Proteome-wide analysis of cysteine oxidation using Stable Isotope Cysteine Labelling with Iodoacetamide (SICyLIA)

Reactive oxygen species \(ROS) are increasingly recognised as important signalling molecules that act through the oxidation of protein cysteine residues.Comprehensive identi cation 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 work ow for thiol oxidation analysis.SICyLIA does not require enrichment steps and achieves unbiased proteome-wide sensitivity.We applied SICyLIA to diverse cellular models and primary tissues and generated the most indepth thiol oxidation pro les 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 bio uids, through which cellular redox stress can affect whole-body physiology.Obtaining accurate peptide oxidation pro les from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies.Reagents • Sodium dodecyl sulfate \(SDS) • Iodoacetamide light \( 12 C 2 H 4 INO, Sigma-Aldrich \(Merck)) • Iodoacetamide heavy \( 13 C 2 D 2 H 2 INO, Sigma-Aldrich \(Merck)) • Phosphate buffered saline \(PBS) • Bicinchoninic acid \(BCA) assay kit \(Thermo Scienti c) • Ammonium bicarbonate \(Ambic) • Dithiothreitol \(DTT) • N-ethylmaleimide \(NEM) • Trichloroacetic acid \(TCA) • Urea • Endoproteinase Lys-C \(mass spectrometry grade, Alpha laboratories) • Trypsin \(mass spectrometry grade, Promega) • Tri uoroacetic acid \(TFA) • Acetic acid • Acetonitrile \(ACN) • Formic acid • LC-MS grade water **Reagent setup** • Lysis buffer 1: 100 mM Tris-HCl pH 7.5, 4% SDS • Ambic: ammonium bicarbonate, 0.1 M stock solution in water, pH 7.0 • DTT: dithiothreitol, 1 M stock solution in water • NEM: N-ethylmaleimide, 0.2 M stock solution in water • TCA: trichloroacetic acid, 100% and 10% stock solutions in water • Urea buffer: 8 M stock solution in water • TFA: tri uoroacetic acid, 50% stock solution in water • Reversed phase \(RP)solvent A: 0.6% \(vol/vol) acetic acid in water • Reversed phase \(RP) solvent B: 0.6% \(vol/vol) acetic acid and 80% \(vol/vol) acetonitrile in water • Light formaldehyde/cyanoborohydride solution, 5 ml per sample: 4.5 ml of 50 mM sodium phosphate buffer pH 7.5 \(1 ml of 50 mM NaH 2 PO 4 with 3.5 ml of 50 mM Na 2 HPO 4 ) with 250z μl of 4% \(v/v) formaldehyde in water \(light, CH 2 O) and 250 μl of 0.6 M cyanoborohydride in water \(light, NaBH 3 CN) • Heavy formaldehyde/cyanoborohydride solution, 5 ml per sample: 4.5 ml of 50 mM sodium phosphate buffer pH 7.5 \(1 ml of 50 mM NaH 2 PO 4 with 3.5 ml of 50 mM Na 2 HPO 4 ) with 250 μl of 4% \(vol/vol) formaldehyde in water \(heavy, 13 CD 2 O) and 250 μl of 0.6 M cyanoborohydride in water \(heavy, NaBD 3 CN).• Elution buffer 1: acetonitrile with 2.5% TFA • HPLC solvent A: 98% water, 2% acetonitrile, adjusted to pH 10 using ammonium hydroxide • HPLC solvent B: