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Endogenous S-nitrosocysteine proteomic inventories identify a core of proteins in heart metabolic pathways.

ABSTRACT: Protein cysteine residues are essential for protein folding, participate in enzymatic catalysis, and coordinate the binding of metal ions to proteins. Enzymatically catalyzed and redox-dependent post-translational modifications of cysteine residues are also critical for signal transduction and regulation of protein function and localization. S-nitrosylation, the addition of a nitric oxide equivalent to a cysteine residue, is a redox-dependent modification. In this study, we curated and analyzed four different studies that employed various chemoselective platforms coupled to mass spectrometry to precisely identify S-nitrosocysteine residues in mouse heart proteins. Collectively 1974 S-nitrosocysteine residues in 761 proteins were identified and 33.4% were identified in two or more studies. A core of 75 S-nitrosocysteine residues in 44 proteins were identified in all four studies. Bioinformatic analysis of each study indicated a significant enrichment of mitochondrial proteins participating in metabolism. Regulatory proteins in glycolysis, TCA cycle, oxidative phosphorylation and ATP production, long chain fatty acid β-oxidation, and ketone and amino acid metabolism constitute the major functional pathways impacted by protein S-nitrosylation. In the cardiovascular system, nitric oxide signaling regulates vasodilation and cardiac muscle contractility. The meta-analysis of the proteomic data supports the hypothesis that nitric oxide signaling via protein S-nitrosylation is also a regulator of cardiomyocyte metabolism that coordinates fuel utilization to maximize ATP production. As such, protein cysteine S-nitrosylation represents a third functional dimension of nitric oxide signaling in the cardiovascular system to ensure optimal cardiac function.

SUBMITTER: Lau B

PROVIDER: S-EPMC8497991 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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Endogenous S-nitrosocysteine proteomic inventories identify a core of proteins in heart metabolic pathways.

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