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Global, in situ, site-specific analysis of protein S-sulfenylation

Nature Protocols volume 10, pages 1022–1037 (2015)Cite this article

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Abstract

Protein S-sulfenylation is the reversible oxidative modification of cysteine thiol groups to form cysteine S-sulfenic acids. Mapping the specific sites of protein S-sulfenylation onto complex proteomes is crucial to understanding the molecular mechanisms controlling redox signaling and regulation. This protocol describes global, in situ, site-specific analysis of protein S-sulfenylation using sulfenic acid–specific chemical probes and mass spectrometry (MS)-based proteomics. The major steps in this protocol are as follows: (i) optimization of conditions for selective labeling of cysteine S-sulfenic acids in intact cells with the commercially available dimedone-based probe, DYn-2; (ii) tagging the modified cysteines with a functionalized biotin reagent containing a cleavable linker via Cu(I)-catalyzed azide-alkyne cycloaddition reaction; (iii) enrichment of the biotin-tagged tryptic peptides with streptavidin; (iv) liquid chromatography-tandem MS (LC-MS/MS)-based shotgun proteomics; and (v) computational data analysis. We also outline strategies for quantitative analysis of this modification in cells responding to redox perturbations and discuss special issues pertaining to experimental design of thiol redox studies. Our chemoproteomic platform should be broadly applicable to the investigation of other bio-orthogonal chemically engineered post-translational modifications. The entire analysis protocol takes ∼1 week to complete.

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Figure 1: Selective labeling and detection of protein S-sulfenic acid in cells.
Figure 2: Two workflows for proteome-wide identification of targeted sites of alkyne-tagged probes.
Figure 3: Identification of Cys238 of NAA15 as the target of S-sulfenylation in RKO cells.
Figure 4: Identification of two cysteine residues of GAPDH as the targets of S-sulfenylation in RKO cells.
Figure 5: Quantitative S-sulfenylome analysis.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grants U24CA159988 and R01GM102187.

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Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Jing Yang & Daniel C Liebler

  2. Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Jing Yang & Daniel C Liebler

  3. Department of Chemistry, The Scripps Research Institute, Jupiter, Florida, USA

    Vinayak Gupta & Kate S Carroll

  4. Department of Chemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Keri A Tallman & Ned A Porter

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  1. Jing Yang
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  2. Vinayak Gupta
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  3. Keri A Tallman
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  4. Ned A Porter
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  5. Kate S Carroll
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  6. Daniel C Liebler
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Contributions

J.Y. designed the protocol, optimized experimental conditions for applications and wrote the manuscript; V.G. synthesized the DYn-2-d6; K.A.T. synthesized Az-UV-biotin; and N.A.P., K.S.C. and D.C.L. supervised the project and edited the manuscript.

Corresponding authors

Correspondence to Jing Yang or Daniel C Liebler.

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Competing interests

D.C.L. is a stockholder in Protypia, Incorporated.

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Yang, J., Gupta, V., Tallman, K. et al. Global, in situ, site-specific analysis of protein S-sulfenylation. Nat Protoc 10, 1022–1037 (2015). https://doi.org/10.1038/nprot.2015.062

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