Two-dimensional electrophoretic profiling of normal human kidney glomerulus proteome and construction of an extensible markup language (XML)-based database.
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.
Yoshida
Yutaka
Y
Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan. yyoshi@med.niigata-u.ac.jp
Miyazaki
Kenji
K
Kamiie
Junichi
J
Sato
Masao
M
Okuizumi
Seiji
S
Kenmochi
Akihisa
A
Kamijo
Ken'ichi
K
Nabetani
Takuji
T
Tsugita
Akira
A
Xu
Bo
B
Zhang
Ying
Y
Yaoita
Eishin
E
Osawa
Tetsuo
T
Yamamoto
Tadashi
T
D002853:Chromatography, Liquid; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D020407:Internet; D007668:Kidney / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism*; D007680:Kidney Neoplasms / Q000378:metabolism; D013058:Mass Spectrometry; D008858:Microscopy, Phase-Contrast; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D016622:Silver Staining; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020543:Proteome