Identification and Quantification of Glutathionylated Cysteines under Ischemic Stress

Ischemia reperfusion injury contributes
to adverse cardiovascular
diseases in part by producing a burst of reactive oxygen species that
induce oxidations of many muscular proteins. Glutathionylation is
one of the major protein cysteine oxidations that often serve as molecular
mechanisms behind the pathophysiology associated with ischemic stress.
Despite the biological significance of glutathionylation in ischemia
reperfusion, identification of specific glutathionylated cysteines
under ischemic stress has been limited. In this report, we have analyzed
glutathionylation under oxygen–glucose deprivation (OGD) or
repletion of nutrients after OGD (OGD/R) by using a clickable glutathione
approach that specifically detects glutathionylated proteins. Our
data find that palmitate availability induces a global level of glutathionylation
and decreases cell viability during OGD/R. We have then applied a
clickable glutathione-based proteomic quantification strategy, which
enabled the identification and quantification of 249 glutathionylated
cysteines in response to palmitate during OGD/R in the HL-1 cardiomyocyte
cell line. The subsequent bioinformatic analysis found 18 glutathionylated
cysteines whose genetic variants are associated with muscular disorders.
Overall, our data report glutathionylated cysteines under ischemic
stress that may contribute to adverse outcomes or muscular disorders.