Abstract
Ultraviolet (UV) radiation is the major environmental factor that causes oxidative skin damage. Keratins are the main constituents of human skin and have been identified as oxidative target proteins. We have recently developed a mass spectrometry (MS)-based non-invasive proteomic methodology to screen oxidative modifications in human skin keratins. Using this methodology, UV effects on methionine (Met) oxidation in human skin keratins were investigated. The initial screening revealed that Met259, Met262, and Met296 in K1 keratin were the most susceptible oxidation sites upon UVA (or UVB) irradiation of human tape-stripped skin. Subsequent liquid chromatography/electrospray ionization-MS and tandem MS analyses confirmed amino acid sequences and oxidation sites of tryptic peptides D290VDGAYMTK298 (P1) and N258MQDMVEDYR267 (P2). The relative oxidation levels of P1 and P2 increased in a time-dependent manner upon UVA irradiation. Butylated hydroxytoluene was the most effective antioxidant for artifactual oxidation of Met residues. The relative oxidation levels of P1 and P2 after UVA irradiation for 48h corresponded to treatment with 100mM hydrogen peroxide for 15min. In addition, Met259 was oxidized by only UVA irradiation. The Met sites identified in conjunction with the current proteomic methodology can be used to evaluate skin damage under various conditions of oxidative stress. Biological significanceWe demonstrated that the relative Met oxidation levels in keratins directly reflected UV-induced damages to human tape-stripped skin. Human skin proteins isolated by tape stripping were analyzed by MS-based non-invasive proteomic methodology. Met259, Met262, and Met296 in K1 keratin were the most susceptible oxidation sites upon UV irradiation. Met259 was oxidized by only UVA irradiation. Quantitative LC/ESI-SRM/MS analyses confirmed a time-dependent increase in the relative oxidation of target peptides (P1 and P2) containing these Met residues, upon UVA irradiation of isolated human skin. The relative oxidation levels of P1 and P2 along with the current proteomic methodology could be applied to the assessment of oxidative stress levels in skin after exposure to sunlight.
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