臺灣博碩士論文加值系統

文獻指出兒茶酚雌激素(Catechol Estrogen, CE)共價修飾是雌激素失衡和癌症的潛在生物標誌，而兒茶酚雌激素代謝物：高活性的醌類(quinone)致癌性物質，已被證實能夠與血清白蛋白上的離胺酸 (Lysine, K)、半胱胺酸 (Cysteine, C)和組胺酸(Histidine, H)進行共價鍵結形成雌激素修飾蛋白，由於蛋白具有較長的半生期，因此測量蛋白上的加成物更能反應出這些化學致癌物的水平。
本篇論文開發利用平行反應監測模式(PRM)來去偵測雌激素化蛋白，並定量血清白蛋白上兒茶酚雌激素的共價修飾程度，並應用在臨床樣品上評估其可行性。在此，我們證明了雌激素與HSA的共價修飾程度可以用來測定人類血液中循環兒茶酚雌激素水平。透過加入一系列不同濃度的兒茶酚雌激素與血清一起活化，產生各種共軛-CE/HSA比例，透過完整蛋白質的測量與分析，可以得到兩個線性良好的濃度範圍: 0-19μg/mL (R2=0.97)與57-1900 μg/mL (R2=0.99)；接著用胰凝乳蛋白酶與胰蛋白酶的消化，並利用nano-LC Q-Orbitrap平行反應模式(精確的MS2質量〈0.05Da)靶向定量C34、K20、K378位點片段(胰凝乳蛋白酶)；K20、C34、K73、K281、H338、K378位點片段(胰蛋白酶)的修飾與未修飾片段。透過胰凝乳蛋白酶的消化，透過兩種共價程度位點之合併的計算(共價程度/Site、共價程度/HSA)，皆會得到良好的線性且RSD下降，但與完整蛋白斜率趨勢相反；而與胰凝乳蛋白酶相比，胰蛋白酶能夠得到更低的定量極限，且RSD較小(C34：2.96-28.90%)，與共軛-CE/HSA相比，在低濃度下數據趨近吻合(斜率=1.9)，主要為C34之貢獻，且R2〉0.99。最後，我們將此方法應用在代謝異常病患與健康者的血清臨床樣品上，在完整蛋白分析，病患平均皆比健康者高，p值為0.000379，顯示兩者有顯著差異；使用胰蛋白酶部分，雖然經T.Test測試目前並無顯著差異，但病患平均可以看到比健康者高。我們證明了雌激素與HSA的共價修飾程度可以用來測定人類血液中循環兒茶酚雌激素水平。

Recent study points out that covalent modification of Catechol Estrogen (CE) is an estrogen imbalance and a potential biomarker for cancer. In this study, a method developed to detect and relative quantitative CEs-adducted serum proteins based on LC-MS/MS using parallel reaction monitoring (PRM). Instead of free estrogen molecules, the protein has a longer half-life, CEs-adducted serum proteins are more able to reflect these chemical carcinogen levels in blood.
Blood serum was added a series of concentrations of CEs to yield various conjugated-CE/HSA ratios’ serum standards. Through the measurement and analysis of intact proteins, exhibited two good linearity could be obtained: 0-19 μg/mL (R2=0.97) and 57-1900 μg/mL (R2=0.99). These serum standards were cleaned up by trichloroacetic acid (TCA) and digested by chymotrypsin and trypsin. In bottom-up analysis three conjugated and non-conjugated peptide pairs covering the site of K20、C34、K378 for chymotrypsin and six conjugated and non-conjugated peptide pairs covering the site of K20、C34、K73、K281、H338、K378 for trypsin were identified and targeted by PRM with accurate fragment mass (〈0.05 Da), using nanoLC QE-Orbitrap. In the two- different enzymes system, the conjugation level/ site and conjugation level/ HSA derived from 18 fragments (chymotrypsin) and 63 fragments (trypsin) were revealed to have good linearity and RSD. However, the limit of quantification and the RSD calculated in the trypsin system were lower than it was in the chymotrypsin system.
Compared with the conjugated-CE/HSA, the data was close to the agreement at the low concentration (slope=1.9), mainly contribution was the site of C34 and had good linearity (R2=0.99). The method was applied to clinical samples to detect conjugation level in serum on 10 healthy individuals and 10 patients. In the intact protein analysis, patient conjugation level could see the significant difference (p=0.000379). In the bottom-up analysis, we can not see the significant difference between the T.Test but the average of patient can be seen to be higher than healthy.

摘要 I
Abstract II
誌謝 VIII
目錄 IX
表目錄 XI
圖目錄 XII
簡寫表 XV
第一章 文獻回顧 1
1.1 蛋白質體學 1
1.2 雌激素與4-羥基雌二醇(4-hydroxyestradiol) 2
1.2.1 雌激素與其代謝途徑 2
1.2.2 雌激素的定量分析 4
1.2.3 雌激素與肥胖的關係 5
1.3 人類血清白蛋白簡介 7
1.3.1 人類血清白蛋白的加合物 9
1.4 質譜儀於蛋白質體上的應用 11
1.4.1 使用質譜儀進行蛋白質定量分析 12
1.4.2 靈敏度、偵測極限、檢量線 14
1.5 質譜儀簡介 15
1.6 質譜掃描模式 19
第二章 實驗動機 21
第三章 實驗方法 22
3.1 實驗藥品與儀器 22
3.1.1 實驗藥品 22
3.1.2 實驗耗材及儀器 23
3.2 樣品前處理 24
3.2.1 血液檢體 24
3.2.2 蛋白質的定量 24
3.2.3 血清樣品雌激素活化 - 檢量線製作 24
3.2.4 蛋白質水解 – 三氯乙酸沉澱法 25
3.2.5 奈升級高效能液相層析儀參數設定 26
3.2.6 質譜方法設定 28
3.2.7 資料庫分析軟體 29
第四章 結果與討論 30
4.1 完整人類血清白蛋白分析 30
4.2 自下而上(Bottom-up)的蛋白質分析 33
4.2.1胰凝乳蛋白檢量線製作 33
4.2.2 胰蛋白酶位點分析 41
4.2.3平行反應監測模式產物離子的選擇 43
4.2.4 平行反應監測模式母離子碰撞能量優化 57
4.2.5 胰蛋白酶檢量線製作 60
4.3血清樣品鑑定 67
4.4胰蛋白酶與胰凝乳蛋白酶之比較 75
第五章 結論 78
第六章 參考文獻 80
第七章 附錄 84

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