臺灣博碩士論文加值系統

蛋白質磷酸化在轉譯後修飾的領域中扮演很重要的角色，如細胞生長、代謝及訊息的傳遞，許多文獻指出，酪胺酸磷酸化的蛋白質癌細胞的生成有關。但由於蛋白質磷酸化含量低，過程不穩且可逆，磷酸修飾帶負電而質譜使用正電模式，所以較容易受到非磷酸化胜肽抑制，因此需開發一個有效率且靈敏具有特異性的純化技術，來得到此方面的更多資訊。本實驗利用共沉澱法合成四氧化三鐵再包覆上二氧化鈦利用功能性單體-3-氮基丙基三乙氧基矽烷和交聯劑-苯基三甲氧基矽烷與模板-苯基磷酸合成分子印模聚合物，再利用乙腈與氨水溶液將模板移除，並在5% 三氟乙酸的條件下鍵結目標分子。Fe3O4@TiO2磁性奈米粒子對模板的最大承載量為60 μg/mg，且有效的純化ß酪蛋白及磷酸化胜肽標準品，其磷酸化胜肽訊號分別為m/z 2061和3122，而標準品的部分其m/z 813,863,1368 和1448可被有效的純化並利用基質輔助雷射脫附飛行時間式質譜儀分析。本研究利用分子印模聚合物之方法成功合成一個簡單且便宜的材料且能有效純化的技術。

Protein phosphorylation is an important post-translational modification which plays a critical role in the regulation of many cellar processes, such as signal transduction, metabolic homeostasis, cell division and growth. Protein phosphorylation on tyrosine has emerged as a vital part in diagnosing and curing cancer due to commonly observed in tumor proteomics. However, the identification of tyrosine-phosphorylated protein remains a challenge due to poor ionization efficiency in mass spectrometer, ion suppression by non-phosphotyrosine peptides. Therefore, it is essential to develop a sensitive and selective method for the specific enrichment of tyrosine-phosphorylated proteins and peptides. The magnetic nanoparticles were coated by titanium butoxide. The functional monomer (3-Aminopropyl) triethoxysilane and crosslinker phenyltrimethoxysilane interacted with template molecules and formed co-polymerization. Subsequently, the template molecules were removed by adding ammonium hydroxide mixed acetonitrile solution to form the phosphotyrosine imprinting polymers. In 5% TFA under the condition, polymers binding to target. The maximum binding capacity of phenylphosphonic acid onto Fe3O4@TiO2 magnetic nanoparticles was 60 μg/mg. The β-casein digests was utilized for the enrichment of phosphopeptides. The signals of β-casein phosphopeptide at m/z 2061 and 3122 could be detected and the standard phosphopeptide at m/z 813, 863, 1368 and 1448 were be observed by MALDI-TOF MS (Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry). MIPs (Molecularly Imprinted Polymers) were successfully synthesized by using the epitope imprinting method which were inexpensive and effective technique.

謝誌 i
摘要 ii
Abstract iii
圖目錄 vii
表目錄 ix
第一章　緒論 1
1.1　蛋白質體學介紹 1
1.2　轉譯後修飾 2
1.3　磷酸化轉譯後修飾的分析 4
1.4　純化磷酸化蛋白質與磷酸化胜肽 5
1.5　奈米粒子 7
1.5.1小尺寸效應 (Small size effect) 7
1.5.2表面效應 8
1.5.3量子尺寸及量子穿隧效應 8
1.6　磁性奈米粒子 8
1.7　分子印模聚合物 11
1.7.1原理 11
1.7.2非共價鍵結 (non-covalent assembly) 11
1.7.3共價鍵結 (covalent modification) 13
1.7.4金屬螯合 (ligand exchange) 13
1.7.5功能性單體 (functional monomer) 14
1.7.6交聯劑 (crosslinker) 14
1.7.7起始劑 (initiator) 15
1.8　質譜儀簡介 15
1.8.1原理 15
1.8.2基質輔助雷射脫附飛行時間式串聯式質譜儀 16
1.8.3飛行時間質量分析器 (Time-of-fight，TOF) 19
1.9　高效液相層析法（High Performance Liquid Chromatography, HPLC） 20
1.10　紫外-可見分光吸收光度法（Ultraviolet–visible spectroscopy，UV-Vis） 23
1.11　研究目的 23
第二章　藥品器材與實驗流程 25
2.1　藥品 25
2.2　實驗器材 26
2.3　藥品前處理 27
2.3.1藥品配置 27
2.3.2牛血清蛋白水解 28
2.3.3 ß牛乳酪蛋白水解 28
2.4　分子印模聚合物合成 29
2.4.1磁性奈米粒子四氧化三鐵製備 29
2.4.2 Fe3O4@TiO2之合成 31
2.4.3甲酸溶解包覆不完全之Fe3O4 31
2.4.4 MIP (Molecular Imprinted Polymers) 與NIP (Non-Molecular Imprinted Polymers) 之合成 32
2.5　純化ß-牛乳酪蛋白之磷酸化胜肽 32
2.6　純化磷酸化胜肽標準品 33
2.7　HPLC-UV數據 34
2.8　模板鑑定 34
2.8.1傅立葉轉換紅外線光譜儀 34
2.8.2穿透式電子顯微鏡 37
2.8.3.超導量子干涉震動磁量儀 37
第三章　結果與討論 39
3.1　分子印模聚合物鑑定 39
3.1.1分子模板聚合物大小鑑定 39
3.1.2傅立葉轉換紅外線光譜儀鑑定 43
3.1.3奈米粒子磁性鑑定 47
3.2　條件探討 50
3.2.1 Fe3O4@TiO2磁性奈米粒子最大吸附量 50
3.2.2 Fe3O4@TiO2磁性奈米粒子吸附時間 52
3.2.4 MIP與NIP的elute量比較 54
3.2.5 MIP與standard phosphoeptide 反應時間 56
3.3　MALDI-TOF MS質譜圖分析 58
3.3.1純化ß-casein 58
3.3.2純化磷酸化胜肽標準品 60
3.3.3磷酸化胜肽標準品純化之層析質譜圖 62
第四章　結論 64
第五章　參考文獻 65





 
圖目錄
圖一、蛋白質體學分析流程圖 3
圖二、分析磷酸化蛋白質之流程圖6
圖三、合成分子印模聚合物流程圖12
圖四、飛行時間質量分析器內部及構造21
圖五、本研究合成分子印模聚合物流程圖30
圖六、磷酸化胜肽純化流程圖35
圖七、Fe3O4磁性奈米粒子之TEM圖40
圖八、不同Titanium(IV) butoxide反應劑量下的Fe3O4@TiO241
圖九、Molecularly imprinted polymers (MIPs) 之TEM圖42
圖十、Non-Molecularly Imprinted Polymers (NIPs) 之TEM圖42
圖十一、四氧化三鐵 (Fe3O4) 磁性奈米粒子之FT-IR圖44
圖十二、Fe3O4@TiO2磁性奈米粒子之FT-IR圖45
圖十三、分子印模聚合物之FT-IR圖46
圖十四、Fe3O4與銣鐵硼磁石圖48
圖十五、Fe3O4@TiO2與銣鐵硼磁石圖48
圖十六、Fe3O4與Fe3O4@TiO2磁滯曲線圖49
圖十七、Fe3O4、Fe3O4@TiO2與MIP、NIP磁滯曲線49
圖十八、Fe3O4@TiO2磁性奈米粒子最大吸附量圖51
圖十九、Fe3O4@TiO2磁性奈米粒子吸附時間0.5至8分鐘53
圖二十、不同沖提溶劑與MIP/NIP關係圖55
圖二十一、 MIP與standard phosphoeptide 反應時間57
圖二十二、ß-casein以Fe3O4@TiO2純化前後MALDI質譜圖59
圖二十三、磷酸化胜肽標準品以MIP純化前後MALDI-MS圖61
圖二十四、 磷酸化胜肽純化前後之層析質譜圖63







表目錄
表一、合成奈米粒子之方法比較10
表二、常見的MALDI基質18
表三、HPLC-UV沖堤梯度表36
 

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