臺灣博碩士論文加值系統

質譜儀具有良好的靈敏度和快速的分析的優點，利用串聯質譜儀更可以分析磷脂的結構，但是不同的離子化法所得到的結果會有所差異。我們利用電灑離子阱質譜儀分析磷脂時，負離子模式[M–H]－碰撞活化解離所得到的離子並不是很穩定，在正離子模式下， [M+H]+ 碰撞活化解離得到脫sn-1和sn-2脂肪酸比例，相較於電灑四極棒質譜儀的結果，極不穩定，為了解決這個問題，我們發現利用磷脂醯膽鹼(phosphatidylcholine)加入0.1 %三氟醋酸會有[ M+K+TFA ]+的訊號，經碰撞活化解離後除了脫去polar head的訊號外，更可以得到穩定的脫sn-1和sn-2脂肪酸的離子訊號，而且從sn-1位置脫脂肪酸大於從sn-2位置脫脂肪酸。另外我們也發現鈷 (Co+2) 金屬離子對磷脂醯乙醇胺(phosphatidylethanolamine) 和磷脂醯甘油 (phosphatidylglycerol) 產生的[M–H+Co]+ 和磷脂醯絲胺酸 (phosphatidylserine) 產生的[M–2H+Na+Co]+碰撞活化解離除了脫去polar head的訊號外，也可以得到穩定的脫sn-1和sn-2脂肪酸離子訊號，對於磷脂醯乙醇胺和磷脂醯絲胺酸從sn-1脫脂肪酸大於從sn-2脫脂肪酸，而磷脂醯甘油則是從sn-2脫脂肪酸大於從sn-1脫脂肪酸，因此我們已成功找出解出上述四種磷脂結構的方法。

When collisionally activiated dissociation (CAD) of glycerophospholipid species is examined using quadrupole ion trap mass spectrometry (QITMS) , the spectral patterns differ from those obtained using sector or quadrupole mass spectrometry. Methods employed in the structure analysis of glycerophospholipids using a sector or quadrupole mass spectrometer are not necessarily useful for an ion trap mass spectrometer. The goal of this work is to enhance the fragmentation of lipids such that the structural determinations of those lipids are feasible. Several classes of glycerophospholipids. A novel method is presented for structurally analyzing glycerophosphocholines (GPC) that involves the CAD of trifluoroacetic acid (TFA) adducts of kaliated GPCs. Solutions of GPC in 0.1% TFA/methanol were electrosprayed to produce precursor ions by attaching a trifluoroacetic acid (TFA) molecule to a kiliated GPC molecule. The CAD-MS/MS spectra obtained by QITMS revealed a dramatic increase in the abundance of fragment ions, corresponding to the loss of sn-1 and sn-2 fatty acyl substituents. A preferential loss of the sn-1 fatty acyl group over the loss of the sn-2 fatty acyl group was observed among the GPC standards examined. A GPC extract from egg yolk was directly analyzed by this method without prior separation. The identities and positions of fatty acyl substituents of over 20 GPC species were identified. The effect of metal cationization on CAD of several glycerophospholipid classes including glycerophosphoethanolamines, glycerophosphoglycerol and glycerophosphoserines was investigated by electrospray ionization tandem mass spectrometry. The metal ions include Li+, Na+, K+, and several first transition metal series. The enhanced fragments of lipid complexes with Ni2+ and Co2+ give valuable structural information regarding the structure of phospholipids.

目錄
圖目錄…………………………………………………………………3
表目錄…………………………………………………………………5
摘要……………………………………………………………………6
Abstract……………………………………………………………7

壹、 緒論
1-1. 質譜儀的發展………………………………………………8
1-2. 質譜儀游離法的發展………………………………………11
1-3. 游離法的發展………………………………………………13
1-4. 電噴灑電噴灑游離法中離子形成機制與原理……………15
1-4-1.帶電液滴的生成……………………………………………15
1-4-2.帶電液滴上溶劑的揮發與液滴再爆裂……………………16
1-4-3.氣相多價電荷離子的生成…………………………………17
1-5. 磷脂的結構與命名…………………………………………20
1-5-1. 磷脂的命名……………………………………………………20
1-5-2. 脂肪酸的命名…………………………………………………21
1-5-3. 磷脂的極性官能基 (polar head) …………………………23
1-6. 磷脂的分析…………………………………………………24
1-7. 研究目標……………………………………………………29
貳、 實驗部份
2-1. 藥品…………………………………………………………30
2-2. 分析溶液的配製……………………………………………31
2-3. 質譜儀的條件………………………………………………32

參、 結果與討論
3-1. 磷脂醯膽鹼分析……………………………………………33
3-2. 磷脂醯乙醇胺分析…………………………………………50
3-3. 磷脂醯甘油的分析…………………………………………59
3-4. 磷脂醯絲胺酸的分析………………………………………75

肆、 結論…………………………………………………………84

伍、 參考文獻……………………………………………………86










圖目錄
圖1. 泰勒錐示意圖……………………………………………………16
圖2. Gomez 和Tang 觀察的電灑液滴分裂示意圖…………………18
圖3. 磷脂的簡單結構式………………………………………………20
圖4. C16:0 /C18:1-GPC的正離子模式質譜圖………………………..34
圖5. C16:0 /C18:1-GPC 的[M+H]＋碰撞活化解離的譜圖…………35
圖6. C16:0 /C18:1-GPC的[M+Na]＋碰撞活化解離的譜圖…………37
圖7. 16:0 /18:1-GPC的[M–H+Cl]－二次碰撞活化解離的譜圖……40
圖8. C18:0/C20:4-GPC的[M+K+AcOH]＋ 碰撞解離的譜圖…………42
圖9. C16:0 /C18:1-GPC在0.1 %三氟醋酸中的譜圖…………………44
圖10A. C16:0 /C18:1-GPC的[M+K+TFA]＋碰撞活化解離的譜圖……45
圖10B. C18:1 /C16:0-GPC的[M+K+TFA]＋碰撞活化解離的譜圖……45
圖11. [Egg+K+TFA]＋的910 m/z碰撞解離譜圖………………………49
圖12. C16:0 /C18:1-GPE的正離子模式質譜圖………………………51
圖13A. C16:0 /C18:1-GPE的[M+Na]+碰撞活化解離的譜圖…………53
圖13B. C16:0 /C18:1-GPE的[M+K]+碰撞活化解離的譜圖…………53
圖14A. C16:0 /C18:1-GPE的負離子模式譜圖………………………55
圖14B. C16:0 /C18:1-GPE [M–H]－碰撞活化解離的譜圖…………55
圖15. C16:0 /C18:1-GPE [M–H+Ni]+ 碰撞活化解離的譜圖………58
圖16. C16:0 /C18:2-GPE加鈷金屬的質譜圖…………………………60
圖17. C16:0 /C18:2-GPE [M–H+Co]+ 碰撞活化解離的譜圖………61
圖18. C16:0 /C18:1 -GPE和C18:1/ C16:0-GPE [M–H+2Na]+碰撞解離的譜……………………………………………………………………63
圖19. C16:0 /C18:1-GPG的正離子模式質譜圖…………………………………………………………………………64
圖20. C16:0 /C18:1-GPG [M+Na]+碰撞活化解離的譜圖……………67
圖21A. C16:0 /C18:1-GPG的負離子模式質譜圖……………………69
圖21B. C16:0 /C18:1-GPG [M–H]－碰撞活化解離的譜圖…………69
圖22. C16:0 /C18:1-GPG [M–H+Ni]+ 碰撞活化解離的譜圖………72
圖23. C18:0 /C18:1-GPG 加鈷金屬的質譜圖…………………………………………………………………………73
圖24 . C18:0 /C18:1-GPG [M–H+Co]+ 碰撞活化解的質圖………74
圖25. C16:0 /C18:1-GPS的正離子模式質譜圖………………………76
圖26. C16:0 /C18:1-GPS [M+Na]＋碰撞活化解離的譜圖…………77
圖27A. C16:0 /C18:1-GPS的負離子模式質譜圖……………………80
圖27B. C16:0 /C18:1-GPS [M–H]－碰撞活化解離的譜圖…………80
圖27C. C16:0 /C18:1-GPS的[M–H]－二次碰撞活化解離的譜圖…80
圖28. C16:0 /C18:1-GPS加鈷、鈉金屬的質譜圖……………………82
圖29為C16:0 /C18:1-GPS的[M–2H+Na+Co]+ 碰撞活化解離的譜圖83



表目錄
表1.常見的飽和脂肪酸………………………………………………21
表2.常見不飽和脂肪酸………………………………………………22
表3.常見的磷脂的polar head………………………………………23
表4. GPC加鈉金屬經碰撞活化解離的主要離子和相對百分比……38
表5. GPC負離子模式下碰撞活化解離的主要離子和相對百分比…41
表6. GPC加TFA後，碰撞活化解離的主要離子和相對百分比………47
表7.蛋黃中鑑定出來的GPC種類……………………………………48
表8. GPE和鈉金屬結合經碰撞活化解離的主要離子和相對百分比…54
表9. C16:0 /C18:1-GPE和不同金屬結合的碰撞活化解離的主要離子和相對百分比…………………………………………………………57
表10. 磷脂醯乙醇胺、磷脂醯甘油和磷脂醯絲胺酸和鈷金屬結合經碰撞
活化解離脫脂肪酸的比值…………………………………………62
表11. GPG和鈉金屬結合經碰撞活化解離的主要離子和相對百分比66
表12. C16:0 /C18:1-GPG和不同金屬結合的碰撞活化解離的主要離子和
相對百分比………………………………………………………………70
表13. GPS和鈉金屬結合經碰撞活化解離的主要離子和相對百分比…………………………………………………………………………78

1.J. J. Thomson, Philosophical Magazine 1899, 48, 547.
2.J. J. Thomson, Longmans, Green and Co. Ltd. London, 1913.
3.A. J. Dempster, Phys. Rev. 1918, 11, 316.
4.F. W. Aston, Nature 1920, 105, 617.
5.W. Stephens, Phys. Rev. 1946, 69, 691.
6.J. A. Hipple, H. Sommer , H. A. Thomas, Phys. Rev.
1949,76, 1877.
7.D. Wobschall, J. R. Graham, D. P. Malone, Phys. Rev.
1965, 131, 1565.
8.M. B. Comisarow, A. G. Marsball, Chem. Phys. Lrtt. 1974,
25, 282.
9.W. Paul, H. Steinwedel, Z. Naturforsch. 1953, 8A, 448.
10. M. S. B. Munson, F. H. Field, J. Am. Chem. Soc. 1960,
88, 2621.
11. H. D. Beckey, Pergamon, Oxford. 1977
12.M. Barber, R. S. Bordoli, G. J. Elliott, R. D. Sedgwick,
A. N. Tyler,Anal. Chem. 1982, 54, 645A.
13.A. Benninghoven, F. G. Rudenauer, H. W. Werner, Wiley
NewYork 1987.
14.R. J. Cotter, Anal. Chem. 1988, 60, 817A.
15.E. Unsold, F. Hillenkamp, R. Nitsche, Analysis. 1990, 4,
115.
16.M. Yamashita, J. B. Fenn, J. Phys. Chem. 1984, 88, 4451.
17. M. Karas, F. Hillenkamp, Anal. Chem. 1988, 60, 2299.
18.J. Zeleny, Phys. Rev. 1917, 10, 1.
19. M. Dole, L. L. Mack, R. L. Hines, J. Chem. Phys. 1968,
49, 2240.
20.L. L. Mack, P. Kralic, A. Rheude, M. Dole, J. Chem.
Phys. 1970, 52,4977.
21.A. P. Bruins, T. R. Covey, J. D. Henion, Anal. Chem.
1987, 59, 2642.
22.J. A. Olivares, N. T. Nguyen, C. R. Yonker, R. D. Smith,
Anal. Chem. 1987, 59, 1232.
23.J. A. Wahl, D. R. Goodlett, H. R. Useth, R. D. Smith,
Anal. Chem. 1992, 64, 3194.
24. D. C. Gale, R. D. Smith, Rapid Commum. Mass Spectrom.
1993, 7, 1017.
25. S. Wilm, M. Mann, J.Mass Spectrom. Ion Process. 1994,
136, 167.
26.A. Gomez, K. Tang, Phys. Fluids. 1994, 6, 2317.
27.M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D.
Ferguson, M.B. Alice, Chem. Phys. 1968, 49, 2240.
28.J. V. Iribarne, B. A. Thormson, J. Chem. Phys. 1976,
64,15.
29.P. Kebarleand, L. Tang, Anal. Chem. 1993, 65, 972A.
30.F. W. Röllgen, E. Bramer-Wegner, L. Butfering, J. Phys.
1987, 48, 253.
31.J. B. Fenn, J. Am. Soc. mass spectrum. 1992, 4, 524.
32. 余傳韜和蘇仲卿主編，生物化學概論，國立編譯館出版。
33.潘福、李詩慶、張固剛、蕭明熙和尹士俊編著，生物化學，台灣
商務印書館發行。
34. Phili B. W. Smith, Anal. Chem. 1995, 67, 1824.
35. Gavin E. Black, A. Peter Snyder, Karen S. Heroux,
Journal of Microbiological Methods 1997, 28, 187.
36.R.J. Hanmilton, LIPID ANALYSIS IN OILS and FATS 1998
37. H. Y. Kim, N. Salem, Jr. Anal. Chem. 1986, 58, 9.
38.H. Y. Kim, N. Salem, Jr. Anal. Chem. 1987, 59, 722.
39.A. Valeur, P. Michelsen, G. Odham, LIPIDS, 1993, 28, 255.
40.Gary Dobson, and Nigel Deighton, Chemistry and Physics
of Lipids 2001, 111,1.
41. J. L. Kerwin, A. R.Tuininga, L. H. Ericsson, J. Lipid
Res. 1994, 35, 1102-1114.
42. A. Å. Karlsson, P. Michelsen, Å. Larsen, G. Odham,
Rapid Commun.Mass Spectrom. 1996, 10, 775.
43.X. Han, R. A. Gubitosi-Klug, B. J. Collins, R. W. Gross,
Biochemistry 1996, 35, 5822.
44.C. Hoischen, W. IHN, K. Gura, J. Gumpert, J. Bacteriol.
1997, 179,3437.
45. J. Fang, J. B. Michael, Journal of Microbiological
Methods 1998, 33,23.
46. E. Hvattum, Å. Larsen, S. Uran, P. M. Michelsen, T.
Skotland, J.Chrom. B. 1998, 716, 47.
47. W. C. Byrdwell, Rapid Commmun. Mass Spectrom. 1998, 12,
256.
48. E. A. A. M. Vernooij, J. J. Kettenes-van den Bosch, D.
J. A.Crommelin, Rapid Commmun. Mass Spectrom. 1998, 12,
83.
49. S. Ramanadham, F. F. Hsu, S. Zhang, A. Bohrer, Z. Ma,
J. Turk,Biochem. Biophys. Acta 2000, 1484, 251.
50.E. Hvattum, G. RØsjØ, T. GjØen, G. Rosenlund, B.
Ruyter, J.Chrom. B 2000, 748, 137.
51. S. Uran, Å. Larsen, P. B. Jacobsen, T. Skotand,
J.Chrom. B 2001, 758, 265.
52.N. J. Jensen, K. B. Tomer, M. L. Gross, Lipids 1986, 21,
580.
53.N. J. Jensen, K. B. Tomer, M. L. Gross, Lipids 1987, 22,
480.
54.D. N. Heller, C. M. Murphy, R. J. Cotter, C. Fenselau,
O. M. Uy,Anal. Chem. 1988, 60, 2787.
55. K. Kayganich, R. C.Murphy, J. Am. Soc. Mass Spectrom.
1991, 2, 45.
56.J. A. Zirrolli, K. L. Clay, R. C. Murphy, Lipids 1991,
26, 1112.
57. M. J. Cole, C. G. Encke, Anal. Chem. 1991, 63, 1032.
58. Z. H. Huang, D. A. Gage, C. C. Sweeley, J. Am. Soc.
Mass Spectrom.1992, 3, 71.
59.R. C. Murphy, K. A. Harrison, Mass Spectrometry Reviews
1994, 13,57.
60. C. Li, J. A. Yergey, J. Mass Spectrom. 1997, 32, 314.
61.Y. H. Kim, J. S. Yoo, M. S. Kim, Bull. Korean Chem. Soc.
1997, 18,874
62.P. Domingues, M. R. M. Domingues, F. M. L. Amado, A. J.
F. Correia,Rapid Commun. Mass Spectrom. 2001, 15, 799.
63.E. Hvattum, G. Hagelin, Å. Larsen, Rapid Commun. Mass
Spectrom.1998, 12, 1405.
64.F. F. Hsu, J. Turk, J. Am. Soc. Mass Spectrom. 2000, 11,
892.
65. F. F. Hsu, J. Turk, J. Am. Soc. Mass Spectrom. 2001,
12, 1036.
66.F. F. Hsu, J. Turk, J. Am. Soc. Mass Spectrom. 2000, 11,
797.
67.F. F. Hsu, J. Turk, J. Am. Soc. Mass Spectrom. 2000, 11,
986.
68. F. F. Hsu, J. Turk, J. Am. Soc. Mass Spectrom. 1997, 9,
516.
69.F. F. Hsu, J. Turk, J. Mass Spectrom. 2000, 35, 596.
70.Å. Larsen, S. Uran, P. B. Jacobsen, T. Skotand, Rapid
Commun. Mass Spectrom. 2001, 15, 2393.
71.N. J. Jensen, M. L. Gross, Mass Spectrom. Reviews 1988,
7, 41.
72.X. Han, R. W. Gross, J. Am. Soc. Mass Spectrom. 1995, 6,
1202.
73.X. Han, R. W. Gross, J. Am. Soc 1996, 118, 451.
74.F. F. Hsu, A. Bohrer, J. J. Turk, J. Am. Soc. Mass
Spectrom. 1998, 9,516.
75.R. C. Murphy, Handbook of Lipid Research Vol. 7, Plenum:
New York, 1993, Chap 7.
76.S. Ramanadham, F. F. Hsu, A. Bohrer, W. Nowatzke, Z. Ma,
J. Turk,Biochemistry 1998, 37 4553.