本研究主要是利用胰蛋白酶水解蛋白質，以液相層析儀(HPLC)、毛細管電泳儀(CE)和MALDI-TOF質譜儀對蛋白質水解產物進行純化和分析。以HPLC的分離純化與MALDI的胜肽序列鑑定，我們完成了α-酪蛋白和β-酪蛋白經胰蛋白酶水解所得胜肽樣品在毛細管電泳圖譜中的析出順序。根據此CE圖譜，我們分析了在不同離子強度的緩衝溶液中，以及添加不同濃度的不同種類金屬離子於電泳緩衝溶液中，α-酪蛋白胜肽片段的電泳遷移行為。其中添加適當濃度Ca2+、Ba2+離子會對含磷酸化胜肽有較大的電泳遷移變化。另外，改變蛋白質水解反應條件，包括反應緩衝溶液的離子強度、金屬離子的添加、有機溶劑的添加、以及反應時間的長短…...等，會影響所得胜肽產物的正確切和錯誤切比例和分布情形，也會影響以MALDI進行蛋白質鑑定的結果。
另外，我們發現利用銨鹽與酸混合添加於樣品，可以顯著的改善以MALDI質譜儀偵測磷酸化胜肽片段時，所得訊號偏低的現象，其中以100 mM 亞甲基二磷酸(Methylenediphosphonic acid, MDPNA)與25 mM 磷酸二氫銨混合後添加於樣品之偵測效果最佳。我們也發現此類添加劑不但對磷酸化胜肽片段的偵測有幫助，也可有效提高親水性胜肽片段質譜訊號。2,5-二羫基苯甲酸(2,5-Dihydroxy benzoic acid, DHB)基質對於胜肽有良好的偵測效果，但其所形成的針狀結晶之均勻度不佳，會影響定量分析的再現性。我們開發以冷凍抽氣(Ice vacuum)的方式進行MALDI樣品製備，可以大幅提高樣品的均勻度以及質譜訊號的再現性。不過，冷凍抽氣後樣品的訊號值通常都比室溫空氣乾燥訊號值小，我們使用添加劑以及小幅提高雷射強度，可以有效提高MALDI訊號，並維持良好的再現性。此外，我們也分別以C18和幾丁聚醣(Chitosan)對蛋白質水解之胜肽片段進行固相微萃取實驗，兩者皆可達到純化、濃縮胜肽樣品的效果。

In this thesis we mainly utilize high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and matrix-assisted laser desorption/ionization-time of flight mass spectrometer (MALDI-TOF MS) to purify and analyze the hydrolysis products of proteins digested by trypsin. With the sample purification by HPLC and sequence identification of peptides by MALDI, we have established the electrophoretic elution orders of the tryptic digests in the respective electropherograms of α-casein and β-casein. Based on the resulting electropherograms we are able to analyze the effects of ionic strength and metal ion addition in CE buffer on the electrophoretic migration behaviors of the peptide fragments generated from the tryptic digests of α-casein and β-casein. We found that the addition of adequate concentrations of Ca2 + and Ba2 + ions would result in larger electrophoretic mobility shifts for the phosphopeptides. Besides, varying protein digestion conditions such as ionic strength of reaction buffer, addition of metal ions and organic solvents, and reaction duration would alter the product ratio of the correct cut to the wrong cut peptide fragments, and thus influence the results of protein identification by MALDI.
Moreover, we found that with the addition of certain ammonium salt and acid in peptide sample, the usually inferior MALDI signals of phosphopeptides can be largely enhanced. Among several additives investigated, the mixture of 100 mM methylenediphosphonic acid (MDPNA) and 25 mM ammonium dihydrogen phosphate (ADP) has the best detection results for phosphopeptides. We also observed signal enhancement for the hydrophilic peptides by this MDPNA-ADP additive. 2,5-dihydroxy benzoic acid (DHB) is a commonly used matrix and provides relatively good MALDI detection for peptide fragments. However, its needle-like crystals formed after air-drying sample would generate an inhomogeneous sample spot, making quantitative analysis unfeasible. In order to prepare a homogeneous sample spot for MALD measurement, we developed an ice-vacuum method for MALDI sample preparation and greatly improve the reproducibility of MALDI signals for peptide and protein samples. However, the MALDI　signal of the　ice-vacuum prepared sample is generally smaller than that of the air-dry prepared sample. Utilizing MDPNA-ADP additive and slightly increasing laser power, we can effectively improve the MALDI signals of ice-vacuum prepared sample and in the same time maintain good reproducibility. In addition, C18 and Chitosan were demonstrated as good absorbents in solid phase micro-extraction experiments for the purification and concentration of peptide samples.

目錄
第一章 緒論..............................................................................................1
1-1	研究動機與目的.................................................................................1
1-2	實驗背景資料……………………………………………………….2
1-2.1	建構蛋白質水解胜肽片段的毛細管電泳圖譜....................2
1-2.2	胰蛋白酶反應之錯誤切產物………………………………3
1-2.3	改變胰蛋白酶反應條件……………………………………5
1-2.4	磷酸化蛋白質及其偵測……………………………………6
1-2.5	MALDI質譜儀偵測………………………………………..9
1-2.6	MALDI樣品之均勻度……………………………………12
1-2.7	以固態微萃取法純化蛋白質水解胜肽樣品......................15
1-3	參考資料...........................................................................................18
第二章 實驗方法....................................................................................23
2-1	實驗儀器...........................................................................................23
2-1.1	毛細管電泳儀 Beckmen P/ACE MDQ……………..…..23
2-1.2	MALDI/TOF-TOF Mass……………………………..…..23
2-1.3	HPLC 儀器 JASCO……………………………………...24
2-1.4	pH meter與去離子水處理器……………………………..24
2-1.5	去離子水處理器…………………………………………..24
2-1.6	真空濃縮機………………………………………………..24
2-1.7	離心機…………………………………………………….24
2-2	實驗藥品…………………………………………………………...25
2-2.1	毛細管塗層藥品…………………………………………..25
2-2.2	配製毛細管電泳緩衝溶液藥品…………………………..26
2-2.3	電泳緩衝溶液添加劑……………………………………..26
2-2.4	蛋白質及聚胜肽樣品……………………………………..27
2-2.5	蛋白質水解及去磷酸化所需試劑………………………..29
2-2.6	蛋白質水解反應添加劑…………………………………..30
2-2.7	MALDI-TOF/MS 樣品之基質及試劑…………………...30
2-2.8	MALDI-TOF/MS 樣品添加劑…………………………...31
2-2.9	固態微萃取填充試劑……………………………………..32
2-3	毛細管塗層方法………………….………………………………..33
2-3.1	細管內壁Polyacrylamide塗層步驟………………….......33
2-3.2	未塗層毛細管內壁酸活化步驟…………………..............34
2-4	實驗方法與步驟…………………...................................................35
2-4.1	實驗藥品配置…………………..........................................35
2-4.2	蛋白質水解步驟(Digestion) …………………..................36
2-4.3	蛋白質去磷酸化步驟………………….............................47
2-4.4	胜肽樣品電泳遷移變動率之計算…………………..........48
2-4.5	MALDI-TOF mass 樣品備製……………………............50
2-4.6	C18固相微萃取實驗方法…………………….................51
2-4.7	Chitosan固相微萃取實驗方法……………………...........52
2-4.8	蛋白質分析軟體的運用及胜肽質譜線上比對(Peptide mass fingerprint, PMF) ……………………..............................53
2-4.9	儀器偵測條件……………………………………………..54
2-5	C18自製管柱....................................................................................56
2-6	參考資料…………………………………………………...............58
第三章 結果與討論................................................................................59
3-1	利用HPLC分離純化酪蛋白(casein)與MALDI-TOF質譜鑑定純化胜肽片段於建構CE酪蛋白(casein)水解圖譜……………………59
3-1.1	改變HPLC移動相組成純化水解後α-酪蛋白(casein)之胜肽片段………………………………………………………….59
3-1.2	以HPLC分析酪蛋白經不同水解反應條件所得胜肽片段……………………………………………………………….62
3-1.3	α-casein(Trypsin digest，in 10mM ABC)毛細管電泳圖譜之建構…………………………………………………………….72
3-2	以電泳遷移變動率(mobility shift)實驗偵測α-casein(Tryptic digest)之磷酸化胜肽……………………………………………………...75
3-2.1	不同電泳緩衝溶液濃度的影響…………………………..75
3-2.2	改變不同金屬離子添加於電泳緩衝溶液.........................81
3-2.3	以去磷酸化樣品進行實驗……………………………….85
3-3	以CE分析casein經胰蛋白酶於不同水解反應條件下之胜肽片段…………………………………………………………………...87
3-3.1	碳酸氫胺(ABC)緩衝溶液之濃度效應..............................87
3-3.2	在不同pH碳酸氫銨緩衝溶液中進行水解反應..............91
3-3.3	在ABC中添加界面活性劑對於蛋白質水解反應的影響................................................................................................96
3-3.4	在ABC中添加有機溶劑對於蛋白質水解反應的影響103
3-3.5	蛋白質水解反應的時間效應…………………………....107
3-4	提高磷酸化胜肽的MALDI-TOF偵測訊號..................................112
3-4.1	利用銨鹽/酸混合添加劑提高磷酸化胜肽片段的MALDI
偵測訊號………………………………………….…………112
3-4.2	添加高分子以提高磷酸化胜肽片段的MALDI-偵測訊號..............................................................................................120
3-5	利用冷凍抽氣法提高由2,5-Dihydroxy benzoic acid (DHB)基質所製備樣品之均勻度……………………………………………..126
3-5.1	製備不同MALDI樣品………………………………….126
3-5.2	添加銨鹽/酸以提高冷凍抽氣法所製備樣品之 MALDI偵測訊號……………………………………………………...130
3-5.3	冷凍於更低溫(-80℃)下進行，提高添加銨鹽/酸後基質均勻度..........................................................................................132
3-6	利用固相微萃取法純化蛋白質經胰蛋白酶水解胜肽樣品.........134
3-6.1	以C18萃取α-casein(Trypsin digest，in 10mM ABC).......134
3-6.2	以Chitosan萃取α-casein(Trypsin digest，in 10mM ABC).........................................................................................142
3-7	參考資料.........................................................................................154
第四章 結論..........................................................................................156
第五章 附錄..........................................................................................158

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1.	簡逸棻，「利用毛細管電泳分析胜肽及其受金屬離子與醣類的影響」，專題研究報告，淡江大學化學系，2009。
2.	Iwase, Y.; Honma, S.; Matsuzaki, M.; Miyakawa, Y.; Kanno, T.; Ishii, K.; Furuichi, N.; Furukawa, K.; Horigome, T., “A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis”, Journal of Biochemistry. 2010, 147, 689-696.
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5.	Brady P. Culver; Jeffrey N. Savas; Sung K. Park; Jeong H. Choi; Shuqiu Zheng; Scott O. Zeitlin; John R. Yates III; Naoko Tanese, “Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis”, J. Biol. Chem .2012,287, 21599-21614.
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