title: Site specific identification of endogenous S-nitrosocysteine proteomes.
abstract: Cysteine S-nitrosylation is a post-translational modification regulating protein function and nitric oxide signaling. Herein the selectivity, reproducibility, and sensitivity of a mass spectrometry-based proteomic method for the identification of endogenous S-nitrosylated proteins are outlined. The method enriches for either S-nitrosylated proteins or peptides through covalent binding of the cysteine sulfur with phenylmercury at pH=6.0. Phenylmercury reacts selectively and efficiently with S-nitrosocysteine since no reactivity can be documented for disulfides, sulfinic or sulfonic acids, S-glutathionylated, S-alkylated or S-sulfhydrylated cysteine residues. A specificity of 97±1% for the identification of S-nitrosocysteine peptides in mouse liver tissue is achieved by the inclusion of negative controls. The method enables the detection of 36 S-nitrosocysteine peptides starting with 5pmolS-nitrosocysteine/mg of total tissue protein. Both the percentage of protein molecules modified as well as the occupancy by S-nitrosylation can be determined. Overall, selective, sensitive and reproducible enrichment of S-nitrosylated proteins and peptides is achieved by the use of phenylmercury. The inclusion of appropriate negative controls secures the precise identification of endogenous S-nitrosylated sites and proteins in biological samples.
  
  
  CONCLUSIONS
  The current study describes a selective, sensitive and reproducible method for the acquisition of endogenously S-nitrosylated proteins and peptides. The acquisition of endogenous S-nitrosoproteomes provides robust data that is necessary for investigating the mechanism(s) of S-nitrosylation in vivo, the factors that govern its selectivity, the dependency of the modification on different isoforms of nitric oxide synthases (NOS), as well as the physiological functions of this protein modification. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.
authors.affiliation: Children's Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA. Electronic address: doulias@chop.email.edu.
authors.forename: Paschalis-Thomas
authors.initials: PT
authors.lastname: Doulias
authors.forename: Margarita
authors.initials: M
authors.lastname: Tenopoulou
authors.forename: Karthik
authors.initials: K
authors.lastname: Raju
authors.forename: Lynn A
authors.initials: LA
authors.lastname: Spruce
authors.forename: Steven H
authors.initials: SH
authors.lastname: Seeholzer
authors.forename: Harry
authors.initials: H
authors.lastname: Ischiropoulos
mesh_terms: D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D010663:Phenylmercury Compounds / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D026403:S-Nitrosothiols / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity
chemical_list: D010663:Phenylmercury Compounds; D020543:Proteome; D026403:S-Nitrosothiols; C021315:S-nitrosocysteine; D019001:Nitric Oxide Synthase; D003545:Cysteine; C008859:phenylmercuric chloride
keywords: Cysteine modification; Mass spectrometry; Nitric oxide; Protein S-nitrosylation