Activity‐Based Ubiquitin Probes Capture the Sulfenylated State of Deubiquitinases

Abstract Activity‐based ubiquitin probes (Ub‐ABPs) are powerful tools for studying the functional landscape of deubiquitinases (DUBs). While most existing Ub probes have focused on examining the native state of DUBs, oxidative stress, especially in cancer and inflammatory contexts, can oxidize the catalytic cysteine of DUBs, significantly altering their activity. Here, we developed three novel ubiquitin‐based activity probes (Ub‐ABPs) to selectively trap the sulfenylated form of deubiquitinases (DUB‐SOH). These probes employ ubiquitin as the recognition element and incorporate distinct warheads: an electrophilic norbornene moiety (Biotin‐Ub‐NMA) or dimedone‐derived cyclic C‐nucleophiles (Biotin‐Ub‐PRD and Biotin‐Ub75‐DYn‐2), enabling covalent capture of oxidized cysteine residues. Of these, Biotin‐Ub‐PRD and Biotin‐Ub75‐DYn‐2 successfully labeled DUB‐SOH, highlighting the importance of proper probe‐substrate interaction for effective trapping. Optimization of the ubiquitin length showed that the Ub74 variant displayed enhanced affinity toward DUB‐SOH. Biotin‐Ub74‐DYn‐2 enabled enrichment and identification of DUB‐SOH targets via immunocapture and label‐free quantitative proteomics. Collectively, these sulfenic acid‐targeting Ub‐ABPs represent versatile tools for elucidating redox‐dependent DUB regulation, with potential applications in understanding redox dysregulation in disease contexts.