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

Abstract

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.

Endothelial NO Synthase-Dependent S-Nitrosylation of β-Catenin Prevents Its Association with TCF4 and Inhibits Proliferation of Endothelial Cells Stimulated by Wnt3a

“…We have previously shown by liquid chromatography-tandem mass spectrometry (LC-MS-MS) analyses that cysteine residues 300, 381, 439, 466, 520, and 619 of ␤-catenin are possible substrates for SNO by NO (26). In addition, Cys213 was identified as a potential SNO site in another proteomics study (28). To determine which cysteine residue of ␤-catenin the inhibitory effects of NO on the transcriptional activity of ␤-catenin are attributable to, we generated ␤-catenin mutants in which cysteine residues were replaced by nonnitrosylable serine.…”

“…We also identified other Cys residues on ␤-catenin that could be targets for SNO, namely, Cys300, -381, -439, -466, and -520 (26). Furthermore, Cys213 and -520 were identified as potential SNO sites in large-scale proteomics assays (27,28). In addition to its effects at cell-cell junctions, it has been shown that NO can inhibit the transcriptional activity of ␤-catenin in the nucleus and reduce proliferation of cancer cells (29,30).…”

Site-Specific Nitrosoproteomic Identification of EndogenouslyS-Nitrosylated Proteins in Arabidopsis

“…To more precisely identify S-nitrosylated proteins and their modified Cys residues, we used a site-specific proteomics approach to characterize S-nitrosylated peptides. In this method, the biotinylated proteins were first subjected to tryptic digestion before biotin-affinity purification, and the biotinylated peptides were then affinity purified for further analysis (Hao et al, 2006;Chen et al, 2010). Using this method, we analyzed and identified S-nitrosylated peptides from Col-0 and gsnor1-3 seedlings.…”

“…Although the method is relatively simple, the number of S-nitrosylated proteins identified by shotgun proteomics is often few due to various technical limitations (Torta et al, 2008). The identification capacity of nitrosoproteomics was greatly improved by the site-specific strategy, in which biotinylated proteins were first digested by trypsin and the enriched peptides were then characterized by mass spectrometry (Hao et al, 2006;Chen et al, 2010). Moreover, S-nitrosylated Cys residues can also be identified from site-specific nitrosoproteomic analysis.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…Experimentally these algorism were found to be 75% accurate at predicting sites of S-nitrosation [56]. However, these motifs have been criticised due to the high concentrations of NO donor used in the founding experiments to map S-nitrosation sites [57,58], and so provide a platform for the prediction algorithm. Supra-physiological concentrations of NO may have resulted in the identification of motifs that lack biological significance.…”