本研究是對蛋白質水解產物用不同DDA%幾丁聚醣(chitosan)進行吸附/脫附後樣品以毛細管電泳(CE)和MALDI-TOF質譜儀進行分析。脫附出的胜肽片段會藉由MALDI-TOF確認分子量。而在電泳圖剩餘的胜肽會藉由CE計算其面積%定義回收率。再來根據本實驗室之前的實驗結果，我們已經定出α-酪蛋白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，我們使用不同去乙醯化程度(degree of deacetylation,DDA)的幾丁聚醣進行吸附，因幾丁聚醣DDA%值的不同，其所含去乙醯基的比例不同，會與胜肽吸附能力有關，觀察吸附後殘留的胜肽，再用適當的脫附劑，進行脫附，觀察回收後樣品的回收率。
在不同去乙醯化程度的結果中，我們觀察到在吸附時，磷酸根部分是全部被吸附，而脫附回收率在不同DDA%有不同的結果，DDA95%的回收率在60%以下，是三種DDA%脫附百分比最小的。
另外，為了要在三種DDA%中求得最好的脫附效果，本研究將改變鹼性液的濃度(0.15%、1.5%、15%)、極性後(添加ACN/MeOH)，發現0.15%氨水的脫附百分比最差，而對於較低DDA%的chitosan，較高濃度的鹼液有較好的脫附效果，1.5%/15%氨水濃度對大部分的胜肽來說脫附百分比相似，但是親疏水性有些微不同，而改變極性對於DDA95%/DDA54%的脫附行為有提升效果但是對於DDA34%脫附回收沒有明顯幫助

In this thesis we utilize capillary electrophoresis (CE) and MALDI-TOF/MS technique to investigate the adsorption and desorption behaviors of α-casein tryptic digests on chitin and chitosans with degrees of deacetylation (DDA) of 95%, 54%, 34%. The elution orders of the peptide fragments in CE electropherogram were determined by HPLC purification of the tryptic digests and MALDI-TOF/MS identification of the peptide molecular weights. The ratios of residual peptides to original tryptic digests were calculated from the peak areas in CE electropherograms. The effects of concentration and polarity of desorbents on the peptide desorption efficacy were studied for the four adsorbents. Peptide desorption ratios were also obtained from CE electropherograms.
According to our results, the phosphopeptides of the peptide mixtures almost completely adsorbed onto the four chitosan adsorbents. The DDA95% chitosan seemed to have stronger affinity for peptide fragments and thus resulted in lower desorption ratios. In order to achieve better desorption efficacy for the four adsorbent systems, 0.15%, 1.5%, and 15% ammonia aqueous solutions, as well as 1.5% NH3 in 30% ACN and in 30% MeOH solutions were used as desorbents for the peptide desorption reaction. In general, low concentration NH3(aq) would result in poor desorption efficacy. For chitosans with high and medium DDAs, 1.5% and 15% NH3(aq) resulted in similar desorption ratios. For chitosans with high and medium DDAs, the additions of both ACN and MeOH would increase the desorption ratio. However, for low DDA chitosan, no enhancement of desorption ratio was observed with MeOH addition, and lower desorption efficacy was found with ACN addition

目錄
第一章	緒論……………………………………………………………7
1-1	研究動機與目的……………………………………………………8
1-2	實驗背景資料………………………………………………………8
1-2.1蛋白質簡介……………………………………………………8
1-2.2磷酸化蛋白(phosphoprotein)介紹……………………………9
1-2.3 MALDI質譜儀偵測………………………………………….11
1-2.4 MALDI樣品選用基質……………………………………….14
1-2.5以幾丁質和幾丁聚醣進行蛋白質純化實驗………………...16
1-3	參考資料……………………………………………………………17
第二章	實驗方法……………………………………………………..19
2-1 實驗儀器…………………………………………………………...19
2-1.1 毛細管電泳儀 Beckmen P/ACE MDQ…………………….19
2-1.2 MALDI/TOF MASS………………………………………....19
2-1.3 HPLC 儀器 JASCO…………………………………………20
2-1.4 pH meter ……………………………………………………..20
2-1.5 去離子水處理器…………………………………………….20
2-1.6 真空濃縮機………………………………………………….21
2-1.7 離心機……………………………………………………….21
2-2 實驗藥品…………………………………………………………...21
	2-2.1 毛細管塗層藥品……………………………………………21
	2-2.2 配置毛細管電泳緩衝溶液藥品……………………………21
	2-2.3 電泳緩衝溶液添加劑………………………………………21
	2-2.4 蛋白質及聚胜肽樣品………………………………………22
	2-2.5 蛋白質水解所需試劑………………………………………22
	2-2.6 MALDI-TOF/MS樣品之基質及試劑………………………21
	2-2.7 MALDI-TOF/MS樣品添加劑………………………………22
2-3 毛細管內壁塗層方式…………………………………………26
	2-3.1 細管內壁Polyacryamide塗層步驟…………………………27
	2-3.2 未塗層毛細管活化步驟…………………………………….28
	2-3.3毛細管內壁Polybrene塗層步驟……………………………29
2-4 實驗方法與步驟…………………………………………………...29
	2-4.1實驗藥品配置………………………………………………..29
	2-4.2蛋白質水解步驟……………………………………………..30
	2-4.3 MALDI-TOF MASS樣品備製………………………………39
	2-4.4 chitosan固相微萃取實驗方法………………………………40
	2-4.5儀器操作條件以及chitosan製備..........................................41
2-6 參考資料…………………………………………………………...46


第三章	結果與討論
3-1實驗變因的控制組電泳圖譜……………………………..………..47
3-2α-casein(tryptic digests，in 10 mM ABC)經不同去乙醯化程度幾丁聚醣吸附後的毛細管電泳圖和MALDI-TOF質譜圖.………………52
3-3α-casein(tryptic digests，in 10 mM ABC)經不同去乙醯化程度幾丁聚醣脫附後的毛細管電泳圖和MALDI-TOF質譜圖………………..66
3-4不同脫附劑作用下，胜肽片段的毛細管電泳圖譜以及MALDI-TOF質譜圖比較……………………………………………………………..73
3-5不同時間吸附/脫附毛細管電泳圖譜以及MALDI-TOF質譜圖比較……………………………………………………............................100
3-6改變chitosan的使用量，並觀察其造成的差異性.........................119
3-7EB添加對胜肽的MALDI訊號影響增..........................................122
3-810mM ABC/50mM HAC與10mM ABC/10mM HAC切出來的αCN差異........................................................................................................126
3-9 參考資料………………………………………………………..127
第四章 結果與討論..............................................................................128

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.	薛仁瑋，「利用毛細管電泳及MALDI-TOF/MS技術分析蛋白質」，碩士論文，淡江大學化學系，2011。
3.	楊承熹，「利用毛細管電泳結合表面修飾劑分析不同方式合成的金奈米粒子」，博士論文，淡江大學化學系，2007。
4.	Sven, K.; Norregaard, J. O., “Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins”, Analytical chemistry, 2004, 76, 5109-5117.