臺灣博碩士論文加值系統

血液中存在許多游離的小分子，比較常見的小分子像是糖或是雌激素。血液中的4-OHE2(4-hydrosyestradiol)在體內會受到細胞色素P450或是過氧化酶催化成具有遺傳毒性的醌類代謝產物E2-3,4-Q，此類代謝物會與DNA上的鳥嘌呤及腺嘌呤形成鍵結而形成脫嘌呤的加成物，使得DNA序列產生缺陷進而產生突變使得癌症發生。
糖化血紅蛋白(HBA1c)主要源自於血紅蛋白與單糖產生非酶促結合，高比例的HBA1c除了被認為是糖尿病的評斷標準外，對於與急性心肌梗塞、心力衰竭、急性冠脈綜合症等心血管疾病也具有正相關的風險。
小分子的含量具有高變化性，在檢測上比較困難也具比較高的不確定性。其中血紅蛋白(Hemoglobin,Hb)是紅血球中含量最多的的蛋白，同時也是一種生命周期較長的蛋白，而這些小分子會與血紅蛋白形成加成物，透過檢測蛋白加成物的比例去判斷小分子長時間暴露在血液中的情形，能更準確的評估患病的情況。
傳統的蛋白質分析中進行在進行由下而上的蛋白質組學分析時往往需要花費兩天以上的時間進行樣品前處理以及分析，在微滴反應中很多反應都會被大幅加速，本次實驗會利用離線微滴裝置研究血紅蛋白的葡萄糖、雌激素加成反應以及酵素水解反應將兩天以上的樣品製備時間縮短為數分鐘內，以及透過串聯液相層析後分析特定片段。除此之外我們使用雙管微滴裝置發現酵素水解反應能夠在液滴-液滴之間進行反應，其中Hb-α和Hb-β的序列覆蓋率透過串聯液相層析後更是達到了100%, 91.7%，以及嘗試改變各種參數後觀察微滴在不同微滴尺寸或是pH值以及具有無電壓情況下的反應程度。或是將雌激素加成反應以及酵素水解分成兩管後讓反應有順序的發生外，在極短的時間內進行兩種不同的反應，並且透過與液相層析設備串聯分析出具有修飾的胜肽片段。

Blood contains numerous free small molecules, with sugars and estrogens being common examples.The catechol estrogen metabolite of 4-hydroxyestradiol (4-OHE2), E2-3,4-Q,can form adducts with adenine and guanine on DNA, leading to DNA depurination and cause cancer.Glycated hemoglobin (HbA1c) levels can elevate not only serve as diagnostic criteria for diabetes but also carry a positive correlation with cardiovascular diseases.
Small molecules exhibit high variability in their abundance, making them challenging to detect. Hemoglobin (Hb), which is the most abundant protein in red blood cells and has a longer lifetime, forms adducts with these small molecules. By evaluating the proportion of protein adducts, it becomes possible to more accurately assess the prolonged exposure of small molecules in the blood and better evaluate disease conditions.
Traditional protein analysis, particularly bottom-up proteomics, often requires over two days for sample preparation and analysis. In microdroplet reactions, many processes can be significantly accelerated. In this experiment,offline microdroplet device is used to study the estrogen adduction reaction and enzymatic digestion of hemoglobin. This enables the reduction of sample preparation time from over two days to a few minutes. Additionally, specific fragments are analyzed through tandem liquid chromatography. Furthermore, using a dual-spray microdroplet device, it was discovered that enzymatic digestion reactions can occur between droplets, achieving a sequence coverage of 100% for Hb-α and 91.7% for Hb-β through tandem liquid chromatography. Various parameters are adjusted to observe the extent of microdroplet reactions under different conditions. By dividing the estrogen adduction reaction and enzymatic digestion into two separate channels, the reactions occur in a sequential manner, allowing for two different reactions to take place in an extremely short time.

摘要 i
Extend Abstract ii
致謝 vi
目錄 vii
表目錄 x
圖目錄 xi
第一章 文獻回顧 1
1-1 蛋白質轉譯後修飾 1
1-1-1 雌激素 1
1-1-2 糖化血紅蛋白 5
1-2 血紅蛋白 7
1-3 加成物的定量 8
1-3-1 雌激素定量 8
1-3-2 糖化血紅蛋白定量 9
1-4 質譜技術 11
1-4-1 質譜基本原理 11
1-4-2 離子源 12
1-4-3 質量分析器 13
1-5 蛋白質組學 15
1-6 微滴化學 18
第二章 材料與實驗方法 20
2-1 實驗藥品與儀器 20
2-1-1 藥品 20
2-1-2 實驗儀器與耗材 21
2-2 實驗方法 22
2-2-1 血液檢體 22
2-2-2 體相樣品製備(Bulk Phase) 23
2-2-3 微滴樣品製備 25
2-2-4 四極桿 –飛行式串聯質譜儀分析 28
第三章 結果與討論 29
3-1 體相反應-完整蛋白分析 29
3-1-1 血液中的血紅蛋白-雌激素加成物分析 29
3-1-2 血液中的血紅蛋白-葡萄糖加成物分析 30
3-2 體相反應-MS1片段分析 35
3-2-1 酵素水解血紅蛋白加成物分析 35
3-3 微滴反應-完整蛋白分析 37
3-3-1 雌激素加成反應 37
3-3-2 葡萄糖加成反應 44
3-4 微滴反應- MS1片段分析 46
3-4-1 酵素水解血紅蛋白 46
3-4-2 酵素水解血紅蛋白加成物分析 48
3-5 微滴反應-雙噴頭微滴反應 50
3-5-1 酵素水解反應 50
3-5-2 微滴加成-水解反應 57
第四章 結論 61
第五章 參考文獻 63
第六章 附錄 68

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