Proteomics Study On Atherosclerosis Models And Related Methodology

Presently proteomic research focuses on differential proteome analysis of diseases, which is designed to find key proteins with potentials to be used as markers for diagnosis or targets for meditation. Proteomics is also becoming the spotlight of analytical chemistry. The fast development of proteomics relies on breakthroughs for high throughput analytical technologies which include two-dimensional electrophoresis, biological mass spectrometry and bioinformatics.The main contributions of this dissertation are: proteomics study on Atherosclerosis models which based on two-dimensional electrophoresis and mass spectrometry; a new platform of stepwise protein extraction of human lliver has been established; a new method of protein in-solution digestion after gel-separated was developed. The results were described as follows.1. Atherosclerosis (AS) is a disease with high mortality, and has a great mass of patients all over the world. Atherosclerosis is a process which is the principal contributor to the pathogenesis of myocardial and cerebral infarction, gangrene, and loss of function in the extremities. However, the pathogenic mechanisms underlying AS, along with most other heart diseases, have not yet been identified. It is likely, though, that significant alterations in myocardial gene and protein expression underlie these diseases processes and determine their progression and outcome.Rat was considered suitable for many cardiovascular model establishment including cardiac hypertrophy, hypertension, heart failure, but not easy in atherosclerosis for its hyperlipidemia-resistant character. In this work, we combined the high fat /cholesteroldiets containing sodium cholate and propyl-thyracil with injection of vitamin D3, which led a new way to establishment of an experiment atherosclerotic model in rats. The different expression kinetics of growth factor and adhesion molecule led a major initiative in our laboratory to determine the expression patterns in this atherosclerotic rat model to gain further evidences in vivo.Despite the worldwide occurrence of AS, the pathogenic mechanism underlying this disease remains to a great exact unknown. In this study, the experimental model of atherosclerosis in rat (AS rat) was established by the injection of vitamin D3 associated with high fat diet for 8 weeks. By using the proteomic approach, a comparative study on the proteome of the control and AS rat aorta and heart muscle tissues was carried out. The methods and technology including two dimensional electrophoresis gel (2DE-gel), Commasie brilliant blue (CBB) staining, image scanning and analysizing; protein in-gel digestion, peptide extraction and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS/MS) identification have been uutilized. Separation of the proteins was performed by using 2-DE at pH range of 3-11. For rat aorta, 1878±100 protein spots were detected, and 1239 spots were matched between different gels. The average matching rate was achieved 66%. 26 protein spots were detected in normal rat aorta but were not detected in AS rat aorta, 224 spots, including 161 down-regulated and 63 up-regulated from normal to AS rat, were detected. For rat heart muscle, 2626±100 protein spots were detected, and 1841 spots were matched between different gels. The average matching rate was achieved 70.1%. 33 protein spots were detected in normal rat heart muscle but were not detected in AS rat heart muscle, 70 spots, including 31 down-regulated and 39 up-regulated from normal to AS rat, were detected. There had a good reproducibility of spot position of 2DE map, with average deviation in IEF direction of 1.52±0.22 mm, while in SDS-PAGE direction it was 1.97±0.13mm.The proteins with different expression in control and AS rat tissue show that many components in cell are changed with the AS appearing. In order to know the functionof the proteins with different expression in the AS, 266 proteins in rat aorta and 129 proteins in rat heart muscle were identified by MALDI-TOF-MS/MS after in-gel digestion. These proteins play a very important role on knowing AS mechanism. The identified proteins were classified to five, namely, structure protein, enzymes, cell signaling proteins, transport proteins, and others. The present study provided new insight in pharmacoproteomic study which would be very important for the understanding of the mechanism of the pharmacological process and possible discovery of novel diagnosis markers and therapeutic approaches.2. Protein phosphorylation is a key event in signal transduction. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this dissertation, phosphorylation protein in macrophage-derived foam cells was separated and detected by antibody. The needed methods contained 2DE-gel, immublotting, antibody detection, MS identification and bioinformatics. The results show that phosphorylation protein in macrophage-derived foam cells were centralized in Mr 40 kDa -70 kDa and acidic region. Six phosphorylation proteins were identified by MS and two of them, GAPDH and Stress-induced-phosphoprotein were verified to play a key role in formation and proliferation of cells for AS disease.3. It is an important international project to research the total map of human liver protein. A stepwise method for protein extraction of human lliver has been established. 18 extract fractions were collected and then separated by 2DE-gel and identified by MALDI-TOF-MS/MS. Most proteins of human liver have been extracted with the established platform up to now. In addition, the stepwise extraction reduced interference in the detection.4. A new method involving electroblotting of the gel-separated proteins to a poly (vinylidene difluoride, PVDF) membrane, extracting proteins from the membrane using a solution of 1% trifluoroacetic acid in 70% acetonitrile and lyophilized,...