A protein microarray-based analysis of S-nitrosylation

Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):18948-53. doi: 10.1073/pnas.0900729106. Epub 2009 Oct 28.


The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of alpha-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amide Synthases / metabolism
  • Cysteine / metabolism*
  • Humans
  • Nitric Oxide / metabolism*
  • Protein Array Analysis / methods
  • Protein Tyrosine Phosphatases / metabolism
  • Proteins / metabolism*
  • Proteomics / methods
  • S-Nitrosothiols / metabolism*
  • Substrate Specificity
  • Thiosulfate Sulfurtransferase / metabolism
  • Ubiquitin Thiolesterase / metabolism
  • Yeasts


  • Proteins
  • S-Nitrosothiols
  • Nitric Oxide
  • Thiosulfate Sulfurtransferase
  • Protein Tyrosine Phosphatases
  • Ubiquitin Thiolesterase
  • Amide Synthases
  • NAD+ synthase
  • Cysteine