| Abstract: | ["Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules that act through the oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Identifying cysteine oxidation on a whole-proteome scale remains a technical challenge due to the low abundance of oxidised thiols. Redox proteomics techniques therefore use multistep enrichment protocols, but these have inherent limitations and inform only on the enriched proteome. We developed stable isotope cysteine labelling with iodoacetamide (SICyLIA), a simple, unbiased, and robust mass spectrometry-based workflow for thiol oxidation analysis. We applied SICyLIA to diverse cellular models and primary tissues and generated the most in-depth thiol oxidation profiles to date. 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 showed 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."] |
