Identification of S-glutathionylated cellular proteins during oxidative stress and constitutive metabolism by affinity purification and proteomic analysis

• Published in: Archives of biochemistry and biophysics 2002-10, Vol.406 (2), p.229-240
• Main Authors: Lind, Christina, Gerdes, Robert, Hamnell, Ylva, Schuppe-Koistinen, Ina, von Löwenhielm, Helena Brockenhuus, Holmgren, Arne, Cotgreave, Ian A.
• Format: Article
• Language: English
• Subjects: Affinity chromatography; Cell Line; Cells, Cultured; Chromatography, Affinity; Cysteine - metabolism; Electrophoresis, Gel, Two-Dimensional; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Ethylmaleimide - pharmacology; Glutaredoxins; Glutathione; Glutathione - metabolism; Humans; Oxidation-Reduction; Oxidative Stress - physiology; Oxidoreductases; Polymerase Chain Reaction; Protein thiols; Proteins - genetics; Proteins - isolation & purification; Proteins - metabolism; Proteome; Proteomics; Redox modification; S-glutathionylation; Umbilical Veins - drug effects; Umbilical Veins - metabolism
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/S0003-9861(02)00468-X

Redox modification of proteins is proposed to play a central role in regulating cellular function. However, high-throughput techniques for the analysis of the redox status of individual proteins in complex mixtures are lacking. The aim was thus to develop a suitable technique to rapidly identify proteins undergoing oxidation of critical thiols by S-glutathionylation. The method is based on the specific reduction of mixed disulfides by glutaredoxin, their reaction with N-ethylmaleimide–biotin, affinity purification of tagged proteins, and identification by proteomic analysis. The method unequivocally identified 43 mostly novel cellular protein substrates for S-glutathionylation. These include protein chaperones, cytoskeletal proteins, cell cycle regulators, and enzymes of intermediate metabolism. Comparisons of the patterns of S-glutathionylated proteins extracted from cells undergoing diamide-induced oxidative stress and during constitutive metabolism reveal both common protein substrates and substrates failing to undergo enhanced S-glutathionylation during oxidative stress. The ability to chemically tag, select, and identify S-glutathionylated proteins, particularly during constitutive metabolism, will greatly enhance efforts to establish posttranslational redox modification of cellular proteins as an important biochemical control mechanism in coordinating cellular function.