Research On The CUL4B By Comparative Proteomics

Zou Yongxin has identified CUL4B gene caused XLMR, and then analyzed the expression pattern, localization of CULAB and its role on the cell proliferation and cell cycle regulation. CUL4B as a member of Cullin family, which is the significant moity of ubiquitin ligase enzymes E3, plays an important role in protein polyubiquitinated and degradative regulation. Because there is rare progress in the mechanism of the mental retardation, we have used the proteomic methods to analyze the role of CUL4B during those biological processes.Because of the characteristics, such as high flux, sensitivity and repetition, proteomic technologies have become more popular in research on bioscience. On the base of routine technologies, we explored the effects of different conditions on two-dimensional gel electrophoresis and made them do better in our research on CUL4B. Those improvements include sample preparation, two-dimensional gel electrophoresis, the staining of 2-D gels, in-gel digestion and so on, and furthermore, we applied them to our research.PART ONE The Optimization of Proteomic Procedures about Two-Dimensional Gel Electrophoresis Combined Mass SpectrometerWe optimized some flow sheet of proteomic process about two-dimensional gel electrophoresis combined mass spectrometer. The procedures of optimization mainly content sample preparation and in-gel digestion. Sample preparation is the critical component element in two-dimensional gel electrophoresis. During this part, we explored washing buffer, a economical and feasible method that included lysis buffer containing EDTA, Tris-base or not, containing thiourea or not, concentration of DTT in lysis buffer, sample purification or not and purified methods. The results showed that to the cell lines PC12 and HEK293, the isoelectric focusing pattern of treated with 250mM Sorbitol/10mM Tris-base (pH7.0-7.4) was better than its control group; lysis buffer containing EDTA and Tris-base or not, did not effect isoelectric focusing in our samples; Lysis buffer containing 7M urea and 2M thiourea could improve the solubility of basic proteins effectually compared to just only 8M urea; Lysis buffer containing 65mM DTT had an advantage to containing lOmM DTT that recommended; We also compared the trichloroacetic acid-acetone (TCA-acetone), 2-D cleanup kit (Bio-Rad) and no extra purified treatment, from the results, we could see that 2-D cleanup kit (Bio-Rad) treated can reduce the background of the gel, but it also makes some protein spots disappeared on the gel.Then, we also contrived a high-performance method of in-gel digestion on the basis of other conventional methods for silver staining and Coomassie brilliant blue staining, which could enhanced the achievement ratio of protein identification, In this part, we separated 30ng,50ng and 100ng BSA (Bovine Serum Albumin) by 1-D SDS-PAGE and visualized by silver staining, at the same time we separated 1μg,3μg, 5μg and 10μg BSA by 1-D SDS-PAGE and visualized by Coomassie brilliant blue staining. The BSA from every 1-D gel were in gel digested by the method A (Andrej Shevchenko,2007) which is used generally before and method B that we contrived respectively, then analysed these peptide mixtures by MALDI-TOF/TOF mass spectrometer(Matrix-Assisted-Laser-Desorption-Ionization-Time-Of-Flight-Mass Spectrometry); whereafter, we searched the protein information with NCBI bovine database, then identified the proteins. Those results showed that method B had excellent performance in No. of queries matches, sequencing coverage and mascot score comparing with method A. We demonstrated that proteins on the 2-DE gel pattern were in gel digested by the method B, had better performance in matched peptide number, sequence coverage and protein score. We also got all-right results when we used method B to treat the protein spots from two-dimensional gel electrophoresis of cell line PC12.PART TWO Research on the CUL4B by Comparative Proteomics in Cell Line HEK293We used two-dimensional gel electrophoresis to separate respectively the proteins of the HEK293 cells which CUL4B-specific RNAi to stably knock down endogenous CUL4B expression (miCUL4B HEK293) and the control cellline miNeg-HEK293 cells, then we have visualized by silver staining and gained the 2-D patterns, the gels were analyzed by software 2-D PDQuest to search for the expression level different protein spots between each other. Those different protein spots were in-gel digestion by method B respectively, then analysed by MALDI-TOF/TOF mass spectrometer and searched the protein information with human-own database using Mascot, and then identified the proteins. In this section, we chosed different expression level protein spots between miCUL4B HEK293 and miNeg HEK293 cells; During those spots, identified succesfully was 23, whose protein score lied in 95% confidence interval, bioinformation like isoelectric point and molecular weight were suitable for its position on the gels.To check the results during CUL4B stably down expression HEK293 cells and miNeg HEK293 cells, we constructed CUL4B stably over expression cell line pcDNA3.1 A-CUL4B-HEK293 and the control cell line pcDNA3.1A-HEK293.We gained the two-dimensional gel electrophoretograms of cell line pcDNA3.1A-CUL4B-HEK293, pcDNA3.1A-HEK293 and wild type HEK293 with silver staining. The gels were analyzed by software 2-D PDQuest. After the in-gel digestion with trypsin, the peptide mass analysis was performed using AB4800 MALDI-TOF/TOF mass spectrometer. Mass spectra were externally calibrated with peptide standard and searched the protein information with human-own database using Mascot, then identified the proteins. In this section, it showed 15 different protein spots; During those spots, identified succesfully was 9, whose protein score lied in 95% confidence interval, and bioinformation like isoelectric point and molecular weight were suitable for its position on the gels. Some of those proteins were selected for validation by western blotting and Real time-PCR in those cells.Taken together, this study established a new proteomic protocol which was suitable for the cell lines (PC12 cells and HEK293 cells) used in our research. In addition, we investigated the diversity of proteome when CUL4B gene stably down expression and stably over expression. These results advanced our understanding of the etiology of XLMR, the molecular mechanism of CUL4B in the Mental Retardation, the functions of CUL4B gene and the other relevant biological processes.