Proteomic measurement away from all over the world cysteine oxidization

Control and oxidatively stressed cells or tissue samples were extracted separately in the presence of either light ( 12 C₂H_cuatroINO) or stable isotope-labelled heavy ( 1step three C₂D₂H₂INO) IAM to alkylate reduced cysteine thiols (SH), coupling a carbamidomethyl (CAM) group to the Henderson NV escort sites cysteine residue. After labelling, equal amounts of protein extracts were mixed using a label-swap replication strategy and treated with dithiothreitol (DTT) to reduce reversibly oxidised thiols, which were subsequently blocked with n-ethylmaleimide (NEM). Proteomes were then digested and peptides fractionated using off-line high pH reversed phase chromatography prior to UHPLC-MS/MS analysis on a Q-Exactive HF. Cysteine oxidation ratios are calculated using the MaxQuant computational platform 20 based on the abundance of light and heavy CAM-modified peptide pairs for each cysteine-containing unique peptide. As IAM reacts with reduced cysteine thiols, a -modification for a given peptide indicates increased cysteine oxidation. Whereas changes in the levels of reduced cysteine between samples undergoing a short-term treatment can be compared immediately (Fig. 1a ), different cell lines or tissues derived from different mice have distinct proteomes and require normalisation for protein levels. For relative protein quantification, stable isotope dimethyl labelling 26 was used in con labelling (Fig. 1b ). This method follows a comparable workflow as described above, streamlining these parallel procedures. As shown in Fig. 1b , a fraction of the lysates used to prepare IAM-labelled samples are digested and dimethylated with either light (H 12 CHO/NaBH₃CN) or heavy (D 13 CDO/NaBD₃CN) formaldehyde/sodium cyanoborohydride, mixed in equal ratios using a label-swap replication strategy for independent replicates, and subjected to high pH reversed phase chromatography fractionation before UHPLC-MS/MS analysis. Protein abundance was then determined using MaxQuant. Finally, peptide oxidation ratios are normalised for protein abundance, providing the normalised oxidation ratio for each cysteine-containing peptide in the analysed proteome.

Schematic overview of the Stable Isotope Cysteine Labelling with IodoAcetamide (SICyLIA) methodology. a Samples are extracted in presence of either light ( 12 C₂H₄INO) or heavy ( 13 C₂D₂H₂INO) 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 (H 12 CHO/NaBH₃CN) or heavy (D 13 CDO/NaBD₃CN) formaldehyde/sodium cyanoborohydride, peptides are fractioned, and analysed using UHPLC-MS/MS similarly to IAM-modified peptides

Overall performance

The SICyLIA workflow was applied to diverse biological models of acute (H₂O₂ treatment) and chronic (Ftitle deficiency) oxidative stress to assess its performance (Fig. 2 ). SICyLIA enabled quantification of 18,022 unique cysteine-containing peptides in mouse cells and 13,112 in kidney tissues (Fig. 2a ). To achieve accurate cysteine oxidation ratios, stringent quality control (QC) criteria were applied. First, only peptide oxidation ratios quantified in at least three replicates were considered for the analysis. Additionally, the coefficient of variation (CV%) between replicates was used to filter out extreme outlier ratios (see Methods). After QC, the median oxidation ratio between replicates was used for further analysis. Oxidation ratios for up to 9479 peptides in the cells and 4415 in tissues were included for further analysis, which corresponds to 3563 and 2168 proteins, respectively. Overall, variability across our data sets was low (Fig. 2b ) and replicates showed high reproducibility, with median CV% of 15.1 (H₂O₂ model), 17.7 (Ftitle cell model), and 16.0 (Ftitle tissue model) (Fig. 2c ). To define which cysteine-containing peptides were significantly oxidised or reduced, the Significance B algorithm in the Perseus software platform was used 27 . This algorithm was specifically developed for the analysis of mass spectrometry-based protein/peptide quantification using log protein/peptide ratios 20 (see Methods). For an extended substantiation of QC strategy and discussion regarding suitability of different statistical analysis strategies for the evaluation of peptide oxidation ratios generated with SICyLIA, see Supplementary Note 1 . In the analysed models, we identified 333 (H₂O₂ model), 252 (Ftitle cell model), and 150 (Ftitle tissue model) significantly oxidised or reduced peptides, corresponding to 3.5, 2.9, and 3.4% of the respective data sets (Fig. 2a ).