The Comparing Study Of Hydrophobic Proteome In Mouse Hepatocarcinoma Cells H22

objective: To explore further the contribution of hydrophobic proteins, including membrane proteins, in the processes of development, invasion and migration of tumor cells according to the alternation of expression levels of hydrophobic proteins in mouse hepatocarcinoma cells H22 (HCC H22) from the comparative analyses of hydrophobic and hydrophilic proteomes in HCC H22 with in normal hepatocytes by the technologies of proteomics, bioinformatics and MS peptide fingerprinting for analyzing and identifying the differential expression proteins. Methods: Mouse hepatocarcinoma cells H22 were cultured by routine and normal hepatocytes as contrast were cultured with"the cutting separation method". By the exponential growth period, the cells were disrupted by two distinct cell lysis procedures. The first procedure was a method in which a conventional cell lysis buffer (8mol/L urea, 4%CHAPS, 1%TBP, 0.2%BioLyte) was applied to cell lysis and collection of soluble proteins. The second procedure was a method in which a modified step-extraction lysing buffer was applied to extract hydrophobic proteins, by freezing and thawing repeatedly with application of a strengthen cell lysis buffer (7mol/L urea, 2mol/L thiourea, 4%CHAPS, 50mmol/L DTT, 0.2%BioLyte) to harvest them from mouse hepatocarcinoma cells and normal hepatocytes. The protein concentrations were determined with Bradford method. The proteins were separated by two-dimensional electrophoresis, which were run by isoelectric focusing on IPG Drystrip pH 4-7 linear gels, following by SDS-PAGE (10% gel concentration), and then stained by silver staining. The 2-DE maps were captured by scanning and then analyzed by PDQuest 2D software. The pI and MW values of protein spots were determined by conferring to the linear gradient of IPG and standard MW marker. By compared with in control group, thirteen protein spots with differential expression level in experimental group were found by means of PDQuest 2D software and then they were cut out from gels. The identification of hydrophobic protein spots expressed differentially in hepatocarcinoma cells H22 was performed through detecting their peptide fingerprinting by MALDI-TOF-MS and matching them to Swiss-Prot protein database with experimental pI and MW data by Aldente software. Results:â‘ There was obvious differentiation between the 2-DE maps of mouse hepatocarcinoma cells H22 and normal hepatocytes by a modified step-extraction cell lysis method or a conventional cell lysis method.â‘¡By the technologies of mass spectrogram with bioinformatics, thirteen hydrophobic proteins had been identified, in which five were membrane proteins and others were dissoluble in cellular plasma or nucleus The physiological functions of them were involved in cellular metabolism, proliferation, signaling transduction, framework structure and so on. Conclusions:â‘ The modified step-extraction cell lysis method can be applied successfully to extraction of hydrophobic proteins from mouse hepatocarcinoma cells and hepatocytes.â‘¡By the researching technology of comparative proteome, thirteen hydrophobic protein spots in the 2-DE map of mouse hepatocarcinoma cells H22 have been identified, which are probably related to the processes of development, invasion and migration of this tumor.