title: Identifying the proteins to which small-molecule probes and drugs bind in cells.
abstract: 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.
authors.affiliation: Proteomics Platform, The Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. song@broad.mit.edu
authors.forename: Shao-En
authors.initials: SE
authors.lastname: Ong
authors.forename: Monica
authors.initials: M
authors.lastname: Schenone
authors.forename: Adam A
authors.initials: AA
authors.lastname: Margolin
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Li
authors.forename: Kathy
authors.initials: K
authors.lastname: Do
authors.forename: Mary K
authors.initials: MK
authors.lastname: Doud
authors.forename: D R
authors.initials: DR
authors.lastname: Mani
authors.forename: Letian
authors.initials: L
authors.lastname: Kuai
authors.forename: Xiang
authors.initials: X
authors.lastname: Wang
authors.forename: John L
authors.initials: JL
authors.lastname: Wood
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Tolliday
authors.forename: Angela N
authors.initials: AN
authors.lastname: Koehler
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Marcaurelle
authors.forename: Todd R
authors.initials: TR
authors.lastname: Golub
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Gould
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Schreiber
authors.forename: Steven A
authors.initials: SA
authors.lastname: Carr
mesh_terms: D002227:Carbazoles / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020104:Immunophilins / Q000737:chemistry / Q000378:metabolism; D026121:Indole Alkaloids / Q000378:metabolism; D007553:Isotope Labeling; D008024:Ligands; D008863:Microspheres; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D015335:Molecular Probes / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics; D012995:Solubility
chemical_list: D002227:Carbazoles; D026121:Indole Alkaloids; D008024:Ligands; D008869:Microtubule-Associated Proteins; D015335:Molecular Probes; D004364:Pharmaceutical Preparations; D047428:Protein Kinase Inhibitors; D011506:Proteins; C049985:staurosporine aglycone; D011494:Protein Kinases; D020104:Immunophilins