臺灣博碩士論文加值系統

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。雖然目前已發展數種鑑定蛋白質磷酸化的方法，但要觀察到細胞內所有的蛋白質的磷酸化仍有一段很長的距離。這是由於蛋白質的磷酸化是一個動態現象，在細胞內的含量低，並且位置也會有所不同。為了要解決這樣的困難，發展更有效的分析蛋白質磷酸化的方法仍是一個重要的課題。
在本論文中，我們結合次細胞分離法(subcellular fractionation),凝膠電泳（SDS-PAGE）,金屬親和層析 ( immobilized metal affinity chromatography,IMAC),質譜 (mass spectrometry, MS)等技術分析複雜蛋白質樣品中所含的磷酸化蛋白，並針對其中IMAC 對於磷酸化胜的容量，專一性，沖提體積以及條件作細部的探討以及最佳化。為了證明此分析平台的可用性，我們將其應用在分析Ｔ細胞中的磷酸蛋白上。實驗中在細胞質中在603 個蛋白質中發現了782 個磷酸化胜及891個磷酸化的位置，在所有鑑定出的胜中，磷酸化胜佔百分之九十。此平台能廣泛應用在分析大量蛋白質磷酸化的研究上。

The protein phosphorylation is key step in cellular signaling to initiate various cellular functions. Despite the advances of various powerful analytical methods is
available, global characterization of the site specific phosphorylation remains far from routine practice. The characterization of phosphorylation has been challenged by the technical difficulty associated with their abundance in cell, dynamic modification pattern, and heterogeneous forms of phosphoproteins. The challenge warrants the need to develop methods capable of accurately elucidating sites of protein phosphorylation.

In this study, a high throughput platform combining subcellular fractionation, SDS-PAGE, immobilized metal affinity chromatography (IMAC) and mass spectrometry
(MS) was introduced for the analysis of phosphoprotein form complex protein mixture.The loading capacity, binding specificity, sample recovery, elution volume and elution
reagent of the IMAC were optimized and studied in detail. To demonstrate the feasibility for the analysis of complex protein mixture, the phosphopeptide from the crude protein
extract of the human jurkat T-cell was subjected to the SDS-PAGE separation, IMAC purification, followed by the nLC-ESI-MS analysis. The preliminary result identified 782
phosphopeptides with 891 phosphorylation sites from 603 proteins in cytosolic fraction, about 90% of the identified peptides were found to be phosphorylated using optimized
IMAC parameters. This technology platform can be applied to profiling protein phosphorylation on the proteome level.

Chinese Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III
Table of Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI
List of Figures and Tables . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . X
Nonstandard Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIII
Chapter 1: Introduction
1-1 Phosphoproteomics
1-1.1 From Genomics to Proteomics
1-1.2 Post Translational Modification
1-1.3 Protein Phosphorylation
1-2 Analysis of Protein Phosphorylation
1-2.1 Analytical Challenges of Protein Phosphorylation
1-2.2 Analytical Techniques for Protein Phosphorylation
1-2.3 Mass Spectrometry Analysis of Protein Phosphorylation
1-2.4 Challenges Associated with Mass Spectrometry Analysis
1-3 Enrichment Strategies to Analyze Phosphopeptides
1-3.1 Phosphospecific Antibodies
1-3.2 Chemical Modification
1-3.3 Immobilized Metal Affinity Chromatography
1-3.4 Strong Cation Exchange
1-4 Objectives of Current Study
VII
Chapter 2: Experimental Section
2-1 Materials
2-1.1 Chemicals and Materials
2-1.2 Standard Proteins
2-2 Instruments
2-2.1 High Performance Liquid Chromatography
2-2.3 MALDI-TOF MS (Voyager)
2-2.3 Hybrid Quadrupole TOF MS (QSTAR)
2-3 Methods
2-3.1 Preparation of Human Jurkat T Cell
2-3.1.1 Cell Culture
2-3.1.2 Subcellular Fractionation
2-3.2 BCA Protein Assay
2-3.3 SDS-PAGE Electrophoresis
2-3.3.1 Preparation of Mini-Gel
2-3.3.2 Preparation of Large-Gel
2-3.3.3 Stain of SDS-PAGE
2-3.4 Trypsin Digestion
2-3.4.1 In-Solution Digestion
2-3.4.2 In-Gel Digestion
2-3.5 Desalting and Concentration
2-3.5.1 Zip Tip
2-3.5.2 Macrotrap
2-3.6 Immobilized Metal Affinity Chromatography
2-3.6.1 Preparation of IMAC Column
VIII
2-3.6.2 IMAC Procedures
2-3.7 Methylation of Carboxylic Acid Groups
2-3.8 HPLC-UV Quantification
2-3.9 Mass analysis
2-3.8.1 MAlDI-TOF MS Analysis
2-3.8.2 Capillary nLC-ESI-MS/MS Analysis
2-3.10 Database Search
Chapter 3: Results and Discussion
3-1 Immobilized Metal Affinity Chromatography
3-1.1 Phosphopeptides Purification with MALDI–TOF MS Analysis
3-1.2 Optimization of IMAC Procedures
3-1.2.1 Elution Volume
3-1.2.2 Elution Solution
3-1.2.3 Reusability
3-1.2.4 Sensitivity
3-1.2.5 Column Capacity
3-1.2.6 Sample Recovery
3-1.2.7 Specificity
3-1.3 Methylation of Phosphopepides
3-1.4 Identification of Phosphorylation sites of β-Caseins Using nLCMS/
MS
3-2 Phosphoproteomic Analysis of Human Jurkat T Cell
3-2.1 Strategic Separated Jurkat Human T Cell
3-1.2.1 Subcellular Fractionation of Human Jurkat T cell
IX
3-1.2.1 Separation of Subcellular Fractionation Using SDS-PAGE
3-2.2 Analysis Human Jurkat T cell Using IMAC with nLC-MS/MS
3-2.3 The Effect of pH on the IMAC Selectivity
3-2.4 Strategic Separated Jurkat Human T Cell Using SDS-PAGE with
IMAC-nLC-MS/MS Analysis
Chapter 4: Conclusion and Prospects
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