Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database
Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database
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DOI：
10.1002/pmic.200401075
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发表时间：
2005-03-01
期刊：
影响因子：
3.4
通讯作者：
Yamamoto, T
中科院分区：
文献类型：
--
作者：
Yoshida, Y;Miyazaki, K;Yamamoto, T
为了对人类肾小球的生理学和病理生理学做出贡献，我们开展了人类肾小球蛋白质组学研究，通过双向凝胶电泳（2-DE）和基质辅助激光解吸/电离飞行时间质谱（matrixassisted laser desorption/ ionization-time of flight mass spectrometry，MALD-TOF）鉴定，建立了正常人肾小球蛋白质表达谱质谱（MALDI-TOF MS）和/或液相色谱-串联质谱（LC-MS/MS）。具有正常外观的肾皮质从由于肾肿瘤而进行手术肾切除的患者获得，并且肾小球通过标准筛分方法高度纯化，然后在相差显微镜下拾取。用24 cm固定化pH梯度条带在3-10范围内进行第一维双向电泳，用26 × 20 cm 12.5%十二烷基硫酸钠聚丙烯酰胺凝胶进行第二维双向电泳。对凝胶进行银染色，并通过由点选择器、凝胶内消化器和MALDI-TOF MS和/或LC-MS/MS组成的集成机器人系统对有效点进行处理以进行识别。根据四名无明显病理表现的受试者的肾小球的2-DE凝胶图像，创建了正常肾小球蛋白的合成凝胶图像。合成凝胶图像包含1713个有效点，其中1559个点通常在相应的2-DE凝胶中观察到。在这1559个蛋白质点中，迄今已鉴定出347个蛋白质点，代表212种蛋白质，并用于构建基于可扩展标记语言（XML）的数据库。该数据库存放在一个网站（http：//www.example.com hgldb/index.html）上，研究人员可以访问该数据库，以促进健康和疾病中人类肾小球的蛋白质组学研究。www.sw.nec.co.jp/bio/rd/
To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 X 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and/or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/ hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.