| 摘要: | 在蛋白質和胜肽的分析工作上，質譜儀是一項非常重要的技術，而它也是分析蛋白質體的一項最主要方法，對於離子源為 ESI的使用，經常是結合層析的裝置，並將蛋白質樣品溶於溶劑當中，可用來分析大部分的一些胜肽，可是液相層析方法的各項層析條件，卻是影響液相層析質譜儀靈敏度及辨識率的最重要因素，這些因素有（1）移動相的性質，例如：各種溶劑混合比率、有機修飾劑的百分率、緩衝溶液的pH、鹽類的濃度、流速。（2）逆相管柱靜相的形式。基於這些原因，在本論文研究工作中係採用甲醇取代習用之乙腈作為沖提溶劑，以研發出一種新型的LC/MS蛋白質體分析方法。在其中，藉由使用甲醇以取代對環境傷害性較大的乙腈，以符合目前環保趨勢的要求，並且也將採用一只C18前濃縮管柱，選擇不同流速以及不同修飾劑，來進行比較，找出最佳的分析條件，以期能夠同時提昇LC/MS蛋白質體分析方法的解析度與靈敏度。並在真實樣品方面，依據以上實驗條件，選擇大腸桿菌所表現產生的人類血小板生長因子（PDGF）與唾液經2-D電泳分離後所切下蛋白質點進行測試，均有良好的結果。所以此本新型LC/MS蛋白質體分析方法已經能夠成它a取代較不環保的舊有LC/MS蛋白質體分析方法，並且也能夠成它a應用於真實樣品的蛋白質定序分析工作中。
Mass spectrometry has become the most important technique for the analysis of proteins and peptides. In ionization meyhod, ESI requires elution of a peptide sample in a solvent, and therefore, it can be easily coupled to a chromatographic device that will separate most of the components in the sample. But in the chromatographic analysis, the analytical of data will be affected by some important parameters, that influence the sensitivity and the identification of LC/MS analysis, there are these parameters :(1) the properties of mobile phases, for example: the slope of the gradient, the percent of organic modifier, pH of the buffer solution, the concentration of the salts, and the flow rate, (2) type of stationary phase of reverse phase column. For the reason, this research work is used the methanol to replace the common solvent, acetonitrile, in order to develop a novel LC/MS proteome method. We hope by using the methanol to replace acetonitrile that has the more environmental injury in order to conform to the request of the environmental protection trend at present, and we use a C18 pre-concentration column, Choose different of flow rate, and the several kinds of modifiers, come to compare, find out the best analysis condition, we expect to promote the resolution and sensitivity of LC/MS proteome method. From the result, these conditions are applied in the study of detection limit and the real sample analysis. In this research work, we apply this novel LC/MS proteome method to the detection of the real sample, the growth factor of human blood platelet produced by E. Coli and saliva of human of the 2-D gel detection of the band, too. From the result we can understand that this proteome method can identify real samples. This novel LC/MS proteome can successfully replace the old LC/MS proteome method that has environmental injury, and can be successfully applied to the protein identification of the true sample. |
