Quantitative profiling of reversible cysteome modification under nitrosative stress

Yue Ting Wang, Sujeewa C. Piyankarage, Gregory R.J. Thatcher

Research output: Chapter in Book/Report/Conference proceedingChapter


Reversible modifications of protein cysteine residues via S-nitrosylation and S-oxidation via disulfide formation are posttranslational modifications (PTM) regulating a broad range of protein activities and cellular signaling. Dysregulated protein nitrosothiol and disulfide formation have been implicated in pathogenesis of neurodegenerative disorders. Under nitrosative or nitroxidative stress, both nitrosylation and oxidation can theoretically occur at redox-sensitive cysteine residues, mediating thiol-regulated stress response. However, few detection strategies address both modifications. Nonquantitative approaches used to observe S-nitrosylation, regardless of unmodified and oxidized thiol forms, may lead to causal conclusions about the importance of protein nitrosothiol in NO-mediated signaling, regulation, and stress response. To observe quantitatively the modification spectrum of the cysteome, we developed a mass spectrometry-based approach, denoted as d-SSwitch, using isotopic labeling and shotgun proteomics to simultaneously identify and quantify different modification states at individual cysteine residues. Both recombinant protein and intact neuroblastoma cells were analyzed by d-SSwitch after treatment with nitrosothiol or NO. In proteins identified to be modified after nitrosothiol treatment, S-oxidation was always observed concomitant with S-nitrosylation and was quantitatively dominant. Herein, we describe the detailed procedures of d-SSwitch and important notes in practice.

Original languageEnglish (US)
Title of host publicationNeuromethods
PublisherHumana Press Inc.
Number of pages18
StatePublished - Feb 23 2016
Externally publishedYes

Publication series

ISSN (Print)0893-2336
ISSN (Electronic)1940-6045


  • D-SSwitch
  • Disulfide formation
  • MS
  • Nitrosative stress
  • Posttranslational modification (PTM)
  • S-Nitrosylation

ASJC Scopus subject areas

  • General Neuroscience
  • General Biochemistry, Genetics and Molecular Biology
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Psychiatry and Mental health


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