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系統識別號 U0026-3108201723015600
論文名稱(中文) 利用質譜結合蛋白質體學技術來分析牛血清蛋白包覆金奈米團簇的結構特徵
論文名稱(英文) Structural Characterization of Bovine Serum Albumin Encapsulated Gold Nanoclusters using Mass Spectrometry-Based Proteomics Technique
校院名稱 成功大學
系所名稱(中) 化學系
系所名稱(英) Department of Chemistry
學年度 105
學期 2
出版年 106
研究生(中文) 陳怡諳
研究生(英文) Yi-An Chen
電子信箱 jelly601095@gmail.com
學號 L36044197
學位類別 碩士
語文別 中文
論文頁數 78頁
口試委員 指導教授-陳淑慧
口試委員-徐睿良
口試委員-梁世欣
中文關鍵字 牛血清蛋白  金奈米團簇  雙硫鍵 
英文關鍵字 BSA  gold nanoclusters  disulfide linkage 
學科別分類
中文摘要 蛋白質包覆金奈米團簇 ( Protein-gold nanoclusters ) 在許多不同的領域下是非常受到矚目的一種螢光奈米材料,由於金奈米團簇在近紅外光區具有良好的螢光特性、生物相容性佳以及功能化,主要是因為蛋白質上的多個胺基酸提供了可修飾各種分子的優點,根據上述這些優點讓蛋白質包覆金奈米團簇廣泛的應用於生物感測器、生物顯影以及生物標記,雖然在許多文獻上已經發表出不同的和成方法,例如使用氧化還原試劑,pH 和溫度來改善金奈米團簇的放光強度或是提升檢測的靈敏度,但蛋白質包覆金奈米團簇 ( Protein-gold nanoclusters ) 的分子結構仍尚未被研究出。在本研究中,我們以液相層析質譜法( LC-MS )為基礎結合蛋白質體學的技術來了解牛血清蛋白 ( BSA ) 在形成金奈米團簇時的結構變化。
在實驗室先前的研究,已經成功的合成出牛血清蛋白包覆金奈米團簇 ( BSA-AuNCs ),首先因為先前的合成有殘留很強的 BSA 訊號,為了提升金奈米團簇的均一性,所以使用兩步驟法 ( two-step method ) 去合成紅光金奈米團簇 ( BSA-AuNCs_red ),同時改變 pH 值以及還原劑,合成出發出藍光的金奈米團簇 ( BSA-AuNCs_blue ),合成出來的樣品以透析、不連續性蔗糖濃度梯度以及超離心分子篩去分離後,再以螢光光譜儀、可見光/紫外光光譜儀、大小排除管柱、基質輔助雷射脫附離子化飛行時間質譜儀 ( MALDI-TOF ) 作性質的鑑定,再來以圓二色偏光光譜儀分析二級結構的變化,以化學分析電子能譜 ( ESCA ),由下而上的方法 ( Bottom –up method ) 主要是以將 BSA-AuNCs 在水溶液下酵素水解 ( In solution digestion ) 以及原態酵素水解 ( Native digestion ) 下來觀察且鑑定胺基酸的變化。
紅光金奈米團簇主要放光在 640 nm 左右;藍光的金奈米團簇則是在 440 nm左右。從 MALDI-TOF 來看,紅光金奈米團簇的半高寬約為 3 kDa 左右並且質量範圍為 71 kDa ,大約由 25 顆金所組成的;反之藍光金奈米團簇質量範圍約在 74 kDa,且由 40 顆金所組成。
從由下而上的方法 ( Bottom –up method ) 可以得知,在形成金奈米團簇時,半胱胺酸扮演重要的角色,因為半胱胺酸會被氧化形成二氧化硫 ( sulfur dioxide ) 或三氧化硫 ( sulfur trioxide )。從 ESCA 同時也可以發現藍光金奈米團簇的氧化程度也遠高於紅光金奈米團簇,就代表在形成藍光金奈米團簇的時候雙硫鍵大部分都斷掉並且氧化。此外,Cys 125 的位置不管在紅光還是藍光的金奈米團簇都是主要氧化的目標,ESCA 也指出紅光金奈米團簇 Au(I) 的比例高於藍光金奈米團簇。
將上述的結果綜合起來,金奈米團簇的生成可能是因為半胱胺酸的特殊位點氧化而造成雙硫鍵的斷裂,在使用抗壞血酸這個還原劑下會加速半胱氨酸的氧化和雙硫鍵的斷裂,即使我們在相對溫和的 pH(pH = 8)合成環境下。所以藍色金奈米團簇的尺寸會比較大就可能是由於雙硫鍵的斷裂,然而後續要再繼續深度研究的部分在於氧化的半胱胺酸硫酸鹽是否為主要穩定金奈米團簇的作用力,以及是否會影響金奈米團簇的光學以及化學性質,這對分析合成的機制上將是一大的幫助。
英文摘要 Nowadays, the use of protein encapsulated gold nanoclusters is of high interest in many fields. Their particular properties such as luminescence in the visible range, nontoxicity, biocompatibility and feasible functionalization made them attractive for applications in biosensing, bioimaging and biolabelling. Although many synthetic strategies such as the use of redox reagents, pH, and temperature have been shown to improve the luminescence or the sensing sensitivity of the nanoclusters, molecular structure of protein protected gold nanoclusters remains un-explored. In this study, we applied liquid chromatography-mass spectrometry (LC-MS) based proteomics technique to study the structural changes of BSA by forming gold nanoclusters.
In our previously study, we have synthesized bovine serum albumin protected gold nanoclusters (BSA-AuNCs). In order to improve their monodispersity, we tried to change our synthesis methods and purification methods. First, we used two-step method to synthesize red fluorescent gold nanoclusters (BSA-AuNCs_red). Secondly, we used ascorbic acid under slightly basic condition (pH =8) to form the blue fluorescent gold nanoclusters (BSA-AuNCs_blue).The products, BSA-AuNCs were first purified by dialysis membrane, and then by non-continuous sucrose gradient or ultracentrifugefilter and characterized by Fluorescent spectroscopy, Ultraviolet-visible spectroscopy, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), Circular Dichroism spectrum (CD spectrum) and Electron spectroscopy for chemical analysis (ESCA). The bottom-up proteomics technique was then applied to identify the changes of amino acid residues of BSA under native and denatured condition.
The red nanoclusters exhibited luminescence mainly around 640 nm; the blue one mainly around 440 nm. MALDI-MS data show a broad range of mass distribution (3Da) with an average around 71 kDa for the red nanoclusters (about 25 Au atom) and 74 kDa for the blue nanoclusters (about 40 Au atom).The data derived from bottom-up proteomics indicated cysteine is the major species been oxidized to sulfur dioxide or sulfur trioxide by nanoclusters formation. The oxidized percentage was much higher for the blue than for the red one, resulting in more disulfide linkages were cleaved for the blue nanoclusters. These observations were consistent with the observations of ESCA. In addition, proteomics data revealed the cleaved disulfide linkage sites, among which Cys 125 appeared to be main target of oxidization for both the red and the blue nanocluster. Moreover, ESCA data indicated a higher percentage of Au(I)/Au(0) for the red nanoclusters than the blue nanoclusters.
Taken together, our study indicated that nanoclusters formation may be driven by oxidation of site-specific cysteine residues, causing some disulfide linkages to break. The use of reducing agents such as ascorbic acid appeared to accelerate cysteine oxidation and disulfide cleavages even under a relatively mild pH (pH=8) environment. The bigger size of blue nanoclusters may be attributed to disulfide cleavages. It remains to study whether the oxidized cysteineor sulfate play a role in stabilizing nanoclusters and whether it affects the optical or chemical properties of the nanoclusters.
論文目次 Abstract I
中文摘要 III
誌謝 X
目錄 XI
圖目錄 XIV
表目錄 XVII
簡稱用與對應表 XVIII
第一章 研究內容 1
1.1 研究動機 1
1.2 研究方向與策略 1
第二章 文獻回顧 2
2.1 金奈米團簇簡介 2
2.1.1 金奈米團簇放光原理 2
2.1.2 金奈米團簇製備方法 4
2.1.3 蛋白質金奈米團簇的應用 5
2.2. 蛋白質結構 7
2.2.1 蛋白質的結構分析 9
2.2.2 蛋白質金奈米團簇結構分析方法 10
2.3 牛血清蛋白 ( Bovine Serum Albumin, BSA )[46, 47] 11
第三章 實驗方法 12
3.1 實驗藥品 12
3.2 實驗耗材與儀器 12
3.3 合成蛋白質包覆金奈米團簇 ( Protein-AuNCs ) 13
3.3.1 合成紅光牛血清蛋白包覆金奈米團簇 ( BSA-AuNCs ) 13
3.3.2 合成藍光牛血清蛋白包覆金奈米團簇[52] 14
3.3.3 純化蛋白質金奈米團簇 14
3.4 基質輔助雷射脫附游離飛行質譜儀 ( MALDI ) 樣品配製方法 17
3.5 牛血清蛋白包覆金奈米團簇由下而上的步驟 18
3.5.1 在水溶液下酵素水解法 ( In solution digestion ) 18
3.5.2 原態酵素水解法( Native digestion ) 19
3.6 液相層析-電噴灑線性離子阱式軌道阱質譜儀 ( LTQ-Orbitrap XL MS ) 20
3.7 分析數據軟體– PEAKS 21
3.7. 圓偏光二色光譜 (Circular Dichroism spectrum,CD) 22
第四章 實驗結果與數據討論 23
4.1 牛血清蛋白包裹金奈米團簇的光學性質鑑定 23
4.1.1 紫外光/可見光光譜 (Ultraviolet-Visible spectroscopy) 24
4.1.2 螢光光譜儀 (Photoluminescence Spectrometer) 24
4.2 牛血清蛋白包裹金奈米團簇的分析 27
4.2.1 合成條件優化測試 27
4.2.2 溶劑的穩定性 28
4.2.1 樣品純化 32
4.2.2 質譜分析 36
4.3 牛血清蛋白包裹金奈米團簇的結構分析 39
4.3.1 化學分析電子能譜 39
4.3.2 圓偏光二色光譜 43
4.4 牛血清蛋白包覆金奈米團簇由下而上的方法 ( Bottom-up method ) 的分析 45
第五章 結論 54
第六章 參考文獻 55
附錄 60

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