Non-invasive proteomic analysis of human skin keratins: Screening of methionine oxidation in keratins by mass spectrometry
Non-invasive proteomic analysis of human skin keratins: Screening of methionine oxidation in keratins by mass spectrometry
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人体皮肤角蛋白的非侵入性蛋白质组学分析：通过质谱法筛选角蛋白中的蛋氨酸氧化
DOI：
10.1016/j.jprot.2011.08.010
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Lee;S.H.;Miyamoto;K.;Goto;T.;Oe;T.
角蛋白是人体皮肤的主要成分，是主要的氧化靶蛋白。然而，由于角蛋白的不溶性和处理困难，缺乏旨在确定角蛋白氧化位点的研究。在这里，我们介绍了一种基于质谱（MS）的蛋白质组学方法来筛选人类皮肤角蛋白的氧化修饰。采用胶带剥离法无创提取人体皮肤蛋白，SDS缓冲液溶解后，采用改进的过滤辅助制样方法纯化消化。采用MALDI-TOF/MS、LC-ESI /MS和MS/MS分析色氨酸。PMF分析已经确定角蛋白K1和K10是人类皮肤的主要蛋白质。Met259, Met262， Met296和Met469位于K1 α-螺旋棒结构域，是过氧化氢诱导的最易感位点。我们的研究结果表明，鉴定的蛋氨酸残基作为氧化性皮肤损伤的生物标志物具有潜在的用途。目前的方法是第一个基于质谱的方法来检测直接从人体皮肤中获得的角蛋白的氧化修饰，并且可以很容易地应用于监测各种皮肤状况下的其他角蛋白修饰。
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. Met259, Met262, Met296, and Met469, 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.