Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling.
Loss of renal function is often associated with the injury of kidney glomeruli. It is therefore necessary to understand the mechanisms leading to progressive glomerular diseases; this may be addressed using proteomics. Until now, however, analysis of the glomeruli proteome using 2-DE has been technically hampered by low protein yields from scarce samples. To circumvent this problem, we developed a procedure which allows the human and mouse glomeruli proteome to be analysed. In this study, two different approaches were used to isolate mouse and human glomerular protein from kidney cortex. Mouse glomeruli were extracted by embolisation magnetic beads into the glomerular capillaries. Laser capture microdissection (LCM) was utilised to harvest glomeruli from human biopsy material. Human and murine samples were analysed using a fluorescence saturation labelling technique. Using 3 microg mouse glomerular protein a total of 2900 spots were resolved for differential proteome analysis. Moreover, it was also demonstrated for the first time that only ten glomeruli (0.5 microg) picked by LCM from a slide of a human kidney biopsy material were sufficient to visualise 900 spots. This novel strategy paves the way for future experiments aimed at investigating functional proteomics of glomerular diseases in humans and in mice.
Sitek
Barbara
B
Medical Proteom-Center, Ruhr-University, Bochum, Germany.
Potthoff
Sebastian
S
Schulenborg
Thomas
T
Stegbauer
Johannes
J
Vinke
Tobias
T
Rump
Lars-Christian
LC
Meyer
Helmut E
HE
Vonend
Oliver
O
Stühler
Kai
K
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007672:Kidney Cortex / Q000378:metabolism; D007678:Kidney Glomerulus / Q000378:metabolism*; D051379:Mice; D042282:Microdissection / Q000379:methods; D008863:Microspheres; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000295:instrumentation / Q000379:methods
D005456:Fluorescent Dyes; D011506:Proteins