Harnessing Redox Cross-Reactivity To Profile Distinct Cysteine Modifications

Cysteine <i>S</i>-nitrosation and <i>S</i>-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous <i>S</i>-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of <i>S</i>-nitrosation. In parallel, <i>S</i>-nitrosothiol-linked probes enable enrichment and detection of endogenous <i>S</i>-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases <i>S</i>-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both <i>S</i>-nitrosation and <i>S</i>-sulfination. Quantitative proteomic analysis demonstrates that both <i>S</i>-nitrosation and <i>S</i>-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids