Redox proteomics methods for identifying glutathionylated proteins have been developed, including labelling of GSH either by 35S 16 or biotinylation 17, 19, 20. Interestingly, a number of intracellular proteins that can be released and act as endogenous inflammatory signalling molecules (also known as ‘damage-associated molecular patterns’) can undergo glutathionylation, including high mobility group box 1 protein (HMGB-1) 14, proteins of the S100 family 15, galectin-1 16, peroxiredoxin-2 (Prdx2) 17 and heat-shock protein 70 (HSP70) 18. We and others previously showed that protein glutathionylation can be regulated by macrophage activators 12 and HIV infection 13. Key signalling proteins in infection and immunity that can be regulated by glutathionylation include p50 9, STAT3 10 and HIV protease 11. It is now well accepted that glutathionylation, which is a reversible process, plays important roles in the redox regulation of protein activity and cell signalling 4, 5, 6, 7, 8. A molecular mechanism for the regulatory action of GSH is protein glutathionylation, a post-translational modification in which glutathione (GSH) forms a disulphide bond with a cysteine of a protein. Glutathione (GSH) has a key signalling role in redox regulation 1, 2, 3. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. ![]() We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance.
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