Application of a Thioredoxin-Trapping Mutant for Analysis of the Cellular Nitrosoproteome

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Nitric oxide influences a wide range of cellular functions through S-nitrosylation, a redox-dependent posttranslational protein modification that involves attachment of a nitroso moiety to a reactive thiol group. Over the past two decades, S-nitrosylation has emerged as a ubiquitous mechanism for controlling the activity, subcellular localization, and molecular interactions of proteins, thereby influencing many cellular processes. In addition, recent studies have indicated that aberrant S-nitrosylation may lead to cellular dysfunction and damage. Despite significant advances in the field, progress has been hindered by challenges related to the analysis of S-nitrosylation by large-scale proteomic approaches. This chapter describes the application of a thioredoxin-trapping mutant for proteomic analysis of S-nitrosylation. Thioredoxin is a ubiquitous oxidoreductase directly involved in denitrosylation reactions. The presented method relies upon mechanism-based trapping, whereby a recombinant thioredoxin trap mutant captures nitrosylated proteins, which are subsequently isolated and identified by mass spectrometry. This nitrosothiol-trapping procedure can expand upon and complement currently available methods for the analysis of the nitrosoproteome.

Original languageEnglish
Title of host publicationProteomics in Biology
EditorsArun K. Shukla
Chapter16
Pages285-294
Number of pages10
VolumePart A
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume585

Keywords

  • Nitrosylation
  • Proteomics
  • Thioredoxin

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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