Fig. 1

Schematic overview of the Stable Isotope Cysteine Labelling with IodoAcetamide (SICyLIA) methodology. a Samples are extracted in presence of either light (¹²C₂H₄INO) or heavy (¹³C₂D₂H₂INO) iodoacetamide (IAM) to alkylate free cysteine thiols, introducing a carbamidomethyl (CAM) group. Equal amounts of modified protein extracts are mixed, reversibly oxidised thiols are reduced with DTT and subsequently alkylated with NEM. Protein extracts are digested and peptides are fractionated prior to UHPLC-MS/MS analysis. b In parallel, labelled proteome extracts are trypsin digested and dimethylated using light (H¹²CHO/NaBH₃CN) or heavy (D¹³CDO/NaBD₃CN) formaldehyde/sodium cyanoborohydride, peptides are fractioned, and analysed using UHPLC-MS/MS similarly to IAM-modified peptides