Abstract
Chemical proteomics offers a unique approach for target identification of small molecule inhibitors directly from cell extracts, thus enabling characterization of target proteins under close to physiological conditions. Here, we describe a competition binding procedure that is based on affinity enrichment of potential target proteins on a probe matrix in the presence of increasing amounts of free test compound in solution. Reduced binding of target proteins to the probe matrix as a function of test compound concentration can be measured and thus, enables calculation of IC50 values. The method employs quantitative mass spectrometry using isobaric mass tags which enables determination of potency for a large number of target proteins in a single analysis.
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Acknowledgments
We would like to thank Frank Fischer, Sonja Ghidelli-Disse, and Thilo Werner for contributions to the development of the protocol, Frank Weisbrodt for help with the figures and Gerard Drewes for discussions and advice.
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Hopf, C., Eberhard, D., Boesche, M., Bastuck, S., Dümpelfeld, B., Bantscheff, M. (2012). Determination of Kinase Inhibitor Potencies in Cell Extracts by Competition Binding Assays and Isobaric Mass Tags. In: Drewes, G., Bantscheff, M. (eds) Chemical Proteomics. Methods in Molecular Biology, vol 803. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-364-6_10
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DOI: https://doi.org/10.1007/978-1-61779-364-6_10
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