Harnessing Redox Cross-Reactivity To Profile Distinct Cysteine Modifications

Abstract

Cysteine S-nitrosation and S-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 S-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 S-nitrosation. In parallel, S-nitrosothiol-linked probes enable enrichment and detection of endogenous S-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 S-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 S-nitrosation and S-sulfination. Quantitative proteomic analysis demonstrates that both S-nitrosation and S-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.

A proteomic approach for the analysis of <i>S</i>-nitrosylated proteins using a fluorescence labeling technique

“…Recently, Majmudar et al reported a new method for the detection of S-nitrosylated proteins and S-sulfinated proteins using fluorescent dye 25) . This method involves direct and selective labeling of S-nitrosylated proteins using biotin-SO 2 H, which reacts with S-nitrosothios.…”

Activity-based protein profiling for mapping and pharmacologically interrogating proteome-wide ligandable hotspots

“…Yang et al used the dimedone-based probe for selective labeling of S-sulfenic acids in intact cells and isoTOP-ABPP to map proteome-wide specific sites of S-sulfenylation [36]. In another study, Majmudar et al showed that sulfinate-linked probes could be used to enrich and annotate hundreds of endogenous S-nitrosated proteins [37]. Conversely, these authors also showed that S -nitrosothiol-linked probes could be used to enable enrichment and detection of endogenous S -sulfinated proteins.…”

“…Conversely, these authors also showed that S -nitrosothiol-linked probes could be used to enable enrichment and detection of endogenous S -sulfinated proteins. Using these probes, the authors demonstrated that hydrogen peroxide increased S -sulfination of human DJ-1 at Cys106, but that Cys46 and Cys53 underwent full oxidation to sulfonic acids [37]. …”

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

“…53, 54 Chemical labeling approaches based on specific cross-reactions between SOH and SNO have also been applied for selective enrichment of protein SOHs and SNOs for proteomic analysis. 55, 56 In addition to SOH and SNO, detection of SSH-modified proteins has been realized through a tag-switch approach. 57, 58 Both protein persulfides and free protein thiols were first tagged with an electrophilic reagent, methylsulfonyl benzothiazole (MSBT), to generate benzothiazole-containing disulfides (Fig.…”