S-Alkylating Labeling Strategy for Site-Specific Identification of the S-Nitrosoproteome

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