臺灣博碩士論文加值系統

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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