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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 彭旭明 | |
dc.contributor.author | Yung-Yu Kuo | en |
dc.contributor.author | 郭永宇 | zh_TW |
dc.date.accessioned | 2021-06-15T03:54:00Z | - |
dc.date.available | 2011-08-03 | |
dc.date.copyright | 2010-08-03 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-06-30 | |
dc.identifier.citation | 1. Aebersold, R. and M. Mann, Mass spectrometry-based proteomics. Nature, 2003. 422(6928): p. 198-207.
2. Domon, B. and R. Aebersold, Mass spectrometry and protein analysis. Science, 2006. 312(5771): p. 212-7. 3. Mann, M., R.C. Hendrickson, and A. Pandey, Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem, 2001. 70: p. 437-73. 4. Welch, S., Management of substance misuse problems in the general hospital. Clin Med, 2002. 2(6): p. 513-5. 5. Yu, J., et al., Human embryonic stem cells reprogram myeloid precursors following cell-cell fusion. Stem Cells, 2006. 24(1): p. 168-76. 6. Reid, G.E. and S.A. McLuckey, 'Top down' protein characterization via tandem mass spectrometry. J Mass Spectrom, 2002. 37(7): p. 663-75. 7. McLafferty, F.W., et al., Top-down MS, a powerful complement to the high capabilities of proteolysis proteomics. FEBS J, 2007. 274(24): p. 6256-68. 8. Loo, J.A., et al., High-resolution tandem mass spectrometry of large biomolecules. Proc Natl Acad Sci U S A, 1992. 89(1): p. 286-9. 9. Han, X., et al., Extending top-down mass spectrometry to proteins with masses greater than 200 kilodaltons. Science, 2006. 314(5796): p. 109-12. 10. Ge, Y., et al., Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry. J Am Chem Soc, 2002. 124(4): p. 672-8. 11. Du, Y., et al., Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry. Anal Chem, 2006. 78(3): p. 686-94. 12. Breuker, K., et al., Top-down identification and characterization of biomolecules by mass spectrometry. J Am Soc Mass Spectrom, 2008. 19(8): p. 1045-53. 13. Bogdanov, B. and R.D. Smith, Proteomics by FTICR mass spectrometry: top down and bottom up. Mass Spectrom Rev, 2005. 24(2): p. 168-200. 14. Loo, J.A., C.G. Edmonds, and R.D. Smith, Primary sequence information from intact proteins by electrospray ionization tandem mass spectrometry. Science, 1990. 248(4952): p. 201-4. 15. Zabrouskov, V., et al., Stepwise deamidation of ribonuclease A at five sites determined by top down mass spectrometry. Biochemistry, 2006. 45(3): p. 987-92. 16. Whitelegge, J.P., et al., Protein-Sequence Polymorphisms and Post-translational Modifications in Proteins from Human Saliva using Top-Down Fourier-transform Ion Cyclotron Resonance Mass Spectrometry. Int J Mass Spectrom, 2007. 268(2-3): p. 190-197. 17. Roth, M.J., et al., 'Proteotyping': population proteomics of human leukocytes using top down mass spectrometry. Anal Chem, 2008. 80(8): p. 2857-66. 18. Garcia, B.A., et al., Tissue-specific expression and post-translational modification of histone H3 variants. J Proteome Res, 2008. 7(10): p. 4225-36. 19. Thomas, C.E., N.L. Kelleher, and C.A. Mizzen, Mass spectrometric characterization of human histone H3: a bird's eye view. J Proteome Res, 2006. 5(2): p. 240-7. 20. Roth, M.J., et al., Precise and parallel characterization of coding polymorphisms, alternative splicing, and modifications in human proteins by mass spectrometry. Mol Cell Proteomics, 2005. 4(7): p. 1002-8. 21. Meng, F., et al., Detection and localization of protein modifications by high resolution tandem mass spectrometry. Mass Spectrom Rev, 2005. 24(2): p. 126-34. 22. Forbes, A.J., et al., Targeted analysis and discovery of posttranslational modifications in proteins from methanogenic archaea by top-down MS. Proc Natl Acad Sci U S A, 2004. 101(9): p. 2678-83. 23. Fridriksson, E.K., et al., Heterogeneous glycosylation of immunoglobulin E constructs characterized by top-down high-resolution 2-D mass spectrometry. Biochemistry, 2000. 39(12): p. 3369-76. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/44745 | - |
dc.description.abstract | 攝護腺癌在台灣癌症死因中,已爬升至第七名,在美國更是高居第二名!近年來,台灣攝護腺癌發生率及死亡率急速升高,但仍然沒有一種真正有特異性的生物標記來偵測分辨攝護腺癌與攝護腺肥大。攝護腺特異抗原prostate specific antigen (PSA),是一種攝護腺產生的蛋白質,是目前唯一但並非為有效之標記蛋白,臨床現階段利用血液中攝護腺特異抗原的濃度來辦別,若攝護腺有病變則PSA數值會升高。臨床上有許多人因PSA升高,或其他證據疑似罹有前列腺癌,此時是否要作切片,常困擾泌尿科醫師及病人,若切片顯示為陰性,則是否要做第二次或第三次切片,也沒有一定之準則。若能利用非侵入性採集尿液檢體樣本,利用液相層析質譜儀,以前列腺癌為模式建立疾病相關標記蛋白質。利用online 2D system及offline 2D system等方法來相互比較,建立一個完整的前列腺癌的分析方法,進而開發ELISA Kit來定量尿中的蛋白質,此研究將為人類帶來極大的科學價值。
此論文主要利用二維online與offline高效層析串聯電噴灑質譜儀來建立尿液中的蛋白質資訊。實驗過程中,我們改變了流動相濃度梯度、鹽類濃度以及烘箱的溫度等等。在online的裝置裡,我們可以使儀器自動化操作得到大量的數據,但實驗結果所得到的結果,並不理想。雖然在offline的裝置,我們必須使用人力才能完成一系列的分析,雖然耗時費工,但得到的結果和靈敏度卻比online得到的好上許多! | zh_TW |
dc.description.abstract | Prostate cancer ranks the seventh cause of cancer-related death in Taiwan and the second in United States. The incidence and mortality rates of prostate cancer have been rising rapidly in the past decades. Currently, there are no accurate and specific biomarkers which could discriminate clinically relevant from clinically benign disease. The better indicators and progression are needed to avoid unnecessary treatment. Clinically, prostate-specific antigen (PSA) screening still seems the only way to diagnose the carcinoma of prostate, but lately researches showed that PSA is not prostate cancer specific. Finding novel biomarkers that are sensitive, specific and can be examined by non-invasive means are imperative.
In this thesis, the on-line 2D LC-MS and off-line 2D LC-MS methods are the main approaches to unravel the protein profiling information from urine samples. In order to acquire the moderate experimental results and establish the complete urine sample analysis for identifying prostate cancer, we use these two approaches and tested the effects of modifying several parameters (oven temperature, mobile phase gradient, salt concentration and so on). In online cases, though it can be automatically operated, the information obtained turned out to be unsatisfactory. In offline cases, it demanded much manpower for sample preparation and operating the LC-MS system. The results in offline cases not only show higher intensity and sensitivity than online cases, but provide more useful information. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T03:54:00Z (GMT). No. of bitstreams: 1 ntu-99-R96223146-1.pdf: 3032630 bytes, checksum: 823390f408456395b0cc1be349d22e78 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 目 錄
口試委員會審定書……………………………………………………………… i 誌謝………………………………………………………………………………. ii 中文摘要………………………………………………………………………… iii 英文摘要…………………………………………………………………………. iv Chapter 1 Introduction………………………………………………………………1 1.1 Prostate cancer………………………………………………………………3 1.1.1 Fuction………………………………………………………………..3 1.1.2 Secretions…………………………………………………………….3 1.1.3 Regulation……………………………………………………………4 1.1.4 Structure……………………………………………………………...5 1.2 Liquid Chromatographic background…........................................................7 1.2.1 Brief introduction………………………………………………….…7 1.2.2 The separation mechanism……………………………………...……8 1.3 Ion exchange chromatography…………………………………………….12 1.3.1 Principle……………………………………………………….….....12 1.3.2 The material of resin………………………………………….…......13 1.3.3 The five steps of ion exchange column process…………………….14 1.4 Size exclusion chromatography…………………………………….……..15 1.5 Introduction of electrospray ionization………..…………………………..16 1.6 Ion trap……………………………………………………………….……20 Chapter 1 Reference…………………………………………………………..……24 Chapter 2 Sample preparation…………………………………………..……….…26 2.1 Sample processing…………………………………………………………26 2.2 General…………………………………………………………………….26 2.3 Suggested urine collection practices…………………………………...….27 2.4 Urine sampling protocol for LC-MS analysis……………………………..28 2.5 Size Exclusion Chromatography of Urinary Protein Concentrate………...29 Chapter 2 Reference………………………………………………………………..30 Chapter 3…………………………………………………………………….……..33 3.1 Introduction of software interface…………………………………………33 3.2 Principle of deconvolution…………………………………………...……36 3.3Introduction of dissect method……………………………………………..43 3.4 Compounds and compound spectra…………………………………….....47 3.5 Dissect results of standard proteins………………………………..………49 Chapter 4 On line 2D LC-MS……………………………………………….…......66 4.1 On-line 2D LC-MS…………………………………………………...……66 4.1.1 Experimental section…………………………………...……...........68 4.1.2 Data…………………………………………..…………….………..73 4.2 On-line 2D LC-MS with parallel columns………………………………...77 4.2.1 Experimental section………………………………………………..77 Chapter 4 Reference………………………………………………………………..84 Chapter 5 Off line 2D LC-MS……………………………………………..……....86 5.1 Experimental section…………………………………………………...….87 5.2 Data……………………………………………………………….……….89 Chapter 6 Summary…………………………………………………….………….97 6.1 Bottom-up strategy……………………………………………...…………97 6.2 Two dimensional chromatography………………………………………...98 6.3 Mass spectrometry…………………………………………………………99 6.4 Mobile phase….. ……………………………………………………...…100 6.5 Future work………………………………………………..……………..105 Chapter 6 Reference……………………………………………………..………..108 圖目錄 Figure 1.1 The prostate and nearby organs……………………..……….…………..6 Figure 1.2 Prostate with seminal vesicles and seminal ducts…………………….....6 Figure 1.3 The schematic of an ESI source……………………………….……….17 Figure 1.4 The schematic of the mechanism of ion formation in ESI……………..18 Figure 1.5 Comparative diagram, roughly to scale, of a quadrupole mass filter and a cutaway view of a quadrupole ion trap mass spectrometer. Both instruments may be interfaced to an ion source and a detector………………………………………….22 Figure 3.1 The interface of ESI DataAnalysis software. (A) Analysis List window, (B) Survey View window, (C) Chromatogram window, (D) Spectrum View window and (E) Compound Spectra window…………………………………………………....33 Figure 3.2.a Resolved-isotope deconvolution …………………..…………….....37 Figure 3.2.b Related-ion deconvolution……………...…………….....…….…......38 Figure 3.3 The operation of deconvolution parameters at DataAnalysis………….39 Figuer 3.4 Without the Include shifted spectrum…………………………...……41 Figuer 3.5 With the Include shifted spectrum……………………………………41 Figure 3.6 With the Include component details…………………………………..42 Figure 3.7 The operation of Copmounds-Disset within DataAnalysis…………….44 Figure 3.8 Results of Compounds - Dissect performed on an MS, positive polarity analysis; one compound spectrum is calculated for each compound…………...….48 Figure 3.9 The original TIC diagram of LC-MS. Ubiquitin elute out at 13.5 min, carbonic anhydrase at 15.7 min and gramicidin S at 18.5 min………………….…49 Figure 3.10 The TIC diagram after Compounds-Dissect……………………....…..49 Figure 3.11 Zoom out the region of 12 to 14 mins……………………...…....……50 Figure 3.12 Spectrum of compound 1, 2 and 3. Compound 1 dissect at 12.8-13.2 min, compound 2 dissect at 13.1-13.5 min and compound 3 dissect at 13.2-13.8 min....51 Figure 3.13 The parameters of dissect function. Set internal S/N threshold is 3 and max. number of overlapping compounds is 10………………………………….....51 Figure 3.14 The deconvoluted spectrum of compound 1, 2 and 3. The mass of compound 1 dissect at 12.8-13.2 min is 8567.5 m/z, compound 2 dissect at 13.1-13.5 min is 8566.7 and 8581.9 m/z and compound 3 dissect at 13.2-13.8 min is 8580.8 m/z…………………………………………………………………………….……52 Figure 3.15 The parameters of deconvolution. The parameters of low mass is 1000 and high mass is 100000 at Proteins/ large molecules mode………………....….53 Figure 3.16 TIC diagram of 2μg urine sample……………………………..……...54 Figure 3.17 Compare two TIC diagram……………………………………………54 Figure 3.18 The dissect TIC diagram……………………………………………...55 Figure 3.19 The dissect compound………………………...………………………55 Figure 3.20 The deconvolution compound………………………………..……….61 Figure 4.1 Change valve switching position of the Switchos device for the analysis of a complex proteins mixture………………………………………………….…..…70 Figure 4.2 The flow through TIC diagram with 0% buffer B………………….…..73 Figure 4.3 Change valve switching position of the Switchos device for the analysis of a complex proteins mixture…………………………………………………...……78 Figure 4.4 The 30 Celsius degree TIC compared with 70 Celsius degree…………79 Figure 4.5 The normal person TIC diagram compared with patient sample………81 Figure 5.1 The 1st fraction patient sample A, B and C………………………….….89 Figure 6.1 The TIC diagram shows the effect of flow rate with 3 and 15μL/ min…………………………………………………………………………...…...100 Figure 6.2 The ion pairing agent is 0.1% TFA (red) and 0.1% FA (blue) detected by Agilent DAD…………………………………………………………….………..102 Figure 6.3 (a) The blue line is 0.1% TFA. (b) The green one is 0.02% TFA. (c) The red one is 0.03%.....................................................................................................103 Figure 6.4 Results of comparison of two protein sample preparation methods: (left) MARS-14 column and (right) ProteoMiner protein enrichment kit. Data provided by AstraZeneca……………………………………………………………………....105 Figure 6.5 The bead capacity limits binding abundance, so the high abundance proteins quickly saturate their ligands (red and yellow beads) and excess proteins are washed out. In contrast, low abundance proteins are concentrated on their specific ligands (pink and teal beads)………………………………………………….….106 表目錄 Table 6.1 The information of intensity with different flow rate……………....….100 | |
dc.language.iso | en | |
dc.title | 以前列腺癌為模式建立疾病相關標記蛋白質之LC-MS分析方法 | zh_TW |
dc.title | Profiling novel urinary markers for prostate cancer by 2D HPLC-ESI-MS | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.coadvisor | 韓肇中 | |
dc.contributor.oralexamcommittee | 林震煌 | |
dc.subject.keyword | 攝護腺癌,高效液相層析,攝護腺特異抗原,生物標記,電噴灑質譜儀, | zh_TW |
dc.subject.keyword | prostate cancer,high performance liquid chromatography (HPLC),prostate-specific antigen (PSA),bio-marker,electron spray ionization mass spectrometer (ESI-MS), | en |
dc.relation.page | 110 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-06-30 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
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