Ubiquitin Chloromethylketone Probe Enables Activity-based, Selective Protein Profiling of E2 Ubiquitin Conjugating...

Ubiquitin (Ub) conjugating enzymes (E2s) are central to Ub signaling, yet their systematic activity-based profiling remains challenging due to the weak nucleophilicity and elevated pKa of their catalytic cysteines. Existing Ub probes primarily target deubiquitinases (DUBs) and the only reported E2-targeting probe requires E1-dependent activation to capture limited E2s. To profile E2s broadly, here Ub chloromethylketone (UbCMK) is reported as a standalone activity-based probe. Density functional theory calculations identified CMK as a highly electrophilic warhead with a low activation barrier for reaction with weakly nucleophilic thiolates. UbCMK was synthesized via activated cysteine-based protein ligation and irreversibly labeled multiple E2s and cysteine DUBs. Activity-based protein profiling and quantitative proteomics in HEK293T cell lysates revealed broad enrichment of E2 enzymes, including many previously inaccessible to other probes. UbCMK furthermore enables activity-dependent quantification of endogenous E2 mobilization across oxidative, proteotoxic, inflammatory, metabolic, lipid oxidative, and genotoxic stress conditions. In addition, UbCMK engages both E1s and DUBs as well, indicating its broad utility as a probe. Collectively, these results establish UbCMK as a powerful chemical tool that expands activity-based protein profiling coverage across the Ub-proteasome system and enables functional interrogation of E2 enzymes under physiological and pathological conditions.