跳到主要內容

臺灣博碩士論文加值系統

(3.236.124.56) 您好!臺灣時間:2021/07/31 03:40
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:陳映婷
研究生(外文):Ying-Ting Chen
論文名稱:多維分離技術與標定策略用於蛋白質身分鑑定
論文名稱(外文):Multidimensional Separation and Labeling Strategies for Protein Identification
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Shu-Hui Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:111
中文關鍵詞:HILIC 管柱蛋白質身分鑑定蛋白質分離
外文關鍵詞:protein separationprotein identificationHydrophilic interaction liquid chromatography
相關次數:
  • 被引用被引用:0
  • 點閱點閱:265
  • 評分評分:
  • 下載下載:44
  • 收藏至我的研究室書目清單書目收藏:0
生物體之蛋白質組成成分十分複雜,若要分析全部組成之蛋白質,樣品之分離步驟便非常重要。由於最終要經由質譜分析完之胜肽片段資訊做蛋白質身分鑑定,所以每次分析的胜肽樣品成分越單純,就越能得到完整之胜肽片段訊息。我的研究目標便是利用多維分離技術來將胜肽樣品的複雜度降低,以提升蛋白質身分鑑定之結果,並在蛋白質階段做分離,搭配穩定同位素二甲基標定策略來對蛋白質做身分鑑定。

在本實驗中,運用散彈槍式蛋白質體學的方法來建立人類乳腺癌細胞MCF-7之蛋白質資料庫。利用不同之多維分離技術來分離MCF-7全細胞蛋白質溶解物,發現具有正交性之多維分離系統-ZIC-HILIC親水性管柱連接奈流C18疏水性管柱,對於蛋白質身分鑑定之結果優於C18疏水性管柱連接奈流C18疏水性管柱系統,且回收率也能大大提升。在ZIC-HILIC親水性管柱連接奈流C18疏水性管柱系統總共可以鑑定到1030個蛋白質、3059個胜肽片段;而C18疏水性管柱連接奈流C18疏水性管柱系統只鑑定到615個蛋白質、1094個胜肽片段,而兩個系統共同鑑定到的數量為蛋白質457個、胜肽片段有902個。

在蛋白質分離部分,發現利用C18管柱分離蛋白質時,有很嚴重的樣品殘留現象,因此選擇C4管柱來分離蛋白質樣品,並且能夠用C4管柱將目標蛋白-雌激素受體純化,並用西方墨點法證明之;再搭配蛋白質標定策略-穩定同位素二甲基標定法,直接在蛋白質層面做標定,可做為研究標靶蛋白質體學的新策略。
The sample separation is a critical step in proteome analysis due to the limitation of mass spectrometry. The more simplicity in sample composition, the more peptide information we can get. In this study, we try to reduce the sample complexity by using multidimensional separation method to obtain a good result of protein identification. Also, we separate the total proteins of human breast cancer cells (MCF-7) at protein level to purify our target protein- estrogen receptor. We also combined ”stable isotope dimethyl labeling” to do the modification at protein level followed with trypsin digestion and mass spectrometry identification.

In multidimensional system, we compared the number of identified proteins using two separation platforms: ZIC-HILIC-nano C18 and C18-nano C18, which only differed in first dimension. 500 μg MCF-7 whole cell lysates were digested into peptides and both systems were used for separation before mass spectrometry identification. Total of 1030 proteins with 3059 peptides were identified using ZIC-HILIC-nano C18 system and 615 proteins with 1094 peptides were identified using C18-nano C18 system. Among these results, there were 457 proteins with 902 peptides existed in both platforms. The HILIC-nano C18 system showed greater separation power since the orthogonality of two separation dimensions.

We also compared the protein separation ability by C4 and C18 columns. We chose C4 column for separation because the mobile phase cannot elute all proteins from C18 and lots of samples retained on beads. After separating MCF-7 whole cell lysate using C4 column, all fractions were collected and ERα were detected using western blotting. The result showed that separation at protein level followed with stable isotope dimethyl labeling is a potential strategy in targeting proteomics.
中文摘要 I
英文摘要 III
誌謝 V
表目錄 XI
圖目錄 XII
第一章 序論 1
1.1 蛋白質的基本結構 1
1.2 蛋白質體學的發展 3
1.3 蛋白質體學方法及技術 5
1.3.1 質譜技術在蛋白質體研究中的角色 6
1.3.1.1 質譜儀的構造與原理 6
1.3.1.2 串聯式質譜儀(tandem mass spectrometry,MS/MS) 14
1.3.2 蛋白質身分鑑定(protein identification) 15
1.3.2.1 高效液相層析(High-performance liquid chromatography, HPLC) 分離原理 16
1.4 實驗架構 18
第二章 利用多維液相層析結合質譜技術建立人類乳腺癌細胞(MCF-7)蛋白質資料庫 20
2.1 研究動機 20
2.2 管柱介紹 20
2.2.1 C18管柱 21
2.2.2 Hydrophilic interaction liquid chromatography (HILIC)管柱 21
2.3 人類乳腺癌細胞(MCF-7)介紹 23
2.4 材料與實驗方法 24
2.4.1 細胞培養 24
2.4.2 MCF-7全細胞萃取 24
2.4.3 蛋白質沉澱及消化 25
2.4.4 胜肽溶液之去鹽 25
2.4.5 高效能液相層析儀之分離 26
2.4.5.1 C18管柱分離 26
2.4.5.2 HILIC管柱分離 27
2.4.6 液相層析結合四極柱-飛行時間質譜儀之偵測 27
2.4.7 蛋白質資料庫鑑定 28
2.5 結果與討論 29
2.5.1 HILIC-nano C18 32
2.5.2 C18-nano C18 32
2.5.3 綜合探討 33
2.6 結論 38
第三章 利用高效液相層析儀分離人類乳腺癌細胞(MCF-7)之雌激素受體 39
3.1 研究動機 39
3.2 雌激素與雌激素受體 39
3.3 材料與實驗方法 40
3.3.1 標準蛋白質溶液之配製 40
3.3.2 MCF-7全細胞萃取 41
3.3.2.1 PBS 溶液萃取細胞法 41
3.3.2.2 RIPA 溶液萃取細胞法 41
3.3.3 高效能液相層析儀之分離 42
3.3.3.1 C18管柱之標準蛋白質分離 42
3.3.3.2 C4管柱之標準蛋白質分離 43
3.3.3.3 HILIC管柱之標準蛋白質分離 43
3.3.3.4 C4管柱之MCF-7雌激素受體純化 44
3.3.4 一維膠電泳之分離 44
3.3.5 西方點墨法 (Western blotting) 分析ERα 45
3.4 結果與討論 46
3.4.1 不同管柱對標準蛋白質之分離效果探討 46
3.4.2 C4管柱純化MCF-7之ERα 48
3.4.2.1 PBS溶液萃取結果 48
3.4.2.2 RIPA溶液萃取結果 49
3.5 綜合探討 49
3.6 結論 53
第四章 穩定同位素二甲基標定於蛋白質階段之分析 54
4.1 研究動機 54
4.2 標定策略 54
4.3 材料與實驗方法 56
4.4 數據與結果 56
第五章 總結與未來展望 61
5.1 總結 61
5.2 未來展望 61
參考文獻 63
附錄 70
1.Hong, T.; Nakagawa, T.; Pan, W.; Kim, M. Y.; Kraus, L.; Ikehara, T.; Yasui, K.; Aihara, H.; Takebe, M.; Muramatsu, M.; Ito, T. Isoflavones Stimulate Estrogen Receptor-mediated Core Histone Acetylation. Biochemical and Biophysical Research Communications 2004, 317, (1), 259–264.

2.Osborne, C. K.; Schiff, R.; Estrogen-Receptor Biology: Continuing Progress and Therapeutic Implications. Journal of Clinical Oncology 2005, 23, (8), 1616-1622.

3.Marabotti, A.; Colonna, G.; Facchiano, A. New Computational Strategy to Analyze the Interactions of ERa and ERb with Different ERE Sequences. Journal of Computational Chemistry 2007, 28, (6), 1031–1041.

4.Wilkins, M. R.; Sanchez, J. C.; Gooley, A. A.; Appel, R. D.; Humphery-Smith, I.; Hochstrasser, D. F.; Williams, K. L. Progress with Proteome Projects: Why All Proteins Expressed by a Genome should be Identified and How to do it. Biotechnology and genetic engineering reviews 1996, 13, 19-50.

5.Kahn, P. From Genome to Proteome: Looking at a Cell’s Protein. Science 1995, 270, (20), 369-370.

6.Swinbanks, D. Government Back Proteome Proposal. Nature 1995, 378, (14), 378.

7.Fenn, J.B.; Mann, M.; Meng, C.K.; Wong, S.F.; Whitehouse, C.M. Electrospray Ionization for Mass Spectrometry of Large Biomolecules. Science 1989, 246, (4926), 64-71.

8.Karas, M.; Hillenkamp, F. Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons. Analytical Chemistry 1988, 60, (20), 2299-2301.

9.Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida Y.; Yoshida, T,. Protein and Polymer Analysis up to m/z 100000 by Laser Ionization Time-of-flight Mass Spectrometry . Rapid communications in mass spectrometry 1988, 2, (8), 151-153.

10.Cleveland, D.W.; Fischer, S.G.; Kirschner, M.W.; Laemmli, U.K. Peptide Mapping by Limited Proteolysis in Sodium Dodecyl Sulfate and Analysis by Gel Electrophoresis. Journal of Biological Chemistry 1997, 252, (3), 1102-1106.

11.Henzel, W. J.; Billeci, T. M.; Stults, J. T.; Wong, S. C.; Grimley, C.; Watanabe, C. Identifying Proteins from Two-dimensional Gels by Molecular Mass Searching of PeptideFragments in Protein Sequence Databases. Proceedings of the National Academy of Sciences of the United States of America 1993, 90, (11), 5011-5015.

12.李美貴;凌永健, 液相層析質譜術介面發展與應用.科儀新知 1992,13,(5),38.

13.Yamashita, M.; Fenn, J.B. Electrospray ion source. Another Variation on the Free-jet Theme. The Journal of Physical Chemistry 1984, 88, (20), 4451-4459.

14.Yamashita, M.; Fenn, J.B. Negative Ion Production with the Electrospray Ion Source. The Journal of Physical Chemistry 1984, 88, (20), 4671-4675.

15.McDonald, W.H.; Yates, J.R. Shotgun Proteomics and Biomarker Discovery. Disease Markers 2002, 18, 99-105.

16.Wolters, D.A.; Washburn, M.P., Yates, J.R. An Automated Multidimensional Protein Identification Technology for Shotgun Proteomics. Analytical Chemistry 2001, 73, (23), 5683-5690.

17.Hu, L.; Ye, M.; Jiang, X; Feng, S.; Zou, H. Advances in Hyphenated Analytical Techniques for Shotgun Proteome and Peptidome Analysis—A Review. Analytica Chimica Acta 2007, 598, (2), 193-204.

18.Fournier, M. L.; Gilmore, J. M.; Martin-Brown, S. A.; Washburn, M. P. Multidimensional Separations-Based Shotgun Proteomics. Chemical Review 2007, 107, (8), 3654–3686.

19.Nesvizhskii, A.I. Protein Identification by Tandem Mass Spectrometry and Sequence Database Searching. Methods in Molecular Biology 2007, 367, 87-119.
20.Shibue, M., Mant, C.T., Hodges, R. S. Effect of Anionic Ion-Pairing Reagent Hydrophobicity on Selectivity of Peptide Separations by Reversed-Phase Liquid Chromatography. Journal of Chromatography A 2005, 1080, (1), 68-75.

21.Gygi, S. P., Rist, B., Gerber, S.A., Turecek, F., Gelb, M.H., Aebersold, R. Quantitative Analysis of Complex Protein Mixtures Using Isotope-Coded Affinity Tags. Nat.Biotechnol 1999, 17, 994-999.

22.Nuwaysir, L. M., Stults, J. T. Electrospray Ionization Mass Spectrometry of Phosphopeptides Isolated by On-Line Immobilized Metal-Ion Affinity Chromatography. Journal of the American Society for Mass spectrometry 1993, 4, 662-669.

23.Posewitz, M. C., Tempst, P. Immobilized Gallium (III) Affinity Chromatography of Phosphopeptides. Analytical Chemistry 1999, 71, (14), 2883-2892.

24.Stensballe, A., Andersen, S., Jensen, O, N. Characterization of Phosphoproteins from Electrophoretic Gels by Nanoscale Fe (III) Affinity Chromatography with Off-Line Mass Spectrometry Analysis. Proteomics 2001, 1, (2), 207-222.

25.Washburn, M. P., Wolters, D., Yates, J. R., 3rd. Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology. Nature Biotechnology 2001, 19, (3), 242-247.

26.Wagner, K., Miliotis, T., Marko-Varga, G., Bischoff, R.,Unger, K. K. An Automated On-Line Multidimensional HPLC System for Protein and Peptide Mapping with Integrated Sample Preparation. Analytical Chemistry 2002, 74, (4), 809-820.

27.Peng, J., Elias, J. E., Thoreen, C. C., Licklider, L. J., Gygi, S. P. Evaluation of Multidimensional Chromatography Coupled with Tandem Mass Spectrometry (LC/LC-MS/MS) for Large-Scale Protein Analysis: The Yeast Proteome. Journal of Proteome Research 2003, 2, (1), 43-50.
.
28.Kachman, M. T., Wang, H.; Schwartz, D. R., Cho, K. R., Lubman, D. M. Multiplexed Gene Expression Analysis Using the Invader RNA Assay with MALDI-TOF Mass Spectrometry Detection. Analytical Chemistry 2002, 74, (8), 1779-1791
29.Silva, J. C., Denny, R., Dorschel, C. A., Gorenstein, M., Kass, I. J., Li, G. Z., McKenna, T., Nold, M. J., Richardson, K., Young, P., Geromanos, S. Quantitative Proteomic Analysis by Accurate Mass Retention Time Pairs Analytical Chemistry 2005, 77, (7), 2187-2200.

30.Guzzetta, A. Reverse Phase HPLC Basics for LC/MS.

31.Hemström, P., Irgum, K. Hydrophilic interaction chromatography. Journal of Separation Science 2006, 29, (12), 1784 – 1821.

32.Alpert, A. J., Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. Journal of Chromatography A 1990, 499, (19), 177-196.

33.Boersema, P. J., Mohammed, S. (et al.). Hydrophilic Interaction Liquid Chromatography (HILIC) in Proteomics. Analytical and Bioanalytical Chemistry 2008, 391, (1), 151–159.

34.Gilar, M., Olivova, P., Daly, A. E., Gebler, J. C. Orthogonality of Separation in Two-Dimensional Liquid Chromatography. Analytical Chemistry 2005, 77, (19), 6426-6434.

35.Boersema, P. J., Divecha, N., Heck, A. J. R., Mohammed, S. Evaluation and Optimization of ZIC-HILIC-RP as an Alternative MudPIT Strategy. Journal of Proteome Research 2007, 6, (3), 937-946.

36.Alpert, A. J. Electrostatic Repulsion Hydrophilic Interaction Chromatography for Isocratic Separation of Charged Solutes and Selective Isolation of Phosphopeptides. Analytical Chemistry 2007, 80, (1), 62–76.

37.Thaysen-Andersen M., Thogersen I. B., Nielsen H. J., Lademann U., Brunner N., Enghild J. J., Hojrup P. Rapid and Individual-specific Glycoprofiling of the Low Abundance N-Glycosylated Protein Tissue Inhibitor of Metalloproteinases-1.Molecular and Cellular Proteomics 2007, 6, 638–647.

38.Garcia, B. A., Pesavento, J. J., Mizzen, C. A., Kelleher, N. L. Pervasive Combinatorial Modification of Histone H3 in Human Cells. Nature Methods 2007, 4, 487-489.
39.Dickson, R. B., Bates, S. E., Mc Manaway, M. E., Lippman, M. E. Characterization of Estrogen Responsive Transforming Activity in Human Breast Cancer Cell Lines. Cancer research 1986, 46, 1707-1713.

40.http://www.abcam.com/MCF-7-Human-breast-adenocarcinoma-cell-line-Whole-cell-lysate-ab3871.html

41.Mukhopadhyay, R., Theriault, R. L., Price, J. E. Increased levels of alpha 6 integrins are associated with the metastatic phenotype of human breast cancer cells. Clinical & experimental metastasis 1999, 17 325-332.

42.http://www.matrixscience.com

43.Shou, W. Z., Naidong, W. Simple Means to Alleviate Sensitivity Loss by Trifluoroacetic acid (TFA) Mobile Phases in the Hydrophilic Interaction Chromatography–Electrospray Tandem Mass Spectrometric (HILIC–ESI/MS/MS) Bioanalysis of Basic Compounds. Journal of Chromatography B 2005, 825, (2), 186 – 192.

44.Jiang, W., Fischer, G., Girmay, Y., Irgum, K. Zwitterionic Stationary Phase with Covalently Bonded Phosphorylcholine Type Polymer Grafts and its Applicability to Separation of Peptides in the Hydrophilic Interaction Liquid Chromatography Mode. Journal of Chromatography A 2006, 1127, (1-2), 82 – 91.

45.Ikegami, T., Horie, K., Jaafar, J., Hosoya, K., Tanaka, N. Preparation of Highly Efficient Monolithic Silica Capillary Columns for the Separations in Weak Cation-Exchange and HILIC Modes. Journal of Biochemical and Biophysical Methods 2007, 70, (1), 31 – 37.

46.Godejohann, M. Hydrophilic Interaction Chromatography Coupled to Nuclear Magnetic Resonance Spectroscopy and Mass Spectroscopy—A New Approach for the Separation and Identification of Extremely Polar Analytes in Bodyfluids. Journal of Chromatography A 2007, 1156, (1-2), 87 – 93.

47.Xu, Y., Sprung, R., Won Kwon, S., Chan Kim, S., Zhao. Y. Isolation of Phosphopeptides by pI-Difference-Based Electrophoresis. Journal of Proteome Research 2007, 6, 1153-1157.
48.林峰賢, Fluorescein Affinity Enrichment of Cystein-containing Peptides for MS-based Proteomics.國立成功大學碩士論文,2007.

49.Giddings, J. C. Concepts and Comparisons in Multidimensional Separation. Journal of High Resolution Chromatography 1987, 10, (5), 319-323.

50.Jandera, P. Stationary Phases for Hydrophilic Interaction Chromatography, their Characterization and Implementation into Multidimensional Chromatography Concepts. Journal of separation science 2008, 31, (9), 1421 – 1437.

51.Hochstrasser, D. F. Proteome in Perspective. Clinical Chemistry and Laboratory Medicine 1998, 36, 825-836.

52.Aebersold, R., Goodlett, D. R. Mass Spectrometry in Proteomics. Chemical Review 2001, 101, 269-295.

53.Giguére, V., Tremblay, A., Tremblay. G. B. Estrogen Eeceptor ß: Re-Evaluation of Estrogen and Antiestrogen Signaling. Steroids 1998, 63, (5-6), 344-348.

54.Julie, M. H., John, F., Kenneth, S. et al. The Multifaceted Mechanisms of Estradiol and Estrogen Receptor Signaling. Journal of biological chemistry 2001, 276, (40), 36869 - 36872.

55.Katzenellenbogen, B. S., Montano, M. M., Ekena, K. et al. Antiestrogens:Mechanisms of Action and Resistance in Breast Cancer . Breast Cancer Research and Treatment 1997 , 44, 23 - 38.

56.Dotzlaq, H., Leygue, E., Watson, P. H., Murphy, L. C.Expression of Estrogen Receptor-ß in Human Breast Tumors. Journal of clinical endocrinology and metabolism 1997, 82, 2371 – 2374.

57.Nunno, L. D., Larsson, L. G., Rinehart, J. J. et al. Estrogen and Progesterone Receptors in nor Small Cell Lung Caner in 248 Consecutive Patient s who Underwent Surgical Resection . Archives of pathology and laboratory medicine 2000, 124, (10), 1467 - 1468.

58.Deroo, B. J., Korach, K. S. Estrogen Receptors and Human Disease. Journal of clinical investigation 2006, 116, 561-570.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top