title: Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry.
abstract: Keratins are the main constituent of human skin and have been identified as major oxidative target proteins. However, there has been a lack of studies aimed at identifying the oxidation sites of keratins because of the difficulties associated with their insolubility and handling. Here, we introduce a mass spectrometry (MS)-based proteomic methodology to screen oxidative modifications in human skin keratins. Human skin proteins were obtained non-invasively by tape stripping and solubilized in SDS buffer, followed by purification and digestion using the modified filter-aided sample preparation method. The tryptic peptides were then analyzed by MALDI-TOF/MS, LC-ESI/MS, and MS/MS. PMF analyses have identified keratins K1 and K10 as the major proteins of human skin. Met(259), Met(262), Met(296), and Met(469), located in the α-helical rod domain of K1, were the most susceptible sites to oxidation induced by hydrogen peroxide in vitro and in vivo. Our results indicate a potential use of the identified methionine residues as biomarkers of oxidative skin damage. The present methodology is the first MS-based approach to detecting oxidative modifications in keratins obtained directly from human skin and can be easily applied to the monitoring of other keratin modifications in various skin conditions.
authors: Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan. sh-lee@mail.pharm.tohoku.ac.jp
authors: Seon Hwa
authors: SH
authors: Lee
authors: Kohei
authors: K
authors: Miyamoto
authors: Takaaki
authors: T
authors: Goto
authors: Tomoyuki
authors: T
authors: Oe
mesh_terms: D000595:Amino Acid Sequence; D016477:Artifacts; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D053556:Keratin-1 / Q000737:chemistry; D053550:Keratin-10 / Q000737:chemistry; D053557:Keratin-2 / Q000737:chemistry; D053534:Keratin-9 / Q000737:chemistry; D007633:Keratins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D040901:Proteomics / Q000379:methods; D012867:Skin / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism
chemical_list: C507493:KRT10 protein, human; C507440:KRT9 protein, human; D053556:Keratin-1; D053557:Keratin-2; D053534:Keratin-9; D053550:Keratin-10; D007633:Keratins; D008715:Methionine; D006861:Hydrogen Peroxide; D014357:Trypsin