Cysteine sulfinic acid or S-sulfinylation is an oxidative post-translational modification (OxiPTM) that is known to be involved in redox-dependent regulation of protein function but has been historically difficult to analyze biochemically. To facilitate the detection of S-sulfinylated proteins, we demonstrate that a clickable, electrophilic diazene probe (DiaAlk) enables capture and site-centric proteomic analysis of this OxiPTM. Using this workflow, we revealed a striking difference between sulfenic acid modification (S-sulfenylation) and the S-sulfinylation dynamic response to oxidative stress, which is indicative of different roles for these OxiPTMs in redox regulation. We also identified >55 heretofore-unknown protein substrates of the cysteine sulfinic acid reductase sulfiredoxin, extending its function well beyond those of 2-cysteine peroxiredoxins (2-Cys PRDX1–4) and offering new insights into the role of this unique oxidoreductase as a central mediator of reactive oxygen species–associated diseases, particularly cancer. DiaAlk therefore provides a novel tool to profile S-sulfinylated proteins and study their regulatory mechanisms in cells.
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This work was supported by the National Key R&D Program of China (2016YFA0501303 to J.Y.), the National Natural Science Foundation of China (31770885 to J.Y.), the Beijing Nova Program (Z171100001117014 to J.Y.) and the US National Institutes of Health (R01 GM102187 and R01 CA174864 to K.S.C. and R01 GM072866 to W.T.L.). This work was also supported by the Wake Forest Baptist Comprehensive Cancer Center (P30CA012197 to W.T.L.). We thank Q. Zhou and W. Leng (National Center for Protein Sciences–Beijing) for expert technical assistance, C. Liu and H. Chi (Institute of Computing Technology, CAS) for helpful discussions in proteomic informatics, K. Tallman and N. Porter (Vanderbilt University) for providing light and heavy Az–UV–biotin reagents, P. Wu (The Scripps Research Institute) for providing the BTTP click ligand, M. Wilson (University of Nebraska, Lincoln) for providing recombinant DJ-1, and S. G. Rhee (Yonsei University College of Medicine) and M. Toledano (Institut des Science du Vivant Frédérique Joliot) for providing Srx+/+ and Srx–/– MEFs.
The authors declare no competing interests.
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Electronic supplementary material
Supplementary Figures 1–22
S-Sulfinylated cysteines identified and quantified in response to exogenous oxidants in A549 and HeLa cells as shown in Fig. 4
S-Sulfenylated cysteines identified and quantified in response to exogenous oxidants in A549 and HeLa cells as shown in Fig. 5
S-Sulfinylated cysteines identified and quantified in Srx+/+ and Srx–/– MEF cells as shown in Fig. 6
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Akter, S., Fu, L., Jung, Y. et al. Chemical proteomics reveals new targets of cysteine sulfinic acid reductase. Nat Chem Biol 14, 995–1004 (2018). https://doi.org/10.1038/s41589-018-0116-2
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