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研究生:林佳穎
研究生(外文):Chia-Ying Lin
論文名稱:脂族羥基酸修飾的金屬氧化物層析法(HAMMOC)搭配液相層析串聯式質譜分析技術(LC-MS/MS)之磷酸化蛋白質體研究
論文名稱(外文):Phosphoproteomic Analysis by HAMMOC Enrichment and Liquid Chromatography Tandem Mass Spectrometry
指導教授:陳頌方
指導教授(外文):Sung-Fang Chen
學位類別:碩士
校院名稱:國立臺灣師範大學
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:2014
畢業學年度:102
語文別:中文
論文頁數:126
中文關鍵詞:磷酸化胜肽脂族羥基酸修飾的金屬氧化物層析法液相層析串聯式質譜儀等電點聚焦電泳強陽離子交換層析法親水性作用層析無標記定量方法
外文關鍵詞:phosphopeptidesHAMMOCLC-MS/MSsIEFSCXHILIClabel-free
相關次數:
  • 被引用被引用:1
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蛋白質磷酸化是一種主要的後轉譯修飾,它調節許多生物系統,如基因表現、訊號傳遞與代謝路徑。但磷酸化修飾為可逆反應且含量低,故需要高靈敏度與具特異性的分析方法。本研究結合脂族羥基酸修飾的金屬氧化物層析法(HAMMOC)與液相層析串聯式質譜分析技術(LC-MS/MS)研究磷酸化蛋白質體學。首先選用丙酮沉澱法與離心過濾法,以比較不同去鹽方法之磷酸化研究;接著於HAMMOC純化後,以等電點聚焦電泳(sIEF)、強陽離子交換層析法(SCX)與親水性層析法(HILIC)降低樣品複雜度,並比較不同分離方法之磷酸化研究;最後,採用無標記定量方法(label-free)進行磷酸化蛋白質體定量。取小鼠巨噬細胞株(RAW 264.7)裂解液,經離心過濾法以及丙酮沉澱法去鹽,毫克含量的蛋白質水解後搭配HAMMOC-sIEF與質譜分析,在兩種方法共鑑定到1231個磷酸化蛋白質與2953段磷酸化胜肽。以離心過濾法搭配HAMMOC與三種分離方法,結果顯示單一HAMMOC-SCX可以鑑定到1747段磷酸化胜肽,再搭配HAMMOC-sIEF和HAMMOC-HILIC可額外鑑定1517段(86.8%)磷酸化胜肽,由此可知三種分離方法具良好的互補性。從小鼠巨噬細胞株之控制組與脂多醣刺激組取200微克的蛋白質,以HAMMOC與LC-MS/MS利用無標記定量方法進行相對定量分析,從中挑選出114個差異表現的磷酸化蛋白質。以上結果顯示選用HAMMOC搭配LC-MS/MS適合深入研究磷酸化蛋白質體學的定性和定量分析。
Protein phosphorylation is one of major post-translational modifications, which regulates many biological intracellular systems, including gene expression, signal transduction pathways and metabolic processes. Phosphorylation, a transient modification and low abundance, requires highly sensitive and specific strategies for its analysis. The combination of aliphatic hydroxyl acid-modified metal oxide chromatography (HAMMOC) enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis were employed for the phosphoproteome analysis in this study. Two desalting methods, including acetone precipitation and centrifugal filter, were used separately before HAMMOC treatment. Moreover, three fractionation strategies, solution isoelectric focusing electrophoresis (sIEF), strong cation exchange chromatography (SCX) and hydrophilic interaction liquid chromatography (HILIC), were utilized to reduce the sample complexity after HAMMOC enrichment. Finally, label-free strategy was implemented for the phosphoproteomic quantification. A total of 2953 phosphopeptides and 1231 phosphoproteins were found in milligram proteins of RAW264.7 cell lysate by combining two desalting methods coupled with HAMMOC-sIEF approach. The use of centrifugal filter and HAMMOC coupled with three various fractionation strategies had shown good complementarity (86.8% more phosphopeptide identifications than SCX only). One hundred and fourteen phosphoproteins were found differentially expressed between control and lipopolysaccharide-treated RAW264.7 from 200 microgram proteins by using label-free comparative approach after HAMMOC and LC-MS/MS. These results indicate that HAMMOC followed by LC-MS/MS analysis is suited for the in-depth analysis of qualitative and quantitative phosphoproteome profiling.
目錄 I
圖目錄 IV
表目錄 VI
英文縮寫檢索表 VII
Abstract IX
摘要 X
第一章 序論 1
一、蛋白質磷酸化 1
二、偵測磷酸化蛋白質 2
2.1 放射性標記法 2
2.2 免疫染色標記法 2
2.3 質譜法 3
三、純化磷酸化蛋白質與磷酸化胜肽方法 4
3.1 化學衍生法(Chemical Derivatization Strategies) 4
3.2 親和性層析法(Affinity Chromatography) 5
四、液相層析分離技術 8
4.1 等電點聚焦電泳(Solution Isoelectric Focusing Electrophoresis, sIEF) 8
4.2 離子交換層析法(Ion Exchange Chromatography, IEX) 9
4.3 親水性作用層析法(Hydrophilic Interaction Liquid Chromatography, HILIC) 10
4.4 靜電排斥親水性作用層析法(Electrostatic Repulsion Hydrophilic Interaction Chromatography, ERLIC) 11
五、質譜儀技術 12
5.1 質譜儀(Mass Spectrometry, MS) 12
5.2 蛋白質身分鑑定(Protein Identification) 14
5.3 質譜應用在磷酸化蛋白質序列鑑定 16
5.4 差異蛋白質體學(Differential Proteomics) 19
六、研究動機 21
第二章 實驗材料 22
一、樣品 22
二、藥品與試劑 22
三、材料 23
四、儀器 23
第三章 實驗方法 24
一、樣品 24
二、蛋白質濃度測定 25
三、樣品前處理 26
3.1 離心過濾法(Centrifugal Filter) 26
3.2 丙酮沉澱法(Acetone Precipitation) 26
四、蛋白質水解(In Solution Digestion) 27
五、聚苯乙烯二乙烯基苯去鹽(Polystyrenedivinylbenzene Copolymer Stage Tip Desalting, SDB Desalting) 27
六、脂族羥基酸修飾的金屬氧化物層析法(HAMMOC) 28
七、聚苯乙烯二乙烯基苯去鹽(SDB Desalting) 29
八、第一維分離策略 30
8.1 等電點聚焦電泳(Solution Isoelectric Focusing Electrophoresis, sIEF) 30
8.2 強陽離子交換層析法(Strong Cation Exchange Chromatography, SCX) 31
8.3 親水性作用層析法(Hydrophilic Interaction Liquid Chromatography, HILIC) 32
九、碳18離心管柱去鹽(C18 Spin Column Desalting) 33
十、液相層析參數設定 34
10.1 ACUQITY Ultra Performance LC system 34
10.2 Dionex UltiMate 3000 Rapid Separation LC system 34
十一、串聯式質譜參數設定 35
11.1 LTQ XL 35
11.2 LTQ Orbitrap XL 36
11.3 應用於無標記(Label-Free)之LTQ Orbitrap XL 37
十二、蛋白質資料庫搜尋(Data Analysis) 38
第四章 實驗結果與討論 39
一、蛋白質濃度測定 39
二、不同去鹽策略對於蛋白質濃度測定與磷酸化胜肽鑑定之影響 39
2.1 以離心過濾法與丙酮沉澱法去鹽後之各別蛋白質濃度測定 40
2.2 以離心過濾法與丙酮沉澱法去鹽後之各別鑑定磷酸化胜肽 40
2.3 去鹽策略之互補性(Complementarity)分析 42
三、不同分離策略對於磷酸化胜肽鑑定之影響 43
3.1 等電點聚焦電泳(Solution Isoelectric Focusing Electrophoresis, sIEF) 43
3.2 強陽離子交換層析法(Strong Cation Exchange Chromatography, SCX) 44
3.3 親水性作用層析法(Hydrophilic Interaction Liquid Chromatography, HILIC) 45
3.4 分離策略之互補性(Complementarity)分析 46
四、無標記(Label-free)方法分析脂多醣刺激小鼠巨噬細胞(RAW264.7)之差異磷酸化蛋白質體研究 46
4.1 磷酸化蛋白質身分鑑定 47
4.2 磷酸化蛋白質定量分析 48
4.3 生物資訊分析 48
第五章 結論與未來展望 49
附圖 50
附表 97
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