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研究生:李信標
研究生(外文):Hsin-Piao Li
論文名稱:電噴灑串聯質譜法應用在分析氣相中金屬離子與胜肽鍵結位置的研究
論文名稱(外文):Probing the interaction of metal ions with peptides by electrospray ionization tandem mass spectrometry
指導教授:何彥鵬
指導教授(外文):Yen-Peng Ho
學位類別:碩士
校院名稱:國立東華大學
系所名稱:化學系
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:2002
畢業學年度:90
語文別:中文
論文頁數:111
中文關鍵詞:電噴灑金屬離子胜肽串聯質譜儀
外文關鍵詞:electrospraymetal iontandem mass spectrometry
相關次數:
  • 被引用被引用:1
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  • 下載下載:25
  • 收藏至我的研究室書目清單書目收藏:0
在生物系統中,胜肽或蛋白質和金屬離子的鍵結是個重要的研究方向,由於質譜儀離子源的進步,使得利用質譜分析生物分子和金屬之間的反應更為方便。本實驗是運用電噴灑串聯質譜術(ESI/MS/MS) 來研究胜肽 (M) 和金屬離子 (Cat) 之間的鍵結,經由電灑游離法,胜肽和金屬離子Na+、K+、Cs+、Ag+、Cu2+、Ca2+、Sr2+、 Ba2+、Co2+、Ni2+、Fe2+、Mn2+、Zn2+、Sc3+、La3+產生[M+Cat+]+、[M-H++Cat2+]+ 或[M-2H++Cat3+]+的複合物。經碰撞活化解離 (CAD) 可獲得金屬鍵結位置的訊息。胜肽DH16 (C2OH3-DKDGDGYISAAEAAAQ-NH2) 實驗結果發現Na+、K+、Cs+離子是鍵結在aspartic acid (D) 側鏈羧基的氧原子上,而glutamic acid (E) 側鏈的氧原子也會與 Na+、K+、Cs+離子形成鍵結。Ca2+、Sr2+、Ba2+離子與胜肽形成的複合物比Na+、K+、Cs+來的穩定。La3+離子鍵結的位置為aspartic acid (D) 側鏈上的氧,不過形成的複合物比Ca2+、Sr2+、Ba2+複合物的結構更穩定。Co2+、Ni2+、Fe2+、Mn2+、Zn2+、Sc3+有著類似鍵結的位置。Ag+、Cu2+離子會與aspartic acid (D) 側鏈上的氧形成鍵結,但並無選擇性,卻可得到整串序列的訊息。而胜肽GH11 (C2OH3-GYISAAEAAAQ-NH2) 的實驗中我們了解在沒有aspartic acid (D) 情況下,Na+、K+、Cs+、Ca2+、Sr2+及Ba2+離子會鍵結在glutamic acid (E) 的位置,此結果告訴我們Na+、K+、Cs+、Ca2+、Sr2+及Ba2+離子對於aspartic acid (D) 的鍵結力大於和glutamic acid (E) 的鍵結力。另外glutamine (Q) 的側鏈氧原子也會與金屬離子形成鍵結,Ag+、Cu2+離子一樣不具有選擇性。在鈣調素 (Calmodulin) 消化實驗中,鈣離子 (Ca2+) 與鈣調素 (CaM) 第三個loop鍵結位置,有aspartic acid (D)、asparagine (N)、glutamic acid (E) 和tyrosine (Y) 側鏈上的氧原子,所有結果顯示我們可利用MS/MS來決定金屬離子與胜肽鍵結的位置。另外,我們亦利用MS/MS來研究胰島素鏈A (ICA)+金屬的構形,由實驗知,K+離子與ICA形成的複合物,解離只會發生在某些側鏈及C端上。Co2+、Fe2+、Mn2+、Zn2+和ICA形成的複合物,其解離之圖譜彼此間極為類似,顯示有類似的結構,但與ICA+K的結構是不同的。Ni2+、Pd2+離子與ICA前四個胺基酸殘基形成很強的鍵結,而Cu2+離子所形成的複合物至少有兩個不同的結構。在胰島素鏈A (ICA) 消化實驗 (Digest) 中我們得知,消化後的胜肽(1~13)+Cu2+、胜肽 (1~13)+Ni2+的複合物CAD的譜圖極為相似,跟胜肽 (1~13)+Mn2+的CAD譜圖差異較大,結果與胰島素鏈A (ICA)實驗相符。
Abstract
Interactions between metal ions and proteins are related to a variety of biomolecular functions. The advances in ion sources for mass spectrometry have made the study of metal-biomolecule interaction possible. Our long-term goal is to study the intrinsic interaction
of metals and real biomolecules in the gas phase. Toward this
goal, we started with a known calcium-binding system, DH16 (C2OH3-DKDGDGYISAAEAAAQ-NH2), which is analogous to the third calcium-binding loop of calmodulin. Tandem mass spectrometry of [DH16+Ca2+-H]+ shows that the strongest calcium binding sites are aspartic acids. The third loop of calmodulin was obtained by trypsin digestion. The Ca2+ ion complex with this loop was subjected to tandem mass spectrometry. The results show that the aspartic acid, asparagines, glutamic acid and tyrosine are involved in the interaction between Ca2+ and the loop. This approach to investigate the interactions between metal ions and polypeptides by tandem mass spectrometry was extended to the oxidized insulin chain A (ICA), which is of relatively large size (21 amino acid residues). We investigated the interactions between alkali, alkaline-earth, the first and second series transition metal ions and ICA in the negative ion mode. Alkali, alkaline-earth metal ions, Mn2+, Fe2+, Co2+ and Zn2+ bind with ICA in a specific configuration such that dissociation occurs only at some side chains and the C-terminus. Ni2+ and Pd2+ ions, which have d6 configuration, are bound to part of the first four amino acid residues of ICA. Cu2+-ICA complex seems to have at least two structures, with one similar to Ni2+-ICA and the other similar to Mn2+-ICA. To the best of our knowledge, this is the largest system that has been directly analyzed by the tandem mass spectrometry approach.
論文摘要

壹、緒論.................................................1

1.1質譜游離法的發展......................................1
1.2電噴灑游離質譜法的發展歷史............................5
1.3電噴灑游離法中離子形成機制與原理......................8
1.4串聯質譜術 (MS/MS) 的發展.............................15
1.5金屬離子與胜肽形成複合物的文獻探討....................16
1.6研究目標..............................................19


貳、實驗部份.............................................23

2.1藥品..................................................23
2.2儀器裝置..............................................24
2.3實驗方法..............................................26
2.3.1藥品的配製.......................................................26
2.3.2實驗步驟............................................28
2.4理論碎片離子的比較....................................30


參、結果與討論...........................................31

3.1碎片離子的命名........................................31
3.2胜肽 (DH16) 與金屬離子鍵結位置的探討..................31
3.3胜肽 (C2OH3-GYISAAEAAAQ-NH2) 與金屬離子
鍵結位置的探討.......................................................53
3.4使用電噴灑串聯質譜術探討胰島素鏈A與金屬離子 之間的作用.......................................................71

肆、 結論.......................................97

伍、 參考文獻………………………………………………….100



圖目錄

圖1-1.電噴灑介面示意圖…………………………………………….8
圖1-2.電噴灑帶電液滴離子形成過程……………………….………10
圖1-3.毛細管尖端形成帶電離子的模型………………………….…11
圖1-4.胜肽DH16的序列及結構...............................20
圖1-5.胜肽GH11的序列及結構...............................21
圖1-6.胰島素鏈A的胺基酸序列,其中6個用正方形框起來的為
酸性胺基酸...............................................22
圖3.1.胜肽碎片離子片段的命名圖解.........................32
圖3.2.胜肽 (DH16) 的質譜全圖.............................33
圖3.3.[DH16+H]+在碰撞能量為28%的CAD譜圖..................35
圖3.4a-b.[DH16+Na]+和[DH16+K]+的CAD譜圖..................37
圖3.5a-b.[DH16+Ca]+和[DH16+Sr]+的CAD譜圖.................41
圖3.6.[DH16+La]+CAD譜圖..................................43
圖3.7.[DH16+Zn]+、[DH16+Fe]+、[DH16+Mn]+、[DH16+Sc]+、 [DH16+Ni]+和[DH16+Co]+的CAD譜圖..........................46
圖3.8. [DH16+Ag]+的CAD譜圖...............................47
圖3.9.[VR16+H]+在碰撞能量為28%的CAD譜圖..................51
圖3.10.胜肽 (VR16) 消化反應後加鈣離子的CAD譜圖...........52
圖3.11.胜肽 (GH11) 的質譜全圖............................55
圖3.12.[GH11+H]+在碰撞能量為28%的CAD譜圖.................56
圖3.13a-c.[GH11+Na]+、[GH11+K]+和[GH11+Cs]+的CAD譜圖.....59
圖3.14.[GH11+Ca]+、[GH11+Sr]+及[GH11+Ba]+的CAD譜圖.......61
圖3.15.[GH11+La]+的CAD譜圖...............................62
圖3.16.[GH11+Fe]+、[GH11+Mn]+及[GH11+Co]+的CAD譜圖.......64
圖3.17.[GH11+Sc]+、[GH11+Ni]+及[GH11+Zn]+的CAD譜圖.......66
圖3.18.[GH11+Ag]+的CAD譜圖...............................67
圖3.19.胰島素鏈A (ICA) 的質譜全圖........................72
圖3.20.[ICA-4H]4-在碰撞能量為30%的CAD譜圖................73
圖3.21.[ICA+Na]4和[ICA+K]4--的CAD譜圖....................75
圖3.22.[ICA+Mn]4-的CAD譜圖...............................76
圖3.23.[ICA+Mn]4-、[ICA+Fe]4-、[ICA+Co]4-和[ICA+Zn]4-
的CAD譜圖................................................78
圖3.24.[ICA+Ni]4-和[ICA+Pd]4-的CAD譜圖...................79
圖3.25.[ICA+Cu]4-的CAD譜圖...............................81
圖3.26.[ICA+2Pd]4-的CAD譜圖..............................83
圖3.27.[ICA+Cu]5-的CAD譜圖...............................84
圖3.28.胰島素鏈A (ICA) 經胃蛋白酶 (pepsin) 消化 (digest) 後的質譜全圖...............................................87
圖3.29.胜肽 (1~13) 在碰撞能量為28 %的CAD譜圖.............88
圖3.30.胜肽 (14~21) 在碰撞能量為28 %的CAD譜圖............89
圖3.31.[胜肽 (1~13)+Cu]1-及[胜肽 (1~13)+Ni]1-的CAD譜圖...90
圖3.32.[胜肽 (1~13)+Mn]1-的CAD譜圖.......................91
圖3.33.[胜肽 (14~21)+Cu]1-的CAD譜圖......................94
圖3.34.[胜肽 (14~21)+Ni]1-的CAD譜圖......................95
圖3.35.[胜肽 (14~21)+Mn]1-的CAD譜圖......................96
圖解1.[y15+K]+碎片離子形成機制...........................38
圖解2a-b.[y15+Ca-H]+碎片離子形成機制.....................42
圖解3.[b12+Ag]+碎片離子形成機制..........................48



表目錄

表1.胜肽 (DH16) 和各種金屬鍵結的整理......................49
表2.胜肽 (GH11) 和各種金屬鍵結的整理......................70
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