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Chemical proteomics reveals new targets of cysteine sulfinic acid reductase


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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Fig. 1: Development of electrophilic nitrogen species for labeling sulfinic acids.
Fig. 2: Reactivity of electron-deficient diazenes toward recombinant-protein models.
Fig. 3: Detection of protein S-sulfinylation from cells with DiaFluo.
Fig. 4: Site-centric and quantitative chemoproteomic profiling of protein S-sulfinylation.
Fig. 5: Comparison of S-sulfenylome and S-sulfinylome sites and dynamic fold changes.
Fig. 6: Proteome-wide analysis of SRX-regulated changes in S-sulfinylation.


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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.

Author information




K.S.C. and J.Y. designed the experiments, analyzed data and wrote the manuscript, and all authors provided input. K.S.C. and M.L. devised the ENS concept for sulfinic acid detection. M.L. performed reactions of diazonium salts and diazenes with model sulfinic acids and fluorescence imaging. J.Y. developed the chemoproteomic method. L.F. and R.S. performed chemoproteomic experiments and validation of SRX substrates. S.A. verified and maintained cell lines; analyzed redox, time and dose dependence of DiaAlk proteome labeling; developed BioDiaAlk and SRX luminescence-based ATPase assays; and performed validation of SRX substrates. Y.J. synthesized probes; performed rate and adduct-stability studies; and characterized the N-Fmoc cysteine sulfur acid–DiaAlk adducts. J.R.L. and W.T.L. purified recombinant SRX and PRDX2. K.L. performed computational and bioinformatics analyses.

Corresponding authors

Correspondence to Jing Yang or Kate S. Carroll.

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Supplementary Text and Figures

Supplementary Figures 1–22

Reporting Summary

Supplementary Note

Synthetic procedures

Supplementary Dataset 1

S-Sulfinylated cysteines identified and quantified in response to exogenous oxidants in A549 and HeLa cells as shown in Fig. 4

Supplementary Dataset 2

S-Sulfenylated cysteines identified and quantified in response to exogenous oxidants in A549 and HeLa cells as shown in Fig. 5

Supplementary Dataset 3

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).

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