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

Mol Cell. 2023 May 18;83(10):1725-1742.e12. doi: 10.1016/j.molcel.2023.03.026. Epub 2023 Apr 20.


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.

Keywords: activity-based protein profiling; chemical probe; covalent; cysteine; proteasome; protein complexes; proteomics; size-exclusion chromatography; spliceosome.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Cysteine / metabolism
  • Humans
  • Ligands
  • Proteomics* / methods
  • Transcription Factors*


  • Transcription Factors
  • Cysteine
  • Ligands