Abstract
Nitroxidative stress in cells occurs mainly through the action of reactive nitrogen and oxygen species (RNOS) on protein thiol groups. Reactive nitrogen and oxygen species-mediated protein modifications are associated with pathophysiological states, but can also convey physiological signals. Identification of Cys residues that are modified by oxidative stimuli still poses technical challenges and these changes have never been statistically analyzed from a proteome-wide perspective. Here we show that GELSILOX, a method that combines a robust proteomics protocol with a new computational approach that analyzes variance at the peptide level, allows a simultaneous analysis of dynamic alterations in the redox state of Cys sites and of protein abundance. GELSILOX permits the characterization of the major endothelial redox targets of hydrogen peroxide in endothelial cells and reveals that hypoxia induces a significant increase in the status of oxidized thiols. GELSILOX also detected thiols that are redox-modified by ischemia-reperfusion in heart mitochondria and demonstrated that these alterations are abolished in ischemia-preconditioned animals.
Highlights
Plays an important role in several pathologies, including endothelial dysfunction [3] and ischemia-reperfusion injury [4], one of the most common cause of death in the world
Many studies have analyzed the nature of proteins that are oxidized in conditions of oxidative stress, the systematic, high-throughput characterization of the specific cysteine thiol groups (Cys) sites that are modified in these situations has been addressed in a more limited number of reports
Gel-based Stable Isotope Labeling of Oxidized Cys acterization of free thiols (i.e. Cys sites that were in reduced form) [20], but by blocking the free thiols and performing a subsequent treatment with a reductant, Cys residues that were reverted to the free thiol state could be tagged, purified, and characterized [14, 17, 19]
Summary
Pablo Martínez-Acedo‡‡‡, Estefanía Nun ̃ ez‡§, Francisco J. There is increasing evidence that dysfunctions like atherosclerosis, hypertension, diabetes and heart failure are in part caused by oxidative damage produce by RNOS [5], and mitochondria are thought to play a relevant role in the generation of these species [6] For these reasons, identification of redox targets and quantification of redox damage in vascular endothelial cells and mitochondria is essential to develop therapeutic tools in the treatment of cardiovascular diseases. We demonstrate the performance of the new method by identifying changes in protein abundance together with the exact Cys sites that increase their oxidation state in several cellular and animal models of oxidative stress that have only scarcely been studied before by high-throughput redox proteomics
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