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

Lazear, M.; Remsberg, J.; Jaeger, M.; Rothamel, K.; Her, H.-l.; DeMeester, K.; Njomen, E.; Hogg, S.; Rahman, J.; Whitby, L.; Won, S. J.; Schafroth, M.; Ogasawara, D.; Yokoyama, M.; Lindsey, G.; Li, H.; Germain, J.; Barbas, S.; Vaughan, J.; Hanigan, T.; Vartabedian, V.; Reinhardt, C.; Dix, M.; Koo, S. J.; Heo, I.; Teijaro, J.; Simon, G.; Ghosh, B.; Abdel-Wahab, O.; Ahn, K.; Saghatelian, A.; Melillo, B.; Schreiber, S.; Yeo, G.; Cravatt, B.

doi:10.1101/2022.12.12.520090

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bioRxiv · 10.1101/2022.12.12.520090

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

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Abstract

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.

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Lazear, M., Remsberg, J., Jaeger, M., Rothamel, K., Her, H.-l., DeMeester, K., Njomen, E., Hogg, S., Rahman, J., Whitby, L., Won, S. J., Schafroth, M., Ogasawara, D., Yokoyama, M., Lindsey, G., Li, H., Germain, J., Barbas, S., Vaughan, J., Hanigan, T., Vartabedian, V., Reinhardt, C., Dix, M., Koo, S. J., Heo, I., Teijaro, J., Simon, G., Ghosh, B., Abdel-Wahab, O., Ahn, K., Saghatelian, A., Melillo, B., Schreiber, S., Yeo, G., Cravatt, B.. 2022-12-13. Proteomic discovery of chemical probes that perturb protein complexes in human cells. https://doi.org/10.1101/2022.12.12.520090

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