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.
Ong
Shao-En
SE
Proteomics Platform, The Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA. song@broad.mit.edu
Schenone
Monica
M
Margolin
Adam A
AA
Li
Xiaoyu
X
Do
Kathy
K
Doud
Mary K
MK
Mani
D R
DR
Kuai
Letian
L
Wang
Xiang
X
Wood
John L
JL
Tolliday
Nicola J
NJ
Koehler
Angela N
AN
Marcaurelle
Lisa A
LA
Golub
Todd R
TR
Gould
Robert J
RJ
Schreiber
Stuart L
SL
Carr
Steven A
SA
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
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