Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection

Heaseung Sophia Chung; Christopher I Murray; Vidya Venkatraman; Erin L Crowgey; Peter P Rainer; Robert N Cole; Ryan D Bomgarden; John C Rogers; Wayne Balkan; Joshua M Hare; David A Kass; Jennifer E Van Eyk

doi:10.1161/CIRCRESAHA.115.307336

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Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection

Journal article

Open access

Peer reviewed

Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection

Heaseung Sophia Chung, Christopher I Murray, Vidya Venkatraman, Erin L Crowgey, Peter P Rainer, Robert N Cole, Ryan D Bomgarden, John C Rogers, Wayne Balkan, Joshua M Hare, …

Circulation research, Vol.117(10), pp.846-857

2015-10-23

DOI: https://doi.org/10.1161/CIRCRESAHA.115.307336

PMCID: PMC4979977

PMID: 26338901

Abstract

Molecular Probes

Reproducibility of Results

Humans

Mice, Inbred C57BL

Male

Nitrosation

Mice, Knockout

Tandem Mass Spectrometry

Animals

Nitroso Compounds - metabolism

Myocardium - metabolism

Alcohol Dehydrogenase - genetics

HEK293 Cells

Biotin - metabolism

Female

Alcohol Dehydrogenase - deficiency

Cysteine - metabolism

Protein Processing, Post-Translational

Proteomics - methods

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. 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. 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. 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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Collaboration types: Industry collaboration; Domestic collaboration; International collaboration
Citation topics: 1 Clinical & Life Sciences; 1.127 Molecular & Cell Biology - Pharmacology; 1.127.87 Nitric Oxide
Web Of Science research areas: Cardiac & Cardiovascular Systems; Hematology; Peripheral Vascular Disease
ESI research areas: Clinical Medicine

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Title: Dual Labeling Biotin Switch Assay to Reduce Bias Derived From Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection
Creators: Heaseung Sophia Chung - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Christopher I Murray - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Vidya Venkatraman - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Erin L Crowgey - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Peter P Rainer - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Robert N Cole - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Ryan D Bomgarden - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
John C Rogers - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Wayne Balkan - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Joshua M Hare - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
David A Kass - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.)
Jennifer E Van Eyk - 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 Spectrometry and Proteomic Core Facility (R.N.C.), Johns Hopkins University School of Medicine, Baltimore, MD; Thermo Fisher Scientific, Rockford, IL (R.D.B., J.C.R.); Advanced Clinical Biosystems Research Institute, Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (V.V., E.L.C., J.E.V.E.); Department of Medicine, Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, FL (W.B., J.M.H.); Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada (C.I.M.); and Division of Cardiology, Medical University of Graz, Austria (P.P.R.). Jennifer.VanEyk@cshs.org
Publication Details: Circulation research, Vol.117(10), pp.846-857
Publisher: United States
Grant note: NHLBI-HV-05(2) / NHLBI NIH HHS P01 HL077180 / NHLBI NIH HHS R01 HL119012 / NHLBI NIH HHS P01 HL10026 / NHLBI NIH HHS P01 HL107153 / NHLBI NIH HHS 1P01HL112730-01A1 / NHLBI NIH HHS P01HL77189-01 / NHLBI NIH HHS R01 HL101235 / NHLBI NIH HHS P01 HL112730 / NHLBI NIH HHS R01HL119012 / NHLBI NIH HHS
Academic Unit: Leadership Department; Miller School of Medicine; Interdisciplinary Stem Cell Institute
Language: English
Resource Type: Journal article
PMID: 26338901
PMCID: PMC4979977
Record Identifier: 991031559376102976