SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures

Proc Natl Acad Sci U S A. 2006 Jan 24;103(4):1012-7. doi: 10.1073/pnas.0508412103. Epub 2006 Jan 17.

Abstract

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.

Publication types

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

MeSH terms

  • Animals
  • Binding Sites
  • Cerebellum / metabolism
  • Chromatography, Liquid
  • Cysteine / chemistry*
  • Databases, Protein
  • Dose-Response Relationship, Drug
  • Mass Spectrometry
  • Models, Chemical
  • Nitrogen / chemistry
  • Oxidation-Reduction
  • Peptides / chemistry
  • Protein Conformation
  • Proteins / chemistry*
  • Proteomics / methods*
  • Rats
  • Sulfhydryl Compounds / chemistry
  • Time Factors
  • Tubulin / chemistry

Substances

  • Peptides
  • Proteins
  • Sulfhydryl Compounds
  • Tubulin
  • Cysteine
  • Nitrogen