本研究旨在探討利用共沉澱法與溶膠−凝膠法，搭配高溫爐鍛燒來合成的具磁性之Ce/Fe複合奈米材料以用於純化複雜生物基質中的多磷酸化胜肽。吾人首先合成四氧化三鐵(Fe3O4)奈米材料，藉由溶膠−凝膠法合成二氧化矽包覆之鐵奈米粒子後，最後利用高溫鍛燒法合成Ce/Fe合金包覆之方形磁性奈米材料(Fe3O4@Ce/Fe)。實驗結果顯示Fe3O4@Ce/Fe奈米材料，可於調整溶液裡的麩胺酸(Glutamic acid)濃度下，對於多磷酸化胜肽有良好的靈敏度和專一性。當利用Fe3O4@Ce/Fe奈米材料作為多磷酸化胜肽選擇性探針時，最低可偵測到20 femtomole之低濃度β-酪蛋白(β-casein)之多磷酸化胜肽外，亦可於牛血清蛋白(BSA)和β-casein以莫爾數比5000:1的比例混合下，有效偵測屬於-casein的多磷酸化胜肽片段。此外，Fe3O4@Ce/Fe奈米材料也能從β-酪蛋白(β-casein)和α-酪蛋白(α-casein)的胰蛋白酶消化胜肽富集多磷酸化胜肽，並能有效真實樣品如脫酯奶粉的分析時，亦可選擇性的分離9條多磷酸化胜肽。

Herein we provide a straightforward strategy for the synthesis of cubic Ce/Fe magnetic nanomaterials (Fe3O4@Ce/Fe) using a sol-gel method under hydrothermal treatment for the enrichment of multi-phosphpeptides in complex biological matrices. Under optimized glutamic acid concentration, Fe3O4@Ce/Fe nanomaterials display superuor affinity towards muti-phosphopeptides with good detection limit (20 fmol for β-casein tryptic lysates), immoderate selectivity (5000:1 molar ratio of bovine serum albumin digest vesus β-casein digest), and reasonable repeatability. In addition, the improved performance of muti-phosphopeptides enrichment by Fe3O4@Ce/Fe has been shown by serial dilution of sample matrix composed of α-casein and b-casein digests. Moreover, the Fe3O4@Ce/Fe also demonstrate their feasibility by efficiently capturing 9 muti-phosphopeptides from a milk powder digest.

總目錄
謝誌 I
中文摘要 III
英文摘要 V
總目錄 VII
圖目錄 XI
表目錄 XV
一、 緒論 1
1.1 奈米材料和磁性奈米材料的基本特性 1
1.2 利用質譜法進行蛋白質體學或磷酸化蛋白質體學分析 3
1.3 固定化金屬離子層析法(immobilized metal-ion affinity chromatography，IMAC) 5
1.4 金屬氧化物親和層析法(metal oxide affinity chromatography，MOAC) 6
1.5 以溶膠−凝膠法(sol-gel method)製備二氧化矽包覆之鐵奈米粒子 7
1.6 富集磷酸化胜肽的方法 9
二、 研究目標 13
三、 研究內容 15
3.1 藥品 15
3.2 儀器 16
3.3 實驗方法 17
3.3.1 Fe3O4奈米材料的合成 17
3.3.2 Fe3O4@MPS的合成 17
3.3.3 Fe3O4@Ce/Fe(1:0.3)的合成 17
3.3.4 Fe3O4@Ce/Fe(1:0.6)的合成 17
3.3.5 Fe3O4@Ce/Fe(1:0.9)的合成 18
3.3.6 Fe3O4@Ce/Fe(1:1.2)的合成 18
3.3.7 製備α-casein經胰蛋白酶酵素消化之胜肽樣品 18
3.3.8 製備β-casein經胰蛋白酶酵素消化之胜肽樣品 19
3.3.9 製備BSA經胰蛋白酶酵素消化之胜肽樣品 19
3.3.10 製備脫脂牛奶經胰蛋白酶酵素消化之胜肽樣品 19
3.3.11 製備人類血清(human serum)經胰蛋白酶酵素消化之胜肽樣品 20
3.3.12 利用所合成之磁性奈米材料進行磷酸化胜肽富集 20
3.3.13 利用Fe3O4@Ce/Fe(1:0.6)奈米材料富集脫脂奶粉中經由胰蛋白酶消化後產生之磷酸化胜肽 20
3.3.14 利用Fe3O4@Ce/Fe(1:0.6)奈米材料富集人類血清中經由胰蛋白酶消化後產生之磷酸化胜肽 20
3.4 儀器設定參數 21
3.4.1 MALDI-MS參數設定 21
3.4.2 MALDI-MS校正 21
四、 實驗結果 23
4.1 合成並鑑定所合成之Ce/Fe修飾之鐵奈米粒子 23
4.2 量測不同奈米材料的FT-IR圖譜 26
4.3 不同磁性奈米材料的磁性能力分析 28
4.4 不同奈米材料的熱重分析儀之結果分析 30
4.5 不同奈米材料的X光繞射分析圖譜分析 32
4.6 Fe3O4@Ce/Fe奈米材料的能量色散X射線譜分析 34
4.7 研究不同奈米材料的富集效果 35
4.8 合成不同莫爾比例的Fe3O4@Ce/Fe奈米材料 37
4.9 不同奈米材料的Zeta-potential圖譜分析 39
4.10 不同莫爾比例Fe3O4@Ce/Fe奈米材料的ICP-MS圖譜 41
4.11 不同莫爾比例Fe3O4 @Ce/Fe奈米材料的XRD圖譜 43
4.12 不同莫爾比例Fe3O4@Ce/Fe奈米材料的富集效果 45
4.13 不同奈米材料的X射線光電子能譜學圖譜分析 47
4.14 Loading Buffer中不同濃度的Glutamic acid對於多磷酸化胜肽之富集效果 51
4.15 不同Glutamic acid濃度對於α-casein與-casein多磷酸化胜肽之富集效果 53
4.16 研究不同奈米材料對於α-casein與-casein富集單磷酸和多磷酸的效率 56
4.17 Fe3O4@Ce/Fe(1:0.6)富集不同濃度下α-casein與-casein磷酸化胜肽之效率 58
4.18 利用最佳化Fe3O4@Ce/Fe奈米材料富集含有不同數量磷酸根之磷酸化胜肽 60
4.19 利用最佳化Fe3O4@Ce/Fe(1:0.6)奈米材料做重複性的測試 61
4.20 以Fe3O4@Ce/Fe(1:0.6)富集β-casein磷酸化胜肽之最低濃度的測試 62
4.21 以Fe3O4@Ce/Fe(1:0.6)奈米材料不同複雜的基質中富集β-casein磷酸化胜肽 63
4.22 利用最佳化Fe3O4@Ce/Fe奈米材料和相對較小的Fe3O4@Ce/Fe奈米材料比較β-casein富集效果 64
4.23 利用最佳化Fe3O4@Ce/Fe奈米材料富集脫酯牛奶之磷酸化胜肽 66
4.24 利用最佳化Fe3O4@Ce/Fe奈米材料富集人類血清之磷酸化胜肽 67
五、 結論 69
六、 參考文獻 71

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