Summary
Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such “binding-first” assays affect protein function, nonetheless, often remains unclear. Here, we describe a “function-first” proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.
Competing Interest Statement
G.M Simon V.F. Vartabedian and L.R. Whitby are employees of Vividion Therapeutics. B.F. Cravatt is a founder and advisor to Vividion Therapeutics. G.W.Y. is a co-founder and advisor for Locanabio and Eclipse BioInnovations. G.W.Y. is a visiting professor at the National University of Singapore. G.W.Y. interests have been reviewed and approved by the University of California San Diego in accordance with its conflict-of-interest policies.
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