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研究生： 葉婷婷
Yeh, Ting-Ting
論文名稱： 比較不同分離策略搭配液相層析串聯式質譜儀之磷酸化蛋白體學研究
Comparison of Different Fractionation Strategies for In-depth Phosphoproteomics by Liquid Chromatography Tandem Mass Spectrometry
指導教授： 陳頌方
Chen, Sung-Fang
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2016
畢業學年度： 104
語文別： 中文
論文頁數： 85
中文關鍵詞： 磷酸化胜肽 、羥基脂肪酸修飾之金屬氧化物層析法 、強陽離子交換層析法 、靜電排斥親水交互作用層析法 、液相等電點聚焦分離法 、液相層析串聯式質譜儀
英文關鍵詞： fractionation, ERLIC
DOI URL： https://doi.org/10.6345/NTNU202203937
論文種類： 學術論文
相關次數： 點閱：145  下載：8 
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    • 磷酸化蛋白體學為近年來許多生化分析學家致力研究的領域，原因是蛋白質的磷酸化對於調控蛋白質活性、細胞間訊息傳遞等生理機制扮演著極為重要的角色。然而，具有磷酸化修飾的蛋白質僅占總體蛋白質的一部份，經水解後所形成的磷酸化胜肽含量偏低，在一般的質譜鑑定流程中容易被含量較高的一般胜肽遮蔽；為了降低樣品的複雜度，並有效提升磷酸化胜肽的鑑定機率，在本研究中使用了羥基脂肪酸修飾之金屬氧化物層析法 （aliphatic hydroxy acid-modified metal oxide chromatography, HAMMOC），先純化出小鼠巨噬細胞株（RAW 264.7）當中的磷酸化胜肽，再選用三種不同的分離方法（fractionation）進行比較。方法分別為強陽離子交換層析法（strong cation chromatography, SCX）、靜電排斥親水性交互作用層析法（electrostatic repulsion hydrophilic interaction chromatography, ERLIC）及液相等電點聚焦分離法（solution isoelectric focusing electrophoresis, sIEF），最後將經三種不同方法分離出來的磷酸化胜肽送入液相層析串聯式質譜儀（LC-MS/MS）進行分析。在蛋白質樣品起始量皆為1毫克的情況下，鑑定到的磷酸化胜肽數目分別如下： SCX-LC-MS/MS 可鑑定到 4336 條磷酸化胜肽、ERLIC-LC-MS/MS為 2064 條磷酸化胜肽，sIEF-LC-MS/MS 則是 2424 條磷酸化胜肽。三種方法總共鑑定到5744條不重複的磷酸化胜肽及 2159 個磷酸化蛋白質，且僅在單一方法中單獨出現的磷酸化胜肽個數依序為 SCX （2430）、ERLIC （438）、sIEF （751）。由此可知，本研究中所選用的三種分離方法具有良好的互補性，能有效提升對磷酸化胜肽的鑑定能力，以期應用於分析磷酸化蛋白體學的研究中。

    Phosphorylation is one of the major post-translational modifications that plays a significant role in protein activity and cell signaling. However, it is difficult to detect protein phosphorylation because its low abundance and the analysis can be hindered by high abundant non-phosphoproteins. In order to improve the identification efficiency of phosphopeptides and reduce the complexity of sample, aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) is applied for phosphopeptides enrichment from RAW 264.7 cell lysates. Moreover, strong cation chromatography (SCX), electrostatic repulsion hydrophilic interaction chromatography (ERLIC) and solution isoelectric focusing (sIEF) were investigated for phosphopeptides fractionation strategies following by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Total 5744 non-redundant phosphopeptides and 2159 phosphoproteins were identified from 1 mg RAW 264.7 cell lysates combining three fractionation approach. Besides, 4336, 2064 and 2424 phosphopeptides were identified by SCX-LC-MS/MS, ERLIC-LC-MS/MS and sIEF-LC/MS-MS, including 2430, 438 and 751 phosphopeptides only found in SCX, ERLIC and sIEF fractionations specifically. In conclusion, the strategies of three different fractionations have great orthogonality, which improve identification efficiency of phosphopeptides and be suitable for in-depth phosphoproteomic research.

    摘要 I ABSTRACT II 目錄 III 表目錄 VI 圖目錄 VII 第一章 緒論 1 第一節 研究背景 1 第二節 研究動機 2 第三節 研究流程 3 第二章 文獻探討 5 第一節 偵測磷酸化蛋白質 5 第二節 純化磷酸化蛋白質與磷酸化胜肽之技術 6 2.2.1 免疫沉澱法（Immunoprecipitation, IP） 7 2.2.2 固定相金屬親和性層析法（Immobilized Metal Affinity Chromatography, IMAC） 7 2.2.3 金屬氧化物親和性層析法（Metal Oxide Affinity Chromatography, MOAC） 8 第三節 分離技術 10 2.3.1 離子交換層析法（Ion Exchange Chromatography, IEX） 11 2.3.2 親水性交互作用層析法（Hydrophilic Interaction Chromatography, HILIC） 13 2.3.3 靜電排斥親水性交互作用層析法（Electrostatic Repulsion Hydrophilic Interaction Chromatography, ERLIC ） 14 2.3.4 電泳法（Electrophoresis） 15 第四節 質譜儀技術 17 2.4.1 電噴灑游離法（Electrospray Ionization, ESI） 18 2.4.2 四極桿（Quadruple） 19 2.4.3 離子阱（Ion Trap, IT） 20 2.4.4 軌道阱（Orbitrap） 20 2.4.5 質譜鑑定磷酸化蛋白質之流程 21 第五節 蛋白質身份鑑定 22 2.5.1 胜肽碎片指紋比對（Peptide Fragment Fingerprinting, PFF） 23 第六節 分離策略比較 24 第三章 研究方法 26 實驗設計 26 藥品 26 一、 樣品（Sample） 27 二、 前處理（Sample Preparation） 28 三、 蛋白質酵素水解（In Solution Digestion） 30 四、 聚苯乙烯二乙烯基苯去鹽一（Polystyrene divinylbenzene Copolymer Stage Tip Desalting, SDB Desalting 1） 31 五、 羥基脂肪酸修飾金屬氧化層析法（Aliphatic Hydroxyl Acid-Modified Metal Chromatography, HAMMOC） 32 六、 聚苯乙烯二乙烯基苯去鹽二（Polystyrene divinylbenzene Copolymer Stage Tip Desalting, SDB Desalting 2） 34 七、 分離方法（Fractionation Strategies） 37 A. 強陽離子交換層析法 37 B. 靜電排斥親水性交互作用層析法 39 C. 液相等電點聚焦分離法 40 八、 碳18離心管柱去鹽（C18 Spin Column Desalt） 42 九、 液相層析串聯式質譜儀分析（Analysis by Liquid Chromatography–Tandem Mass Spectrometry） 43 十、 搜尋引擎與資料庫鑑定（Database Searching） 45 十一、 數據處理軟體暨資料分析（Data Analysis by Software） 47 第四章 結果與討論 51 第一節 以SCX分離磷酸化胜肽之質譜鑑定結果（12管） 51 第二節 以SCX分離磷酸化胜肽之質譜鑑定結果（24管） 54 第三節 以ERLIC分離磷酸化胜肽之質譜鑑定結果 58 第四節 以sIEF分離磷酸化胜肽之質譜鑑定結果 63 第五節 比較兩次SCX層析法之鑑定結果 66 第六節 三種不同分離方法互補性比較（一）- SCX（12）、ERLIC及sIEF之比較 68 第七節 三種不同分離方法互補性比較（二）- SCX（24）、ERLIC及sIEF之比較 70 第八節 不同分離方法之解析度比較 71 第九節 不同分離方法鑑定之磷酸化胜肽模組（motif）分析 73 第五章 結論與未來展望 75 參考文獻 76 附圖 79 附表 80

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