Ong, Shao-EnSchenone, MonicaMargolin, Adam A.Li, XiaoyuDo, KathyDoud, Mary K.Mani, D. R.Kuai, LetianWang, XiangWood, John L.Tolliday, Nicola J.Koehler, Angela N.Marcaurelle, Lisa A.Golub, Todd R.Gould, Robert J.Schreiber, Stuart L.Carr, Steven A.2019-10-032009Ong, S.-E., M. Schenone, A. A. Margolin, X. Li, K. Do, M. K. Doud, D. R. Mani, et al. 2009. “Identifying the Proteins to Which Small-Molecule Probes and Drugs Bind in Cells.” Proceedings of the National Academy of Sciences 106 (12): 4617–22. https://doi.org/10.1073/pnas.0900191106.0027-84240744-28311091-6490http://nrs.harvard.edu/urn-3:HUL.InstRepos:41461195Most small-molecule probes and drugs alter cell circuitry by interacting with 1 or more proteins. A complete understanding of the interacting proteins and their associated protein complexes, whether the compounds are discovered by cell-based phenotypic or target-based screens, is extremely rare. Such a capability is expected to be highly illuminating-providing strong clues to the mechanisms used by small-molecules to achieve their recognized actions and suggesting potential unrecognized actions. We describe a powerful method combining quantitative proteomics (SILAC) with affinity enrichment to provide unbiased, robust and comprehensive identification of the proteins that bind to small-molecule probes and drugs. The method is scalable and general, requiring little optimization across different compound classes, and has already had a transformative effect on our studies of small-molecule probes. Here, we describe in full detail the application of the method to identify targets of kinase inhibitors and immunophilin binders.en-USJournal Article2019-10-0310.1073/pnas.0900191106