Fig 1. Schematic overview of isotopic-labeling strategy to identify and quantify S-glutathionylated proteins.

NEM and IAM were used to alkylate free thiols. A cocktail of Grx, GR, GSH and NADPH was used to selectively reduce glutathionylated cysteines. 126 Da, 127 Da and 131 Da iodoTMT reagents were used to label groups WT, AB, and T, respectively. DTT was used to reduce disulfide bonds and other thiol oxidative modifications such as S-nitrosylation. Anti-TMT antibody-conjugated resins were used for specifically enrich the iodoTMT labeled peptides. The eluted peptides were subjected to LC-MS/MS analysis.