氫氘交換（Hydrogen/Deuterium Exchange）是研究蛋白質結構的方法之一，其原理是利用蛋白質結構中某些位置的氫（hydrogen）與含有氘（deuterium）的溶液進行H/D交換，使得蛋白質分子上的氫被置換成氘，而這些發生氫氘交換的位置是在NH, NH2, OH, COOH, SH等官能基。而由於蛋白質結構的不同，因此在不同位置的交換速度也不同，當蛋白質分子其中一個氫交換成氘之後其分子量也將增加1 Da，因此近年來這項技術也逐漸應用質譜技術做為分析其交換程度的工具。與NMR分析H/D交換比較，以質譜進行H/D交換的分析具有分析物需要量較少，分析時間較短及分析物質量無上限等優點。
融合微滴電噴灑游離法（Fused Droplet/Electrospray Ionization, FD/ESI）是將傳統的電噴灑分爲兩個部分進行的游離方式，其與傳統電噴灑游離法(ESI)主要的不同之處在於融合微滴電噴灑游離法將分析物溶液與電噴灑溶液分別進行霧化與電噴灑，使之産生含分析物的微滴與帶電液滴，而兩種液滴融合後並且持續電噴灑的溶劑揮發與液滴爆裂的過程，最後産生帶有多價電荷的分析物離子。
本研究即是結合融合微滴電噴灑游離質譜法與H/D交換的技術，將含有氘的電噴灑溶液（D2O/CH3OD 50:50, 1% CH3COOD）以電噴灑的方式產生帶電荷的微小液滴，而分析物則是配置於水溶液中再以霧化器霧化成微小的液滴送至反應區，讓兩種液滴產生融合，並在融合之後繼續電噴灑的過程最後產生帶有多價電荷的分析物，因此分析物在液滴融合至產生多價電荷離子的游離過程中將進行H/D交換，由於融合至游離的過程所需要的時間相當短暫，推測是在毫秒的時間範圍內，因此可以觀察到分析物中最容易交換位置的H/D交換情形。
由實驗結果發現，胜肽鍵上的氫、胺基酸側鏈帶電荷或有氫鍵形成時，其交換速度較慢。此外，蛋白質分子則因其三度空間結構的影響，使不同構形的蛋白質在交換數與帶電荷數上產生差異，本篇報告將針對這些分析物在毫秒氫氘交換技術中氫氘交換的情形與影響其交換的因素進行探討。

none

目 錄

論文摘要………………………………………………………………….i
目錄…………………………………………………………………….iii
圖目錄…………………………………………………………………….v
壹、緒論………………………………………………………………….1
一、前言…………………………………………………………….1
二、融合微滴電噴灑游離質譜法發展與原理簡介……………….3
1.電噴灑游離質譜法的歷史………………………………….3
2.電噴灑游離質譜法的原理………………………………….4
3.融合微滴電噴灑游離質譜法的發展與原理……………….6
三、電噴灑游離質譜法結合氫氘交換在蛋白質結構的研究…….7
1.蛋白質的組成與結構……………………………………….7
2.影響蛋白質構形的因素…………………………………..13
3.電噴灑游離質譜法於蛋白質結構的研究………………..14
四、氫氘交換原理………………………………………………..20
五、論文目標……………………………………………………..25
貳、實驗………………………………………………………………..27
一、儀器裝置……………………………………………………..27
1.質譜儀……………………………………………………..27
2.超音波霧化器……………………………………………..28
3.微量注射幫浦……………………………………………..28
二、實驗試劑與配置方法………………………………………..29
三、實驗方法……………………………………………………..31
1.分析物直接配置在氘溶液中進行電噴灑………………..31
2.分析物配置在水溶液中進行毫秒氫氘交換……………..32
參、結果與討論…………………………………………………………33
一、毫秒氫氘交換於有機分子與胜肽分子之研究……………..33
1. reserpine進行氫氘交換實驗……………………………33
2. leucine-enkephalin進行氫氘交換實驗……………….36
3. bradykinin進行氫氘交換實驗………………………….39
4. angiotensin I進行氫氘交換實驗………………………41
二、毫秒氫氘交換於蛋白質分子之研究…………………………44
1.細胞色素c進行氫氘交換實驗…………………………….44
2.溶菌脢進行氫氘交換實驗………………………….…….48
肆、結論…………………………………………………………………56
伍、參考文獻……………………………………………………….….58

1. Thomson, J. J. 1913, Longmans, London. “Rays of Positive Electriity and Their Application to Chemical Analysis”
2. Nier, A. O. Rev. Sci. Instrum. 1947, 18, 415. “Electron Impact Mass Spectrometry”
3. Munson, M. S. B., Field, F. H. J. Am. Chem. Soc. 1960, 88, 2621. “Chemical Ionization Mass Spectrometry”
4. Beckey, H. D. 1977, Pergamon, Oxford. “Principles of Field Ionization and Field Desorption Mass Spectrometry”
5. Barber, M, Bordoli, R. S., Elliott, G. J., Sedgwick, R. D., Tyler, A. N. Anal. Chem. 1982, 54, 645A. “Fast Atom Bombardment Mass Spectrometry”
6. Benningghoven, A., Rudenauer, F. G., Werner, H. W. 1987, Wiley, New York. “Secondary Ion Mass Spectrometry-Basic Concepts Instrumental Aspects Applications and Trends”
7. Cotter, R. J. Anal. Chem. 1988, 60, 781A. “Plasma Desorption Mass Spectrometry : Coming of Age”
8. Unsold, E, Hillenkamp, F., Nitsche, R. Analysis, 1990, 4, 115. “Mass Group Spectrometry of Peptides and Proteins by Matrix Assisted Ultraviolet Laser Desorption/ Ionization in Methods in Enzymology”
9. Yamashita, M., Fenn, J. B. J. Phys. Chem. 1984, 88, 4451. “Electrospray Ion Source : Another Variation on The Free-Jet Theme”
10.Karas, M., Hillenkamp, F. Anal. Chem. 1988, 60, 2299. “Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons”
11.Zeleny, J. Phys. Rev. 1917, 10, 1. “Industry Application of Electrospray Technology”
12.Dole, M., Mack, L. L., Hines, R. L. J. Chem. Phys. 1968, 49, 2240. “Molecular Beams of Macroions”
13.Mack, L. L., Kralic, P., Rheude, A., Dole, M. J. Chem. Phys. 1970, 52, 4977. “Molecular Beams of Macroions part II”
14.Aleksandrov, M. L., Gall, L. N., Shkurov, V. A., Pavlenko, V. A., Krasnov, N. V., Nikolaev, V. I. J. Anal. Chem. USSR 1984, 39, 1268. “Mechanism of Ion Formation During the Electrohydrodynamic Sputtering of a Liquid into a Vacuum”
15.Olivares, J. A., Nguyen, N. T., Yonker, C. R., Smith, R. D. Anal. Chem. 1987, 59, 1232. “On-Line Mass Spectrometry Detection for Capillary Zone Electrophoresis”
16.Feng, R., Konishi, Y., Bell, A. W. J. Am. Soc. Mass Spectrom. 1991, 2, 297. “High Accuracy Molecular-Weight Determination and Variation Characterization of Protein Up to 80-ku by Ionspray Mass Spectrometry”
17.Loo, R. R. O., Udseth, H. R., Smith, R. D. J. Phys. Chem. 1991, 95, 6412. “ Evidence of Charge Inversion in the Reaction of Singly Charged Anions with Multiple Charged Macroions”
18.Wang, C. S., Shiea, J. J. Mass Spectrom. 1997, 32, 247. “Application of Multiple-Channel Electrospray Ionization Sources for Biological Sample Analysis”
19.Lee, C. Y., Shiea, J. Anal. Chem. 1998, 70, 1757. “Gas Chromatography Connected to Multiple-Channel Electrospray Ionization Mass Spectrometry for the Detection of Volatile Organic Compounds”
20.Hong, C. M., Tsai, F. C., Shiea, J. Anal. Chem. 2000, 72, 1175. “A Multiple-Channel Electrospray Source Used to Detect Highly Reactive Ketenes from a Flow Pyrolyzer”
21.Shiea, J., Chang, D. Y., Lin, C. H., Jiang, S. J. Anal. Chem. 2001, 73, 4983. “Generating Multiply Charged Protein Ions by Ultrasonic Nebulization/Multiple Channel-Electrospray Ionization Mass Spectrometry”
22.Stryer, L. 4th ed., W. H. Freeman and Company, 1995. “Biochemistry”
23.Creighton, T. E. 2nd ed., W. H. Freeman and Company, 1993. “Proteins Structures and Molecular Properties”
24.Garrett, R. H., Grisham, C. M. 2nd ed., Saunders College Publishing, 1995. “Biochemistry”
25.Loo, J. A., Edmonds, C. G., Smith, R. D. Anal. Chem. 1991, 63, 2488. “Collisional Activation and Dissociation of Large Multiply Charged Proteins Produced by Electrospray Ionization”
26.Wanger, G., Wüthrich, K. J. Mol. Biol. 1982, 160, 343. “Amide proton exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution: studies with two-dimensional nuclear magnetic resonance”
27.Meng, C. K., Fenn, J. B. Organic Mass Spectrum., 1991, 26, 542. “Formation of Charged Cluster During Electrospray Ionization of Organic Solute Species”
28.Loo, J. A., Loo, R. R. O., Light, K. L., Edmonds, C. G., Smith, R. D. Anal. Chem. 1992, 64, 81. “Multiply Charged Negative-Ions by Electrospray Ionization of Polypeptides and Protein”
29.Mann, M., Meng, C. K., Fenn, J. B. Anal. Chem. 1989, 61, 1702. “Interpreting Mass Spectra of Multiply Charged Ions”
30.Loo, J. A., Edmonds, C. G., Udseth, H. R., Smith, R. D. Anal. Chem. 1990, 62, 693. “Effect of Reducing Disulfide-Containing Proteins on Electrospray Ionization Mass Spectra”
31.Helden, G. V., Wyttenbach, T., Bowers, M. T. Science 1995, 267, 1483. “Conformation of Macromolecules in the Gas-Phase - Use of Matrix-Assisted Laser-Desorption Methods in Ion Chromatography”
32.Helden, G. V., Wyttenbach, T., Bowers, M. T. Int. J. Mass Spectrom. Ion Processes 1995, 146/147, 349. “Inclusion of a Maldi Ion-Source in the Ion Chromatography Technique - Conformational Information on Polymer and Biomolecular Ions”
33.Wyttenbach, T., Helden, G. V., Bowers, M. T. J. Am. Chem. Soc. 1996, 118, 8355. “Gas-Phase Conformation of Biological Molecules – Bradykinin”
34.Clemmer, D. E., Hudgins, R. R., Jarrold, M. F. J. Am. Chem. Soc. 1995, 117, 10141. “Naked Protein Conformations - Cytochrome-C in the Gas-Phase”
35.Shelimov, K. B., Jarrold, M. F. J. Am. Chem. Soc. 1996, 118, 10313. “Denaturation and Refolding of Cytochrome-C in-Vacuo”
36. Shelimov, K. B., Clemmer, D. E., Hudgins, R. R., Jarrold, M. F. J. Am. Chem. Soc. 1997, 119, 2240. “Protein Structure in Vacuo: Gas-Phase Conformations of BPTI and Cytochrome c”
37.Miranker, A., Robinson, C. V., Radford, S. E., Aplin, R. T., Dobson, C. M. Science 1993, 262, 896. “Detection of Transient Protein Folding Populations by Mass Spectrometry”
38.Zhang, Z., Post, C. B., Smith, D. L. Biochemistry 1996, 35, 779. “Amide Hydrogen Exchange Determined by Mass Spectrometry: Application to Rabbit Muscle Aldolase”
39.Yang, H., Smith, D. L. Biochemistry 1997, 36, 14992. “Kinetics of Cytochrome c Folding Examined by Hydrogen Exchange and Mass Spectrometry”
40.Deng, Y., Smith, D. L. Biochemistry 1998, 37, 6256. “Identification of Unfolding Domains in Large Proteins by Their Unfolding Rates”
41.Engen, J. R., Gmeiner, W. H., Smithgall, T. E., Smith, D. L. Biochemistry 1999, 38, 8926. “Hydrogen Exchange Shows Peptide Binding Stablizes Motions in Hck SH2”
42.Dharmasiri, K., Smith, D. L. Anal. Chem. 1996, 68, 2340. “Mass Spectrometric Determination of Isotopic Exchange Rates of Amide Hydrogens Located on the Surfaces of Proteins”
43.Deng, Y., Smith, D. L. J. Mol. Biol. 1999, 294, 247 “Hydrogen Exchange Demonstrates Three Domains in Aldolase Unfold Sequentially”
44.Suckau, D., Shi, Y., Beu, S. C., Senko, M. W., Quinn, J. P., Wampler III, F. M., McLafferty, F. W. Proc. Natl. Acad. Sci. USA 1993, 90, 790. “Coexisting Stable Conformations of Gaseous Protein Ions”
45.Wood, T. D., Chorush, R. A., Wampler III. F. M., Little, D. P., O’Connor, P. B., McLafferty, F. W. Proc. Natl. Acad. Sci. USA 1995, 92, 2451. “Gas-Phase Folding and Unfolding of Cytochrome cCations”
46.Freitas, M. A., Hendrickson, C. L., Emmett, M. R., Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 1012. “High-Field Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Simultaneous Trapping and Gas-Phase Hydrogen/ Deuterium Exchange of Peptide Ions”
47.Cambell, S., Rodgers, M. T., Marzluff, E. M., Beauchamp, J. L., J. Am. Chem. Soc. 1995, 117, 12840. “Deuterium-Exchange Reactions as a Probe of Biomolecule Structure- Fundamental-Studies of Gas Phase H/D Exchange- Reactions of Protonated Glycine Oligomers with D2O, CD3OD, CD3CO2D, and ND3”