Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 12, Pages 4617-4622Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0900191106
Keywords
SILAC; small molecules; target identification
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Funding
- National Cancer Institute's Initiative for Chemical Genetics [N01-CO-12400]
- National Institutes of Health Genomics Based Drug Discovery-Target ID Project [RL1HG004671]
- National Institutes of Health [RL1CA133834, RL1GM084437, UL1RR024924]
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Most 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.
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