Interdisciplinary Nanoscience Center (iNANO) and Department of Molecular Biology, Aarhus University, and Department of Ophthalmology, Aalborg Hospital, Denmark.
J Proteome Res. 2012 Aug 3;11(8):4231-9. doi: 10.1021/pr300358k. Epub 2012 Jul 10.
Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.
角膜疾病很常见，是指感染、外伤和遗传缺陷等疾病。从形态学上看，许多角膜疾病仅影响角膜的某些层，因此对各层进行单独分析有助于探索与角膜健康和疾病相关的基本分子机制。在这项研究中，我们从健康人眼角膜中分离出了包括上皮、基质和内皮在内的三个主要层。在通过 LC-MS/MS 进行分析之前，我们对不同层的蛋白质进行了以下处理：(i) 通过 SDS-PAGE 分离，然后进行胶内胰蛋白酶消化；(ii) 无需事先进行蛋白质分离，直接在溶液中消化；或 (iii) 在溶液中消化后进行阳离子交换层析。我们在人眼角膜中总共鉴定出了 3250 种独特的 Swiss-Prot 注释蛋白，其中上皮层有 2737 种，基质层有 1679 种，内皮层有 880 种。在这些蛋白中，有 1787 种以前没有通过质谱法在人角膜中鉴定过。总共定量了 771 种蛋白，其中 157 种基于溶液消化，770 种基于 SDS-PAGE 分离，然后对切取的凝胶块进行胶内消化。蛋白质分析表明，许多鉴定出的蛋白是参与防御反应的血浆蛋白。
