Abstract
Identifying cellular targets of bioactive small molecules from large-scale screening campaigns can be a significant bottleneck in developing novel therapeutics. Our rapid small-molecule target profiling protocol combines affinity enrichment and SILAC for proteomic identification of small molecule-protein interactions. Selective interactions are easily discernable from nonspecific protein binding by quantitative ratios. Using kinase inhibitors as an example, we provide an optimized protocol featuring on-bead protein digestion and single nano-flow liquid chromatographic-mass spectrometric (LC-MS) analyses, consequently increasing analytical throughput and sensitivity over gel-based sample preparation methods for rapid profiling of kinase inhibitor targets.
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Acknowledgments
We gratefully acknowledge the input and helpful discussions from members of the Ong Laboratory. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number R21CA177402. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Golkowski, M., Maly, D.J., Ong, SE. (2017). Proteomic Profiling of Protein Kinase Inhibitor Targets by Mass Spectrometry. In: Tan, AC., Huang, P. (eds) Kinase Signaling Networks. Methods in Molecular Biology, vol 1636. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7154-1_8
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DOI: https://doi.org/10.1007/978-1-4939-7154-1_8
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