Isotopically Labeled Clickable Glutathione to Quantify Protein S-Glutathionylation.

ABSTRACT: Protein S-glutathionylation is one of the important cysteine oxidation events that regulate various redox-mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S-glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido-Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification strategy with clickable glutathione by using isotopically labeled heavy and light derivatives of azido-Ala, which provides the relative quantification of glutathionylated peptides in mass spectrometry-based proteomic analysis. We applied isotopically labeled clickable glutathione to HL-1 cardiomyocytes, quantifying relative levels of 1398 glutathionylated peptides upon addition of hydrogen peroxide. Importantly, we highlight elevated levels of glutathionylation on sarcomere-associated muscle proteins while validating glutathionylation of two structural proteins, ?-actinin and desmin. Our report provides a chemical proteomic strategy to quantify specific glutathionylated cysteines.

SUBMITTER: VanHecke GC 

PROVIDER: S-EPMC7078011 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature