title: Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents.
abstract: S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography-tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease.
authors.affiliation: Department of Pathology and Anatomical Sciences, ‡Department of Biochemistry, and §Center for Translational Neuroscience, University of Missouri School of Medicine , Columbia, Missouri, United States.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Qu
authors.forename: Fanjun
authors.initials: F
authors.lastname: Meng
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Bomgarden
authors.forename: Rosa I
authors.initials: RI
authors.lastname: Viner
authors.forename: Jilong
authors.initials: J
authors.lastname: Li
authors.forename: John C
authors.initials: JC
authors.lastname: Rogers
authors.forename: Jianlin
authors.initials: J
authors.lastname: Cheng
authors.forename: C Michael
authors.initials: CM
authors.lastname: Greenlief
authors.forename: Jiankun
authors.initials: J
authors.lastname: Cui
authors.forename: Dennis B
authors.initials: DB
authors.lastname: Lubahn
authors.forename: Grace Y
authors.initials: GY
authors.lastname: Sun
authors.forename: Zezong
authors.initials: Z
authors.lastname: Gu
mesh_terms: D000818:Animals; D002460:Cell Line; D007461:Iodoacetates / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D058977:Molecular Sequence Annotation; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D013194:Staining and Labeling
chemical_list: D007461:Iodoacetates; D008070:Lipopolysaccharides