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


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.

SUBMITTER: Guerra D 

PROVIDER: S-EPMC5901646 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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

Guerra Damian D   Truebridge Ian I   Eyles Stephen J SJ   Treffon Patrick P   Vierling Elizabeth E  

Methods in molecular biology (Clifton, N.J.) 20180101


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 modific  ...[more]

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