for 2004; PubMed Scopus Google Scholar) as protein detection and for the of protein fraction the large of the up to of protein on on a for more than and more than proteins protein and in to four from the different by small to and as described (11Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. Cotten M. Daub H. An efficient proteomics method to identify the cellular targets of protein kinase inhibitors..Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15434-15439Crossref PubMed Scopus (324) Google Scholar). The in and a of more than on the of protein at with of with and using the and in in a phosphopeptide enrichment using a phosphopeptide enrichment all or in This to be for efficient binding of phosphopeptides a and for in of binding of and up to to the The for and this by the up to using of binding for with 2 of with of The combined and in a and the by in of by a purification using to LC-MS/MS. For analysis of the of combined The of the using a of up to of and and on a at a of in on an at a of 200 The at and at or MS and data using a and by the and for a of MS data and for protein identification by using with and in the of with with only as identified at an which or using the 2; phosphorylation Because described kinase purification have used inhibitors as selective for affinity chromatography with we that further method and the combined use of inhibitor with distinct might permit the enrichment of a fraction of cellular protein kinases D. Greff Z. Godl K. Blencke S. Kurtenbach A. Weber M. Müller S. Klebl B. Cotten M. Kéri G. Wissing J. Daub H. Cellular targets of gefitinib..Cancer Res. 2005; 65: 379-382PubMed Google Scholar, J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, D. Godl K. B. Wissing J. Daub H. identification of cellular targets by protein kinase inhibitors..Mol. Cell. Proteomics. 2004; 5: Full Text Full Text PDF Scopus Google Scholar, M. P. K. D. J. M. K. P. L. targets of cyclin-dependent kinase identification by affinity chromatography using Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. U. S. A. S. S. D. M. S. A approach to the for targets of small kinase 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). we the potential of different kinase inhibitor resins for this type of and using total cell extracts inhibitor that the of protein in the of as immobilized affinity not on these we the four different inhibitor in with to their protein binding for example, protein kinases with affinity to of the other immobilized be to the in a of the protein and a more kinase fractionation to the four different To this prefractionation total cell from on the four different inhibitor in The to the efficient of protein kinases to the or to various inhibitors of and tyrosine protein included to cellular protein phosphorylation cell and to the four from and by of with the inhibitor and the in to procedures D. Greff Z. Godl K. Blencke S. Kurtenbach A. Weber M. Müller S. Klebl B. Cotten M. Kéri G. Wissing J. Daub H. Cellular targets of gefitinib..Cancer Res. 2005; 65: 379-382PubMed Google Scholar, J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google from the to potential in phosphorylation events the of the protein kinase of the total cell and by and distinct of proteins for of the four because to the cell and the of the by the proteins subfractions of the cellular To the fractionation of known protein kinase targets of the immobilized we further the and to with selective As in most of by the and not in the from the which also known to these protein kinases K. J. U. S. A. S. S. D. M. S. A approach to the for targets of small kinase 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). for RIPK2, which from the by immobilized and not detected in the from the with binding D. Greff Z. Godl K. Blencke S. Kurtenbach A. Weber M. Müller S. Klebl B. Cotten M. Kéri G. Wissing J. Daub H. Cellular targets of gefitinib..Cancer Res. 2005; 65: 379-382PubMed Google Scholar, J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). As further in the protein kinases and PAK4, which are known protein kinase targets of and purB, by the inhibitor (14Wissing J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, K. J. U. S. A. S. S. D. M. S. A approach to the for targets of small kinase 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). these a strategy permitting the fractionation of kinase proteins four subfractions from the total cell kinase fractionation and enrichment by affinity chromatography of from cells. To the proteins by the different inhibitor of the from the bisX, and compared with of the total and by and In of the from specific with of the and to with specific GSK3, RIPK2, and as factor factor kinase; factor kinase; kinase; factor tyrosine kinase; tyrosine kinase with and we used this enrichment technique to total cell from different cell to analysis by As we used total extracts from and cells. chromatographic the the majority of protein for of the four inhibitor and the from the bisX, and resins by different molecular weight and to and the further with to and analysis of all and of the by the led to the identification of more than 140 different members of the protein kinase superfamily in the cell Many protein kinases by more than inhibitor a higher often the fractionation to of the in most In 200 other proteins different enzymes and regulatory of protein kinases also detected in the with the four inhibitor resins As in the majority of proteins from the and protein kinases, whereas the and the resins to higher numbers of other However, because in to the protein of the to protein kinases, the fractionation procedure to be kinase-selective in of protein As for the protein kinases and detected in this the more than 140 identified members of the human protein kinase superfamily in the by (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome..Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6259) Google of prefractionation procedure for a specific of protein kinases as for the tyrosine kinase in analysis of the affinity led to the identification of different sets of protein kinases from the and cells. Although the procedures used in this study do not the of kinase a variety of tyrosine kinases specific for and only in cell whereas certain tyrosine kinases expressed in factor and only detected in the from the cell and data that the procedure permitted the detection of more members of the human kinome than in previous proteomics study using immobilized kinase inhibitors (14Wissing J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In the also provide of kinase different cell The efficient enrichment of 140 protein kinases from the vast majority of other cellular proteins an for the characterization of their phosphorylation For further phosphopeptide we of the from of the different kinase affinity and of the identified in total protein kinase-derived phosphopeptides with identification higher than The phosphopeptide enrichment than and further chemical of could be different phosphopeptides from the of for to their and to the phosphorylation of these to be as a potential and the of could not be in a further The analysis of cells, which with inhibitors to cellular phosphorylation led to the identification of different phosphopeptides from protein kinases. In and which in protein and different protein kinase-derived phosphopeptides could be In we could identify different phosphorylation sites that could be at different phosphopeptides from protein kinases; of in more than cell that with of phosphorylation sites more than have not been described The from kinases phosphorylation sites are in using the and the B. B. P. M. in and phosphorylation in signaling 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). A full of all protein kinase all their from the in this study in this dataset the in-depth analysis of a variety of members of the protein kinase superfamily such as for calcium/calmodulin-dependent protein kinase microtubule-associated protein-regulating kinase 2 and and to represents of the largest of phosphorylation sites on protein kinases reported to Furthermore we used phosphopeptides to the of phosphorylation sites of As in and of in the for phosphopeptides the the of of to by the of the phosphopeptides a substrate for phosphorylation in which in to the binding domain of this kinase and identified in all cell The other the for extracellular signal-regulated kinase phosphorylation and on a phosphopeptide identified from all cell the identification of on protein kinases can provide for their by other members of this enzyme superfamily and further of these potential phosphorylation sites identified on protein kinase kinase 3; kinase; protein kinase protein kinase protein kinase; and interacting kinase; kinase kinase to the by (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome..Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6259) Google of the protein in the identified in the phosphopeptides are of the identified phosphorylation sites are in also in and in a kinase kinase 3; kinase; protein kinase protein kinase protein kinase; and interacting kinase; kinase kinase to the by (1Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome..Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6259) Google Scholar). of the protein in the identified in the phosphopeptides are of the identified phosphorylation sites are in also in and to their key regulatory functions in cell the members of the protein kinase superfamily of the most of a cellular The biochemical characterization of protein kinases usually an efficient enrichment to their analysis by MS affinity purification used to study protein kinases either on a or in the of tyrosine cellular proteins S. L. G. P. analysis of the factor by reveals Cell. Proteomics. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, R. B.A. Gygi S.P. J. The tumor kinase a target of survival 2005; 15: Full Text Full Text PDF PubMed Scopus Google Scholar, B.A. S.A. J. Gygi S.P. Quantitative phosphorylation of the and the tumor Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar, B. M. analysis of signaling networks by 2004; PubMed Scopus Google Scholar, J. A. A. H. of tyrosine phosphorylation in 2005; PubMed Scopus Google Scholar). However, these do not permit the analysis of large numbers of protein kinases from different and of the human To address this we have developed a prefractionation method that uses different immobilized kinase inhibitors as for kinase affinity and up to 113 protein kinases cell could be identified by their from the majority of other cellular The prefractionation procedure resulted in a substantially increased coverage of the expressed kinome compared with previous chemical proteomics studies with affinity resins D. Greff Z. Godl K. Blencke S. Kurtenbach A. Weber M. Müller S. Klebl B. Cotten M. Kéri G. Wissing J. Daub H. Cellular targets of gefitinib..Cancer Res. 2005; 65: 379-382PubMed Google Scholar, J. Godl K. Brehmer D. Blencke S. Weber M. Habenberger P. Stein-Gerlach M. Missio A. Cotten M. Müller S. Daub H. Chemical proteomic analysis reveals novel cellular modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors..Mol. Cell. Proteomics. 2004; 3: 1181-1193Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, D. Godl K. B. Wissing J. Daub H. identification of cellular targets by protein kinase inhibitors..Mol. Cell. Proteomics. 2004; 5: Full Text Full Text PDF Scopus Google Scholar). the immobilized kinase inhibitor potential as an efficient prefractionation Although it used as the capture for kinases not by of the other inhibitor from the to more protein kinases than identified in previous studies M. P. K. D. J. M. K. P. L. targets of cyclin-dependent kinase identification by affinity chromatography using Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. U. S. A. S. S. D. M. S. A approach to the for targets of small kinase 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Although the described in this study represents a further method by additional or more kinase inhibitors as affinity can be and further protein kinase for specific to the analysis of specific kinase by a the can be by affinity chromatography on a For example, chromatography be for the phosphoproteomics analysis of the CDK family members expressed in a cell or whereas immobilized might be useful for the analysis of the ERK, and protein kinases as as of their upstream as the kinases or the dataset a for the and of kinase-selective prefractionation strategies to the specific As in this protein kinase prefractionation combined with selective phosphopeptide enrichment permitted the identification of both known and novel phosphorylation sites on the protein kinases by analysis. The phosphorylation sites on protein kinases specific for further studies to potential regulatory functions with to cellular kinase activity In we that the phosphoproteomics analysis of protein kinases reported in study to have a potential combined with MS based for example, with distinct of and or with such as for and A. S. P. M. Quantitative phosphoproteomics to the signaling Cell. Proteomics. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, B. M. M. M. of factor in cell 2005; PubMed Scopus Google Scholar, A. J. of tyrosine phosphorylation sites in the factor signaling reveals Cell. Proteomics. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. Quantitative analysis of signaling networks by and in 2006; PubMed Scopus Google Scholar). Quantitative and phosphorylation a in signal transduction studies as by for HeLa B. B. P. M. in and phosphorylation in signaling 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of and to such as or and are in both and In this the of study affinity purification strategies the of of the signal transduction that this of phosphorylation events on the central of cellular signal transduction networks and provide the signaling of biological are for the by that to this by The kinome in with of Signaling