Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.
J Proteomics. 2011 Dec 21;75(2):435-49. doi: 10.1016/j.jprot.2011.08.010. Epub 2011 Aug 22.
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.
角蛋白是人类皮肤的主要组成部分，已被确定为主要的氧化靶蛋白。然而，由于角蛋白的不溶性和处理困难，缺乏针对角蛋白氧化位点的鉴定研究。在这里，我们介绍了一种基于质谱（MS）的蛋白质组学方法，用于筛选人皮肤角蛋白的氧化修饰。通过胶带剥离非侵入性地获得人皮肤蛋白质，并在 SDS 缓冲液中溶解，然后使用改良的过滤辅助样品制备方法进行纯化和消化。然后通过 MALDI-TOF/MS、LC-ESI/MS 和 MS/MS 分析胰蛋白酶肽。PMF 分析鉴定角蛋白 K1 和 K10 为人皮肤的主要蛋白质。位于 K1 的α-螺旋杆域中的 Met(259)、Met(262)、Met(296)和 Met(469)是体外和体内过氧化氢诱导氧化的最易受影响的位点。我们的结果表明，鉴定的蛋氨酸残基可能作为氧化皮肤损伤的生物标志物。本方法是第一种基于 MS 的方法，可直接从人皮肤中检测角蛋白的氧化修饰，并且易于应用于各种皮肤状况下角质素修饰的监测。
