Abstract
Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Here, we present stable isotope cysteine labelling with iodoacetamide (SICyLIA), a mass spectrometry-based workflow to assess proteome-scale cysteine oxidation. SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity. Applying SICyLIA to diverse cellular models and primary tissues provides detailed insights into thiol oxidation proteomes. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys identifies oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies.
Highlights
Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues
Titrating different concentrations of hydrogen peroxide showed that this was a dosedependent effect (Supplementary Fig. 2d). These results indicate that acute oxidative stress causes metabolic adaptation through the oxidation of metabolic and mitochondrial proteins to amplify the production of reducing equivalents in the phosphate pathway (PPP), and potentially to minimise the production of endogenous ROS by inhibition of mitochondrial respiration
Despite the elimination of enrichment steps, which requires extensive sample manipulation and can introduce error and bias, the stable isotope cysteine labelling with iodoacetamide (SICyLIA) workflow achieves a proteomic depth that enabled the identification of a large number of previously unknown redoxsensitive peptides
Summary
Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules through oxidation of protein cysteine residues. Specific or insufficient enrichment can introduce bias To mitigate these issues, we developed a simple, unbiased, and robust quantitative proteomic approach (SICyLIA) to sensitively detect and accurately measure proteome-wide cysteine oxidation dynamics under conditions of acute and chronic oxidative stress. As reduced protein thiols are the more abundant cysteine species in cells, we achieve proteome-wide sensitivity without the need for enrichment steps. This high sensitivity allows for the detection of smaller, but biologically relevant, changes in oxidation states. Analysis of kidney proteomes suggests that chronic intracellular oxidative stress may have profound effects on tissue remodelling and whole-body physiology through oxidation of proteins in circulating biofluids
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