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研究生： 陳偉豪
Chen, Wei-Hao
論文名稱： 整合不同液相層析技術與質譜平台進行蛋白質複合體的自下而上分析
Bottom-up analysis of protein complexes through the integration of multiple liquid chromatography techniques and mass spectrometry platforms
指導教授： 賴思學
Lai, Szu-Hsueh
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系
Department of Chemistry
論文出版年： 2025
畢業學年度： 114
語文別： 中文
論文頁數： 63
中文關鍵詞： 尺寸排阻層析法 、電噴灑發射器 、毛細管管柱製備 、液相層析串聯質譜 、蛋白質體學
外文關鍵詞： Size exclusion chromatography, Electrospray emitter, Packing Capillary column, Liquid chromatography–tandem mass spectrometry, Proteomics
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    • 蛋白質體學為鑑定和定量細胞、組織或生物體內總蛋白質含量的技術應用，以了解蛋白質的結構與功能。基於蛋白質體學的技術有著廣泛的應用，例如診斷標記物、理解致病機制和研究蛋白質。我們將整合高效液相層析尺寸排阻法(HPLC-SEC)與奈流液相層析聯用質譜(nLC-MS/MS)建立一套比較由上至下(Top-down)與由下至上(Bottom-up)的蛋白體學分析方法，首先初步處理樣品後，取得的混合蛋白質以酶消化作用後，得到特定位點胜肽片段經由nLC-MS/MS分離及質量分析後，以軟體比對蛋白質數據庫得到樣品中蛋白質的種類和覆蓋度。本研究以完善 HPLC-SEC 結合Native-MS與 nLC-MS/MS 系統， HPLC-SEC 中以牛血清蛋白作為標準進行儀器測試後將用於大腸桿菌核醣體的分離與純化；在 nLC-MS/MS 系統中建立 Bottom-up 的蛋白體學流程，工作包含蛋白質樣品前處理、毛細管管柱的設備建立和製備、優化奈流液相層析與質譜系統、數據分析處理等等，並進一步改善 nLC-MS/MS 平台，例如層析流速及梯度、奈流管柱製備、電噴灑發射器的開口尺寸、質譜參數等等。在自製毛細管柱上也取得初步的成效，理論板數可達15000，並搭配質譜儀可以做到蛋白質分析。我們主要使用消化的牛血清蛋白確立系統的穩定性和重複性。因此對於複雜樣品有良好的分離效果和訊號強度。最後我們操作本平台應用於更大的蛋白質複合體，以核醣體為例其蛋白質種類和數量皆有在軟體中被比對出。我們在本研究中整合了 HPLC-SEC 方法與Native-MS以 Top-down分析與一套可行的 nLC-MS/MS ，以由下至上質譜法進行複雜樣品中胜肽的定性分析。

    This study successfully established a robust and integrated proteomic workflow combining high-performance liquid chromatography–size exclusion chromatography (HPLC-SEC) and nano-liquid chromatography tandem mass spectrometry (nLC-MS/MS). This platform enables the comprehensive structural characterization of protein complexes through both top-down and bottom-up analytical approaches.
    Initially, HPLC-SEC was meticulously optimized using bovine serum albumin (BSA) as a standard to rigorously assess column performance, dead volume, and achieve precise flow rate control. The optimized SEC step was then critically applied to the purification of intact E. coli ribosomal complexes, which were subsequently subjected to high-resolution Native-MS analysis.
    For the nLC-MS/MS component, we developed custom nano-electrospray emitters and fabricated self-packed capillary columns, significantly enhancing chromatographic resolution and stability. System optimization, including gradient and flow rate refinement, yielded superior separation efficiency with a peak capacity of approximately 200 and narrow peak widths (0.3–0.9 min). This optimized system notably boosted BSA peptide identification from 20 to 30. Ultimately, the platform identified 260 peptides from ribosomal samples, providing extensive coverage of both S30 and S50 ribosomal proteins, proving its high stability, sensitivity, and broad applicability for complex structural analysis.

    第一章 緒論 1 1.1 層析分離技術 1 1.1.1 尺寸排阻層析法 1 1.1.2 逆相層析法(Reversed-Phase Liquid Chromatography, RPLC) 3 1.1.3 層析分離技術在蛋白質體學的應用 5 1.2 毛細管管柱製備 5 1.2.1 融塊篩板技術(Column Frits ) 5 1.2.2 管柱填料方法 6 1.2.3 高壓漿料填料 (high-pressure slurry packing) 7 1.3 質譜儀 7 1.3.1 質譜儀簡介 7 1.3.2 電噴灑發射器 8 1.4 蛋白質體學(Protemics ) 8 1.4.1 蛋白質體學相關技術與方法 9 1.4.2 整合層析與質譜技術 10 1.5 整合不同液相層析技術與質譜平台進行蛋白質複合體的分析 10 第二章 實驗方法 12 2.1 HPLC-SEC靈敏度與蛋白質分離定性 12 2.2 奈流液相層析串聯質譜(nanoLC-MS/MS) 13 2.2.1 製備質譜電噴灑發射器 13 2.2.2 壓力彈設計 13 2.2.3 毛細管管柱測試 14 2.2.4 最佳化奈流液相層析表現 14 2.2.5 質譜的參數優化 14 2.2.6 數據處理 15 2.3 HPLC-SEC 與 nLC-MS 對核醣體複合體的分析 15 2.3.1 由上至下法純化及收集核醣體 15 2.3.2 由下至上法分析核醣體蛋白 15 第三章 實驗結果與討論 16 3.1 尺寸排阻法分離並純化蛋白質 16 3.1.1 UV/Vis檢測極限與 SEC 管柱效能分析 16 3.1.2蛋白質定量與收集 18 3.1.3 HPLC-SEC 之應用不同蛋白複合體 20 3.2 建置奈流液相層析儀串聯質譜儀 21 3.2.1 奈米質譜發射器 21 3.2.3 最佳化奈流液相層析儀聯用質譜儀系統 25 3.2.3 質譜參數優化 29 3.2.4 胜肽層析梯度優化 29 3.3 整合Top-down與Bottom-up方法分析核醣體 31 3.3.1 由上至下法測量分離後核醣體 31 3.3.2 由下至上法分析核醣體蛋白 33 第四章 結論 35 參考文獻 36 附錄 38 S-A 藥品、儀器、耗材與儀器參數 38 S-B實驗方法 40 S-B.1 製備毛細管管柱 40 S-B.2 蛋白質消化 40 S-C Top-down 與 Bottom-up 結合實例 41 S-D 在不同條件下分離與鑑定核醣體 43 S-E 補充資料圖 44

    1. Coskun, O., Separation techniques: chromatography. Northern clinics of Istanbul, 2016. 3(2): p. 156.
    2. Mould, D. and R. Synge, Separations of polysaccharides related to starch by electrokinetic ultrafiltration in collodion membranes. Biochemical journal, 1954. 58(4): p. 571.
    3. Wheaton, R. and W. Bauman, Non‐ionic separations with ion exchange resins. Annals of the New York Academy of Sciences, 1953. 57(3): p. 159-176.
    4. Lindqvist, B. and T. Storgårds, Molecular-sieving properties of starch. Nature, 1955. 175(4455): p. 511-512.
    5. Shi, Y., et al., The role of liquid chromatography in proteomics. Journal of Chromatography A, 2004. 1053(1-2): p. 27-36.
    6. Shan, L. and B.R. Jones, Nano‐LC: An updated review. Biomedical Chromatography, 2022. 36(5): p. e5317.
    7. Sanders, K.L. and J.L. Edwards, Nano-liquid chromatography-mass spectrometry and recent applications in omics investigations. Analytical Methods, 2020. 12(36): p. 4404-4417.
    8. Cheong, W.J., Stainless steel tubing/frit with sintered inorganic particle, the chromatography comprising it, and their manufacturing method. 2006, Google Patents.
    9. Cheong, W.J., Fritting techniques in chromatography. Journal of separation science, 2014. 37(6): p. 603-617.
    10. Perchepied, S., et al., Insights in column packing processes of narrow bore and capillary-scale columns: Methodologies, driving forces, and separation performance–A tutorial review. Analytica Chimica Acta, 2022. 1235: p. 340563.
    11. Blue, L.E. and J.W. Jorgenson, 1.1 μm superficially porous particles for liquid chromatography. Part I: synthesis and particle structure characterization. Journal of Chromatography A, 2011. 1218(44): p. 7989-7995.
    12. Gibson, G.T., S.M. Mugo, and R.D. Oleschuk, Nanoelectrospray emitters: trends and perspective. Mass Spectrometry Reviews, 2009. 28(6): p. 918-936.
    13. El-Faramawy, A., K.M. Siu, and B.A. Thomson, Efficiency of nano-electrospray ionization. Journal of the American Society for Mass Spectrometry, 2005. 16(10): p. 1702-1707.
    14. Al-Amrani, S., et al., Proteomics: Concepts and applications in human medicine. World journal of biological chemistry, 2021. 12(5): p. 57.
    15. Aslam, B., et al., Proteomics: technologies and their applications. Journal of chromatographic science, 2016: p. 1-15.
    16. Lenco, J., et al., Reversed-phase liquid chromatography of peptides for bottom-up proteomics: a tutorial. Journal of Proteome Research, 2022. 21(12): p. 2846-2892.
    17. Petelski, A.A. and N. Slavov, Analyzing ribosome remodeling in health and disease. Proteomics, 2020. 20(17-18): p. 2000039.
    18. Emmott, E., M. Jovanovic, and N. Slavov, Approaches for studying ribosome specialization. Trends in biochemical sciences, 2019. 44(5): p. 478-479.
    19. Satish, L., et al., Thermal aggregation of bovine serum albumin in conventional buffers: an insight into molecular level interactions. Journal of Solution Chemistry, 2017. 46(4): p. 831-848.
    20. Peng, W., et al., Analytical strategies for identification and quantitation of heterodimers in co-formulated monoclonal antibody cocktails by native SEC-MS. Scientific Reports, 2025. 15(1): p. 28042.
    21. Reschke, B.R. and A.T. Timperman, A study of electrospray ionization emitters with differing geometries with respect to flow rate and electrospray voltage. Journal of The American Society for Mass Spectrometry, 2011. 22(12): p. 2115-2124.
    22. Schweiger, S. and A. Jungbauer, Scalability of pre-packed preparative chromatography columns with different diameters and lengths taking into account extra column effects. Journal of Chromatography A, 2018. 1537: p. 66-74.
    23. Berg, H.S., et al., Self-packed core shell nano liquid chromatography columns and silica-based monolithic trap columns for targeted proteomics. Journal of Chromatography A, 2017. 1498: p. 111-119.
    24. Wöhlbrand, L., et al., Influence of NanoLC column and gradient length as well as MS/MS frequency and sample complexity on shotgun protein identification of marine bacteria. Journal of Molecular Microbiology and Biotechnology, 2017. 27(3): p. 199-212.
    25. McKay, A.R., et al., Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. Journal of the American Chemical Society, 2006. 128(35): p. 11433-11442.
    26. Zhang, Y., et al., Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity. Journal of Pharmaceutical Analysis, 2023. 13(1): p. 63-72.

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