S-alkylating labeling strategy for site-specific identification of the s-nitrosoproteome

J Proteome Res. 2010 Dec 3;9(12):6417-39. doi: 10.1021/pr100680a. Epub 2010 Nov 10.


S-nitrosylation, a post-translational modification of cysteine residues induced by nitric oxide, mediates many physiological functions. Due to the labile nature of S-nitrosylation, detection by mass spectrometry (MS) is challenging. Here, we developed an S-alkylating labeling strategy using the irreversible biotinylation on S-nitrosocysteines for site-specific identification of the S-nitrosoproteome by LC-MS/MS. Using COS-7 cells without endogenous nitric oxide synthase, we demonstrated that the S-alkylating labeling strategy substantially improved the blocking efficiency of free cysteines, minimized the false-positive identification caused by disulfide interchange, and increased the digestion efficiency for improved peptide identification using MS analyses. Using this strategy, we identified total 586 unique S-nitrosylation sites corresponding to 384 proteins in S-nitroso-N-acetylpenicillamine (SNAP)/l-cysteine-treated mouse MS-1 endothelial cells, including 234 previously unreported S-nitrosylated proteins. When the topologies of 84 identified transmembrane proteins were further analyzed, their S-nitrosylation sites were found to mostly face the cytoplasmic side, implying that S-nitrosylation occurs in the cytoplasm. In addition to the previously known acid/basic motifs, the ten deduced consensus motifs suggested that combination of local hydrophobicity and acid/base motifs in the tertiary structure contribute to the specificity of S-nitrosylation. Moreover, the S-nitrosylated cysteines showed preference on beta-strand, having lower relative surface accessibility at the S-nitrosocysteines.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Biotinylation / methods*
  • COS Cells
  • Cell Line
  • Chlorocebus aethiops
  • Chromatography, Liquid
  • Cysteine / analogs & derivatives*
  • Cysteine / metabolism
  • Mice
  • Proteome / analysis*
  • Proteome / classification
  • Proteome / metabolism
  • Proteomics / methods*
  • S-Nitroso-N-Acetylpenicillamine / metabolism
  • S-Nitrosothiols / metabolism*
  • Spectrometry, Mass, Electrospray Ionization


  • Proteome
  • S-Nitrosothiols
  • S-Nitroso-N-Acetylpenicillamine
  • S-nitrosocysteine
  • Cysteine