title: Application of a Thioredoxin-Trapping Mutant for Analysis of the Cellular Nitrosoproteome.
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.
authors.affiliation: Rappaport Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel. Electronic address: benhar@tx.technion.ac.il.
authors.forename: M
authors.initials: M
authors.lastname: Benhar
mesh_terms: D015538:Nitrosation; D010084:Oxidation-Reduction; D040901:Proteomics / Q000379:methods*; D013879:Thioredoxins / Q000378:metabolism*
chemical_list: D013879:Thioredoxins
keywords: Nitrosylation; Proteomics; Thioredoxin