臺灣博碩士論文加值系統

傳統上使用電噴灑 (Electrospray Ionization, ESI) 進行質譜儀 (Mass Spectrometer, MS) 分析時，通常是以高電壓直流電場 (Direct Current, DC) 施加於毛細鋼針來達到離化的效果，然而其離化效率與蛋白質電荷分佈容易隨溶液 pH 值與其組成而改變。雖然現在許多學者對於直流電電噴灑已有相當程度的了解，而高頻率 (> 100 kHz) 交流電 (Alternating Current, AC) 電噴灑進行質譜儀分析與探討則是近幾年才開始逐漸發展起來。
交流電電噴灑機制可分為陽極半周期與陰極半周期，兩個半周期變換電場所需的時間非常短，致使低淌度 (low mobility) 的蛋白質離子可以大量聚集在針尖前端。由於交流電電噴灑生成的泰勒錐角度較小，產生的液滴也較小顆，離子可以直接從液滴脫附。我們先前的研究發現，在適當的高頻率下，經交流電電噴灑下所獲致的圖譜比較不隨溶液組成而改變，與直流電電噴灑有顯著的不同，推測此現象應與交流電電噴灑機制所產生的高溫效應有關。本研究利用兩種不同熱穩定性的蛋白質：分別是熱穩定性較高的細胞色素 c (Cytochrome c)，及熱穩定性較低的肌紅蛋白 (Myoglobin) 來觀察蛋白質的電荷分佈以了解直流電電噴灑及交流電電噴灑的差異性。
在擁有較高熱穩定性的細胞色素 c 部分，利用細胞色素 c 在不同pH值、有機溶劑的與否以及不同電壓條件下，觀察其在直流電與交流電電噴灑兩機制下的電荷分佈，並藉此判斷該蛋白質構形有無變化。實驗結果指出，直流電電噴灑會隨著溶液組成的改變而有所不同；相比之下，在交流電電噴灑中，尤其是施加高電壓時，較不會隨著溶液條件的差異而有所改變，並發現此機制下蛋白質的電荷分佈有朝向低電荷 (高m/z) 移動的趨勢，推測原因為交流電電噴灑的高溫效應使得氣相水分子增加，進而與蛋白質離子爭搶質子，造成分析物所帶電荷數降低轉而傾向形成較為緊密且折疊的 native 構形。
熱穩定性較低的肌紅蛋白則是配置在不同 pH 值的醋酸銨溶液中，並選擇於 pH 7 的條件中加入 30 % 的有機溶劑，觀察直流電與交流電電噴灑下的構形變化，可以得知在交流電電噴灑中，不論溶液組成為何，高溫效應都會讓肌紅蛋白完全變性，而直流電電噴灑則是會隨著溶液組成而有所改變。

Traditionally, mass spectrometry (MS) analysis usually used electrospray ionization (ESI) by applying a high voltage direct current (DC) electric field to the capillary needle to achieve the effect of ionization. However, its ionization efficiency and protein charge distribution easily transformed by the solution pH values and compositions. Although many scholars have a considerable degree of understanding of DC ESI, high frequency (> 100 kHz) alternating current (AC) ESI for mass spectrometer analysis and discussions is developed in recent years.
The mechanism can be understood by examining the two AC half cycles, the anodic half cycle and cathodic half cycle, separately. During the complete AC cycle, the time required for two half cycles to convert the electric field is very short, so the low mobility protein ions can be accumulated at the tip. Due to the angle of the Taylor cone formed by AC ESI (~12°) is smaller than DC ESI (~49°), and the AC droplets are also smaller than DC droplets, so the ions can be directly desorbed from the droplets. At the appropriate high frequency, the spectrum obtained by AC ESI does not change with the compositions of the solution, which is quite different from the DC ESI. This phenomenon should be related to the high temperature effect caused by the AC ESI mechanism. In this study, we used two different thermal stability protein including the high thermal stability cytochrome c and the low thermal stability myoglobin to observe the differences of the charge state distribution of protein between DC ESI and AC ESI mechanism.
In the part of the high thermal stability cytochrome c, we investigated the mass spectra of the charge state distributions at three pH values, pH 3.8, pH 2.8, and pH 2.3 with various organic solvent conditions (acetonitrile 0% and 30% (v/v)) and different AC output voltage by using AC ESI at frequencies in the hundreds of kilohertz (> 100 kHz) to observe the differences of the mass spectra between DC ESI and AC ESI and the protein conformation. The experimental results indicated that the DC ESI spectra would change with the various solution compositions. In contrast, the spectra under AC ESI especially applied the high voltage which would not change with the solution conditions and we observed that the charge distribution of the cytochrome c has a tendency to move toward a low charge state (high m/z). We speculated the reason is the high temperature effect increases the gas phase water molecules which with the protein ions to compete the proton that resulted in decreasing the charge of the analyte and the protein conformation transformed to a relatively tight and folded native conformation.
The mass spectra of the low thermal stability myoglobin are at pH 4, pH 7, pH 8 in ammonium acetate buffer solutions and which pH 7 with various organic solvent conditions (acetonitrile 0% and 30% (v/v)) also observed the different myoglobin conformation between DC ESI and AC ESI. The results indicated AC ESI would make myoglobin denature due to the high temperature effect, and DC ESI would let myoglobin conformation change with the solution conditions.

中文摘要 I
英文摘要 III
圖目錄 VII
表目錄 XII
第一章 緒論 1
1-1 前言與研究動機 1
1-2 質譜儀簡介 2
1-3 離子源發展歷史 3
1-4 電噴灑游離法簡介 6
1-4-1 直流電電噴灑游離法之機制及原理 7
1-4-2 交流電電噴灑游離法之機制及原理 11
1-5 泰勒錐產生之液滴大小及高溫效應 13
1-6 離子阱簡介 14
1-7 本研究使用之蛋白質簡介 17
1-7-1 細胞色素 c (Cytochrome c) 18
1-7-2 肌紅蛋白 (Myoglobin) 20
第二章 實驗 23
2-1 實驗藥品與試劑 23
2-2 實驗儀器與裝置 24
2-2-1 直流電電噴灑裝置 24
2-2-2 交流電電噴灑裝置 25
2-2-3 儀器及工具資訊 27
2-3 參數設定 28
2-3-1 直流電電噴灑參數 28
2-3-2 交流電電噴灑參數 29
2-4 藥品配置 30
2-4-1細胞色素 c (Cytochrome c) 30
2-4-2 肌紅蛋白 (Myoglobin) 31
第三章 結果與討論 32
3-1 細胞色素 c (Cytochrome c) 33
3-1-1 未含有機溶劑之不同 pH 值在不同電壓下的電荷分佈情形 33
3-1-2 含有有機溶劑之不同 pH 值在不同電壓下的電荷分佈情形 47
3-2 肌紅蛋白 (Myoglobin) 58
第四章 結論 63
參考文獻 65

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