Novel biomarkers of protein oxidation sites and degrees using horse cytochrome c as the target by mass spectrometry

Biomarkers held both incredible application and significant challenge in probing the oxidation mechanisms of proteins under oxidative stress. Here, mass spectrometry (MS) coupled with liquid chromatography (LC) was applied to establish a new pipeline to probe the oxidation sites and degrees of horse cytochrome c (HCC) with its oxidative products serving as the biomarkers. Samples of native and UV/H(2)O(2) oxidized HCCs were digested by trypsin and subjected to biomarker discovery using LC/MS and tandem mass spectrometry (MS/MS). Experiment results proved that the main oxidation sites were located at Cys(14), Cys(17), Met(65) and Met(80) residues in peptides C(14)AQC(heme)HTVEK(22), C(14)AQCHTVEK(22), E(60)ETLMEYLENPKK(73), M(80)IFAGIK(86) and M(80)IFAGIKK(87). Quantitative analysis on the oxidized peptides showed the oxidation degrees of target sites had positive correlations with extended oxidation dose and controlled by residues types and their accessibility to solvent molecules. Being able to provide plentiful information for the oxidation sites and oxidation degrees, the identified oxidized products were feasibility biomarkers for HCC oxidation, compared with the conventional protein carbonyl assay.