系統識別號 U0026-3108201711054300
論文名稱(中文) 製備線上奈米級液相層析搭配由上而下蛋白質體學策略應用於偵測紅血球中血紅蛋白的修飾
論文名稱(英文) Fabrication of On-Line Nano-Liquid Chromatography-Mass Spectrometry for Top-down Proteomics and Application for the detection of Modified Hemoglobin in Red Blood Cells
校院名稱 成功大學
系所名稱(中) 化學系
系所名稱(英) Department of Chemistry
學年度 105
學期 2
出版年 106
研究生(中文) 賴威彣
研究生(英文) Wei-Wen Lai
學號 L36041123
學位類別 碩士
語文別 英文
論文頁數 49頁
口試委員 指導教授-陳淑慧
中文關鍵字 奈米級液相層析  質譜  由上而下  蛋白質體學 
英文關鍵字 Nano-Liquid Chromatography  Mass Spectrometry  Top-down  Proteomics 
中文摘要 雌激素及其代謝物已經有被報導會和蛋白進行共價鍵結合進而引發疾病。即使傳統的由下而上質譜法可以解析出修飾的位點但是仍然耗時且不便利。,在此研究中嘗試以由上而下策略進行人類紅血球蛋白上雌激素修飾的分析,在由上而下策略中可以較直觀的檢測出蛋白是否有修飾的存在。為了達到這項目的,我們採用自行填充之奈米級管柱應用在液相層析二次質譜法並結合軟體Prosight PTM 2.0進行分析,同時也使用由下而上質譜法進行確認。


英文摘要 Estrogen and its metabolites have been reported to form covalent bonds with proteins, causing changes of protein functions which are related to diseases. Although conventional bottom-up proteomics technique can be used to identify modified proteins, it is still rather time-consuming and inconvenient for biomarker detection. In this research, we tried to develop a top-down method for identifying hemoglobins modified by catechol estrogens. For this purpose, we develop a packing method for fabricating nano columns and apply the self-packed column for top down analysis of modified hemoglobin in red blood cells using LC-MS/MS and Prosight PTM 2.0 for proteomic search. The bottom-up approach was further applied to confirm the modification sites identified by the top-down approach.
Sample used in this research is the lysed red blood cells which were co-incubated with 4-hydroxyestradiol in order to generate modified hemoglobin.
Two packing materials were attempted for packing. Our data indicated that the self-packed nano-column had reasonable flow resistance for 5 μm reversed phase C18 particles but was relatively loose for 20 μm reversed phase particles. But our results showed the 20 um column can still separate hemoglobin α and β chain as well as 4-hydroxyestradiol modified hemoglobin α and β chain at a relatively faster speed. In the MS1 spectrum can observe 4-hydroxyestradiol modification on hemoglobin α and β chain. When the raw data acquired by using the 20 um colume were analyzed with Prosight PTM 2.0 the modification site could not be confirmed due to few fragments by collision induced dissociation. Moreover, the modification site identified by the top-down approach was able to be confirmed by the bottom-up approach. The modified hemoglobin β chain, however, could not be identified by the top-down approach but was identified by the bottom-up approach using chymotrypsin digestion. Our data provide evidences that top-down method may be developed for biomarker detection of estrogenized hemoglobin.
論文目次 摘要 I
Abstract II
Table of Contents IV
List of Figures V
Abbreviation VII
Chapter1 Introduction 1
1-1 Brief introduction about Estrogen and its metabolites 1
1-2 Proteomics 4
1-3 Hemoglobin 6
1-4 Mass spectrometry used in studying proteomics 7
1-5 Nano liquid chromatography 12
Chapter2 Experimental methods and materials 14
2-1 Materials, hardware and software 14
2.1.1 Material 14
2.1.2 Equipement 15
2.1.3 Software 15
2-2 Sample preparation 16
2.2.1 Extraction of red blood cell from blood 16
2.2.2 Concentration of protein 16
2.2.3 Hemoglobin adduct formation 16
2.2.4 Protein digest 17
2-3 Packing Capillary picofrit Columns 17
2.3.1 Tip Fabrication 17
2.3.2 Making column frits 17
2.3.3 Column Packing 18
2-4 LC MS/MS analysis 18
2.4.1 Condition of top down analyze 18
2.4.2 Condition of button up analyze 19
Chapter3 Result 21
3-1 Evaluation of the column efficiency 21
3-2 LC-MS/MS of intact hemoglobin 26
3.2.1 Optimization of the separation gradient 26
3.2.2 Top-down search by Prosight PTM 2.0 26
3-3 LC-MS/MS of hemoglobin peptides 40
Chapter4 Conclusion 46
Chapter5 Reference 47
參考文獻 1. Files, J.A., M.G. Ko, and S. Pruthi, Bioidentical Hormone Therapy. Mayo Clinic Proceedings, 2011. 86(7): p. 673-680.
2. Tsuchiya, Y., M. Nakajima, and T. Yokoi, Cytochrome P450-mediated metabolism of estrogens and its regulation in human. Cancer Lett, 2005. 227(2): p. 115-24.
3. Samavat, H. and M.S. Kurzer, Estrogen Metabolism and Breast Cancer. Cancer letters, 2015. 356(2 0 0): p. 231-243.
4. Gao, N., R.A. Nester, and M.A. Sarkar, 4-Hydroxy estradiol but not 2-hydroxy estradiol induces expression of hypoxia-inducible factor 1alpha and vascular endothelial growth factor A through phosphatidylinositol 3-kinase/Akt/FRAP pathway in OVCAR-3 and A2780-CP70 human ovarian carcinoma cells. Toxicol Appl Pharmacol, 2004. 196(1): p. 124-35.
5. Lee, A.J., et al., Characterization of the oxidative metabolites of 17beta-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms. Endocrinology, 2003. 144(8): p. 3382-98.
6. Belous, A.R., et al., Cytochrome P450 1B1-mediated estrogen metabolism results in estrogen-deoxyribonucleoside adduct formation. Cancer Res, 2007. 67(2): p. 812-7.
7. Cavalieri, E., et al., Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention. Biochim Biophys Acta, 2006. 1766(1): p. 63-78.
8. Nicolis, S., et al., Neuroglobin modification by reactive quinone species. Chem Res Toxicol, 2013. 26(12): p. 1821-31.
9. Ku, M.-C., et al., Site-specific covalent modifications of human insulin by catechol estrogens: Reactivity and induced structural and functional changes. Scientific Reports, 2016. 6: p. 28804.
10. Haaf, H., S.A. Li, and J.J. Li, Covalent binding of estrogen metabolites to hamster liver microsomal proteins: inhibition by ascorbic acid and catechol- O -methyl transferase. Carcinogenesis, 1987. 8(2): p. 209-215.
11. Epe, B., et al., Covalent binding of reactive estrogen metabolites to microtubular protein as a possible mechanism of aneuploidy induction and neoplastic cell transformation. Environmental Health Perspectives, 1990. 88: p. 123-127.
12. Bolton, J.L., et al., Role of quinones in toxicology. Chem Res Toxicol, 2000. 13(3): p. 135-60.
13. Khan, W.A. and M.W.A. Khan, Cancer Morbidity in Rheumatoid Arthritis: Role of Estrogen Metabolites. BioMed Research International, 2013. 2013: p. 748178.
14. Poirier, M.C., R.M. Santella, and A. Weston, Carcinogen macromolecular adducts and their measurement. Carcinogenesis, 2000. 21(3): p. 353-359.
15. Lin, C., et al., Hemoglobin adducts as biomarkers of estrogen homeostasis: elevation of estrogenquinones as a risk factor for developing breast cancer in Taiwanese women. Toxicol Lett, 2014. 225(3): p. 386-91.
16. Wasinger, V.C., et al., Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis, 1995. 16(7): p. 1090-4.
17. Yang, X.J. and E. Seto, Lysine acetylation: codified crosstalk with other posttranslational modifications. Mol Cell, 2008. 31(4): p. 449-61.
18. Casey, P.J., J.A. Thissen, and J.F. Moomaw, Enzymatic modification of proteins with a geranylgeranyl isoprenoid. Proceedings of the National Academy of Sciences, 1991. 88(19): p. 8631-8635.
19. BAÑÓ, M.C., S.C. JACKSON, and I.A. MAGEE, Pseudo-enzymatic S-acylation of a myristoylated Yes protein tyrosine kinase peptide in vitro may reflect non-enzymatic S-acylation in vivo. Biochemical Journal, 1998. 330(2): p. 723-731.
20. Iberg, N. and R. Fluckiger, Nonenzymatic glycosylation of albumin in vivo. Identification of multiple glycosylated sites. J Biol Chem, 1986. 261(29): p. 13542-5.
21. Kraus, L.M. and A.P. Kraus, Carbamoylation of amino acids and proteins in uremia. Kidney International, 2001. 59: p. S102-S107.
22. Aebersold, R. and M. Mann, Mass spectrometry-based proteomics. Nature, 2003. 422(6928): p. 198-207.
23. Yates, J.R., C.I. Ruse, and A. Nakorchevsky, Proteomics by Mass Spectrometry: Approaches, Advances, and Applications. Annual Review of Biomedical Engineering, 2009. 11(1): p. 49-79.
24. Han, X., A. Aslanian, and J.R. Yates, 3rd, Mass spectrometry for proteomics. Curr Opin Chem Biol, 2008. 12(5): p. 483-90.
25. Felitsyn, N., M. Peschke, and P. Kebarle, Origin and number of charges observed on multiply-protonated native proteins produced by ESI. International Journal of Mass Spectrometry, 2002. 219(1): p. 39-62.
26. Fenn, J., et al., Electrospray ionization for mass spectrometry of large biomolecules. Science, 1989. 246(4926): p. 64-71.
27. Catherman, A.D., O.S. Skinner, and N.L. Kelleher, Top Down proteomics: Facts and perspectives. Biochemical and Biophysical Research Communications, 2014. 445(4): p. 683-693.
28. Gregorich, Z.R., Y.-H. Chang, and Y. Ge, Proteomics in heart failure: top-down or bottom-up? Pflügers Archiv - European Journal of Physiology, 2014. 466(6): p. 1199-1209.
29. Toby, T.K., L. Fornelli, and N.L. Kelleher, Progress in Top-Down Proteomics and the Analysis of Proteoforms. Annual Review of Analytical Chemistry, 2016. 9(1): p. 499-519.
30. Zhou, H., et al., Advancements in Top-Down Proteomics. Analytical Chemistry, 2012. 84(2): p. 720-734.
31. Zhurov, K.O., et al., Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis. Chemical Society Reviews, 2013. 42(12): p. 5014-5030.
32. Ettre, L.S. and K.I. Sakodynskii, M. S. Tswett and the discovery of chromatography I: Early work (1899–1903). Chromatographia, 1993. 35(3): p. 223-231.
33. Moseley, M.A., et al., Nanoscale packed-capillary liquid chromatography coupled with mass spectrometry using a coaxial continuous-flow fast atom bombardment interface. Anal Chem, 1991. 63(14): p. 1467-73.
34. Vissers, J.P.C., H.A. Claessens, and C.A. Cramers, Microcolumn liquid chromatography: instrumentation, detection and applications. Journal of Chromatography A, 1997. 779(1): p. 1-28.
35. Kennedy, R.T. and J.W. Jorgenson, Preparation and evaluation of packed capillary liquid chromatography columns with inner diameters from 20 to 50 micrometers. Analytical Chemistry, 1989. 61(10): p. 1128-1135.
36. Karlsson, K.E. and M. Novotny, Separation efficiency of slurry-packed liquid chromatography microcolumns with very small inner diameters. Analytical Chemistry, 1988. 60(17): p. 1662-1665.
37. Fanali, S., An overview to nano-scale analytical techniques: Nano-liquid chromatography and capillary electrochromatography. ELECTROPHORESIS, 2017: p. n/a-n/a.
38. Hernández-Borges, J., et al., Recent applications in nanoliquid chromatography. Journal of Separation Science, 2007. 30(11): p. 1589-1610.
39. Gama, M.R., C.H. Collins, and C.B.G. Bottoli, Nano-Liquid Chromatography in Pharmaceutical and Biomedical Research. Journal of Chromatographic Science, 2013. 51(7): p. 694-703.
40. Zotou, A., An overview of recent advances in HPLC instrumentation. Central European Journal of Chemistry, 2012. 10(3): p. 554-569.
41. Evans, B.C., et al., Ex Vivo Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs. Journal of Visualized Experiments : JoVE, 2013(73): p. 50166.
42. (UWPR), T.U.o.W.s.P.R., .

  • 同意授權校內瀏覽/列印電子全文服務,於2017-09-06起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2017-09-06起公開。

  • 如您有疑問,請聯絡圖書館