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

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Overview

authors

  • Yang, J.
  • Gupta, V.
  • Tallman, K. A.
  • Porter, N. A.
  • Carroll, Kate
  • Liebler, D. C.

publication date

  • 2015

journal

  • Nature Protocols  Journal

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.
  • 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.

subject areas

  • Cell Line
  • Humans
  • Oxidation-Reduction
  • Protein Processing, Post-Translational
  • Proteins
  • Proteomics
  • Sulfenic Acids
  • Tandem Mass Spectrometry
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Identity

PubMed Central ID

  • PMC4608369

International Standard Serial Number (ISSN)

  • 1754-2189

Digital Object Identifier (DOI)

  • 10.1038/nprot.2015.062

PubMed ID

  • 26086405
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Additional Document Info

start page

  • 1022

end page

  • 1037

volume

  • 10

issue

  • 7

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