Responsive image
博碩士論文 etd-0707106-160558 詳細資訊
Title page for etd-0707106-160558
論文名稱
Title
開發微液相層析儀、離子交換樹脂、毛細管電泳及等電點聚焦電泳結合電噴灑輔助雷射脫附游離質譜法的離線偵測技術
none
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
83
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2006-06-17
繳交日期
Date of Submission
2006-07-07
關鍵字
Keywords
液相層析儀、毛細管電泳、離子交換、等電點聚焦電泳、電噴灑輔助雷射脫附游離
ELDI, μ-HPLC, isoelectric focusing, capillary electrophoresis, ion exchange
統計
Statistics
本論文已被瀏覽 5696 次,被下載 31
The thesis/dissertation has been browsed 5696 times, has been downloaded 31 times.
中文摘要
蛋白質體學是現今重要科學研究之一,科學家經常思考如何從一個複雜生物樣品,偵測一特定蛋白質的訊號,選擇一個合適的分離技術是最重要的關鍵,而其中質譜儀在蛋白質體學中也是扮演重要的分析角色,分離技術若能有效結合於質譜儀偵測,對於蛋白質的快速分析與鑑定上會有很大的優勢。
研究第一部份將探討毛細管電泳結合電噴灑質譜儀對胜肽分離的探討,以cytochrome c消化後的胜肽混合物為樣品,進行毛細管區帶電泳(CZE)模式分離直接串聯質譜進行偵測,研究中分別探討緩衝溶液的pH值變化,以及於分離管柱中加入界面活性劑與緩衝溶液中加入有機溶劑,對胜肽混合物分離的探討。實驗結果發現由於胜肽之間的疏水性及帶電性相近而造成分離的效果有限,且這些修飾劑只對含有heme的特定胜肽有選擇性的改變。
實驗第二部份一般離子交換樹脂萃取管柱與等電點聚焦電泳裝置都是在高濃度鹽類的緩衝溶液系統中進行分析,這些高濃度的鹽類在經過質譜儀的界面時容易產生沉澱現象、電噴灑現象不穩定、分析物訊號減弱及分析物分子形成加成離子造成質譜圖判讀困難。因此無法以質譜對這些化合物進行線上偵測,而須在質譜偵測前先進行去除鹽類等干擾物步驟,這些處理過程會消耗多量樣品或成本。另一部份,若在毛細管柱中若加入奈米粒子或高分子溶劑當偽固定相時,也不適用於線上連接質譜方式偵測,因為奈米粒子與高分子溶劑對質譜儀而言都是污染物。
本實驗的研究目的是利用電噴灑輔助雷射脫附游離法(Electrospray Assisted Laser Desorption Ionization, ELDI),以簡單且快速的方式,去除分析樣品中含有鹽類及固體干擾物。ELDI/MS是本實驗室新開發的游離法,透過雷射剝蝕在樣品產生氣態分子、離子混合物(plume),再以電噴灑之帶電液滴與分析物融合產生多價電荷離子進入質譜儀分析,過程中因為鹽類干擾物與有機溶劑之間有極性差異,所以鹽類無法融入電噴灑溶液中,形成帶電的氣相離子,故在實驗中便利用此一重要的特性去除鹽類存在分析物所產生的干擾。
實驗一開始先將離子交換樹脂萃取槽所沖提下來的分析物與等電點電泳所收集分離液,沉積在樣品盤上進行偵測,可發現鹽類的存在並未干擾分析物的訊號。在毛細管電泳與液相層析儀部份,都可成功以離線方式結合ELDI/MS偵測到分析物訊號,而對於填充金奈米粒子的毛細管,仍可得到蛋白質多價電荷訊號。由實驗結果可證明ELDI/MS比傳統ESI/MS高的干擾物容忍極限,可成功簡化樣品的前處理過程。
Abstract
none
目次 Table of Contents
摘要..........................................................................................................Ι
目錄..........................................................................................................Ⅲ
圖目錄......................................................................................................Ⅵ
表目錄......................................................................................................Ⅸ
壹、 緒論..........................................................................................1
一、 前言.............................................................................................1
二、 儀器之發展與原理介紹.............................................................4
1. 毛細管電泳分離結合電噴灑質譜法(CE/ESI/MS)......................4
(1) 電泳發展歷史.........................................................................4
(2) 電滲流.....................................................................................5
(3) 電泳流.....................................................................................6
(4) CE/ESI界面............................................................................6
(5) CZE/ESI/MS..........................................................................10
(6) MEKC/ESI/MS......................................................................10
2. 電噴灑輔助雷射脫附游離質譜法( electrospray-assisted laser desorption ionization, ELDI-MS).................................................12
(1) 雷射脫附法(laser desorption, LD) ......................................13
(2) 電噴灑游離法(electrospray ionization, ESI) .......................14
(3) 融合微滴電噴灑游離法(fused-droplet electrospray ionization, FD-ESI) ...........................................................15
(4) 電噴灑輔助雷射脫附游離法..............................................16
三、 論文目標..................................................................................18
貳、 實驗
第一部份:.............................................................................................20
線上(on-line)毛細管電泳分離法結合質譜偵測(CE/MS)
一、 儀器裝置..................................................................................20
二、 藥品與試劑..............................................................................21
三、 溶液配製...................................................................................22
四、 實驗步驟...................................................................................22
第二部分:................................................................................................24
開發以微液相層析儀離子交換樹脂、毛細管電泳及等電點聚焦裝置結合電噴灑輔助雷射脫附游離質譜法的離線偵測技術
一、 儀器裝置...................................................................................24
二、 藥品與試劑...............................................................................27
三、 溶液配製...................................................................................27
四、 實驗流程...................................................................................30
參、 結果與討論...................................................................................34
第一部份:.............................................................................................34
線上(on-line)毛細管電泳分離法結合質譜偵測(CE/MS)
第二部分:.............................................................................................46
開發以微液相層析儀離子交換樹脂、毛細管電泳及等電點聚焦裝置結合電噴灑輔助雷射脫附游離質譜法的離線偵測技術
肆、 結論..............................................................................................61
伍、 參考資料......................................................................................64
參考文獻 References
1. Quardroni, M.; James, P. “Proteomics and automation” ELECTORPPHORESIS. 1999, 20, 664-677.
2. Hochstrasser, D. F. “Proteome in perspective” Clin. Chem. Lab. Med.. 1998, 36, 825
3. Aebersold, R.; Goodlett, D. R. “Mass spectrometry in peoteomics” Chem. Rev. 2001, 101, 269-296.
4. Anderson, N. L.; Anderson, N. G.. “The Human Plasma Proteome: History, Character, and Diagnostic Prospects” Mol. Cell Proteomics. 2002, 1, 845-867.
5. Haleem, J. I. “The role of separation science in proteomics research” ELECTROPPHORESI. 2001, 22, 3629-3638.
6. Wilson, K.; Walker, J. Principles and Techniques of practial Biochemistry, 5th edition
7. Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R. “Direct analysis of protein complexes using mass spectrometry” Nature Biotechnolog. 1999, 17,676-682.
8.Winnik, W. M. “Continuous pH/Salt Gradient and Peptide Score for Strong Cation Exchange Chromatography in 2D-Nano-LC/MS/MS Peptide Identification for Proteomics” Anal. Chem. 2005, 77, 4991-4998.
9. Anderson, N. L.; Polanski, M.; Pieper, R.; Gatlin, T.; Tirumalai, R. S.; Conrads, T. P.; Veenstra, T. D.; Adkins, J. M.; Pounds, J. G.; Fagan, R.; Lobley A. “The Human Plasma Proteome: A Nonredundant List Developed by Combination of Four Separate Sources” Mol. Cell Proteomics. 2004, 3, 311-326.
10. Shen, Y.; Jacobs, J. M.; Camp, D. G., II; Fang, R.; Moore, R. J.; Smith, R. D.; Xiao, W.; Davis, R. W.; Tompkins, R. G. “Ultra-High-Efficiency Strong Cation Exchange LC/RPLC/MS/MS for High Dynamic Range Characterization of the Human Plasma Proteome” Anal. Chem. 2004, 76, 1134-1144.
11. Sanz-Nebot, V.; Balaguer, E.; Benavente, F.; Barbosa, J. “Comparison of sheathless and sheath-flow electrospray interfaces for the capillary electrophoresis-electrospray ionization-mass spectrometry analysis of peptides” ELECTROPPHORESIS 2005, 26, 1457-1465.
12. Stutz, H. “Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix-assisted laser desorption/ionization and electrospray-mass spectrometry detection” ELECTRPPHORESIS 2005, 26, 1254-1290.
13. Moini, M. “Capillary electrophoresis mass spectrometry and its applicaction to the analysis of biological mixtures” Anal. Bioanal. Chem. 2002, 373, 466-480.
14. Jorgenson, J. W.; Lukacs, K. D.“Zone electrophoresis in open-tubular glass capillaries” Anal. Chem. 1981, 23, 1298-1302.
15. Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. “On-line mass spectrometric detection for capillary zone electrophoresis” Anal. Chem. 1987, 59, 1230-1232.
16. Foret, F.; krivankova , L.; Bocek, P. “Capillary Zone Electrophoresis” VCH, Weinheim, Germany, 1993
17. Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. “Capillary zone electrophoresis-mass spectrometry using an electrospray ionization interface”, Anal. Chem. 1988, 60, 436-441.
18. Cai, J.; Henion, J. “Capillary electrophoresis-mass spectrometry”, J. Chromatogr. A. 1994, 703, 667-692.
19. Fang, L.; Zhang, R. “Online Time-of-Flight Mass Spectrometric Analysis of Peptides Separated by Capillary Electrophoresis”, Anal. Chem. 1994, 66, 3696-3701.
20. Smith, A. D.; Moini, M. “Control of Electrochemical Reactions at the Capillary Electrophoresis Outlet/Electrospray Emitter Electrode under CE/ESI-MS through the Application of Redox”, Anal. Chem. 2001, 73, 240-246.
21. Muijselaar, P. G.; Otsuka, K.; Terabe, S. “On-line coupling of partial-filling micellar electrokinetic chromatography with mass spctrometry”, J. Chromatogr. A. 1998, 802, 3-15.
22..Orth, R. G.; Harry, T.; Jonkman,M. J. “Secondary ion mass spectrometry of molecular solids: a source of cluster ions” , J. Am. Chem. Soc.1981, 103,1564-1565
23. Hillenkamp, F.; Karas, M. “Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons” , Anal. Chem. 1988, 60,2299-2301
24. Takáts, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. “Mass Spectrometry Sampling Under Ambient Conditions with Desorption Electrospray Ionization” ,Science. 2004, 306,471-473.
25. Cody, R. B.; Laramee, J. A.; Durst, H. D. “Versatile New Ion Source for the Analysis of Materials in Open Air under Ambient Conditions” , Anal. Chem. 2005, 77,2297-2302
26. Benazouz, M.; Benazouz, J. L.; Benazouz1, D.; Weber, G. “Study of the Mechanism of Direct Laser Desorption/Ionisation for Some Small Organic Molecules (M < 400 Daltons)”, Rapid Commun. Mass Spectrom. 1999, 13, 2302-2304.
27. Tabet, J. C.; Cotter, R.J. “Laser Desorption Time-of-Flight Mass Spectrometry of High Mass Molecules”, Anal. Chem. 1994,56, 1662-1667.
28. Coon, J. J.; Harrison, W. W. “Laser Desorption-Atmospheric Pressure Chemical Ionization Mass Spectrometry for the Analysis of Peptides from Aqueous Solutions ”, Anal. Chem. 2002,74, 5600-5605.
29. Coon, J. J.; McHale, K. J.; Harrison, W. W. “ Atmospheric pressure laser desorption /chemical ionization mass spectrometry: a new ionization method base on existing themes” , Rapid Commun. Mass Spectrom. 2002, 16,681-685.
30. Wong, S. F.; Meng, C. K.; Fenn, J. B.; Mann, M.; Whitehouse, C. M. “Electrospray ionization for mass spectrometry of large biomolecules” ,Science. 1989, 246,64-71.
31. Chang, C. Y.; Lee, C. Y.; Shiea, J. “Detecting Large Biomolecules from High-Salt Solutions by Fused-Droplet Electrospray Ionization Mass Spectrometry” , Anal. Chem. 2002, 74,2465-2469
32. Shieh, I. F.; Lee, C. Y.; Shiea, J. “Eliminating the Interferences from TRIS Buffer and SDS in Protein Analysis by Fused-Droplet Electrospray Ionization Mass Spectrometry ” , J. Proteome Res. 2005, 40, 606-612
33. Shiea, J.; Huang, M. Z.; Hsu, H. J. Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. “Electrospray-assisted laser desorption/ionization mass spectrometry for direct ambient analysis of solids” , Rapid Commun. Mass Spectrom. 2005, 19,3701-3704
34. Huang, M. Z.; Hsu, H. J.; Lee, J. Y.; Jeng, J.; Shiea, J. “Direct Protein Detection from Biological Media through Electrospray-Assisted Laser Desorption Ionization/Mass Spectrometry” , J. Proteome Res. 2006, 5, 1107-1116
35. Hutchens, T. W.; Yip, T. T. “New desorption strategeies for the mass spectrometric analysis of macromolecules” , Rapid Commun. Mass Spectrom. 1993, 7,576-580
36. Thorslund, S.; Lindberg, P.; Andren, P. E.; Nikolajeff, F.; Bergquist, F. “Electrokinetic-driven microfluidic system in poly(dimethylsiloxane) for mass spectrometry detection integrating sample injection, capillary electrophoresis, and electrospray emitter on-chip ” ELECTRPPHORESIS 2005, 26, 4674-4683
電子全文 Fulltext
本電子全文僅授權使用者為學術研究之目的,進行個人非營利性質之檢索、閱讀、列印。請遵守中華民國著作權法之相關規定,切勿任意重製、散佈、改作、轉貼、播送,以免觸法。
論文使用權限 Thesis access permission:校內公開,校外永不公開 restricted
開放時間 Available:
校內 Campus: 已公開 available
校外 Off-campus:永不公開 not available

您的 IP(校外) 位址是 44.199.225.221
論文開放下載的時間是 校外不公開

Your IP address is 44.199.225.221
This thesis will be available to you on Indicate off-campus access is not available.

紙本論文 Printed copies
紙本論文的公開資訊在102學年度以後相對較為完整。如果需要查詢101學年度以前的紙本論文公開資訊,請聯繫圖資處紙本論文服務櫃台。如有不便之處敬請見諒。
開放時間 available 已公開 available

QR Code