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研究生: 林子鈺
Lin, Tze-Yu
論文名稱: 運用iTRAQ化學標定分析C型肝炎病毒感染從急性到慢性不同階段之差異蛋白質體研究
Quantitative proteomics analysis from acute to chronic stages of hepatitis C virus infection by iTRAQ technology
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: 2017
畢業學年度: 105
語文別: 中文
論文頁數: 66
中文關鍵詞: C型肝炎病毒蛋白質體學同重元素相對與絕對定量串聯式液相層析質譜儀
英文關鍵詞: hepatitis C virus (HCV), proteomics, iTRAQ, LC-MS/MS
DOI URL: https://doi.org/10.6345/NTNU202202041
論文種類: 學術論文
相關次數: 點閱:144下載:16
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  • 肝癌屬於世界上較為普及且致命的癌症之一,約20-30%的慢性C型肝炎(HCV)患者容易病變為肝硬化或肝癌。大多數急性C型患者容易轉變成慢性C型肝炎,長期存在於肝臟中,且無明顯病徵。C型肝炎雖然有藥物治療可以醫治,但因存在較大的遺傳變異性,目前仍無疫苗可以防範。本實驗中使用Huh7.5-SEAP細胞株作為樣品,比較急性到慢性不同天數受到C型肝炎病毒感染的蛋白質和未受到感染的控制組,進行同重元素相對與絕對定量(iTRAQ)搭配串聯式質譜儀進行分析。藉由二維液相分離,等電點聚焦法(sIEF),強陽離子交換層析法(SCX)和鹼性逆相層析法(bRP)分離已被iTRAQ標記的胜肽,降低樣品的複雜性,也可藉由不同分離方法提供互補性和正交性,鑑定到更多的蛋白質。利用Gene Ontology (GO)軟體分析從急性到慢性差異蛋白質的生物路徑及功能,在本次實驗中發現的差異蛋白與RNA結合、胞外外切體、黑素體和核糖核蛋白複合物結合形成生物路徑有關,未來通過西方墨點法進行方法驗證,尋找能辨別從急性到慢性C型肝炎病毒的標記蛋白質。

    Hepatocellular carcinoma (HCC) is one of the most prevalent and mortal cancer in the world, and 20% to 30% of patients with chronically hepatitis C virus (HCV) lead to liver cirrhosis and liver cancer. Most infected acute HCV people develop chronic HCV easily. Due to genetic variability of HCV, the development of antiviral drugs and vaccines becomes a real challenge. In this study, naïve Huh7.5-SEAP cells were established and infected by hepatitis C virus, then isobaric tags for relative and absolute quantitation (iTRAQ) was applied to investigate protein profiles in acute and chronic infections. The iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF), strong cationic exchange chromatography (SCX) and basic reverse phase chromatography (bRP) column, followed by nano-LC tandem mass spectrometric analysis. Two-dimensional liquid chromatography technique that employed on iTRAQ labeled peptides provided results with excellent complementarity and orthogonality. In the study, the differentially expressed proteins were found to be associated with RNA binding, extracellular exosome,
    melanosome and ribonucleoprotein complex binding. Besides, selected correlated proteins will be confirmed and validated by Western blot in the future, they could serve as novel diagnostic biomarkers for hepatitis C virus infection.

    目錄 I 圖目錄 IV 表目錄 VI Abstract VII 中文摘要 VIII 縮寫 IX 第一章 序論 1 第一節 C型肝炎病毒(Hepatitis C virus) 1 壹、 急性C型肝炎(Acute infection) 2 貳、 慢性C型肝炎(Chronic infection) 2 第二節 液相層析分離技術 2 壹、 逆相層析法 3 貳、 液相等電點聚焦法 6 參、 離子交換層析法(Ion exchange chromatography, IEC) 7 第三節 蛋白質身分鑑定 9 壹、 胜肽質量指紋(Peptide mass fingerprinting, PMF) 10 貳、 胜肽碎片指紋(Peptide fragment fingerprinting, PFF) 11 第四節 質譜儀技術 11 壹、 電噴灑游離法(Electrospray Ionization, ESI) 13 貳、 線性離子阱(Linear ion trap, LIT) 13 第五節 差異蛋白質體學(Differential proteomics) 15 壹、 穩定同位素標定(Stable isotope labeling) 16 第六節 西方墨點法(Western blot) 19 第七節 研究動機 19 第二章 實驗材料與方法 23 第一節 樣品來源 23 第二節 樣品純化濃縮 24 壹、 材料與儀器設備 24 貳、 實驗步驟 24 第三節 蛋白質濃度測定(Bradford protein assay) 25 壹、 藥品與試劑 25 貳、 儀器設備 25 參、 實驗步驟 25 第四節 蛋白質水解(In-solution digestion)與化學標定iTRAQTM 26 壹、 藥品與試劑 26 貳、 儀器設備 26 參、 實驗步驟 27 第五節 第一維分離策略 27 壹、 等電聚焦分級分離儀 (Solution isoelectric focusing, SIEF) 27 貳、 強陽離子交換層析法 (Strong cationic exchange chromatography, SCX) 29 參、 鹼性逆相層析法(Basic reverse phase chromatography, bRP) 31 第六節 自製碳18離心管柱去鹽(C18 spin column) 32 壹、 材料與試劑 32 貳、 儀器設備 33 參、 實驗步驟 33 第七節 奈米級液相層析電噴灑游離串聯式質譜儀(nanoLC ESI tandom mass spectrometry) 34 壹、 自製液相層析分析管柱 34 貳、 超高效能液相層析(Ultra-performance liquid chromatography, UPLC,Waters) 35 參、 線性離子阱量譜儀(Linear ion trap mass spectrometer, LTQ XL, Thermo Fisher) 36 第八節 資料分析(Data analysis) 38 壹、 參數設定 38 第三章 結果與討論 39 第一節 蛋白質樣品濃度測定 39 第二節 利用不同分離策略iTRAQ標記胜肽樣品 41 壹、 等電點聚焦分離儀(sIEF) 41 貳、 強陽離子交換法(SCX) 43 參、 鹼性逆相層析法(bRP) 46 第三節 三種分離策略結果比較 49 第四節 蛋白質相關生物途徑 57 第四章 結論與未來展望 61 第五章 參考文獻 62 附表 66

    Uncategorized References
    1. Lozano, R., et al., Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 2012. 380(9859): p. 2095-128.
    2. Muir, A.J., The rapid evolution of treatment strategies for hepatitis C. The American journal of gastroenterology, 2014. 109(5): p. 628-635.
    3. Law, J.L.M., et al., A hepatitis C virus (HCV) vaccine comprising envelope glycoproteins gpE1/gpE2 derived from a single isolate elicits broad cross-genotype neutralizing antibodies in humans. PloS one, 2013. 8(3): p. e59776.
    4. Feld, J.J. and J.H. Hoofnagle, Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature, 2005. 436(7053): p. 967.
    5. Scheel, T.K. and C.M. Rice, Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat Med, 2013. 19(7): p. 837-49.
    6. Wilkins, T., et al., Hepatitis C: diagnosis and treatment. Am Fam Physician, 2010. 81(11): p. 1351-7.
    7. Maheshwari, A., S. Ray, and P.J. Thuluvath, Acute hepatitis C. Lancet, 2008. 372(9635): p. 321-32.
    8. Chen, J.Y. and R.T. Chung, Future classes of hepatitis C virus therapeutic agents. Infect Dis Clin North Am, 2012. 26(4): p. 949-66.
    9. Datta, S., et al., Case-finding for hepatitis C in primary care: a mixed-methods service evaluation. Br J Gen Pract, 2014. 64(619): p. e67-74.
    10. Washburn, M.P., D. Wolters, and J.R. Yates, 3rd, Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol, 2001. 19(3): p. 242-7.
    11. Zhang, X., et al., Multi-dimensional liquid chromatography in proteomics--a review. Anal Chim Acta, 2010. 664(2): p. 101-13.
    12. Sestak, J., D. Moravcova, and V. Kahle, Instrument platforms for nano liquid chromatography. J Chromatogr A, 2015. 1421: p. 2-17.
    13. Peng, J., et al., Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res, 2003. 2(1): p. 43-50.
    14. Xu, P., D.M. Duong, and J. Peng, Systematical optimization of reverse-phase chromatography for shotgun proteomics. J Proteome Res, 2009. 8(8): p. 3944-50.
    15. Zizzo, A.N., et al., A national retrospective study of paediatric end-stage liver disease as a predictor of change to second-line therapy in children with autoimmune hepatitis. Liver Int, 2017.
    16. Yang, F., et al., High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Rev Proteomics, 2012. 9(2): p. 129-34.
    17. Spicer, V., et al., 3D HPLC-MS with Reversed-Phase Separation Functionality in All Three Dimensions for Large-Scale Bottom-Up Proteomics and Peptide Retention Data Collection. Anal Chem, 2016. 88(5): p. 2847-55.
    18. Kirkland, J.J., M.A. van Straten, and H.A. Claessens, High pH mobile phase effects on silica-based reversed-phase high-performance liquid chromatographic columns. Journal of Chromatography A, 1995. 691(1): p. 3-19.
    19. Hubner, N.C., S. Ren, and M. Mann, Peptide separation with immobilized pI strips is an attractive alternative to in-gel protein digestion for proteome analysis. PROTEOMICS, 2008. 8(23-24): p. 4862-4872.
    20. Horth, P., et al., Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Mol Cell Proteomics, 2006. 5(10): p. 1968-74.
    21. Kopaciewicz, W., et al., Retention model for high-performance ion-exchange chromatography. Journal of Chromatography A, 1983. 266: p. 3-21.
    22. Dowell, J.A., et al., Comparison of two-dimensional fractionation techniques for shotgun proteomics. Anal Chem, 2008. 80(17): p. 6715-23.
    23. Lau, E., et al., Combinatorial use of offline SCX and online RP-RP liquid chromatography for iTRAQ-based quantitative proteomics applications. Mol Biosyst, 2011. 7(5): p. 1399-408.
    24. Edman, P., Method for determination of the amino acid sequence in peptides. Acta chem. scand, 1950. 4(7): p. 283-293.
    25. Ma, B., et al., PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid communications in mass spectrometry, 2003. 17(20): p. 2337-2342.
    26. Henzel, W.J., et al., Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proceedings of the National Academy of Sciences, 1993. 90(11): p. 5011-5015.
    27. Thiede, B., et al., Peptide mass fingerprinting. Methods, 2005. 35(3): p. 237-247.
    28. Eng, J.K., A.L. McCormack, and J.R. Yates, An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry, 1994. 5(11): p. 976-989.
    29. Märk, T.D. and G.H. Dunn, Electron impact ionization. 2013: Springer Science & Business Media.
    30. Harrison, A.G., Chemical ionization mass spectrometry. 1992: CRC press.
    31. Kaufmann, R., Matrix-assisted laser desorption ionization (MALDI) mass spectrometry: a novel analytical tool in molecular biology and biotechnology. Journal of biotechnology, 1995. 41(2-3): p. 155-175.
    32. Fenn, J.B., et al., Electrospray ionization for mass spectrometry of large biomolecules. Science, 1989. 246(4926): p. 64-71.
    33. Wilm, M.S. and M. Mann, Electrospray and Taylor-Cone theory, Dole's beam of macromolecules at last? International Journal of Mass Spectrometry and Ion Processes, 1994. 136(2-3): p. 167-180.
    34. Kaltashov, I.A. and R.R. Abzalimov, Do ionic charges in ESI MS provide useful information on macromolecular structure? Journal of the American Society for Mass Spectrometry, 2008. 19(9): p. 1239-1246.
    35. Schwartz, J.C., M.W. Senko, and J.E. Syka, A two-dimensional quadrupole ion trap mass spectrometer. Journal of the American Society for Mass Spectrometry, 2002. 13(6): p. 659-669.
    36. Hager, J.W., A new linear ion trap mass spectrometer. Rapid Communications in Mass Spectrometry, 2002. 16(6): p. 512-526.
    37. Wells, J.M. and S.A. McLuckey, Collision‐induced dissociation (CID) of peptides and proteins. Methods in enzymology, 2005. 402: p. 148-185.
    38. Wu, W.W., et al., Discovery-and target-based protein quantification using iTRAQ and pulsed Q collision induced dissociation (PQD). Journal of proteomics, 2012. 75(8): p. 2480-2487.
    39. Wasinger, V.C., et al., Progress with gene‐product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis, 1995. 16(1): p. 1090-1094.
    40. Anderson, N.L. and N.G. Anderson, Proteome and proteomics: new technologies, new concepts, and new words. Electrophoresis, 1998. 19(11): p. 1853-1861.
    41. Fenselau, C. and X. Yao, Proteolytic Labeling With 18 O for Comparative Proteomics Studies: Preparation of 18 O-Labeled Peptides and the 18 O/16 O Peptide Mixture. Quantitative Proteomics by Mass Spectrometry, 2007: p. 135-142.
    42. Heller, M., et al., Trypsin catalyzed 16 O-to-18 O exchange for comparative proteomics: tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-ion trap mass spectrometers. Journal of the American Society for Mass Spectrometry, 2003. 14(7): p. 704-718.
    43. Gygi, S.P., et al., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature biotechnology, 1999. 17(10): p. 994-999.
    44. Thompson, A., et al., Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Analytical chemistry, 2003. 75(8): p. 1895-1904.
    45. Evans, C., et al., An insight into iTRAQ: where do we stand now? Analytical and Bioanalytical Chemistry, 2012. 404(4): p. 1011-1027.
    46. Choo, Q.-L., et al., Isolation of a cDNA Clone Derived from a Blood-Borne Non-A, Non-B Viral Hepititis Genome. Science, 1989. 244(4902): p. 359.
    47. Kuo, G., et al., An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science, 1989: p. 362-364.
    48. Tsai, P., et al., Viral dynamics of persistent hepatitis C virus infection in high-sensitive reporter cells resemble patient's viremia. Journal of Microbiology, Immunology and Infection, 2017.
    49. Casey, T.M., et al., Analysis of Reproducibility of Proteome Coverage and Quantitation Using Isobaric Mass Tags (iTRAQ and TMT). Journal of Proteome Research, 2017. 16(2): p. 384-392.
    50. Pichler, P., et al., Peptide labeling with isobaric tags yields higher identification rates using iTRAQ 4-plex compared to TMT 6-plex and iTRAQ 8-plex on LTQ Orbitrap. Analytical chemistry, 2010. 82(15): p. 6549-6558.
    51. Chenau, J., et al., Peptides OFFGEL electrophoresis: a suitable pre-analytical step for complex eukaryotic samples fractionation compatible with quantitative iTRAQ labeling. Proteome science, 2008. 6(1): p. 9.

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