76 - Stoichiometric Quantification of S-Glutathionylation and Total Thiol Oxidation in Mouse Macrophages

Abstract

Reversible oxidative modifications of protein thiols represent a fundamental mechanism of redox signaling by modulating enzyme activities and protein functions in a variety of cellular activities, such as signaling, metabolism, gene expression, and apoptosis. The stoichiometry (or occupancy) of oxidative modifications on specific cysteine residue is important for understanding the functional role of the modification; however, this aspect of quantification has been underexplored. Herein we report the stoichiometric quantification of both S-glutathionylation (SSG) and total oxidation in macrophages at the basal physiological conditions by applying resin-assisted quantitative redox proteomics. The stoichiometry information of SSG and total oxidation on a total of ~4,100 Cys sites was quantified. The basal average oxidation level of protein thiols was ~12%, which is consistent with the overall reducing status in vivo. The average percentage of SSG in total oxidation was 32.0% at basal, supporting that SSG is one major type of thiol modifications. Interesting, we observed that the redox potential of a cellular organelle was well correlated with the level of oxidation in the organelle. For example, endoplasmic reticulum and lysosome have much higher oxidation levels than those from reducing organelles, such as nucleus and mitochondrion. In contrast, the reducing organelles have an average higher percentage of SSG in total oxidation, suggesting SSG is the dominant modification and plays an important regulatory role in these organelles. Moreover, the integration of stoichiometric and dynamic data reveals potential evidence of inhibitory and activation sites via redox modifications