簡易檢索 / 詳目顯示

回結果列表
研究生： 黃庭恩
Huang, Ting-En
論文名稱： 利用蛋白質組學與液相層析串聯式質譜儀鑑定兒茶酚雌激素對乳癌細胞MCF-7中的組蛋白修飾
Identification of histone modifications by catechol estrogen in MCF-7 breast cancer cells using proteomics and LC-MS/MS
指導教授： 陳淑慧
Chen, Shu-Hui
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： 2020
畢業學年度： 108
語文別： 英文
論文頁數： 73
中文關鍵詞： 組蛋白修飾 、兒茶酚雌激素 、乳癌 、雙甲基標定
外文關鍵詞： Histone modifications, catechol estrogen, breast cancer, dimethyl labeling
相關次數： 點閱：107  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 細胞內組蛋白轉譯後修飾可以影響染色質的反應，包括轉錄、基因靜默及其穩定性，已有許多文獻指出組蛋白上的修飾失衡與疾病或癌症有所關聯，若在疾病中反復檢測到特定修飾改變，則該異常修飾便可以作為「生物指標 (Biomarker)」。如今，生物指標已被用於監測疾病上，例如癌症或糖尿病，然而目前對於人類癌症生物指標的相關研究仍十分有限。

    雌激素與表觀遺傳的調控息息相關，本研究的目的為利用蛋白質組學與液相層析串聯式質譜儀檢測兒茶酚雌激素對人類乳癌細胞MCF-7之組蛋白修飾的改變，我們透過酸萃取從MCF-7細胞中分離出組蛋白，並進行雙甲基標記 (Dimethyl Labeling)。從質譜分析結果可以發現在核心組蛋白H2A1A Lysine-16位點上不僅發現了單甲基修飾 (Mono-methylation) 程度的改變，還發現了4-羥基雌二醇 (4-hydroxyestradiol, 4-OHE2) 加合物。我們希望探索這些具有功能性位點的目標組蛋白修飾，並研究它們作為疾病生物標記或藥物標靶的作用。

    Histone post translational modifications can affect a series of chromatin-based reactions, including transcription, gene silencing, and stability. It has been proved that unbalanced modifications on histone proteins are associated with diseases and cancers. A specific modification could be used as a biomarker if it is repeatedly detected in certain disease. Nowadays, biomarkers have been applied to monitor disease such as cancers or diabetes. However, current studies on post translational modifications in human cancers are limited.
    Estrogen has been reported to interact with specific regulation of epigenetic modifications. The objective of this study is to detect global alterations of histone modifications in MCF-7 breast cancer cells by catechol estrogen. Histones isolated from MCF-7 by acid extraction were undergone dimethyl labeling strategy coupled with in solution digestion or in gel digestion. In addition, it was interesting to note that not only epigenetic changes but also catechol estrogen-adducts were found at H2A1A Lys-16. The target peptides were further analyzed by quantitative mass spectrometry. We expected to explore functional roles of the identified targets and study their usefulness as disease markers or drug targets.
     

    中文摘要 II ABSTRACT III ACKNOWLEDGEMENTS V TABLE OF CONTENT VI LIST OF TABLES VIII LIST OF FIGURES IX LIST OF APPENDIX XI ABBREVIATIONS XII CHAPTER 1: INTRODUCTION 1 1.1. Catechol Estrogen Metabolism 1 1.2. The Importance of Histone PTMs 4 1.2.1 Brief Introduction of Histone Post-translational Modifications 4 1.2.2. Epigenetics and Histone Biomarker 5 1.3. Quantification Method in Proteomics 9 1.3.1. Dimethyl Labeling 9 1.3.2. Multiple Reaction Monitoring and Parallel Reaction Monitoring 10 1.4. The Applications of Mass Spectrometry in Proteomics 11 1.4.1. Mass Spectrometry for Protein Identification 11 1.4.2. Basic Principles of Mass Spectrometry 14 CHAPTER 2: MATERIAL AND METHOD 17 2.1. Material 17 2.1.1. Reagents 17 2.1.2. Instruments 18 2.2. Method 19 2.2.1. MCF-7 Cell Culture 19 2.2.2. In vivo 4OHE2-adducted Protein Formation 19 2.2.3. Cellular Viability and Activity Test: MTT Assay 20 2.2.4. DNA Fragmentation Test: TUNEL assay 21 2.2.5. MCF-7 Cell Extraction 21 2.2.7. Dimethyl Labeling 23 2.2.8. In-gel and In-solution Protein Digestion 24 2.3. Nano-UPLC System Coupled with LTQ-Orbitrap XL Mass Spectrometer 25 2.4. QE-Orbitrap Mass Spectrometer 27 2.5. Automatic Database Search 27 CHAPTER 3: RESULTS AND DISCUSSION 28 3.1. Cytotoxic Effect of Catechol Estrogen on MCF-7 Cell Line 28 3.1.1. Cellular Viability and Activity Test: MTT Assay 28 3.1.2. DNA Fragmentation Test: TUNEL assay 30 3.2. Non-targeted approach development 35 3.2.1. Acid Extraction of Histone Protein 35 3.2.2. Dimethyl Labeling Strategy Development 37 3.3 Identify CE-adducted on Histone Protein 40 3.4. Quantitative Mass Spectrometry 47 3.4.1. MRM Quantitation for Estrogenized H2A1A Lys-16 47 3.4.2. Quantification for H2A1A K16 mono-methylation 55 CHAPTER 4: CONCLUTION 59 CHAPTER 5: REFERENCE 60 CHAPTER 6: APPENDIX 65 Appendix 6.1. List of PTMs Identification on Histone Protein 65 Appendix 6.2. Internal Control Peptide 72 Appendix 6.3. The Statistical Results for MRM 73 Appendix 6.4. The Statistical Results for PRM 73

    1. Manly, J.; Merchant, C.; Jacobs, D.; Small, S.; Bell, K.; Ferin, M.; Mayeux, R., Endogenous estrogen levels and Alzheimer’s disease among postmenopausal women. Neurology 2000, 54 (4), 833-837.
    2. Carlsen, C. G.; Soerensen, T. H.; Eriksen, E. F., Prevalence of low serum estradiol levels in male osteoporosis. Osteoporosis international 2000, 11 (8), 697-701.
    3. Chen, G. G.; Zeng, Q.; Tse, G. M., Estrogen and its receptors in cancer. Medicinal research reviews 2008, 28 (6), 954-974.
    4. Cavalieri, E.; Chakravarti, D.; Guttenplan, J.; Hart, E.; Ingle, J.; Jankowiak, R.; Muti, P.; Rogan, E.; Russo, J.; Santen, R., Catechol estrogen quinones as initiators of breast and other human cancers: implications for biomarkers of susceptibility and cancer prevention. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 2006, 1766 (1), 63-78.
    5. Cavalieri, E. L.; Rogan, E. G., Etiology and prevention of prevalent types of cancer. Journal of rare diseases research & treatment 2017, 2 (3), 22.
    6. Liehr, J. G.; Wan-Fen, F.; Sirbasku, D. A.; Ari-Ulubelen, A., Carcinogenicity of catechol estrogens in Syrian hamsters. Journal of steroid biochemistry 1986, 24 (1), 353-356.
    7. Liehr, J. G., Is Estradiol a Genotoxic Mutagenic Carcinogen?1. Endocrine Reviews 2000, 21 (1), 40-54.
    8. Ku, M.-C.; Fang, C.-M.; Cheng, J.-T.; Liang, H.-C.; Wang, T.-F.; Wu, C.-H.; Chen, C.-C.; Tai, J.-H.; Chen, S.-H., Site-specific covalent modifications of human insulin by catechol estrogens: reactivity and induced structural and functional changes. Scientific reports 2016, 6, 28804.
    9. Misra, A., Challenges in delivery of therapeutic genomics and proteomics. Elsevier: 2010.
    10. Bode, A. M.; Dong, Z., Post-translational modification of p53 in tumorigenesis. Nature Reviews Cancer 2004, 4 (10), 793-805.
    11. Nørregaard Jensen, O., Modification-specific proteomics: characterization of post-translational modifications by mass spectrometry. Current Opinion in Chemical Biology 2004, 8 (1), 33-41.
    12. Mann, M.; Jensen, O. N., Proteomic analysis of post-translational modifications. Nature Biotechnology 2003, 21 (3), 255-261.
    13. Witze, E. S.; Old, W. M.; Resing, K. A.; Ahn, N. G., Mapping protein post-translational modifications with mass spectrometry. Nature Methods 2007, 4 (10), 798-806.
    14. Simon, M.; North, J. A.; Shimko, J. C.; Forties, R. A.; Ferdinand, M. B.; Manohar, M.; Zhang, M.; Fishel, R.; Ottesen, J. J.; Poirier, M. G., Histone fold modifications control nucleosome unwrapping and disassembly. Proceedings of the National Academy of Sciences 2011, 108 (31), 12711.
    15. Rothbart, S. B.; Strahl, B. D., Interpreting the language of histone and DNA modifications. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 2014, 1839 (8), 627-643.
    16. Bannister, A. J.; Kouzarides, T., Regulation of chromatin by histone modifications. Cell research 2011, 21 (3), 381-395.
    17. Sun, H.; Kennedy, P. J.; Nestler, E. J., Epigenetics of the depressed brain: role of histone acetylation and methylation. Neuropsychopharmacology 2013, 38 (1), 124-137.
    18. Reily, C.; Stewart, T. J.; Renfrow, M. B.; Novak, J., Glycosylation in health and disease. Nature Reviews Nephrology 2019, 1.
    19. Herranz Martín, N.; Dave, N.; Millanes Romero, A.; Morey Ramonell, L.; Díaz, V. M.; Lórenz Fonfria, V.; Gutiérrez Gallego, R.; Jerónimo, C.; Di Croce, L.; García de Herreros, A., Lysyl oxidase-like 2 deaminates lysine 4 in histone H3. Molecular cell. 2012; 46 (3): 369-376 2012.
    20. Karczmarski, J.; Rubel, T.; Paziewska, A.; Mikula, M.; Bujko, M.; Kober, P.; Dadlez, M.; Ostrowski, J., Histone H3 lysine 27 acetylation is altered in colon cancer. Clinical proteomics 2014, 11 (1), 24.
    21. Zippo, A.; De Robertis, A.; Serafini, R.; Oliviero, S., PIM1-dependent phosphorylation of histone H3 at serine 10 is required for MYC-dependent transcriptional activation and oncogenic transformation. Nature cell biology 2007, 9 (8), 932-944.
    22. Kinner, A.; Wu, W.; Staudt, C.; Iliakis, G., γ-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin. Nucleic Acids Research 2008, 36 (17), 5678-5694.
    23. Strimbu, K.; Tavel, J. A., What are biomarkers? Current Opinion in HIV and AIDS 2010, 5 (6), 463.
    24. Wang, J.-Q.; Wu, M.-Z.; Wu, K.-J., Analysis of Epigenetic Regulation of Hypoxia-Induced Epithelial–Mesenchymal Transition in Cancer Cells by Quantitative Chromatin Immunoprecipitation of Histone Deacetylase 3 (HDAC3). In Histone Deacetylases, Springer: 2016; pp 23-29.
    25. Ellinger, J.; Kahl, P.; von der Gathen, J.; Rogenhofer, S.; Heukamp, L. C.; Gütgemann, I.; Walter, B.; Hofstädter, F.; Büttner, R.; Müller, S. C., Global levels of histone modifications predict prostate cancer recurrence. The Prostate 2010, 70 (1), 61-69.
    26. Elsheikh, S. E.; Green, A. R.; Rakha, E. A.; Powe, D. G.; Ahmed, R. A.; Collins, H. M.; Soria, D.; Garibaldi, J. M.; Paish, C. E.; Ammar, A. A., Global histone modifications in breast cancer correlate with tumor phenotypes, prognostic factors, and patient outcome. Cancer research 2009, 69 (9), 3802-3809.
    27. Ellinger, J.; Bachmann, A.; Göke, F.; Behbahani, T. E.; Baumann, C.; Heukamp, L. C.; Rogenhofer, S.; Müller, S. C., Alterations of global histone H3K9 and H3K27 methylation levels in bladder cancer. Urologia internationalis 2014, 93 (1), 113-118.
    28. Rogenhofer, S.; Kahl, P.; Mertens, C.; Hauser, S.; Hartmann, W.; Büttner, R.; Müller, S. C.; von Ruecker, A.; Ellinger, J., Global histone H3 lysine 27 (H3K27) methylation levels and their prognostic relevance in renal cell carcinoma. BJU international 2012, 109 (3), 459-465.
    29. Tamagawa, H.; Oshima, T.; Numata, M.; Yamamoto, N.; Shiozawa, M.; Morinaga, S.; Nakamura, Y.; Yoshihara, M.; Sakuma, Y.; Kameda, Y., Global histone modification of H3K27 correlates with the outcomes in patients with metachronous liver metastasis of colorectal cancer. European Journal of Surgical Oncology (EJSO) 2013, 39 (6), 655-661.
    30. Benard, A.; Goossens‐Beumer, I. J.; van Hoesel, A. Q.; Horati, H.; de Graaf, W.; Putter, H.; Zeestraten, E. C.; Liefers, G. J.; van de Velde, C. J.; Kuppen, P. J., Nuclear expression of histone deacetylases and their histone modifications predicts clinical outcome in colorectal cancer. Histopathology 2015, 66 (2), 270-282.
    31. Gezer, U.; Yörüker, E. E.; Keskin, M.; Kulle, C. B.; Dharuman, Y.; Holdenrieder, S., Histone methylation marks on circulating nucleosomes as novel blood-based biomarker in colorectal cancer. International journal of molecular sciences 2015, 16 (12), 29654-29662.
    32. Messier, T. L.; Gordon, J. A.; Boyd, J. R.; Tye, C. E.; Browne, G.; Stein, J. L.; Lian, J. B.; Stein, G. S., Histone H3 lysine 4 acetylation and methylation dynamics define breast cancer subtypes. Oncotarget 2016, 7 (5), 5094.
    33. McQueen, C., Comprehensive toxicology. Elsevier: 2017.
    34. Dagdemir, A.; Durif, J.; Ngollo, M.; Bignon, Y.-J.; Bernard-Gallon, D., Histone lysine trimethylation or acetylation can be modulated by phytoestrogen, estrogen or anti-HDAC in breast cancer cell lines. Epigenomics 2013, 5 (1), 51-63.
    35. Daujat, S.; Bauer, U.-M.; Shah, V.; Turner, B.; Berger, S.; Kouzarides, T., Crosstalk between CARM1 methylation and CBP acetylation on histone H3. Current Biology 2002, 12 (24), 2090-2097.
    36. Palermo, A.; Botrè, F.; de la Torre, X.; Zamboni, N., Non-targeted LC-MS based metabolomics analysis of the urinary steroidal profile. Analytica chimica acta 2017, 964, 112-122.
    37. Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.; Heck, A. J., Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nature protocols 2009, 4 (4), 484.
    38. Cheng, H. L.; Huang, H. J.; Ou, B. Y.; Chow, N. H.; Chen, Y. W.; Tzai, T. S.; Wu, C. J.; Chen, S. H., Urinary CD14 as a potential biomarker for benign prostatic hyperplasia–discovery by combining MALDI‐TOF‐based biostatistics and ESI‐MS/MS‐based stable‐isotope labeling. PROTEOMICS–Clinical Applications 2011, 5 (3‐4), 121-132.
    39. Hsu, J.-L.; Chen, S.-H., Stable isotope dimethyl labelling for quantitative proteomics and beyond. Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences 2016, 374 (2079), 20150364.
    40. Jhan, S.-Y.; Huang, L.-J.; Wang, T.-F.; Chou, H.-H.; Chen, S.-H., Dimethyl labeling coupled with mass spectrometry for topographical characterization of primary amines on monoclonal antibodies. Analytical chemistry 2017, 89 (7), 4255-4263.
    41. Wu, C.-J.; Hsu, J.-L.; Huang, S.-Y.; Chen, S.-H., Mapping N-terminus phosphorylation sites and quantitation by stable isotope dimethyl labeling. Journal of the American Society for Mass Spectrometry 2010, 21 (3), 460-471.
    42. Shen, P.-T.; Hsu, J.-L.; Chen, S.-H., Dimethyl isotope-coded affinity selection for the analysis of free and blocked N-termini of proteins using LC− MS/MS. Analytical chemistry 2007, 79 (24), 9520-9530.
    43. Hsu, J.-L.; Huang, S.-Y.; Shiea, J.-T.; Huang, W.-Y.; Chen, S.-H., Beyond quantitative proteomics: signal enhancement of the a1 ion as a mass tag for peptide sequencing using dimethyl labeling. Journal of proteome research 2005, 4 (1), 101-108.
    44. Hsu, J.-L.; Huang, S.-Y.; Chow, N.-H.; Chen, S.-H., Stable-isotope dimethyl labeling for quantitative proteomics. Analytical chemistry 2003, 75 (24), 6843-6852.
    45. Ronsein, G. E.; Pamir, N.; von Haller, P. D.; Kim, D. S.; Oda, M. N.; Jarvik, G. P.; Vaisar, T.; Heinecke, J. W., Parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) exhibit comparable linearity, dynamic range and precision for targeted quantitative HDL proteomics. Journal of proteomics 2015, 113, 388-399.
    46. Peterson, A. C.; Russell, J. D.; Bailey, D. J.; Westphall, M. S.; Coon, J. J., Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Molecular & Cellular Proteomics 2012, 11 (11), 1475-1488.
    47. Zhou, J.; Yin, Y., Strategies for large-scale targeted metabolomics quantification by liquid chromatography-mass spectrometry. Analyst 2016, 141 (23), 6362-6373.
    48. Lacorte, S.; Fernandez‐Alba, A. R., Time of flight mass spectrometry applied to the liquid chromatographic analysis of pesticides in water and food. Mass spectrometry reviews 2006, 25 (6), 866-880.
    49. Chait, B. T., Mass spectrometry: bottom-up or top-down? Science 2006, 314 (5796), 65-66.
    50. Banerjee, S.; Mazumdar, S., Electrospray ionization mass spectrometry: a technique to access the information beyond the molecular weight of the analyte. International journal of analytical chemistry 2012, 2012.
    51. Sobott, F.; Watt, S. J.; Smith, J.; Edelmann, M. J.; Kramer, H. B.; Kessler, B. M., Comparison of CID versus ETD based MS/MS fragmentation for the analysis of protein ubiquitination. Journal of the American Society for Mass Spectrometry 2009, 20 (9), 1652-1659.
    52. Van Meerloo, J.; Kaspers, G. J.; Cloos, J., Cell sensitivity assays: the MTT assay. In Cancer cell culture, Springer: 2011; pp 237-245.
    53. SCHERLIEß, R., The MTT assay as tool to evaluate and compare excipient toxicity in vitro on respiratory epithelial cells. International journal of pharmaceutics 2011, 411 (1-2), 98-105.
    54. Virág, L.; Kerékgyártó, C.; Fachet, J., A simple, rapid and sensitive fluorimetric assay for the measurement of cell-mediated cytotoxicity. Journal of immunological methods 1995, 185 (2), 199-208.
    55. Loo, D. T., In situ detection of apoptosis by the TUNEL assay: an overview of techniques. In DNA Damage Detection In Situ, Ex Vivo, and In Vivo, Springer: 2011; pp 3-13.
    56. Kyrylkova, K.; Kyryachenko, S.; Leid, M.; Kioussi, C., Detection of apoptosis by TUNEL assay. In Odontogenesis, Springer: 2012; pp 41-47.
    57. Malvezzi, H.; Sharma, R.; Agarwal, A.; Abuzenadah, A.; Abu-Elmagd, M., Sperm quality after density gradient centrifugation with three commercially available media: A controlled trial. Reproductive biology and endocrinology : RB&E 2014, 12, 121.
    58. Mattiroli, F.; Vissers, J. H.; van Dijk, W. J.; Ikpa, P.; Citterio, E.; Vermeulen, W.; Marteijn, J. A.; Sixma, T. K., RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling. Cell 2012, 150 (6), 1182-1195.

    無法下載圖示 校內：2025-09-01公開
    校外：不公開
    電子論文尚未授權公開，紙本請查館藏目錄
    QR CODE