Ischemia or ischemic reperfusion is well-known to contribute to heart diseases. During ischemia and reperfusion, the flux of nutrients and oxygen to cardiomyocytes is altered, which causes a burst of ROS from mitochondria and other enzymes. Elevated levels of ROS can cause oxidative modifications of cardiac proteins, such as protein glutathionylation. Despite previous extensive studies, proteomic identification of glutathionylated proteins in cardiomyocytes under altered levels of nutrients and oxygens has been relatively limited. In this report, we have applied our clickable glutathione to a HL-1 cardiomyocyte cell line under metabolic alterations of glucose, oxygen, and fatty acids. We found that glucose is important for maintaining a redox homeostasis, while fatty acid oxidation is one of major factors that induce a high level of glutathionylation during reoxygenation. To further determine specific proteins that are vulnerable to glutathionylation, we have established a mass-spectrometry strategy coupled with clickable glutathione in which two azido-Ala derivatives with isotopic mass difference were used to metabolically synthesize clickable glutathione for identification and quantification of glutathionylated proteins. We have applied our isotopic clickable glutathione approach to HL-1 cells upon addition of fatty acids during reoxygenation, identifying and quantifying the level of individual glutathionylated peptides.<br/>

Clickable glutathione approach to identify protein glutathionylation under ischemic stress

2019 Fall National Meeting

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