本研究進行的主要目的，主要發展多種酵素消化方法以及多維的分離系統的搭配，針對真實樣品中整體蛋白質加以研究，希望藉由這些技術，提升我們蛋白質鑑定的種類與數目。蛋白質體學( proteomics )是目前最為熱門的研究課題之一。加上質譜技術的開發與進展，使我們對於生化樣品得以更進一步的深入研究，蛋白質的分析鑑定是我們此篇論文的研究重點，在有限的資料庫輔助之下，嘗試利用新的技術與分離方法，幫助我們在蛋白質上的鑑定工作。
　　質輔助雷射脫附游離-飛行時間質譜法( MALDI-TOF MS )，因為其具備分析速度快、樣品需求量少、操作簡便且靈敏度高等多項優點，是本研究中我們偵測蛋白質所運用的質譜技術之一。但在質譜偵測之前，蛋白質必須先經過消化步驟，成為分子量大小不一的胜肽片斷才能適合於質譜分析。一般消化蛋白質最常用的酵素是胰蛋白酶( trypsin )，但對於較複雜的生化分子，如真實樣品，單一酵素消化有其挑戰存在：可能無法產生適合於儀器偵測範圍的片斷，或是沒有足夠的訊號以供我們分析。因此我們利用其他多種不同的酵素，分別的加入到我們欲分析的樣品中進行消化水解，藉由酵素的特異性不同，質譜可產生更多我們可以判斷的分子量訊號，最後配合蛋白質資料庫的搜尋，可使蛋白質序列的覆蓋率增加，提高鑑定出蛋白質的準確性。這裡我們以β-酪蛋白( β-casein )以及老鼠胎盤樣品為例，都可以明顯的看出多種酵素作用的優點。
　　此外，在多維層析系統實驗方面( Multi-dimensional chromatogra-phy system )，針對於複雜的真實樣品，假使沒有更仔細的分離步驟，經過消化後，相對少量的微量蛋白訊號可能被抑制而無法被偵測鑑定出，因此在這裡我們也建立了一個多維的液相層析系統實驗方式，有別於一般方法在胜肽分子混合物階段才開始進行多維分離，我們在蛋白質階段就先進行初分，經過兩種酵素水解後，再搭配離子交換方法進行二維的分離分析，最後採用胜肽片斷分子量比對技術( peptide mass mapping, PMM )，雖然目前是採用離線( off-line )的階段分離來測試，但要將系統一貫地自動化動作也是指日可待。
　　真實樣品最後經由基質輔助雷射脫附離子化-飛行時間質譜儀( MALDI-TOF MS )的偵測，以線上蛋白質資料庫來鑑定出所含之蛋白質種類與數目，結果顯示，應用多維系統與多種酵素方法對於真實生化樣品的分離鑑定，能夠有效提升所鑑定之蛋白質數量之外，甚至能鑑定出我們有興趣的微量功能性蛋白，這些蛋白也提供我們鑑定結果的正向佐證。期望在各項參數的最佳化、自動化後，此方法可以作為細胞、組織中整體蛋白質研究、鑑定的另一種替代技術。

　　This study is mainly based on developing a multiple enzymatic digestion method and a multi-dimensional chromatography system for the analysis of biological samples. It is hoped that using this technology, further protein analysis can be identified with an increasing variety and quantity of annotated proteins. Nowadays, proteomic research is one of the hot topics in the science research field. The great invention and improvement of mass spectrometric technology allow us to extend to the next level in the research. On the main focuses of this paper is on protein identification and analysis.
　　MALDI-TOF MS is one of the instrument that are used for protein analysis; its advantages include requiring small amounts of samples, short analyzing period, simple and convenient, as well as, high sensitivity. Before performing MALDI on the real sample, the proteins need to be digested into different sizes of molecules. Trypsin is the most common enzyme that is used in digesting proteins. However, due to the complexity of biological molecules, trypsin does have its limitations. If the enzyme has a hard time digesting the proteins, bad signals or even no signal would show on the screen. Consequently, different types of enzymes are added separately into each analyzing samples for digestion. Due to the fact that different enzymes have different characteristics, proteins would be digested at different digesting sites. The samples will then be detected with more signals than before. β-casein as well as rat placenta are both great examples in pointing out the advantages of using multiple enzymatic digestion method.
　　Besides MALDI-TOF MS, a multi-dimensional chromatography system would meet a problem when the quantity of analyzing protein increases with a constant number of fractionations. When this happens, the signals of proteins with tiny amount would be overlapped; therefore, we have come up with a multi-dimensional high performance liquid chromatography method to resolve the problem of overlapping signals. The original multi-D LC method starts its fractionation after the proteins are digested into peptides. The new method, however, performs fractionation at the protein level. The proteins are then digested with two enzymes followed by MALDI-TOF MS. Although an off-line stage is still presently being used for detection, shifting to an automatic system is being looked forward to. The data from MS are then being searched online via protein database by peptide mass mapping (PMM) technology. This multi-dimensional fractionation method is believed to be a useful way in finding more proteins when working with actual biological samples. The method is effective to increase for identifying those proteins in micro-units, even for some low-abundantly functional protein. Hopefully with all the conditions being tuned to optimal and automatic, this method could be used to analyze and identify cells and tissues.

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