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Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection.
Chung, Heaseung Sophia; Murray, Christopher I; Venkatraman, Vidya; Crowgey, Erin L; Rainer, Peter P; Cole, Robert N; Bomgarden, Ryan D; Rogers, John C; Balkan, Wayne; Hare, Joshua M; Kass, David A; Van Eyk, Jennifer E.
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  • Chung HS; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Murray CI; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Venkatraman V; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Crowgey EL; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Rainer PP; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Cole RN; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Bomgarden RD; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Rogers JC; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Balkan W; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Hare JM; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Kass DA; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
  • Van Eyk JE; From the Department of Biological Chemistry (H.S.C., C.I.M., R.N.C., J.E.V.E.), Division of Cardiology, Department of Medicine (V.V., P.P.R., D.A.K., J.E.V.E.), The Johns Hopkins NHLBI Proteomics Innovation Center on Heart Failure (H.S.C., V.V., D.A.K., J.E.V.E.), Department of Medicine, Mass Spectr
Circ Res ; 117(10): 846-57, 2015 Oct 23.
Article en En | MEDLINE | ID: mdl-26338901
RATIONALE: S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. OBJECTIVE: To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. METHODS AND RESULTS: We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. CONCLUSIONS: Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Biotina / Sondas Moleculares / Cisteína / Proteómica / Miocardio / Compuestos Nitrosos Tipo de estudio: Diagnostic_studies Límite: Animals / Female / Humans / Male Idioma: En Revista: Circ Res Año: 2015 Tipo del documento: Article
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Biotina / Sondas Moleculares / Cisteína / Proteómica / Miocardio / Compuestos Nitrosos Tipo de estudio: Diagnostic_studies Límite: Animals / Female / Humans / Male Idioma: En Revista: Circ Res Año: 2015 Tipo del documento: Article