Yamane Ken, Minamoto Atsushi, Yamashita Hidetoshi, Takamura Hiroshi, Miyamoto-Myoken Yuka, Yoshizato Katsutoshi, Nabetani Takuji, Tsugita Akira, Mishima Hiromu K
Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, Japan.
Mol Cell Proteomics. 2003 Nov;2(11):1177-87. doi: 10.1074/mcp.M300038-MCP200. Epub 2003 Sep 15.
Various protein contents such as enzymes, growth factors, and structural components are responsible for biological activities in organs. We have created a map of vitreous proteins and developed a proteome analysis of human vitreous samples to understand the underlying molecular mechanism and to provide clues to new therapeutic approaches in eyes with proliferative diabetic retinopathy (PDR).
Vitreous and serum samples were obtained from subjects with idiopathic macular hole (MH, 26 cases) and PDR (33 cases). The expressed proteins in the samples were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Protein spots were visualized by silver staining, and their expression patterns were analyzed. Some protein spots of concern were excised from the 2-D gels, digested in situ with trypsin, and analyzed by mass spectrometry.
More than 400 spots were detected on 2-D gels of MH cases, of which 78 spots were successfully analyzed. The spots corresponded to peptide fragments of 18 proteins, including pigment epithelium-derived factor, prostaglandin-D2 synthase, and interphotoreceptor retinoid-binding protein. These were not identified in the corresponding serum samples. These proteins were also expressed in PDR samples, with no distinct tendency to increase or decrease compared with the MH samples. More than 600 spots were detected on 2-D gels of PDR cases, of which 141 spots were successfully analyzed. The spots corresponded to peptide fragments of 38 proteins. Enolase and catalase were identified among four detected spots. Neither was found in MH vitreous or in PDR serum samples.
A map of protein expression was made in human vitreous from eyes with MH and PDR. In the PDR eyes, the increased protein expression observed was due to barrier dysfunction and/or production in the eye. Proteome analysis was useful in systematic screening of various protein expression in human vitreous samples.
多种蛋白质成分，如酶、生长因子和结构成分，负责器官中的生物活性。我们绘制了玻璃体蛋白质图谱，并开展了对人玻璃体样本的蛋白质组分析，以了解其潜在分子机制，并为增殖性糖尿病视网膜病变（PDR）患者的眼部新治疗方法提供线索。
从患有特发性黄斑裂孔（MH，26例）和PDR（33例）的受试者中获取玻璃体和血清样本。样本中的表达蛋白通过二维（2-D）聚丙烯酰胺凝胶电泳进行分离。通过银染使蛋白质斑点可视化，并分析其表达模式。从2-D凝胶中切下一些感兴趣的蛋白质斑点，用胰蛋白酶原位消化，然后进行质谱分析。
在MH病例的2-D凝胶上检测到400多个斑点，其中78个斑点成功分析。这些斑点对应于18种蛋白质的肽片段，包括色素上皮衍生因子、前列腺素-D2合成酶和光感受器间类视黄醇结合蛋白。在相应的血清样本中未鉴定出这些蛋白质。这些蛋白质在PDR样本中也有表达，与MH样本相比，没有明显的增加或减少趋势。在PDR病例的2-D凝胶上检测到600多个斑点，其中141个斑点成功分析。这些斑点对应于38种蛋白质的肽片段。在检测到的四个斑点中鉴定出烯醇化酶和过氧化氢酶。在MH玻璃体或PDR血清样本中均未发现。
绘制了来自患有MH和PDR眼睛的人玻璃体蛋白质表达图谱。在PDR眼中，观察到的蛋白质表达增加是由于眼部屏障功能障碍和/或产生。蛋白质组分析有助于系统筛选人玻璃体样本中的各种蛋白质表达。
