Clickable glutathione-based Isotope Coded Affinity Label to quantify glutathionylation

Identification of cysteines with high oxidation susceptibility is important for understanding redox-mediated biological processes associated with health and disease. We developed a chemical proteomic strategy that helps find cysteines with high susceptibility to S-glutathionylation. Our strategy is based on the isotopically labelled clickable glutathione derivatives that can quantify relative levels of glutathionylated and reduced forms (SSG vs. SH) of cysteines in mass spectrometry, which is named 'Clickable Glutathione-based Isotope-coded Affinity-Tag (G-ICAT).' The G-ICAT approach was applied to the mouse C2C12 cell line upon addition of hydrogen peroxide, finding 1,518 glutathionylated cysteines and their relative levels of SSG over SH upon adding hydron peroxide. This chemical proteomic strategy will significantly contribute to identifying functional cysteines regulated by glutathionylation in redox signaling.