Fig. 1

Quantification of protein abundance and cysteine thiol oxidation levels in Rhodococci fed on glucose or lignin as sole carbon sources. A Proteomics sample preparation. Proteins were extracted in the presence of HPE-IAM. Cysteine free thiols (SH) were alkylated with HPE-IAM while oxidized cysteine residues (e.g. SOH, SSM, S–S, SO₂H, SO₃H) were preserved. Then, proteins were digested for MS analysis. B Simultaneous relative quantification of protein abundance and thiol oxidation level. Peptide samples from glucose- or lignin-fed Rhodococci were subjected to MS analysis. The sum of MS1 peak intensities of all peptides assigned to individual proteins were used to compare a given protein’s abundance between two conditions. The peak intensities of HPE-IAM alkylated Cys-containing (HPE-IAM-Cys) peptides were summed for individual protein Cys residues, showing the abundance of protein cysteines at reduced state, which can be used to compare the thiol oxidation level of a given Cys site between two conditions. Note that the assay provides an indirect measurement of thiol oxidation level. The higher intensities of HPE-IAM-Cys peptides indicate lower cysteine thiol oxidation levels and vice versa