Proteomic analyzes of copper metabolism in an in vitro model of Wilson disease using surface enhanced laser desorption/ionization-time of flight-mass spectrometry.
In Wilson disease, mutations in the ATP7B-gene lead to hepatic accumulation of copper that becomes toxic when the hepatic binding capacity is exceeded, leading to oxidative stress and acute liver failure. Several proteins are probably involved in dealing with the excess copper and oxidative stress. As a first step towards biomarker discovery and analyzes of copper metabolism in Wilson disease patients we characterized copper-induced changes in protein expression in cell lysates and culture media from an in vitro copper-overload model using surface enhanced laser desorption/ionization (SELDI) proteomics technology. HepG2 cells were cultured for 48 h with a physiological (0.5 microM) or a pathological (100 microM) copper concentration. Samples were applied to weak cation exchange (WCX) proteinchip arrays and chips were analyzed by time of flight (TOF)-mass spectrometry. Copper-coated IMAC chips were used to detect copper-binding proteins in cell lysate of copper depleted cells using buffers with increasing imidazole concentrations. Data from the 2 to 50 kDa range indicate that high extra-cellular copper substantially altered both intra-cellular protein expression as well as the composition of the secretome. In the lysate 15 proteins were found up-regulated, while 6 proteins were down-regulated. In culture media 21 proteins were increased while 4 proteins were decreased in abundance. Copper-coated protein chips revealed the presence of 18 high-affinity copper-binding proteins. Further identification is necessary to determine the exact cellular roles of the discovered proteins.
Roelofsen
Han
H
Department of Pediatrics, Division of Nutrition and Metabolism, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. j.roelofsen@med.rug.nl
Balgobind
Reshma
R
Vonk
Roel J
RJ
D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D003300:Copper / Q000378:metabolism*; D003470:Culture Media; D006527:Hepatolenticular Degeneration / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D040081:Protein Array Analysis; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002352:Carrier Proteins; D003470:Culture Media; C023548:copper-binding protein; D003300:Copper