Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database.

Abstract

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.

• Authors: Yutaka Yoshida, Kenji Miyazaki, Junichi Kamiie, Masao Sato, Seiji Okuizumi, Akihisa Kenmochi, Ken'ichi Kamijo, Takuji Nabetani, Akira Tsugita, Bo Xu, Ying Zhang, Eishin Yaoita, Tetsuo Osawa, Tadashi Yamamoto
• Journal: Proteomics (Proteomics) Vol. 5 Issue 4 Pg. 1083-96 (Mar 2005) ISSN: 1615-9853 [Print] Germany
• PMID: 15668994 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Proteome

Topics

• Chromatography, Liquid
• Databases, Protein
• Electrophoresis, Gel, Two-Dimensional (methods)
• Electrophoresis, Polyacrylamide Gel
• Gene Expression Regulation
• Humans
• Hydrogen-Ion Concentration
• Image Processing, Computer-Assisted
• Immunohistochemistry
• Internet
• Kidney (metabolism)
• Kidney Glomerulus (metabolism)
• Kidney Neoplasms (metabolism)
• Mass Spectrometry
• Microscopy, Phase-Contrast
• Proteome
• Proteomics (methods)
• Silver Staining
• Software
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization