Dataset Information

Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation.

ABSTRACT: S-nitrosylation, the selective posttranslational modification of protein cysteine residues to form S-nitrosocysteine, is one of the molecular mechanisms by which nitric oxide influences diverse biological functions. In this study, unique MS-based proteomic approaches precisely pinpointed the site of S-nitrosylation in 328 peptides in 192 proteins endogenously modified in WT mouse liver. Structural analyses revealed that S-nitrosylated cysteine residues were equally distributed in hydrophobic and hydrophilic areas of proteins with an average predicted pK(a) of 10.01 ± 2.1. S-nitrosylation sites were over-represented in α-helices and under-represented in coils as compared with unmodified cysteine residues in the same proteins (χ(2) test, P < 0.02). A quantile-quantile probability plot indicated that the distribution of S-nitrosocysteine residues was skewed toward larger surface accessible areas compared with the unmodified cysteine residues in the same proteins. Seventy percent of the S-nitrosylated cysteine residues were surrounded by negatively or positively charged amino acids within a 6-Å distance. The location of cysteine residues in α-helices and coils in highly accessible surfaces bordered by charged amino acids implies site directed S-nitrosylation mediated by protein-protein or small molecule interactions. Moreover, 13 modified cysteine residues were coordinated with metals and 15 metalloproteins were endogenously modified supporting metal-catalyzed S-nitrosylation mechanisms. Collectively, the endogenous S-nitrosoproteome in the liver has structural features that accommodate multiple mechanisms for selective site-directed S-nitrosylation.

SUBMITTER: Doulias PT

PROVIDER: S-EPMC2947911 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description: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.Biological significanceThe 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.

Dataset Information

Structural profiling of endogenous S-nitrosocysteine residues reveals unique features that accommodate diverse mechanisms for protein S-nitrosylation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets