SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures.
Reversible addition of NO to Cys-sulfur in proteins, a modification termed S-nitrosylation, has emerged as a ubiquitous signaling mechanism for regulating diverse cellular processes. A key first-step toward elucidating the mechanism by which S-nitrosylation modulates a protein's function is specification of the targeted Cys (SNO-Cys) residue. To date, S-nitrosylation site specification has been laboriously tackled on a protein-by-protein basis. Here we describe a high-throughput proteomic approach that enables simultaneous identification of SNO-Cys sites and their cognate proteins in complex biological mixtures. The approach, termed SNOSID (SNO Site Identification), is a modification of the biotin-swap technique [Jaffrey, S. R., Erdjument-Bromage, H., Ferris, C. D., Tempst, P. & Snyder, S. H. (2001) Nat. Cell. Biol. 3, 193-197], comprising biotinylation of protein SNO-Cys residues, trypsinolysis, affinity purification of biotinylated-peptides, and amino acid sequencing by liquid chromatography tandem MS. With this approach, 68 SNO-Cys sites were specified on 56 distinct proteins in S-nitrosoglutathione-treated (2-10 microM) rat cerebellum lysates. In addition to enumerating these S-nitrosylation sites, the method revealed endogenous SNO-Cys modification sites on cerebellum proteins, including alpha-tubulin, beta-tubulin, GAPDH, and dihydropyrimidinase-related protein-2. Whereas these endogenous SNO proteins were previously recognized, we extend prior knowledge by specifying the SNO-Cys modification sites. Considering all 68 SNO-Cys sites identified, a machine learning approach failed to reveal a linear Cys-flanking motif that predicts stable transnitrosation by S-nitrosoglutathione under test conditions, suggesting that undefined 3D structural features determine S-nitrosylation specificity. SNOSID provides the first effective tool for unbiased elucidation of the SNO proteome, identifying Cys residues that undergo reversible S-nitrosylation.
Hao
Gang
G
Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Derakhshan
Behrad
B
Shi
Lei
L
Campagne
Fabien
F
Gross
Steven S
SS
D000818:Animals; D001665:Binding Sites; D002531:Cerebellum / Q000378:metabolism; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry*; D030562:Databases, Protein; D004305:Dose-Response Relationship, Drug; D013058:Mass Spectrometry; D008956:Models, Chemical; D009584:Nitrogen / Q000737:chemistry; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013997:Time Factors; D014404:Tubulin / Q000737:chemistry
D010455:Peptides; D011506:Proteins; D013438:Sulfhydryl Compounds; D014404:Tubulin; D003545:Cysteine; D009584:Nitrogen