Proteomic discovery of chemical probes that perturb protein complexes in human cells

• DOI: 10.1016/j.molcel.2023.03.026
• Publication Date: 2023-04-20T14:29:03Z
• Authors: Michael R. Lazear; Jarrett R. Remsberg; Martin G. Jaeger; Katherine Rothamel; Hsuan-lin Her; Kristen E. DeMeester; Evert Njomen; Simon J. Hogg; Jahan Rahman; Landon R. Whitby; Sang Joon Won; Michael A. Schafroth; Daisuke Ogasawara; Minoru Yokoyama; Garrett L. Lindsey; Haoxin Li; Jason Germain; Sabrina Barbas; Joan Vaughan; Thomas W. Hanigan; Vincent F. Vartab...; Christopher J. Re...; Melissa M. Dix; Seong Joo Koo; Inha Heo; John R. Teijaro; Gabriel M. Simon; Brahma Ghosh; Omar Abdel-Wahab; Kay Ahn; Alan Saghatelian; Bruno Melillo; Stuart L. Schreiber; Gene W. Yeo; Benjamin F. Cravatt
• Keywords: Spliceosome; Chemical Biology; Chemical genetics

SummaryMost 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.