Bioorthogonal Probe BTD-Az Enables Sensitive and Rapid In Vivo Profiling of Protein Cysteine Sulfenylation

Cysteine sulfenylation (Cys-SOH) is a transient redox-sensitive post-translational modification that regulates protein activity and cellular stress responses, yet its in vivo dynamics remain difficult to capture due to short half-life and low abundance. Here, we report the design and application of BTD-Az, a cell-permeable probe that incorporates an azide handle into the benzothiazine scaffold, enabling rapid bioorthogonal labeling of Cys-SOH both in vivo and in vitro, followed by efficient enrichment through strain-promoted azide-alkyne cycloaddition. This approach streamlines the detection and analysis of Cys-SOH with exceptional specificity and convenience. BTD-Az exhibited negligible cytotoxicity, efficiently enriched sulfenylated proteins in vitro, and achieved direct in vivo labeling in mouse tissues through SPAAC-mediated pull-down. Coupling BTD-Az with 4D-DIA proteomics allowed global sulfenylome profiling, revealing >5000 labeled proteins across tissues. As a proof-of-concept biological application, we applied BTD-Az to aging cartilage and identified 95 proteins with differential sulfenylation, including the mitochondrial enzyme IDH2, whose Cys-SOH modification promoted proteasomal degradation and exacerbated redox imbalance. Collectively, this study establishes BTD-Az as a robust chemical tool for in vivo sulfenylation profiling, providing a broadly applicable platform for redox proteomics and the discovery of redox-sensitive regulatory mechanisms in health and disease.