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

M Benhar

Methods in enzymology 2017

filter terms:

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. © 2017 Elsevier Inc. All rights reserved.