Development of Substrate-Directed Activity-Based Probes via Disulfide-Trapping for Site-Specific Interrogation of...

Activity-based ubiquitin probes (Ub-ABPs) have been instrumental in interrogating deubiquitinases (DUBs), yet conventional Ub-ABPs lack the substrate-specific and site-specific contextual cues required to investigate nucleosomal DUBs, whose activity is tightly regulated by the chromatin environments. Here, we developed a substrate-directed nucleosomal DUB activity-based probe (SD-NucDUB-ABP) employing a disulfide-trapping mechanism. This probe features a 2,2'-dithiodipyridine-activated 2-mercaptoethyl modification on ubiquitinated histone isopeptide amide, enabling selective covalent capture of nucleosome-interacting DUBs via their catalytic cysteines while remaining resistant to hydrolysis. Using this strategy, we synthesized the H2AK15Ub^AT nucleosome probe and applied it in the activity-based profiling of DUBs in nuclear lysates of DNA-damaged HeLa cells. Subsequent biochemical analyses confirmed USP3 as a bona fide DUB for H2AK15Ub nucleosomes. Cross-linking mass spectrometry (XL-MS) further delineated the spatial interaction network among USP3, Ub, and the nucleosome, providing mechanistic insights into H2AK15 deubiquitination. Furthermore, we extended this strategy to other nucleosomal ubiquitination sites (H2AK119Ub and H2BK120Ub), capturing their cognate DUBs (USP16 and Ubp10, respectively). The SD-NucDUB-ABP platform thus enables integrated proteomic discovery and mechanistic dissection of nucleosome-specific deubiquitination, providing a versatile chemical tool for epigenetic research at the intersection of chemical biology and chromatin biology.