A mass spectrometry (MS)-based proteomic methodology was employed to monitor oxidative modifications in keratins, the main constituents of human skin ("Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry" , "UV irradiation-induced methionine oxidation in human skin keratins: mass spectrometry-based non-invasive proteomic analysis" ). Human skin proteins were obtained non-invasively by tape stripping and solubilized in sodium dodecyl sulfate (SDS) buffer, followed by purification and digestion using the filter-aided sample preparation method. The tryptic peptides were then analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-MS, tandem MS (MS/MS), and LC/ESI-selected reaction monitoring (SRM)/MS. The MS/MS data were generated to confirm amino acid sequences and oxidation sites of tryptic peptides D290VDGAYMTK298 (P1) and N258MQDMVEDYR267 (P2), which contain the most susceptible oxidation sites (Met259, Met262, and Met296 in K1 keratin) upon UVA irradiation . Subsequently, quantitative determination of the relative oxidation levels of P1 and P1  was achieved by LC/ESI-SRM/MS analyses of P1 and P2 together with their oxidized forms after exposure to UVA radiation or treatment with hydrogen peroxide (H2O2).
ASJC Scopus subject areas