Identifying the proteins to which small-molecule probes and drugs bind in cells.
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.
|Year of Publication||
Proc Natl Acad Sci U S A
2009 Mar 24
|PubMed Central ID||
UL1RR024924 / RR / NCRR NIH HHS / United States
RL1HG004671 / HG / NHGRI NIH HHS / United States
RL1CA133834 / CA / NCI NIH HHS / United States
RL1GM084437 / GM / NIGMS NIH HHS / United States
UL1 RR024924 / RR / NCRR NIH HHS / United States
RL1 GM084437 / GM / NIGMS NIH HHS / United States
N01CO12400 / CA / NCI NIH HHS / United States
RL1 HG004671 / HG / NHGRI NIH HHS / United States
RL1 CA133834 / CA / NCI NIH HHS / United States
N01-CO-12400 / CO / NCI NIH HHS / United States