Abstract
We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.
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Acknowledgements
This work was partially supported by a grant from the German Bundesministerium fuer Bildung und Forschung (BMBF BioChancePLUS grant 0313335A). We would like to thank Charles Cohen, Tim Edwards, David Middlemiss, Markus Schirle, David Simmons and Francis Wilson for helpful discussions and support. We are grateful to Birgit Duempelfeld, Eva-Maria Kashammer-Lorenz, Jana Krause, Anja Podszuweit, Tatjana Rudi and Thilo Werner for expert technical assistance, to Svenja Burckhardt and Cyrille Boussard for the synthesis of compounds, to Michael Rinner, Judith Schlegl and Marianna Stabilini for database and IT tool development, and to Frank Weisbrodt for help with the figures. We would also like to thank Oliver Hantschel and Giulio Superti-Furga for stimulating discussions and valuable comments on the manuscript.
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The authors are employees of Cellzome AG or Cellzome, UK, Ltd. These companies funded the work.
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Supplementary Text and Figures
Figure 1–5, Tables 1–11, Methods, Data (PDF 2257 kb)
Supplementary Table 10
Peptide identification data collected in the experiments shown in Fig. 1 and Supplementary Fig. 1 (TXT 5210 kb)
Supplementary Table 11
Peptide identification data collected in the quantitative experiments shown in Fig. 3b and Fig. 3c. (TXT 16370 kb)
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Bantscheff, M., Eberhard, D., Abraham, Y. et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat Biotechnol 25, 1035–1044 (2007). https://doi.org/10.1038/nbt1328
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DOI: https://doi.org/10.1038/nbt1328
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