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研究生： 蔡家烽
CHIA-FENG TSAI
論文名稱： Qualitative and Quantitative Profiling of Protein Phosphorylation Using Immobilized MetalAffinity Chromatography and LC/MS
CONCLUSION
指導教授： 陳玉如
Chen, Yu-Ju
學位類別： 碩士
Master
系所名稱： 化學系
Department of Chemistry
論文出版年： 2007
畢業學年度： 95
語文別： 英文
論文頁數： 76
中文關鍵詞： 質譜儀 、磷酸化
英文關鍵詞： IMAC
論文種類： 學術論文
相關次數： 點閱：204  下載：0 
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    • 蛋白質磷酸化在細胞訊息傳遞及功能調控上扮演關鍵角色。蛋白質的磷酸化是一個動態現象，在細胞內的含量低，並且不同位置的磷酸化可能有不同的功能。對於蛋白質磷酸化進行定性與定量的分析有助於我們了解這些訊息傳遞的複雜過程，所以發展高效能蛋白質磷酸化的分析方法仍是一個重要的課題。
    在本論文中，我們結合凝膠電泳（SDS-PAGE）,金屬親和層析( Immobilized Metal Affinity Chromatography, IMAC),免標記定量法( Label Free)和質譜 (Mass Spectrometry, MS)技術分析磷酸化蛋白體。在定性的實驗方面，我們針對影響金屬親和層析法專一性的因素作最佳化研究。本實驗中，我們發現待測樣品的pH、系統溶液的種類和濃度以及溶液中所含的鹽類的種類都會影響其專一性和回收率。在定量實驗方面，我們以免標記定量法為平臺，利用所加入標準品的層析譜峰面積去校正分析物的層析譜峰，可進行相對定量。我們將此方法分別應用於標準蛋白質和複雜的細胞樣品，為了證明此分析平台的可用性，我們將其應用在分析H1299細胞中的磷酸蛋白上，並以凝膠電泳為輔作細胞蛋白的一維分離，我們可鑑定出1598個磷酸化蛋白質共含有12353個磷酸化胜肽，在所有鑑定出的胜肽中，磷酸化胜肽純化專一性達百分之80。並且其定量的標準誤差可以小到0.3左右。證明此平台能廣泛應用在分析大量蛋白質磷酸化的研究上。

    Qualitative and quantitative analysis of site-specific protein phosphorylation, a key reversible modification in cellular signaling pathways, presents an analytical challenge due to its heterogeneity and low abundance. The most adapted strategy for identification and quantification phosphorylation is by isotope labeling and mass spectrometry analysis is a major recently; however, current methodological are elaborate, relatively expensive and limited to a number of samples. In qualitative analysis, the challenges warrant the need to develop methods capable of accurately elucidating sites of protein phosphorylation. In this study, we aim to develop an efficient and specific platform for high throughput analysis of phosphoprotein form complex protein mixture. The effect of buffer composition, pH value and salt on sample recovery and enrichment specificity will be studies in details. The applicability of this method will be demonstrated for the qualitative profiling of protein phosphorylation in H1299 cell upon stimulation of pervanadate. In quantitative analysis, we present a label-free LC/MS strategy combining SDS-PAGE fractionation, IMAC chromatography and LC-MS/MS for relative quantification of site-specific phosphorylation .The method takes advantage of addition of an internal standard protein before in-gel digestion; integrated chromatographic peak areas of phosphopeptides from analyte proteins are normalized to the phosphopeptide of the internal standard protein. In this method, we can determine phosphorylation level from different stage of p]hosphorylation stoichiometry and simultaneously determine the change in protein level.

    中文摘要.................................................................................................................. ...I Abstract......................................................................................................................II List of figures........................................................................................................... . III List of tables................................................................................................................V Abbreviations............................................................................................................. VI Contents....................................................................................................................VIII CHAPTER 1. INTRODUCTION 1-1 Importance of Protein Phosphorylation 1-1.1 Proteomics and Post-Translational Modification........................................1 1-1.2 Significance of Protein Phosphorylation.....................................................2 1-2 Analytical of Protein Phosphorylation 1-2.1 Analytical Challenges of Protein Phoshporylation......................................3 1-2.2 Traditional Methods for Protein Phosphorylation.......................................3 1-2.3 Phosphoproteomic Study by Mass Spectrometry........................................4 1-2.4 Identificaiton and Characterization of Phosphorylation Sites.....................4 1-2.5 Quantitative Analysis of Phosphorproteome..............................................10 1-3 Objective of Current Study..................................................................................13 CHAPTER 2. MATERIALS AND METHODS 2-1 Materials 2-1.1 Chemical and Apparatus.............................................................................14 2-1.2 Standard Proteins.........................................................................................15 2-2 Instrumentation...............................................................................................16 2-3 Methods 2-3.1 Cell culture and Preparation of Total Cell Extracts......................................17 2-3.2 Protein quantitation by BCA™ protein assay kit.........................................17 2-3.3 Protein quantitation by Bradford assay.........................................................18 2-3.4 SDS-PAGE....................................................................................................18 2-3.5 In-Gel Digestion............................................................................................19 2-3.6 In-Solution Digestion.....................................................................................19 2-3.7 Tube-Gel Digestion........................................................................................20 2-3.8 Preparation of IMAC Column........................................................................20 2-3.9 Phosphopeptides Purification.........................................................................20 2-3.10 Desalting and Concentration.........................................................................21 2-3.11 Mass Analysis...............................................................................................21 2-3.12Data Analysis for Protein Qualitative............................................................22 CHAPTER 3. RESULTS AND DISCUSSION 3-1 Workflow of phosphoproteome analysis using IMAC................................................25 3-1.1 Factor that affect the IMAC Performance...........................................................25 3-1.2 The pH effect on IMAC Performance.................................................................26 3-1.3 The Effect of Acids on IMAC Performance........................................................28 3-1.4 The Effect of Digestion Condition on the IMAC Performance...........................30 3-1.5 Recovery of Optimized IMAC System................................................................31 3-1.6 Profiling of Protein Phosphorylation Using IMAC-LC-MS/MS.........................32 3-2 The Overview of Label Free Quantitative of Phosphoproteom..............................34 3-2.1 Reproducibility of Retention Time in Different LC-MS Runs............................35 3-2.2 Dynamic Range Label Free for the phosphoprotein Quantitation.......................35 3-2.3 Protein Quantification in HI299 Cell lysate Using Tube-Gel Digestion..............36 3-2.4 Protein Quantitation in HI299 Cell Lysate with SDS-PAGE Fractionation.........37 CHAPTER 4. CONCLUSION.............................................................................................39 References..........................................................................

    1. Alaiya, A.; Al-Mohanna, M.; Linder, S., Clinical Cancer Proteomics: Promises and Pitfalls. J. Proteome Res. 2005, 4, (4), 1213-1222.
    2. Mann, M.; Jensen, O. N., Proteomic analysis of post-translational modifications. Nat Biotech 2003, 21, (3), 255-261.
    3. Delom, F.; Chevet, E., Phosphoprotein analysis: from proteins to proteomes. Proteome Science 2006, 4, (1), 15.
    4. Mitchell, K. J.; Tsuboi, T.; Rutter, G. A., Role for Plasma Membrane-Related Ca2+-ATPase-1 (ATP2C1) in Pancreatic {beta}-Cell Ca2+ Homeostasis Revealed by RNA Silencing. Diabetes 2004, 53, (2), 393-400.
    5. Cohen, P., PROTEIN KINASES - THE MAJOR DRUG TARGETS OF THE TWENTY-FIRST CENTURY? Nature Reviews Drug Discovery 2002, 1, (4), 309-315.
    6. Hunter, T., Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling. Cell 1995, 80, (2), 225-236.
    7. Raska, C. S.; Parker, C. E.; Dominski, Z.; Marzluff, W. F.; Glish, G. L.; Pope, R. M.; Borchers, C. H., Direct MALDI-MS/MS of Phosphopeptides Affinity-Bound to Immobilized Metal Ion Affinity Chromatography Beads. Anal. Chem. 2002, 74, (14), 3429-3433.
    8. Blume-Jensen, P.; Hunter, T., Oncogenic kinase signalling. Nature 2001, 411, (6835), 355-365.
    9. Lit-fui Lau, M. A.; Schachter, J. B.; Seymour, P. A.; Sanner, M. A., Tau Protein Phosphorylation as a Therapeutic Target in Alzheimer's Disease. Current Topics in Medicinal Chemistry 2002, 2, (4), 395.
    10. Loyet, K. M.; Stults, J. T.; Arnott, D., Mass Spectrometric Contributions to the Practice of Phosphorylation Site Mapping through 2003: A Literature Review. Mol Cell Proteomics 2005, 4, (3), 235-245.
    11. Peter van der Geer, T. H., Phosphopeptide mapping and phosphoamino acid analysis by electrophoresis and chromatography on thin-layer cellulose plates. Electrophoresis 1994, 15, (1), 544-554.
    12. Sullivan, S.; Wong, T. W., A manual sequencing method for identification of phosphorylated amino acids in phosphopeptides. Analytical Biochemistry 1991, 197, (1), 65-68.
    13. Campbell, D. G.; Morrice, N. A., Identification of protein phosphorylation sites by a combination of mass spectrometry and solid phase Edman sequencing. J Biomol Tech 2002, 13, (3), 119-130.
    14. Mann, M.; Ong, S.-E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A., Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends in Biotechnology 2002, 20, (6), 261-268.
    15. Yan, J. X.; Packer, N. H.; Gooley, A. A.; Williams, K. L., Protein phosphorylation: technologies for the identification of phosphoamino acids. Journal of Chromatography A 1998, 808, (1-2), 23-41.
    16. Ballard, J. N. M.; Lajoie, G. A.; Yeung, K. K. C., Selective sampling of multiply phosphorylated peptides by capillary electrophoresis for electrospray ionization mass spectrometry analysis. Journal of Chromatography A In Press, Corrected Proof.
    17. Steen, H.; Jebanathirajah, J. A.; Rush, J.; Morrice, N.; Kirschner, M. W., Phosphorylation Analysis by Mass Spectrometry: Myths, Facts, and the Consequences for Qualitative and Quantitative Measurements. Mol Cell Proteomics 2006, 5, (1), 172-181.
    18. Seeley, E. H.; Riggs, L. D.; Regnier, F. E., Reduction of non-specific binding in Ga(III) immobilized metal affinity chromatography for phosphopeptides by using endoproteinase glu-C as the digestive enzyme. Journal of Chromatography B 2005, 817, (1), 81-88.
    19. Oda, Y.; Nagasu, T.; Chait, B. T., Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotech 2001, 19, (4), 379-382.
    20. Hitto Kaufmann, J. E. B. M. F., Use of antibodies for detection of phosphorylated proteins separated by two-dimensional gel electrophoresis. PROTEOMICS 2001, 1, (2), 194-199.
    21. Guoan Zhang, T. A. N., Use of detergents to increase selectivity of immunoprecipitation of tyrosine phosphorylated peptides prior to identification by MALDI quadrupole-TOF MS. PROTEOMICS 2006, 6, (2), 571-578.
    22. Rush, J.; Moritz, A.; Lee, K. A.; Guo, A.; Goss, V. L.; Spek, E. J.; Zhang, H.; Zha, X.-M.; Polakiewicz, R. D.; Comb, M. J., Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat Biotech 2005, 23, (1), 94-101.
    23. Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P., Large-scale characterization of HeLa cell nuclear phosphoproteins. PNAS 2004, 101, (33), 12130-12135.
    24. Ballif, B. A.; Villen, J.; Beausoleil, S. A.; Schwartz, D.; Gygi, S. P., Phosphoproteomic Analysis of the Developing Mouse Brain. Mol Cell Proteomics 2004, 3, (11), 1093-1101.
    25. Lim, K. B.; Kassel, D. B., Phosphopeptides enrichment using on-line two-dimensional strong cation exchange followed by reversed-phase liquid chromatography/mass spectrometry. Analytical Biochemistry 2006, 354, (2), 213-219.
    26. Villen, J.; Beausoleil, S. A.; Gerber, S. A.; Gygi, S. P., Large-scale phosphorylation analysis of mouse liver. PNAS 2007, 104, (5), 1488-1493.
    27. Nuhse, T. S.; Stensballe, A.; Jensen, O. N.; Peck, S. C., Large-scale Analysis of in Vivo Phosphorylated Membrane Proteins by Immobilized Metal Ion Affinity Chromatography and Mass Spectrometry. Mol Cell Proteomics 2003, 2, (11), 1234-1243.
    28. Posewitz, M. C.; Tempst, P., Immobilized Gallium(III) Affinity Chromatography of Phosphopeptides. Anal. Chem. 1999, 71, (14), 2883-2892.
    29. Corthals, G. L.; Aebersold, R.; Goodlett, D. R.; Burlingame, A. L., Identification of Phosphorylation Sites Using Microimmobilized Metal Affinity Chromatography. In Methods in Enzymology, Academic Press: 2005; Vol. Volume 405, pp 66-81.
    30. Tornqvist, M.; Mowrer, J.; Jensen, S.; Ehrenberg, L., Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman degradation method. Analytical Biochemistry 1986, 154, (1), 255-266.
    31. Anna Dubrovska, S. S., Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography. PROTEOMICS 2005, 5, (18), 4678-4683.
    32. Ping Cao, J. T. S., Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry. Rapid Communications in Mass Spectrometry 2000, 14, (17), 1600-1606.
    33. Jinglan Wang, Y. Z. H. J. Y. C. X. Q., Phosphopeptide detection using automated online IMAC-capillary LC-ESI-MS/MS. PROTEOMICS 2006, 6, (2), 404-411.
    34. Ndassa, Y. M.; Orsi, C.; Marto, J. A.; Chen, S.; Ross, M. M., Improved Immobilized Metal Affinity Chromatography for Large-Scale Phosphoproteomics Applications. J. Proteome Res. 2006, 5, (10), 2789-2799.
    35. Kokubu, M.; Ishihama, Y.; Sato, T.; Nagasu, T.; Oda, Y., Specificity of Immobilized Metal Affinity-Based IMAC/C18 Tip Enrichment of Phosphopeptides for Protein Phosphorylation Analysis. Anal. Chem. 2005, 77, (16), 5144-5154.
    36. Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q. T.; Ericson, C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.; Peters, E. C., Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. PNAS 2003, 100, (2), 443-448.
    37. Pinkse, M. W. H.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. R., Selective Isolation at the Femtomole Level of Phosphopeptides from Proteolytic Digests Using 2D-NanoLC-ESI-MS/MS and Titanium Oxide Precolumns. Anal. Chem. 2004, 76, (14), 3935-3943.
    38. Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. D., Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns. Mol Cell Proteomics 2005, 4, (7), 873-886.
    39. Chen, C. T.; Chen, Y. C., Fe3O4/TiO2 Core/Shell Nanoparticles as Affinity Probes for the Analysis of Phosphopeptides Using TiO2 Surface-Assisted Laser Desorption/Ionization Mass Spectrometry. Anal. Chem. 2005, 77, (18), 5912-5919.
    40. Chen, C. T.; Chen, W. Y.; Tsai, P. J.; Chien, K. Y.; Yu, J. S.; Chen, Y. C., Rapid Enrichment of Phosphopeptides and Phosphoproteins from Complex Samples Using Magnetic Particles Coated with Alumina as the Concentrating Probes for MALDI MS Analysis. J. Proteome Res. 2007, 6, (1), 316-325.
    41. Lo, C. Y.; Chen, W. Y.; Chen, C. T.; Chen, Y. C., Rapid Enrichment of Phosphopeptides from Tryptic Digests of Proteins Using Iron Oxide Nanocomposites of Magnetic Particles Coated with Zirconia as the Concentrating Probes. J. Proteome Res. 2007, 6, (2), 887-893.
    42. Bodenmiller, B.; Mueller, L. N.; Mueller, M.; Domon, B.; Aebersold, R., Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Meth 2007, 4, (3), 231-237.
    43. Zhang, H.; Zhang, C.; Lajoie, G. A.; Yeung, K. K. C., Selective Sampling of Phosphopeptides for Detection by MALDI Mass Spectrometry. Anal. Chem. 2005, 77, (18), 6078-6084.
    44. Kirkpatrick, D. S.; Gerber, S. A.; Gygi, S. P., The absolute quantification strategy: a general procedure for the quantification of proteins and post-translational modifications. Methods 2005, 35, (3), 265-273.
    45. Ong, S.-E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M., Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Mol Cell Proteomics 2002, 1, (5), 376-386.
    46. 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 Biotech 1999, 17, (10), 994-999.
    47. Yao, X.; Freas, A.; Ramirez, J.; Demirev, P. A.; Fenselau, C., Proteolytic 18O Labeling for Comparative Proteomics: Model Studies with Two Serotypes of Adenovirus. Anal. Chem. 2001, 73, (13), 2836-2842.
    48. Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P., Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. PNAS 2003, 100, (12), 6940-6945.
    49. Fang, R.; Elias, D. A.; Monroe, M. E.; Shen, Y.; McIntosh, M.; Wang, P.; Goddard, C. D.; Callister, S. J.; Moore, R. J.; Gorby, Y. A.; Adkins, J. N.; Fredrickson, J. K.; Lipton, M. S.; Smith, R. D., Differential Label-free Quantitative Proteomic Analysis of Shewanella oneidensis Cultured under Aerobic and Suboxic Conditions by Accurate Mass and Time Tag Approach. Mol Cell Proteomics 2006, 5, (4), 714-725.
    50. Nadja B. Cech, C. G. E., Practical implications of some recent studies in electrospray ionization fundamentals. Mass Spectrometry Reviews 2001, 20, (6), 362-387.
    51. Robert D. Voyksner, H. L., Investigating the use of an octupole ion guide for ion storage and high-pass mass filtering to improve the quantitative performance of electrospray ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 1999, 13, (14), 1427-1437.
    52. Wang, G.; Wu, W. W.; Zeng, W.; Chou, C. L.; Shen, R. F., Label-Free Protein Quantification Using LC-Coupled Ion Trap or FT Mass Spectrometry: Reproducibility, Linearity, and Application with Complex Proteomes. J. Proteome Res. 2006, 5, (5), 1214-1223.
    53. LeBihan, T.; Goh, T.; Stewart, I. I.; Salter, A. M.; Bukhman, Y. V.; Dharsee, M.; Ewing, R.; Wisniewski, J. R., Differential Analysis of Membrane Proteins in Mouse Fore- and Hindbrain Using a Label-Free Approach. J. Proteome Res. 2006, 5, (10), 2701-2710.
    54. Ian I. Stewart, L. Z. T. L. B. B. L. S. S. D. F. G. D. M. O. O. M. D. C. O. R. E. T. G., The reproducible acquisition of comparative liquid chromatography/tandem mass spectrometry data from complex biological samples. Rapid Communications in Mass Spectrometry 2004, 18, (15), 1697-1710.
    55. Lu, X.; Zhu, H., Tube-Gel Digestion: A Novel Proteomic Approach for High Throughput Analysis of Membrane Proteins. Mol Cell Proteomics 2005, 4, (12), 1948-1958.
    56. Steen, H.; Kuster, B.; Mann, M., Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning. Journal of Mass Spectrometry 2001, 36, (7), 782-790.
    57. Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M., Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode. Analytical Chemistry 2001, 73, (7), 1440-1448.
    58. Chernushevich, I. V.; Loboda, A. V.; Thomson, B. A., An introduction to quadrupole-time-of-flight mass spectrometry. Journal of Mass Spectrometry 2001, 36, (8), 849-865.
    59. al, T. C. e., LC/MS and LC/MS/MS screening for the sites of posttranslational modification in proteins. Methods in Protein Sequence Analysis 1991, 249-256.
    60. al, M. J. H. e., Selective detection of phosphopeptides in complex mixtures by electrospray liquid chromatography/mass spectrometry. J. Am. Soc. Mass Spectrom 1993, 4, 710-717.
    61. Schlosser, A.; Pipkorn, R.; Bossemeyer, D.; Lehmann, W. D., Analysis of protein phosphorylation by a combination of elastase digestion and neutral loss tandem mass spectrometry. Anal Chem 2001, 73, (2), 170-6.
    62. Li, X.; Gerber, S. A.; Rudner, A. D.; Beausoleil, S. A.; Haas, W.; Villen, J.; Elias, J. E.; Gygi, S. P., Large-Scale Phosphorylation Analysis of α-Factor-Arrested Saccharomyces cerevisiae. J. Proteome Res. 2007, 6, (3), 1190-1197.
    63. Ueda, E. K. M.; Gout, P. W.; Morganti, L., Current and prospective applications of metal ion-protein binding. Journal of Chromatography A 2003, 988, (1), 1-23.
    64. Cutillas, P. R.; Geering, B.; Waterfield, M. D.; Vanhaesebroeck, B., Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards: Analysis of Phosphoprotein Dynamics in a B Cell Lymphoma Cell Line. Mol Cell Proteomics 2005, 4, (8), 1038-1051.

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