title: Identification and quantification of S-nitrosylation by cysteine reactive tandem mass tag switch assay.
abstract: Redox-switches are critical cysteine thiols that are modified in response to changes in the cell's environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT(6), is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT(6) quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches.
authors: Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland 21224, USA.
authors: Christopher I
authors: CI
authors: Murray
authors: Helge
authors: H
authors: Uhrigshardt
authors: Robert N
authors: RN
authors: O'Meally
authors: Robert N
authors: RN
authors: Cole
authors: Jennifer E
authors: JE
authors: Van Eyk
mesh_terms: D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D013438:Sulfhydryl Compounds; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D001710:Biotin; D003545:Cysteine