Application Of Nanoflow Liquid Reversed-phase Chromatography-tandem Mass Spectrometry For Rice Proteomics Analysis

Proteomics research aimed to discuss and find the law and the nature of life activities at the overall level of protein. The research involving animals, plants, microorganisms and other aspects.Serum proteomic studies help to reveal the pathology of the disease; plant pathogens, microbial proteomics research, which will help to have a clear understanding of the pathogenesis of plants, then we can take timely measures to reduce losses; plant stress proteomics research to find a defense mechanism from the source to ensure that agricultural product quality and safety.Rice is one of the most important cereal crop in the world, it is the source of food for more than half of the world’s population who depends on subsistence, at the same time, it is the Gramineae plant which has the smallest genome. For so many reasons, it has been seem as the model plant for monocots and molecular biology research. So the rice proteomics research has far-reaching significance.A series of treatments should be done before the separation and identification for rice protein, including the extraction of protein, the lysis of protein extracts, the purification of protein (cleaning), proteolysis and the desalination of enzymatic peptides and so on. These processes have significant impact on the results of subsequent separation and identification. There is little study of these, which is specilized for rice proteomics. In this thesis, Nano reversed-phase chromatography-tandem mass spectrometry is used for the separation and identification for the enzymatic peptide of rice protein, and lucubrate the extraction method of rice protein, the selection of lysis modes and the means of protein purification. Ultimately, we established a Nano-RPLC-MS/MS method for rice proteomics research. The main results are as follows:1. Amethod of Nano RPLC-MS/MS was studied and established for rice proteomics research, and many parameters were optimized, such as the flow rate, the time for desalination, the spray voltage,et al The experiment had found that when the ratio of initial aqueous phase and the organic phase was98:2, the initial flow rate was110μL/min, the current flow rate of mobile phase through the column was approximately200-300nL/min, this could met the requirements of separation and identification,and protected the spray needle. After optimization, the desalting time was set to0.5min, and the spray voltage was set to1800V2. The method of rice protein extraction had been studied. Comparing with the extraction efficiency、the purity of the extract, the total number of identified protein、the molecular weight distribution and isoelectric point distribution of extracted proteins by the phenol extraction method、 TCA/acetone method,、TCA/acetone combine with phenol extraction method and SDS direct extraction method. The results showed that the most appropriate extraction method for rice leaf was TCA/acetone method; and for rice grain was SDS direct extraction method.3. The most suitable lysis buffer for rice protein lysis was found. We chose four different rice protein lysis buffer, and compared with the total number of identified protein、the molecular weight distribution and isoelectric point distribution of extracted proteins, we found that the solution contained 4%SDS which was made up with100mmol/L Tris-HCl (pH8.5) had the best ability for protein dissolution.4. We discovered the best assistant mean for rice protein enzymolysis, which can be called with the protein purified way. In this part, three methods were compared and optimized. The comprehensive result was that the best method for removing SDS and other impurities was the one using commercial detergents removal column. This not only had the highest efficiency of removeing impurity, but also could preserved the most proteins.In this thesis, we established the method of Nano RPLC-MS/MS for rice proteomics research. There were319leaves proteins and347grain proteins that was separated and identificated by one time.