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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M800588-MCP200v1 8/7/1751    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Oppermann, F. S. Articles by Daub, H. Search for Related Content PubMed PubMed Citation Articles by Oppermann, F. S. Articles by Daub, H. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:1751-1764, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Large-scale Proteomics Analysis of the Human Kinome

Felix S. Oppermann, Florian Gnad, Jesper V. Olsen, Renate Hornberger, Zoltán Greff^¶, György Kéri^¶,||, Matthias Mann and Henrik Daub^,

From the Cell Signaling Group, Department of Molecular Biology,
Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
¶Vichem Chemie Ltd., Herman Ottó u. 15., Budapest, 1022, Hungary, and
||Pathobiochemistry Research Group of the Hungarian Academy of Science, Semmelweis University, Puskin u. 9., Budapest, 1088, Hungary

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 pyrido[2,3-d]pyrimidine-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.

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