| Background: though it has been just ten yeas since the "proteomics" was presented by Australian scientists, the early proteomics based on the protein analysis of tissue or cell has come further into the subcellular level.More and more attention is paid to the analysis of organellar proteomes which can not be circumvented for the fundamental exploration of life mechanism, because the magnificent complexity of organism has evolved on the base of intracellular compartmentalization, which provides cells distinct and suitable environments for biochemical processes. In addition, the 2-DE resolution of proteins can not match the complexity of protein composition of cell, so the targeting of subcellular proteomes is a practicable strategy to gain more information about low-abundant proteins and about their transfer between organelles and interaction. Obiective: In the present work, the subproteomic approach is used for the analysis of hepatoma cells.the fractions of mitochondrial, nucleus and cytosol are subject to proteomic analysis for the express-altered proteins when the normal hepatocyte is used as a control, in the meantime the effect of epirubicin on the expression of proteins of hepatoma cells is valued with the same approach as the above. The results from the subproteomic analysis can be useful for the understanding of the mechanism of carcinogenesis and the action of anticancer drug. Methods: 1.subcellular fractionation: the cell homogenate was subject to diferential centrifugation and four fractions of 1000g pellet,20000g pellet,100000g pellet and cytosol were recovered. Then density gradient centrifugation was applied to the 1000g pellet and 20000g pellet for preparation of nuclei and mitochondrial. So five fractions of nuclei, mitochondrial,20000g pellet,100000g pellet,cytosol were ready for the step 2. 2.two dimensional gel electrophoresis(2-DE) and imaging analysis: 2-DE was applied to the five fractions prepared in step 1 for the separation of protein, imaging analysis of gels was done with ImageMaster 2D(V2002.1)software.protein spots which were differentially expressed between hepatoma cells and normal hepatocyte were cut from the gels for the MS analysis, protein spots which were differentially expressed between hepatoma cells administered with epirubicin and not-administered were also cut from the gels for the MS analysis. 3.MALDI-TOF-MS(matrix-assisted laser desorption/ionization-time of flight) and database search: protein spots from step 2 were subject to in-gel digestion and MALDI-TOF-MS analysis, which resulted in the peptident mass fingerprint spectra(PMF). PMF were search against swiss-protdatabase using two search program of Mascot and PeptIdent, search results from two program could be checked with each other for a high confidence. Results: 1.Afetr didderential centrifugation and two dimensional electrophoresis were applied, 260,464,340,397protein spots were detected in the four fractions of nucleus,20000g pellet,100000g pellet and cytosol respectively, the total number of protein spots of the four fractions was 1461, whereas just 870 spots could be acquired for the cell homogenate. It had been also shown that most mitochondrial and nuclear proteins were not able to present themselves in the 2-DE profile of the cell homogenate. 2.In comparative proteome analysis, 117 expression-altered spots were excised for MS and informatic analysis, 58 spots of which were identified and characterised, they were categorized into 43 sets of proteins. several were found to responded to more than one 2-DE spot, thus revealing different post-translation modifications of proteins. 3.Compared with the normal hepatocytes, 28 proteins were upregulated in the hepatoma cells, 8 proteins were downregulated. Cells have undergone a wide variety of changes in protein expression during carcinogenesis. Firstly, the altered proteins show a high demand for energy by cells due to the rapid preliferation of cancer cells. Sencondly, the altered expression of cytoskeleton proteins may play a role in meta... |
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