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研究生: 宋祖儀
Tsue-Yi Sung
論文名稱: 比較粒子填充式與聚合式分離管柱搭配奈流液相層析串聯質譜儀分析法於蛋白質體學研究
Comparison of SDVB-Monolithic & Bead-Based Columns Used in Nanoflow LC/MS for Proteomic Study
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2014
畢業學年度: 102
語文別: 中文
論文頁數: 94
中文關鍵詞: 奈流液相層析整體聚合苯乙烯二乙烯苯共聚物熔融核粒子質譜儀
英文關鍵詞: nanoHPLC, monolithic, SDVB, fused core, mass spectrometry
論文種類: 學術論文
相關次數: 點閱:165下載:5
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  • 在蛋白質體學的研究領域中,奈米級液相層析連接電噴灑游離源搭配串聯式質譜儀系統是相當重要的分析工具,當中以移動相不含鹽類的逆相層析法具有最好的儀器相容性與極佳的解析能力。本篇研究中,我們將採用shotgun proteomics分析策略,比較實驗室所自製的三種逆相層析毛細管分離管柱,並選用經胰蛋白酶水解之胜肽作為樣品,搭配串聯式質譜儀系統(QTOF)進行分析,藉此研究不同類型管柱對胜肽樣品分離特性。製備的毛細管分離管柱包括了兩種粒子填充式管柱totally porous silica C18 particle (3 μm, 100 Å)、HALO® fused core C18 particle (2.7 μm, 90 Å),與一種聚合式管柱 SDVB monolithic (50 μm X 400 cm);並於實驗中優化monolithic column製備之條件,包括聚合物種類、單體濃度、毛細管內徑、致孔劑組成、進樣量等條件逐一進行測試。實驗結果顯示40% SDVB單體濃度添加5% 1-propanol作為致孔劑配合內徑50 μm的毛細管所製備的管柱具有最佳的分離效能,且monolithic管柱可大幅度的降低系統運作壓力。搭配totally porous C18作為trapping column下,四公尺的SDVB column能夠有效降低co-elute干擾與減緩離子抑制現象,因此可大幅提升整體訊號強度,並增加質譜分析時所得到的譜圖數目;同時於GRAVY value分析中可發現SDVB與HALO® 能明顯提升親水性胜肽整體的鑑定比率;
    同時亦透過萃取離子層析圖(XIC)來分析胎球蛋白(fetuin)的醣基化胜肽,結果顯示SDVB 與C18 相較於HALO®能鑑別較多的醣基化胜肽。這種新型的固定相分離介質具有廣泛的操作便利性與極佳的分離效能,相信能在蛋白質體學的研究中能夠展現出更完備的應用性。

    Nanoflow liquid chromatography coupled with electrospray tandem mass spectrometry (nanoLC-ESI-MS/MS) is a powerful tool in proteomic analysis. The preparation conditions for the SDVB-Monolithic column including the choice of monomer, inside diameter of capillary, and porogen are investigated. The performance of polymeric column was also compared and evaluated with micro particle-filled capillary columns, including a totally porous silica C18 column (75 μm x 100 mm, 3 μm, 100Å) and a HALO® fused core C18 column (75 μm x 100 mm, 2.7 μm, 90 Å). With all optimized conditions, the monolithic capillary column was prepared by in-situ polymerization of styrene and divinylbenzene (SDVB) inside a 4 meter-long, 50 μm i.d. fused silica capillary using 1-propanol as porogen. This continuous unitary porous structure provides more robust and high separation efficiency when comparing with the bead-based columns. Since the meter-long SDVB column could substantially reduce peptides co-elution and abate the ion suppression, the total ion current signal could be significantly enhanced with comparable flow rate and pressure. For the peptide identification in fetuin, the use of SDVB column in shotgun approach can identify more glycopeptides than HALO® column. The characterization of this novel monolithic media will be a promising addition to the stationary phase used in capillary column for proteome research.

    目錄 I 中文摘要 III ABSTRACT IV 縮寫 V 圖目錄 VIII 表目錄 X 第一章導論 1 1.1前言 1 1.2高效能液相層析法 2 1.3管柱填充材料 4 1.3.1 Totally porous silica particle 4 1.3.2 HALO® fused core particle 5 1.3.3 Monolithic 6 1.4質譜儀 6 1.4.1電噴灑游離 10 1.4.2四極柱串聯時間飛行式質量分析器 11 1.5質譜蛋白質鑑定 12 1.5.1 胜肽質量指紋法 12 1.5.2 胜肽碎片指紋鑑定 13 1.5.3 De Novo sequencing 13 1.5.4醣基化修飾鑑定 14 1.6研究動機 14 第二章 實驗材料 16 2.1 樣品 16 2.2 材料 16 2.3 藥品 16 2.4 儀器設備 17 第三章 實驗方法 19 3.1 蛋白質樣品製備 19 3.2 Totally porous silica trapping column preparation 19 3.3 SDVB monolithic trapping column preparation 20 3.4 Totally porous silica analytical column preparation 21 3.5 HALO® fused core analytical column preparation 21 3.6 SDVB monolithic analytical column preparation 22 3.7 液相層析連結電噴灑游離化界面設定 23 3.8樣品進樣 24 3.9 串聯式質譜儀掃描模式設定 24 3.10 資料分析 25 3.11 掃描式電子顯微鏡分析 26 第四章 實驗結果與討論 27 4.1 SDVB monolithic column preparation condition 27 4.1.1聚合物型態及濃度測試 28 4.1.2不同內徑毛細管效能分析 29 4.1.3不同管柱選配與承受進樣樣品量分析 30 4.1.4製備步驟添加致孔劑測試 31 4.1.5 SDVB monolithic管柱製備優化條件 32 4.2 三種管柱應用於Regular protein分析之質譜分析結果 33 4.3三種管柱應用於Glycoprotein分析之質譜分析結果 33 4.4實驗所鑑定的胜肽親疏水性質分布 34 4.5 實驗中所鑑定到的胜肽分子量分布 35 4.6 實驗中不同管柱所鑑定的胜肽互補性差異 35 4.7 三種分離管柱對tryptic BSA分析之全離子層析圖 36 4.8相同胜肽於不同管柱間沖提時間 36 第五章 結論與未來展望 37 圖表說明 38 參考文獻 91

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