Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies

John E. Wiktorowicz, Susan Stafford, Harriet Rea, Petri Urvil, Kizhake Soman, Alexander Kurosky, J. Regino Perez-Polo, Tor C. Savidge

Research output: Contribution to journalArticlepeer-review

31 Scopus citations


Cysteinyl S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins because of physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that uses accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo) and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia/reperfusion model and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique's power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses.

Original languageEnglish (US)
Pages (from-to)5601-5614
Number of pages14
Issue number25
StatePublished - Jun 28 2011

ASJC Scopus subject areas

  • Biochemistry


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