Fig. 1.

Site-resolved, large-scale analysis of the in vivo oxidation status of the yeast proteome. a Yeast cells grown in 2% galactose medium were immediately frozen in 10% TCA (1). Extracted proteins were denatured in 6 M urea in the presence of heavy ICAT (¹³C-ICAT) to label free thiol groups (2). Reversibly oxidised cysteine residues were reduced by TCEP and labelled by light ICAT (¹²C-ICAT) (3). Proteins were digested with trypsin and ICAT-labelled peptides were enriched by streptavidin affinity chromatography (4). Following cleavage of the biotin tag, peptides were analysed in duplicate by LC-MS/MS. b For the determination of the in vivo oxidation status of cysteine residues, peptides were identified based on fragment ions observed in MS2 spectra using MaxQuant (v.1.4.1.2) and then quantified by extracting MS1 ion chromatograms using Skyline (v.2.5.0). Following the integration of peak areas of heavy and light peptide variants, the proportion of reversibly oxidised cysteine residues (% oxidation) was calculated. c Overlap of unique cysteine-containing peptides (top) and proteins (bottom) identified in three biological replicates and the proportion of peptides/proteins quantified in least two of the three biological replicates