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系統識別號 U0026-3108201711054300
論文名稱(中文) 製備線上奈米級液相層析搭配由上而下蛋白質體學策略應用於偵測紅血球中血紅蛋白的修飾
論文名稱(英文) Fabrication of On-Line Nano-Liquid Chromatography-Mass Spectrometry for Top-down Proteomics and Application for the detection of Modified Hemoglobin in Red Blood Cells
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) 賴威彣
研究生(英文) Wei-Wen Lai
學號 L36041123
學位類別 碩士
語文別 英文
論文頁數 49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 奈米級液相層析  質譜  由上而下  蛋白質體學 
英文關鍵字 Nano-Liquid Chromatography  Mass Spectrometry  Top-down  Proteomics 
學科別分類
中文摘要 雌激素及其代謝物已經有被報導會和蛋白進行共價鍵結合進而引發疾病。即使傳統的由下而上質譜法可以解析出修飾的位點但是仍然耗時且不便利。,在此研究中嘗試以由上而下策略進行人類紅血球蛋白上雌激素修飾的分析,在由上而下策略中可以較直觀的檢測出蛋白是否有修飾的存在。為了達到這項目的,我們採用自行填充之奈米級管柱應用在液相層析二次質譜法並結合軟體Prosight PTM 2.0進行分析,同時也使用由下而上質譜法進行確認。

在本實驗中實驗樣品為分離自血液中的血紅蛋白(Hemoglobin)和4-羥基雌二醇(4-OHE2)反應。

實驗中共使用兩種不同粒徑的填充粒子,分別為5微米及20微米,在測試自行製備之管柱的結果時,20微米填充粒子製成的管柱經由公式計算出管柱阻力因子並不良好,但是仍能使用此管柱進行蛋白次單元及修飾後蛋白的分離。在以由上而下策略鑑定蛋白質時,一次質譜圖可以看出血紅蛋白亞基α及β上具有4-羥基雌二醇的修飾。再經由二次質譜圖輸入軟體鑑定時由於產生的碎片離子資訊太少並不能很準確地確定修飾所在的位置,僅能鑑定出該母離子為目標蛋白,但經由手動分析後可以確認血紅蛋白亞基α上的修飾位置,且符合由下而上策略使用胰蛋白酶水解分析之結果。在血紅蛋白亞基β中並無法以由上而下策略鑑定修飾位點必須在由下而上策略搭配胰凝乳蛋白酶水解才可得知。由此資料中可以證明由上而下質譜法未來可以發展出針對雌激素化的紅血球蛋白的生物標記物。
英文摘要 Estrogen and its metabolites have been reported to form covalent bonds with proteins, causing changes of protein functions which are related to diseases. Although conventional bottom-up proteomics technique can be used to identify modified proteins, it is still rather time-consuming and inconvenient for biomarker detection. In this research, we tried to develop a top-down method for identifying hemoglobins modified by catechol estrogens. For this purpose, we develop a packing method for fabricating nano columns and apply the self-packed column for top down analysis of modified hemoglobin in red blood cells using LC-MS/MS and Prosight PTM 2.0 for proteomic search. The bottom-up approach was further applied to confirm the modification sites identified by the top-down approach.
Sample used in this research is the lysed red blood cells which were co-incubated with 4-hydroxyestradiol in order to generate modified hemoglobin.
Two packing materials were attempted for packing. Our data indicated that the self-packed nano-column had reasonable flow resistance for 5 μm reversed phase C18 particles but was relatively loose for 20 μm reversed phase particles. But our results showed the 20 um column can still separate hemoglobin α and β chain as well as 4-hydroxyestradiol modified hemoglobin α and β chain at a relatively faster speed. In the MS1 spectrum can observe 4-hydroxyestradiol modification on hemoglobin α and β chain. When the raw data acquired by using the 20 um colume were analyzed with Prosight PTM 2.0 the modification site could not be confirmed due to few fragments by collision induced dissociation. Moreover, the modification site identified by the top-down approach was able to be confirmed by the bottom-up approach. The modified hemoglobin β chain, however, could not be identified by the top-down approach but was identified by the bottom-up approach using chymotrypsin digestion. Our data provide evidences that top-down method may be developed for biomarker detection of estrogenized hemoglobin.
論文目次 摘要 I
Abstract II
Table of Contents IV
List of Figures V
Abbreviation VII
Chapter1 Introduction 1
1-1 Brief introduction about Estrogen and its metabolites 1
1-2 Proteomics 4
1-3 Hemoglobin 6
1-4 Mass spectrometry used in studying proteomics 7
1-5 Nano liquid chromatography 12
Chapter2 Experimental methods and materials 14
2-1 Materials, hardware and software 14
2.1.1 Material 14
2.1.2 Equipement 15
2.1.3 Software 15
2-2 Sample preparation 16
2.2.1 Extraction of red blood cell from blood 16
2.2.2 Concentration of protein 16
2.2.3 Hemoglobin adduct formation 16
2.2.4 Protein digest 17
2-3 Packing Capillary picofrit Columns 17
2.3.1 Tip Fabrication 17
2.3.2 Making column frits 17
2.3.3 Column Packing 18
2-4 LC MS/MS analysis 18
2.4.1 Condition of top down analyze 18
2.4.2 Condition of button up analyze 19
Chapter3 Result 21
3-1 Evaluation of the column efficiency 21
3-2 LC-MS/MS of intact hemoglobin 26
3.2.1 Optimization of the separation gradient 26
3.2.2 Top-down search by Prosight PTM 2.0 26
3-3 LC-MS/MS of hemoglobin peptides 40
Chapter4 Conclusion 46
Chapter5 Reference 47
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