Nitric Oxide pp 143-160 | Cite as

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

  • Damian Guerra
  • Ian Truebridge
  • Stephen J. Eyles
  • Patrick Treffon
  • Elizabeth Vierling
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

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.

Key words

S-nitrosation S-nitrosothiol (SNO) GSNOR Mass spectrometry Orbitrap fusion Redox switch Cysteine modification 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Damian Guerra
    • 1
    • 2
  • Ian Truebridge
    • 1
  • Stephen J. Eyles
    • 1
  • Patrick Treffon
    • 1
  • Elizabeth Vierling
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of MassachusettsAmherstUSA
  2. 2.Department of Obstetrics and GynecologyUniversity of ColoradoAuroraUSA

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