Large-scale Proteomics Analysis of the Human Kinome

Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido2,3-dpyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis. Members of the human protein kinase superfamily are the major regulatory enzymes involved in the activity control of eukaryotic signal transduction pathways. As protein kinases reside at the nodes of phosphorylation-based signal transmission, comprehensive analysis of their cellular expression and site-specific phosphorylation can provide important insights into the architecture and functionality of signaling networks. However, in global proteome studies, low cellular abundance of protein kinases often results in rather minor peptide species that are occluded by a vast excess of peptides from other cellular proteins. These analytical limitations create a rationale for kinome-wide enrichment of protein kinases prior to mass spectrometry analysis. Here, we employed stable isotope labeling by amino acids in cell culture (SILAC) to compare the binding characteristics of three kinase-selective affinity resins by quantitative mass spectrometry. The evaluated pre-fractionation tools possessed pyrido2,3-dpyrimidine-based kinase inhibitors as immobilized capture ligands and retained considerable subsets of the human kinome. Based on these results, an affinity resin displaying the broadly selective kinase ligand VI16832 was employed to quantify the relative expression of more than 170 protein kinases across three different, SILAC-encoded cancer cell lines. These experiments demonstrated the feasibility of comparative kinome profiling in a compact experimental format. Interestingly, we found high levels of cytoplasmic and low levels of receptor tyrosine kinases in MV4–11 leukemia cells compared with the adherent cancer lines HCT116 and MDA-MB-435S. The VI16832 resin was further exploited to pre-fractionate kinases for targeted phosphoproteomics analysis, which revealed about 1200 distinct phosphorylation sites on more than 200 protein kinases. This hitherto largest survey of site-specific phosphorylation across the kinome significantly expands the basis for functional follow-up studies on protein kinase regulation. In conclusion, the straightforward experimental procedures described here enable different implementations of kinase-selective proteomics with considerable potential for future signal transduction and kinase drug target analysis. Reversible protein phosphorylation represents the most common type of post-translational modification (PTM) 1The abbreviations used are:AMLacute myelogenous leukemiaFLT3FMS-like tyrosine kinase 3GOgene ontologyIPIInternational Protein IndexLC-MSliquid chromatography-mass spectrometryNEKNIMA-related expressed kinasePTKprotein tyrosine kinasePTMpost-translational modificationSILACstable isotope labeling by amino acids in cell cultureACNacetonitrileFDRfalse-discovery rate1The abbreviations used are:AMLacute myelogenous leukemiaFLT3FMS-like tyrosine kinase 3GOgene ontologyIPIInternational Protein IndexLC-MSliquid chromatography-mass spectrometryNEKNIMA-related expressed kinasePTKprotein tyrosine kinasePTMpost-translational modificationSILACstable isotope labeling by amino acids in cell cultureACNacetonitrileFDRfalse-discovery rate in eukaryotic organisms. A plethora of studies on a large variety of proteins have established that site-specific phosphorylation events fulfill key functions in the activity control of signaling cascades and networks (1Ubersax J.A. Ferrell Jr., J.E. Mechanisms of specificity in protein phosphorylation.Nat. Rev. Mol. Cell Biol. 2007; 8: 530-541Crossref PubMed Scopus (975) Google Scholar). Cellular protein phosphorylation is controlled by more than 500 members of the protein kinase superfamily, which comprises one of the largest enzyme families encoded by the human genome (2Manning 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 (6126) Google Scholar). Protein kinases represent the key elements in phosphorylation-based signal transmission. Aberrant protein kinase expression and/or activity, often because of gene amplification or mutational changes, is involved in pathological processes leading to malignant transformation and tumor development (3Blume-Jensen P. Hunter T. Oncogenic kinase signaling.Nature. 2001; 411: 355-365Crossref PubMed Scopus (3099) Google Scholar). Therefore, protein kinases have emerged as a major class of drug targets for therapeutic intervention (4Strebhardt K. Ullrich A. Paul Ehrlich's magic bullet concept: 100 years of progress.Nat. Rev. Cancer. 2008; 8: 473-480Crossref PubMed Scopus (846) Google Scholar, 5Krause D.S. Van Etten R.A. Tyrosine kinases as targets for cancer therapy.N. Engl. J. Med. 2005; 353: 172-187Crossref PubMed Scopus (1155) Google Scholar, 6Faivre S. Demetri G. Sargent W. Raymond E. Molecular basis for sunitinib efficacy and future clinical development.Nat. Rev. Drug Discov. 2007; 6: 734-745Crossref PubMed Scopus (581) Google Scholar). Given the diversity of molecular mechanisms related to de-regulated kinase function in human cancers, proteomic approaches could significantly enhance our understanding of disease-relevant kinase function and also help to optimize and adjust therapeutic strategies. In addition to assessing protein expression, the analysis of site-specific phosphorylations on protein kinases is of particular relevance, as these PTMs can be indicative of their cellular catalytic activities (7Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 8Rikova K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li J. survey of signaling kinases in 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). Protein kinases can functions and activities site-specific phosphorylation often also site-specific B. S. G. of protein activity Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the comprehensive of can provide important insights into the of these key in enzymes as protein kinases are often expressed at low cellular This can their by in peptide from cell or These analytical are further in experiments to the that species from phosphorylation events H. J.A. J. analysis by mass and the for and quantitative Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). have to be as immobilized affinity or enrichment by of have found in phosphoproteomics Yu L. of protein phosphorylation on a 2007; PubMed Scopus Google Scholar, K. H. phosphoproteomics an key in 2008; 8: PubMed Scopus Google Scholar, B. Mann M. Olsen J.V. and site-specific quantitative and Rev. PubMed Scopus Google Scholar). In to protein by or peptide by is in phosphoproteomics W. J. J.E. phosphorylation analysis of 2007; 6: PubMed Scopus Google Scholar, J.V. B. Gnad F. B. C. P. Mann M. in and site-specific phosphorylation in signaling Full Text Full Text PDF PubMed Scopus Google Scholar, M. J.E. J. Li J. of cell PubMed Scopus Google Scholar). These in with on mass the of of phosphorylation sites from cellular J.V. B. Gnad F. B. C. P. Mann M. in and site-specific phosphorylation in signaling Full Text Full Text PDF PubMed Scopus Google Scholar, J. phosphorylation analysis of 2007; PubMed Scopus Google Scholar, C. J. Bakalarski C.E. A quantitative of 2008; PubMed Scopus Google Scholar). these considerable and the is comprehensive across the of the This the rationale for to high and analytical which is for members of the protein kinase enzyme the pre-fractionation the enrichment of more than a protein kinases are affinity capture on immobilized and kinase-selective inhibitors H. of kinase-selective inhibitors by 2005; PubMed Scopus Google Scholar, K. J. A. P. S. H. K. M. A. M. H. proteomics to the cellular targets of protein kinase PubMed Scopus Google Scholar, O. G. M. P. J. T. G. proteomic of the inhibitors and kinase and 2007; PubMed Scopus Google Scholar). and have demonstrated that of kinase resins pre-fractionate kinases for phosphorylation analysis (7Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, J. L. M. G. R. Kéri G. J. H. analysis of protein kinases by target and mass Cell. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. S. M. S. T. J. M. C. G. A. T. C. G. J. B. G. proteomics mechanisms of of clinical kinase 2007; PubMed Scopus Google Scholar). capture for kinase proteomics have high the kinase protein kinases and other of cellular proteins the of the pre-fractionation our to affinity these we compared a of immobilized pyrido2,3-dpyrimidine-based inhibitors with to their kinase binding Based on an affinity displaying the VI16832 was used as an enrichment for the comparative expression analysis of protein kinases in different cancer cell lines. The VI16832 affinity resin further a phosphoproteomics survey in the and of about 1200 phosphorylation sites on more than 200 distinct protein our we immobilized kinase inhibitors from the class of to affinity resins for the pre-fractionation of protein M. J. E. A. A. P. P. M. F. W. M. S. P. Z. E. inhibitors of Med. PubMed Scopus Google Scholar). Cellular target capture was compared for three immobilized quantitative M. and quantitative proteomics Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). The and VI16832 resins as as these affinity resins of more than distinct protein kinases from a cell represent straightforward and for a considerable of the expressed human kinome. to a of protein kinases are to by In can provide a straightforward for targeted for and analysis is to the molecular the the here a large of protein kinases for signal transduction analysis, which quantitative to different of cell quantitative for the of represents a can be to other kinase-selective capture which subsets of the expressed kinome by the and VI16832 comparative be to further on described which immobilized kinase inhibitors with distinct target for of the expressed kinome (7Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, J. L. M. G. R. Kéri G. J. H. analysis of protein kinases by target and mass Cell. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. S. M. S. T. J. M. C. G. A. T. C. G. J. B. G. proteomics mechanisms of of clinical kinase 2007; PubMed Scopus Google proteomics on a of the which is of high for targeted therapeutic intervention in as human comparative analysis of three different cancer cell lines quantitative profiling of kinase expression in a compact experimental format. three cell lines can be compared by in a further is by from experiments a J.V. B. Gnad F. B. C. P. Mann M. in and site-specific phosphorylation in signaling Full Text Full Text PDF PubMed Scopus Google Scholar). kinase profiling across of cancer cell lines could expression which help to adjust targeted therapeutic to the kinases involved in could also be to the analysis of protein from tumor for by with as an to In a by K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li J. survey of signaling kinases in 2007; Full Text Full Text PDF PubMed Scopus Google cell cancer cell lines and for their survey of the into different with distinct tyrosine kinase with proteomics of can be to provide to tyrosine as as kinases also other Protein expression from the kinase of the human kinome further insights into cancer cell as by that of as A and and can in and in malignant transformation Li the key to and and PubMed Scopus Google Scholar, K. Ullrich A. kinase for cancer Rev. Cancer. 6: PubMed Scopus Google Scholar). The resins described in are for to their to capture these key enzymes as as other kinases. Here, we have further exploited the enrichment of the pyrido2,3-dpyrimidine-based capture ligand VI16832 to phosphorylation sites on protein kinases. from these provide a of for further functional studies on cellular kinase regulation. of kinase-selective quantitative and considerable potential for future studies, as kinome-wide on the protein as as the post-translational across different cancer cell lines or tumor significantly our about kinase drug targets and their activities on a Reversible protein phosphorylation represents the most common type of post-translational modification (PTM) 1The abbreviations used are:AMLacute myelogenous leukemiaFLT3FMS-like tyrosine kinase 3GOgene ontologyIPIInternational Protein IndexLC-MSliquid chromatography-mass spectrometryNEKNIMA-related expressed kinasePTKprotein tyrosine kinasePTMpost-translational modificationSILACstable isotope labeling by amino acids in cell cultureACNacetonitrileFDRfalse-discovery rate1The abbreviations used are:AMLacute myelogenous leukemiaFLT3FMS-like tyrosine kinase 3GOgene ontologyIPIInternational Protein IndexLC-MSliquid chromatography-mass spectrometryNEKNIMA-related expressed kinasePTKprotein tyrosine kinasePTMpost-translational modificationSILACstable isotope labeling by amino acids in cell cultureACNacetonitrileFDRfalse-discovery rate in eukaryotic organisms. A plethora of studies on a large variety of proteins have established that site-specific phosphorylation events fulfill key functions in the activity control of signaling cascades and networks (1Ubersax J.A. Ferrell Jr., J.E. Mechanisms of specificity in protein phosphorylation.Nat. Rev. Mol. Cell Biol. 2007; 8: 530-541Crossref PubMed Scopus (975) Google Scholar). Cellular protein phosphorylation is controlled by more than 500 members of the protein kinase superfamily, which comprises one of the largest enzyme families encoded by the human genome (2Manning 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 (6126) Google Scholar). Protein kinases represent the key elements in phosphorylation-based signal transmission. Aberrant protein kinase expression and/or activity, often because of gene amplification or mutational changes, is involved in pathological processes leading to malignant transformation and tumor development (3Blume-Jensen P. Hunter T. Oncogenic kinase signaling.Nature. 2001; 411: 355-365Crossref PubMed Scopus (3099) Google Scholar). Therefore, protein kinases have emerged as a major class of drug targets for therapeutic intervention (4Strebhardt K. Ullrich A. Paul Ehrlich's magic bullet concept: 100 years of progress.Nat. Rev. Cancer. 2008; 8: 473-480Crossref PubMed Scopus (846) Google Scholar, 5Krause D.S. Van Etten R.A. Tyrosine kinases as targets for cancer therapy.N. Engl. J. Med. 2005; 353: 172-187Crossref PubMed Scopus (1155) Google Scholar, 6Faivre S. Demetri G. Sargent W. Raymond E. Molecular basis for sunitinib efficacy and future clinical development.Nat. Rev. Drug Discov. 2007; 6: 734-745Crossref PubMed Scopus (581) Google Scholar). Given the diversity of molecular mechanisms related to de-regulated kinase function in human cancers, proteomic approaches could significantly enhance our understanding of disease-relevant kinase function and also help to optimize and adjust therapeutic strategies. In addition to assessing protein expression, the analysis of site-specific phosphorylations on protein kinases is of particular relevance, as these PTMs can be indicative of their cellular catalytic activities (7Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, 8Rikova K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li J. survey of signaling kinases in 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). Protein kinases can functions and activities site-specific phosphorylation often also site-specific B. S. G. of protein activity Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). the comprehensive of can provide important insights into the of these key in myelogenous leukemia tyrosine kinase gene Protein chromatography-mass spectrometry expressed kinase protein tyrosine kinase post-translational modification stable isotope labeling by amino acids in cell culture rate myelogenous leukemia tyrosine kinase gene Protein chromatography-mass spectrometry expressed kinase protein tyrosine kinase post-translational modification stable isotope labeling by amino acids in cell culture rate enzymes as protein kinases are often expressed at low cellular This can their by in peptide from cell or These analytical are further in experiments to the that species from phosphorylation events H. J.A. J. analysis by mass and the for and quantitative Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). have to be as immobilized affinity or enrichment by of have found in phosphoproteomics Yu L. of protein phosphorylation on a 2007; PubMed Scopus Google Scholar, K. H. phosphoproteomics an key in 2008; 8: PubMed Scopus Google Scholar, B. Mann M. Olsen J.V. and site-specific quantitative and Rev. PubMed Scopus Google Scholar). In to protein by or peptide by is in phosphoproteomics W. J. J.E. phosphorylation analysis of 2007; 6: PubMed Scopus Google Scholar, J.V. B. Gnad F. B. C. P. Mann M. in and site-specific phosphorylation in signaling Full Text Full Text PDF PubMed Scopus Google Scholar, M. J.E. J. Li J. of cell PubMed Scopus Google Scholar). These in with on mass the of of phosphorylation sites from cellular J.V. B. Gnad F. B. C. P. Mann M. in and site-specific phosphorylation in signaling Full Text Full Text PDF PubMed Scopus Google Scholar, J. phosphorylation analysis of 2007; PubMed Scopus Google Scholar, C. J. Bakalarski C.E. A quantitative of 2008; PubMed Scopus Google Scholar). these considerable and the is comprehensive across the of the This the rationale for to high and analytical which is for members of the protein kinase enzyme the pre-fractionation the enrichment of more than a protein kinases are affinity capture on immobilized and kinase-selective inhibitors H. of kinase-selective inhibitors by 2005; PubMed Scopus Google Scholar, K. J. A. P. S. H. K. M. A. M. H. proteomics to the cellular targets of protein kinase PubMed Scopus Google Scholar, O. G. M. P. J. T. G. proteomic of the inhibitors and kinase and 2007; PubMed Scopus Google Scholar). and have demonstrated that of kinase resins pre-fractionate kinases for phosphorylation analysis (7Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, J. L. M. G. R. Kéri G. J. H. analysis of protein kinases by target and mass Cell. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. S. M. S. T. J. M. C. G. A. T. C. G. J. B. G. proteomics mechanisms of of clinical kinase 2007; PubMed Scopus Google Scholar). capture for kinase proteomics have high the kinase protein kinases and other of cellular proteins the of the pre-fractionation In our to affinity these we compared a of immobilized pyrido2,3-dpyrimidine-based inhibitors with to their kinase binding Based on an affinity displaying the VI16832 was used as an enrichment for the comparative expression analysis of protein kinases in different cancer cell lines. The VI16832 affinity resin further a phosphoproteomics survey in the and of about 1200 phosphorylation sites on more than 200 distinct protein kinases. our we immobilized kinase inhibitors from the class of to affinity resins for the pre-fractionation of protein M. J. E. A. A. P. P. M. F. W. M. S. P. Z. E. inhibitors of Med. PubMed Scopus Google Scholar). Cellular target capture was compared for three immobilized quantitative M. and quantitative proteomics Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). The and VI16832 resins as as these affinity resins of more than distinct protein kinases from a cell represent straightforward and for a considerable of the expressed human kinome. to a of protein kinases are to by In can provide a straightforward for targeted for and analysis is to the molecular the the here a large of protein kinases for signal transduction analysis, which quantitative to different of cell quantitative for the of represents a can be to other kinase-selective capture which subsets of the expressed kinome by the and VI16832 comparative be to further on described which immobilized kinase inhibitors with distinct target for of the expressed kinome (7Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, J. L. M. G. R. Kéri G. J. H. analysis of protein kinases by target and mass Cell. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. S. M. S. T. J. M. C. G. A. T. C. G. J. B. G. proteomics mechanisms of of clinical kinase 2007; PubMed Scopus Google proteomics on a of the which is of high for targeted therapeutic intervention in as human comparative analysis of three different cancer cell lines quantitative profiling of kinase expression in a compact experimental format. three cell lines can be compared by in a further is by from experiments a J.V. B. Gnad F. B. C. P. Mann M. in and site-specific phosphorylation in signaling Full Text Full Text PDF PubMed Scopus Google Scholar). kinase profiling across of cancer cell lines could expression which help to adjust targeted therapeutic to the kinases involved in could also be to the analysis of protein from tumor for by with as an to In a by K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li J. survey of signaling kinases in 2007; Full Text Full Text PDF PubMed Scopus Google cell cancer cell lines and for their survey of the into different with distinct tyrosine kinase with proteomics of can be to provide to tyrosine as as kinases also other Protein expression from the kinase of the human kinome further insights into cancer cell as by that of as A and and can in and in malignant transformation Li the key to and and PubMed Scopus Google Scholar, K. Ullrich A. kinase for cancer Rev. Cancer. 6: PubMed Scopus Google Scholar). The resins described in are for to their to capture these key enzymes as as other kinases. Here, we have further exploited the enrichment of the pyrido2,3-dpyrimidine-based capture ligand VI16832 to phosphorylation sites on protein kinases. from these provide a of for further functional studies on cellular kinase regulation. of kinase-selective quantitative and considerable potential for future studies, as kinome-wide on the protein as as the post-translational across different cancer cell lines or tumor significantly our about kinase drug targets and their activities on a In our we immobilized kinase inhibitors from the class of to affinity resins for the pre-fractionation of protein M. J. E. A. A. P. P. M. F. W. M. S. P. Z. E. inhibitors of Med. PubMed Scopus Google Scholar). Cellular target capture was compared for three immobilized quantitative M. and quantitative proteomics Rev. Mol. Cell Biol. PubMed Scopus Google Scholar). The and VI16832 resins as as these affinity resins of more than distinct protein kinases from a cell represent straightforward and for a considerable of the expressed human kinome. to a of protein kinases are to by In can provide a straightforward for targeted for and analysis is to the molecular the the here a large of protein kinases for signal transduction analysis, which quantitative to different of cell The quantitative for the of represents a can be to other kinase-selective capture which subsets of the expressed kinome by the and VI16832 comparative be to further on described which immobilized kinase inhibitors with distinct target for of the expressed kinome (7Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Körner R. Greff Z. Kéri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar, J. L. M. G. R. Kéri G. J. H. analysis of protein kinases by target and mass Cell. 2007; 6: Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. S. M. S. T. J. M. C. G. A. T. C. G. J. B. G. proteomics mechanisms of of clinical kinase 2007; PubMed Scopus Google Scholar). Kinase-selective proteomics on a of the which is of high for targeted therapeutic intervention in as human comparative analysis of three different cancer cell lines quantitative profiling of kinase expression in a compact experimental format. three cell lines can be compared by in a further is by from experiments a J.V. B. Gnad F. B. C. P. Mann M. in and site-specific phosphorylation in signaling Full Text Full Text PDF PubMed Scopus Google Scholar). kinase profiling across of cancer cell lines could expression which help to adjust targeted therapeutic to the kinases involved in could also be to the analysis of protein from tumor for by with as an to In a by K. Guo A. Zeng Q. Possemato A. Yu J. Haack H. Nardone J. Lee K. Reeves C. Li Y. Hu Y. Tan Z. Stokes M. Sullivan L. Mitchell J. Wetzel R. Macneill J. Ren J.M. Yuan J. Bakalarski C.E. Villen J. Kornhauser J.M. Smith B. Li J. survey of signaling kinases in 2007; Full Text Full Text PDF PubMed Scopus Google cell cancer cell lines and for their survey of the into different with distinct tyrosine kinase with proteomics of can be to provide to tyrosine as as kinases also other Protein expression from the kinase of the human kinome further insights into cancer cell as by that of as A and and can in and in malignant transformation Li the key to and and PubMed Scopus Google Scholar, K. Ullrich A. kinase for cancer Rev. Cancer. 6: PubMed Scopus Google Scholar). The resins described in are for to their to capture these key enzymes as as other kinases. Here, we have further exploited the enrichment of the pyrido2,3-dpyrimidine-based capture ligand VI16832 to phosphorylation sites on protein kinases. from these provide a of for further functional studies on cellular kinase regulation. of kinase-selective quantitative and considerable potential for future studies, as kinome-wide on the protein as as the post-translational across different cancer cell lines or tumor significantly our about kinase drug targets and their activities on a Ullrich for of the with from the of Molecular of The was by A. The kinome in was with of Cell with with