Abstract LB037: A systematic mass spectrometry-based chemoproteomic platform (chomiXdegradeTM) for molecular glue degrade discovery and mechanistic dissection

Abstract To enable systematic discovery and mechanistic dissection of molecular glue (MG) degraders, we established an integrated mass spectrometry-based chemoproteomic platform, ChomiXDegrade™, which combines global degradation proteomics, ubiquitinomics, and living-cell proximity labeling to support MG discovery, target validation, and mechanism-of-action studies. As a proof of concept, we performed a comparative analysis of two aryl-sulfonamide MGs, E7820 and indisulam, which induce RBM39 degradation via recruitment to the DCAF15 E3 ligase but have not been systematically compared. Global degradation proteomics in AGS cells, using label-free quantification on an ultra-high-resolution Astral mass spectrometer, quantified over 7,000 proteins and revealed robust RBM39 degradation by both compounds together with distinct off-target degradation profiles. Pharmacological inhibition with MG132 or the cullin-RING E3 ligase inhibitor MLN4924 markedly suppressed RBM39 degradation, confirming a proteasome- and CRL-dependent mechanism. Complementary ubiquitinomics profiling quantified thousands of ubiquitination sites, demonstrating pronounced RBM39 ubiquitination at multiple lysine residues and revealing additional previously unrecognized ubiquitinated substrates induced by MG treatment. Furthermore, a living-cell TurboID proximity labeling strategy enabled direct proteomic capture of MG-induced ternary complexes, with reciprocal enrichment of RBM39, DCAF15, and associated interaction partners. Collectively, these results establish ChomiXDegrade™ as a scalable and versatile chemoproteomic framework for molecular glue degrader discovery and systematic characterization of substrate-E3 ligase interactions. Citation Format: Nan Chen, Fengying Zhang, Xin Yuan, Hao Hu. A systematic mass spectrometry-based chemoproteomic platform (chomiXdegradeTM) for molecular glue degrade discovery and mechanistic dissection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr LB037.