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Direct Measurement of S-Nitrosothiols with an Orbitrap Fusion Mass Spectrometer: S-Nitrosoglutathione Reductase as a Model Protein

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Nitric Oxide

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1747))

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Abstract

Recent studies suggest cysteine S-nitrosation of S-nitrosoglutathione reductase (GSNOR) could regulate protein redox homeostasis. “Switch” assays enable discovery of putatively S-nitrosated proteins. However, with few exceptions, researchers have not examined the kinetics and biophysical consequences of S-nitrosation. Methods to quantify protein S-nitrosothiol (SNO) abundance and formation kinetics would bridge this mechanistic gap and allow interpretation of the consequences of specific modifications, as well as facilitate development of specific S-nitrosation inhibitors. Here, we describe a rapid assay to estimate protein SNO abundance with intact protein electrospray ionization mass spectrometry. Originally designed using recombinant GSNOR, these methods are applicable to any purified protein to test for or further study nitrosatable cysteines.

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Acknowledgment

Mass spectral data were obtained at the University of Massachusetts Amherst Mass Spectrometry Core Facility. This work was supported by a Massachusetts Life Sciences Center new faculty award (E.V.), a grant from the National Science Foundation (MCB1517046 to E.V., which funded D.G., I.T., and P.T.), and the National Institutes of Health (S10OD010645 to S.J.E.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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  1. Damian Guerra

    Present address: Department of Obstetrics and Gynecology, University of Colorado, Aurora, CO, USA

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, USA

    Damian Guerra, Ian Truebridge, Stephen J. Eyles, Patrick Treffon & Elizabeth Vierling

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  1. Damian Guerra
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  2. Ian Truebridge
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  3. Stephen J. Eyles
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  4. Patrick Treffon
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  5. Elizabeth Vierling
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Correspondence to Elizabeth Vierling .

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Editors and Affiliations

  1. Helmholtz Zentrum München–German Research GmbH, Center for Environment Health Institute of Biochemical Plant Pathology, Ingolstädter Landstraße 1, Munich-Neuherberg, Germany

    Alexander Mengel

  2. Helmholtz Zentrum München–German Research GmbH, Center for Environment Health Institute of Biochemical Plant Pathology, Ingolstädter Landstraße 1, Munich-Neuherberg, Germany

    Christian Lindermayr

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Guerra, D., Truebridge, I., Eyles, S.J., Treffon, P., Vierling, E. (2018). Direct Measurement of S-Nitrosothiols with an Orbitrap Fusion Mass Spectrometer: S-Nitrosoglutathione Reductase as a Model Protein. In: Mengel, A., Lindermayr, C. (eds) Nitric Oxide. Methods in Molecular Biology, vol 1747. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7695-9_12

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  • DOI: https://doi.org/10.1007/978-1-4939-7695-9_12

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7694-2

  • Online ISBN: 978-1-4939-7695-9

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