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研究生:李威漢
研究生(外文):Wei-Han Lee
論文名稱:線上一次及多次交錯沖堤固相萃取毛細電泳-質譜與二維心切毛細電泳-質譜開發
論文名稱(外文):The Development of On-line Single / Staggered Multi-Step Elution SPE-CE-MS and Heart-cut 2D–CE-MS
指導教授:何國榮何國榮引用關係
指導教授(外文):Guor-Rong Her
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:197
中文關鍵詞:毛細電泳-質譜線上前濃縮固相萃取蛋白質序列涵蓋率毛細電泳二維分離心切式二維分離雙通道毛細電泳質譜
外文關鍵詞:CE-MSon-line preconcentrationsolid phase extractionsequence coverage2D-CEheart-cut 2D-CEdual-channel CE-MS
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本研究進行固相萃取-毛細電泳質譜前濃縮介面之開發,利用PDMS的絕緣材質銜接SPE與CE管柱製作出SPE-CE-MS的線上銜接介面。 在這個介面的設計中,上下層的設計及正交的排列方向能避免有機相導入電泳通道而影響分離。以雙交錯進樣模式在SPE與電泳通道間提供有效的進樣電場,控制SPE的沖堤流速能決定樣品進樣的寬度(時間)。 胜肽標準品 (angiotensin II,Leu-enkephalin, 及 Met-enkephalin)在介面的測試中,可達到基線分離同時並保有毛細電泳的分離效率(~70-90k plates/m)。 使用固相萃取進行分析樣品的前濃縮,可偵測到低濃度(~86 nM)的色素蛋白C (Cytochrome C)蛋白質水解片段,並具有72%的序列涵蓋率。

分析複雜蛋白質水解胜肽時,為了增加毛細電泳分離複雜樣品的能力,我們提出了多次階段沖堤固相萃取毛細電泳的二維分離策略。 此策略是以多次沖堤固相萃取作為第一個維度的分離,而毛細電泳作為第二個維度分離。使用多次階段沖堤SPE-CE-MS/MS進行蛋白質水解胜肽混合物的分析,可比對到更多的胜肽片段(數量上超過一次沖堤的60%),提升了蛋白質分析的序列涵蓋率及鑑定比對分數,增加蛋白質鑑定的可信度。 為了能縮短二維分離的時間,我們進一步提出交錯近樣電泳(staggered CE)的方法,能有效消除分析的空載時間(dead time),節省超過一半的分析時間。

為了能避免耗時的廣泛性二維分析,我們提出心切式的二維電泳分離技術。利用心切介面使第一個分離維度中難以分離的混合物在第二個維度進行分離,並由雙通道電泳質譜達到兩種分離通道的同步分析。 本介面使用區帶電泳 (CZE)及微胞電動毛細層析電泳(MEKC)作為二維毛細電泳的兩種分離系統並以磺胺類的混合物進行心切式二維毛細電泳效能的評估。初步實驗的結果中四個被心切送入MEKC通道進行分離的磺胺類樣品(SDZ、SMR、STZ、SMM)具有相似單一MEKC的順序,初步證實了此系統在心切式二維分離系統上分析的可行性。


A PDMS based two-leveled two cross design interface was proposed for on-line coupling SPE-CE-MS. In this interface, the SPE column and the CE separation column were positioned orthogonally and two crosses were fabricated on the interface. With the two cross design, the operation of SPE could be performed independently without unexpected flow through leakage into the separation column. The performance of the interface was optimized using a peptide mixture. The position of the SPE column related to the CE separation channel was found to be critical to the performance of the system. Under the optimal position, the separation efficiency was similar to a CE-MS experiment without SPE. The peptide signals were enhanced 50 to 100-fold and the repeatability was within 4% RSD for migration time and 10% RSD for peak area. A tryptic digest of cytochrome C was used to demonstrate the feasibility of the interface in protein identification at a level of 1 ng/mL.


In a protein mixture analysis, the identification of proteins usually suffers in low sequence coverage in the single run CZE-ESI-MS/MS. An original concept of on-line coupling multistep elution solid phase extraction (SPE) to CZE-MS/MS was proposed to increase sequence coverage of protein mixture analysis. The multistep elution SPE (the first dimension) provides an additional dimension of separation prior to CZE (the second dimension) and extends the separation capacity for protein mixture analysis. Furthermore, a staggered CZE method was described to increase the throughput of each CZE runs in the second dimension separation and thus to reduce entire analysis time. In this study for protein mixture standards, more than 60% of additional peptides were discovered , and more than 50% was improved in sequence coverage by using multistep elution SPE-CE-MS/MS. By using staggered CZE method, half of the entire analysis time could be saved (54%) in comparison with the sequential CZE method used in multistep elution SPE-CE-MS/MS and thus avoiding the time-consuming analytical procedure in comprehensive 2D separation.
An interface for heart-cut 2D CE-MS was proposed to increase separation selectivity in mixture analysis. Several concepts were adapted to overcome the limitations of heart-cut 2D-CE designed in the present studies. First, the manipulation of chip-based interface provides an isolated buffer system to connect two sets of capillary electrophoresis. Second, the parallel separation of the two dimensional capillary electrophoresis was detected simultaneously by a pulsed electrospray-based duel-channel CE-MS system. In this study, the system was demonstrated by using capillary zone electrophoresis- micellar electrokinetic chromatography (CZE-MEKC) system to analyze sulfonamide mixtures. Under the consideration of correspondence in EOF for fused silica capillary the PDMS based chip channel, 8 sulfonamide standards can be transferred successfully without loss and peak broadening during the heart-cutting operation. The preliminary feasibility of heart-cut CZE-MEKC with dual-channel CE-MS was studied in sulfonamides analysis. Four sulfonamides(SDZ、SMR、STZ、SMM) were transferred into the MEKC channel by the heart-cut interface after separation in the first dimension of CZE. The migration order of four heart-cut sulfonamides was found similar order in the single-run MEKC.


目錄
中文摘要 I
英文摘要 IV
圖目錄 X
表目錄 XIV
第一章 序論
1-1 前言 1
1-2 毛細管電泳 3
1-2-1 電泳的基本原理 6
1-2-2 電滲透流 7
1-2-3 毛細管區帶電泳 8
1-2-4 微胞電動毛細層析 9
1-2-5 毛細電泳-質譜 (CE-MS) 10
1-3 電灑游離法 11
1-3-1 電灑游離法之發展歷史 12
1-3-2 電灑法原理 13
1-4 離子阱質量分析儀 19
1-4-1 三維離子阱 19
1-4-1-1 三維離子阱之構造 19
1-4-1-2 離子阱之分析電壓 21
1-4-1-3 離子阱的分析步驟 24
1-4-2 二維離子阱(線性離子阱) 26
1-5 參考文獻 28
第二章 線上固相萃取-毛細電泳質譜介面之開發及毛細電泳質譜線上濃縮技術之應用
2-1 前言 42
2-2 藥品與實驗方法 47
2-2-1 藥品,材料 47
2-2-2 SPE管柱之製作 47
2-2-3 蛋白質水解消化 49
2-3 結果與討論 50
2-3-1 SPE-CE-MS介面設計之概念 50
2-3-1-1 使用PDMS作為介面銜接的基材 50
2-3-1-2 介面通道的製作與裝置之架設 52
2-3-2 SPE與CE通道獨立流動之設計 52
2-3-2-1 通道獨立流動設計之考量 52
2-3-2-2 介面上下通道滲漏(leakage)之評估 53
2-3-3 樣品進樣 55
2-3-3-1 SPE-CE單交錯(single-cross)及雙交錯(double-cross)進樣 55
2-3-3-2 SPE管柱位置對於進樣之影響 57
2-3-3-3 進樣寬度之操作討論 58
2-3-4 SPE-CE-MS效能之評估 60
2-3-4-1 SPE-CE-MS 分離效率之評估 60
2-3-4-2 胜肽樣品濃縮倍數之評估 61
2-3-5 SPE-CE-MS對於低濃度蛋白質水解胜肽之分析 63
2-4 結論 65
2-5 參考文獻 66
第三章 交錯式多次階段沖堤固相萃取毛細電泳質譜/質譜於二維分離技術之開發及蛋白質分析序列涵蓋率提升之應用
3-1 前言 95
3-2 藥品與實驗方法 103
3-2-1 藥品,材料 103
3-2-2 SPE管柱之製作 103
3-2-3 蛋白質水解消化 105
3-3 結果與討論 106
3-3-1 SPE管柱多次階段沖堤的分離可行性之評估 106
3-3-2一次沖堤SPE-CE-MS/MS進行蛋白質分析 107
3-3-3 多次階段沖堤SPE-CE-MS/MS 進行蛋白質分析 109
3-3-4交錯式多次階段沖堤SPE-CE-MS/MS進行蛋白質分析 113
3-4 結論 117
3-5 參考文獻 118
第四章 雙通道毛細電泳質譜二維分離心切技術之介面開發
4-1 前言 138
4-2 藥品與實驗方法 141
4-2-1 藥品,材料 141
4-2-2 儀器設備 142
4-3 結果與討論 143
4-3-1 雙通道毛細電泳質譜 143
4-3-1-1 雙通道電灑操作與質譜訊號的同步 144
4-3-1-2 PDMS 晶片式雙通道噴頭基座 148
4-3-2 晶片式心切毛細電泳介面 150
4-3-2-1 介面設計之構想與操作 150
4-3-2-2 樣品傳送效率之評估 152
4-3-2-3 樣品心切傳送測試 155
4-3-2-4 二維電泳緩衝溶液之探討 156
4-3-3 心切式二維毛細電泳於磺胺類藥物之分析 157
4-3-3-1 磺胺類混合物於CZE與MEKC上的分離 158
4-3-3-2 心切式二維毛細電泳進行磺胺類藥物的分離 158
4-4 結論 160
4-5 參考文獻 161
總結 195


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