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標題: | 開發整合型醣胜肽分析策略應用於探討血清生物標記蛋白質 Development of an Integrated Glycopeptide Analytical Strategy for Serum Protein Biomarker Analysis |
作者: | Chen-Chun Chen 陳貞均 |
指導教授: | 翁啟惠 |
關鍵字: | 蛋白質醣基化,串聯式質譜技術,醣胜?定量,甲種胎兒蛋白,肝癌, N-linked glycosylation,intact glycopeptide identification,mass spectrometry,targeted quantification,alpha-fetoprotein,hepatocellular carcinoma, |
出版年 : | 2017 |
學位: | 博士 |
摘要: | 蛋白質醣基化為一個相當普遍的後轉譯修飾,並且在生物體中扮演著許多重要的角色,例如:細胞辨識、免疫細胞自我調節功能等,近年來,蛋白質醣基化被廣泛的研究,其中醣基化程度的異常表現被認為與人類癌症發展具有高度關聯性。隨著質譜技術的發展與演進,質譜儀成為研究蛋白質體不可或缺的分析工具,然而,由於醣基化蛋白質在細胞中的低含量及其醣型結構的複雜程度,以質譜技術為基礎的醣蛋白分析仍面臨許多挑戰。基於上述原因,我們發展一整合型的醣胜肽分析策略結合生物資訊的技術平台分析及酵素輔助質譜定量技術,期許此一研究平台可加速我們對於蛋白質醣基化生物意義上的了解。
在本論文第二章,我們藉由高解析度及高靈敏度的串聯式質譜技術結合生物資訊方法發展準確的醣胜肽鑑定軟體,利用搜尋串聯質譜圖中特有的醣胜肽碎片,自動產生可用於蛋白質資料庫作鑑定的檔案格式,最後全面性的對醣胜肽之定序、特定位點的醣結構、及蛋白質作完整分析並依其候選醣型型式提供分數以供使用者參考。我們將此分析工具分別鑑定不同樣品複雜度的串聯式質譜圖以確認其實際應用上的表現,其結果顯示,此一平台提供快速且精準的分析,預期能應用於未來大規模醣蛋白質體的研究。 此外,於本論文第三章,我們開發以質譜為基礎的醣蛋白定量技術。由於岩藻醣化(core-fucosylated)的甲種胎兒蛋白為美國食品藥品管理局(Food and Drug Administration 簡稱FDA)認可的肝癌生物標記分子,因此我們以其此蛋白質作為方法開發的分析標的。然而,醣型結構的多樣性造成醣胜肽游離程度的極大差異,直接定量醣胜肽在實際應用上並不可行,因此,我們利用切醣酵素使醣胜肽統一化做僅一至兩顆醣修飾的胜肽片段,這樣的方法使得其目標分子離子的質荷比變為已知,也使得定量質譜術可充分用於特定位點的醣胜肽的定量分析。此一方法預期在未來可應用於實際臨床樣品的分析。 Protein glycosylation is one of the most common post-translational modifications and has received increased attention for its critical role in cell biology and diseases. Despite its biological significance, the delineation of intact glycopeptides and site-specific glycopeptide quantification still await exploration. In this dissertation, we established an analytical workflow integrating a newly-designed automated tool for glycopeptide profiling and an enzyme-assisted targeted glycopeptide quantification to facilitate the understanding of glycosylation in diseases. First, Mass-spectrometry-based Automated Glyco-peptide IdentifiCation platform (MAGIC), an in-house automated computational tool was developed to accurately characterize the intact glycopeptides without prior knowledge of the proteins and glycans present in the sample. Using a novel “Trident” strategy that detects a triplet pattern corresponding to [Y0, Y1, Y2] or [Y0−NH3, Y0, Y1] from the fragmentation of the common trimannosyl core of N-linked glycopeptides, the Y1 (i.e. peptide+GlcNAc) ion was identified from beam-type collision-induced dissociation tandem mass spectra. After Y1 assignment, the software generates in silico spectra by overwriting the original precursor with the naked peptide m/z and removing all of the glycan-related ions to identify the peptide sequence by common database search engines. Finally, MAGIC computes the glycan compositions and ranks them. Result from several datasets with various sample complexity from single protein to cell lysate implied that MAGIC enables to identify N-linked intact glycopeptide at a high speed and with high accuracy. In the second part, we developed a mass spectrometry based methodology to study the altered core-fucosylation in human sera. In this study, we selected alpha-fetoprotein (AFP) as a model target analyte since its core-fucosylated isoform has been used as a diagnostic and prognostic marker for hepatocellular carcinoma (HCC). To overcome the limitation on quantitative mass spectrometry, we took an advantage of endoglycosidase, which specifically cleaves the glycosidic bond between two GlcNAc residues, to reduce the microheterogeneity of the glycan on peptide backbone. This will result in truncated glycopeptides, e.g. peptide with GlcNAc or peptide with coreFuc GlcNAc, which can be directly analyzed by LC-MS/MS and quantified by extracting the peak area of target m/z. We evaluated the analytical figures of merit of the proposed method by following the guideline of the US Food and Drugs Administration (US FDA) for bioanalytical method validation. The targeted glycopeptide quantitation had good linearity from 15.6-1000 ng/mL (r2=0.9930 for interday experiments) and a limit of detection of 15.6 ng/mL. We anticipate applying the proposed protocol to understand the correlation between glycosylation and liver diseases by calculating the ratio of core-fucosylated to non-core-fucosylated glycopeptides in different patient groups. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59892 |
DOI: | 10.6342/NTU201700296 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
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