Studies On The Quantification Techniques Of Comparative Proteomics And Its Applications To Disease Investigations

Proteomic techniques investigation is the main content of analytical chemistry in the field of Life Sciences, and becomes the leading area in recent years. This dissertation is focused on the investigation of proteomics and quantitative proteomics methodology together with its applications to the human serious diseases in the course of Proteomics Lab foundation.The goal of quantitative proteomics is to examine all of the changes induced by a certain perturbation in a biological system. Quantitative proteomics is the main aspects of comparative proteomics, and can provide major approaches to the elucidation of disease mechanisms and to the identification of new diagnostic markers and therapeutic targets. The main contributions are, Establishment of a technique platform with high throughput involving protein sequential extraction - two dimensional gel electrophoresis (2DE) separation - semi automatic in gel digestion - MS identification; Establishment of a new quantitative proteomic technique based on the acetylation stable-isotope labeling combining with 1D PAGE separation, which has been successfully confirmed by applying it to real bio-samples; Comparative proteomic investigations of several real-bio-samples involving Atherosclerosis, Coronary Heart Disease and metastasis of liver cancer, and evaluation of some kinds of pharmaceutics from the protein level. Meanwhile, investigations on affinity isolation based on the firm combination between biotin and avidin, in order to eventually realize the efficient combination of affinity isolation and acetylation isotope labeling. The main contents can be descried as follows:1. Establishment of a high throughput technique platform including protein sequential extraction -2DE separation - semi automatic in gel digestion - MS identification,found a solid experimental basis for proteomics investigations (1) Sample pre-fractionation is essential to the proteomic investigation because of the complexity of a certain proteome, and sequential extraction holds a great promise on protein fractionation prior to further analysis. Reasonable selection of the extraction solution is the key factor to the successive analysis of proteomics study.A feasible three-step protein extraction protocol was proposed. Almost all the proteins were entered into the related extracts. Briefly, an aqueous solution containing 1 mmol/L PMSF was used as the first extract solution (T1) and only restricted sorts of proteins were extracted because of no denaturant and detergent was contained, so that E1 was suited for the direct separation by chromatography or electrophoresis, etc. because of its clean background. Also, in case a definite quantity of urea and CHAPS were added into the rehydration system of E1, 2DE separation on E1 can be carried out. The mixture of 7 mol/L urea, 2 mol/L thiourea, 2% CHAPS, 2% SB3-10, 50 mmol/L DTT, 1 mmol/L PMSF was used as the second extract solution (T2) which possesses the greatest solubility and can still be used to the 2D gel separation. Moreover, the proteins with relatively low abundance can thus be detected because of the removal of large quantity of cytosolic proteins. At last, 2% SDS substituted 2% SB3-10 of T2 was used as T3, and nearly no residue was remained. E3 obtained can be analyzed via either 1D PAGE-LC/MS or 2D LC/MS. Thus, almost all proteins in a certain proteome can be solubilized by using such a three-step sequential extraction protocol described above.As the linkage of 2DE separation and MS identification, in-gel digestion remains the primary procedure for peptide coverage. The original protocol of manual in-gel digestion has been re-adjusted to some extent, and was applied to the semi-automatic analysis, resulting in high speed and throughput; Many factors during the semi-automatic in-gel digestion, such as trypsin quantity, digestion duration, pre-desalting or not prior to MS, et al, which would interfere with the peptide coverage and definition of MS identification severely are tested.2. Applications of comparative prote...