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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：143  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..........................................................................

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