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Nitric Oxide pp 89-101 | Cite as

A Proteomics Workflow for Dual Labeling Biotin Switch Assay to Detect and Quantify Protein S-Nitroylation

  • Heaseung Sophia Chung
  • Christopher I. Murray
  • Jennifer E. Van EykEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1747)

Abstract

S-nitrosylation (or S-nitrosation, SNO) is an oxidative posttranslational modification to the thiol group of a cysteine amino acid residue. There are several methods to detect SNO modifications, mostly based on the classic biotin-switch assay, where the labile SNO sites are replaced with a stable biotin moiety to facilitate enrichment of the modified proteins. As the technique has evolved, new and more advanced thiol-reactive reagents have been introduced in the protocol to improve the identification of modified peptides or to quantify the level of modification at individual cysteine residues. However, the growing diversity of thiol-reactive affinity tags has not produced a consistent set of protein modifications, suggesting incomplete coverage using a single tag. Here, we present a parallel dual labeling strategy followed by an optimized proteomics workflow, which maximizes the overall detection of SNO by reducing the labeling bias derived from the use of a single tag-capture approach.

Key words

IodoTMT6-switch assay S-nitrosylation S-nitrosation Quantification Redox proteomics Mass spectrometry 

Notes

Acknowledgments

The authors would like to thank Ronald J. Holewinski for his technical expertise in LC/MS/MS separation and acquisition. This work was supported by an American Heart Association MidAtlantic Fellowship Grant (H.S.C.), Canadian Institutes of Health Research Postdoctoral fellowship (C.I.M.), NHLBI R01HL119012 and NHLBI PO1 HL10026, and 5P01HL112730-03 (J.E.V.E.). Funds were also received from the Erika Glazer Endowed Chair and the Barbra Streisand Women’s Heart Center.

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Copyright information

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

Authors and Affiliations

  • Heaseung Sophia Chung
    • 1
  • Christopher I. Murray
    • 1
  • Jennifer E. Van Eyk
    • 1
    Email author
  1. 1.Medicine and Heart InstituteCedars Sinai Medical CenterLos AngelesUSA

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