A systematic mass spectrometry-based chemoproteomic platform (chomiXdegrade TM ) for molecular glue degrade discovery and mechanistic dissection

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.