臺灣博碩士論文加值系統

許多膜蛋白質是重要的藥物分子標的，發展有效方法針對疾病相關膜蛋白質作定性以及定量分析一直是研究的重心。為了達到高靈敏、快速且同時針對多組樣品作膜蛋白質體定量分析的目的，我們發展了一個 SEMI 概念為基礎的免標定 (Label-free) 定量膜蛋白平台。此平台具有以下幾點優點:(1)針對膜蛋白進行大規模定性分析;(2)可同時定量分析多組樣品;(3)可分析少量的樣品量。在論文的第一部分著重在新定量策略的方法學開發及評估執行的可行性，並與同位素標定定量法之一的 iTRAQ 標定法進行比較。在 label-free (免標定定量法) 中，使用 106 個細胞即可高準確的定量 376 個蛋白質 (在 log2 的模式中，mean±SD = -0.03±0.29)。
本研究的第二部分，我們將 label-free 與 iTRAQ 兩種定量法應用到兩種生物系統。在阿拉伯芥系統之應用中，針對阿拉伯芥 pho2 突變植株與 wild-type (野生型) 的植株其根部的膜蛋白進行分析，實驗結果建立目前最大規模的阿拉伯芥膜蛋白質體資料庫。近來有研究指出 PHO2 (UBC24; a ubiquitin-conjugating E2 enzyme) 在阿拉伯芥調控磷酸鹽吸收及運送中扮演關鍵性的角色，然而 PHO2 下游之訊號調控路徑仍然未知。由於 PHO2 的訊號調控路徑中膜蛋白易參與 ubiquitin (泛素) 調控的蛋白質水解反應，在此研究中，我們應用定量膜蛋白體學來探討 PHO2 下游可能作用之標的。利用本實驗室先前所發展之 gel-assisted digestion (膠體輔助蛋白質水解方法) 結合 iTRAQ 定量法、2D LC-MS/MS (二維液相層析串聯質譜) 技術的定量策略，針對兩種不同的植物生長系統，總計定量了 2795 個蛋白質，而在其中相對於 wild-type 植株的蛋白質，有 245 個蛋白質在阿拉伯芥 pho2 突變植株上表現量有變化。相較於文獻所載，在阿拉伯芥中，我們所鑑定到的 1519 個膜蛋白質是目前最豐富的一組數據。值得探討的是在此 245 個有變化的蛋白質中，其中有 65 個蛋白質為 transporter (轉運蛋白)。其中，Pi transporter traffic facilitator 1 (PHF1) 與許多 PHT1 家族的 Pi transporters (PHT1;1, PHT1;2, PHT1;3 and PHT1;4) 在 pho2 突變植株的根中表現量是上升的，顯示其可能在 pho2 突變植株中扮演增加磷酸鹽吸收及運送的角色。此外，在西方墨點法之實驗結果顯示 PHF1、PHT1;1、PHT1;2、PHT1;3 和 PHT1;4 此五種蛋白質之表現與質譜分析鑑定結果吻合，且從 phf1 基因突變的研究結果也可證實 PHF1 與 PHT1 家族在 PHO2 下游的角色。此研究有助於探討植物之吸收作用及其調控磷酸鹽的分子機制。
在幹細胞系統之應用中, 本研究著重於對人類胚胎幹細胞的膜蛋白質體分化過程之探討。同時進行利用 SEMI 概念為基礎的 label-free 與 iTRAQ 定量法分析，比對胚胎幹細胞分化前及分化後之膜蛋白表現量。結果顯示，在 label-free 定量法中，共計鑑定出 981 個蛋白質，其中可定量 867 個蛋白質；而在 iTRAQ 定量法中，則可鑑定出 3369 個蛋白質，當中可定量 2734 個蛋白質。在不同表現量之蛋白質中， label-free 與 iTRAQ 定量法的結果進行分析比較後發現其變化的趨勢為一致，顯示本研究發展之定量方法與定量策略確實能有效的尋找出幹細胞的細胞表面標的蛋白，而此些細胞表面標的蛋白可能應用在人類胚胎幹細胞以分離其特定分化時期之細胞，並幫助釐清幹細胞自我更新及分化之機制。

Many membrane proteins are implicated in particular diseases states and often are attractive therapeutic targets. Comprehensive and quantitative profiles of membrane proteins facilitate our understanding of their roles in regulating biological processes and in cellular signaling. Towards highly sensitive, multiplexed, and robust quantitation of the membrane proteome, we aimed to develop a SEMI-based label-free quantitative membrane proteomics strategy capable of (1) comprehensive identification of large number of membrane proteins, (2) multiplexed analysis of multiple samples, and (3) requirements for limited amounts of starting material. The first part of study focuses on the methodology development and performance evaluation of the new strategy and comparison with isotope labeling method. Using this quantitation approach, 376 proteins can be quantified from 106 cells with high quantitation accuracy and reasonable standard deviation (-0.03±0.29 in log2 scale).
In the second part of thesis, we applied this new label-free strategy and our previously developed iTRAQ-based quantitation method to two biology systems. The first example presents the most comprehensive proteomics delineation of membrane proteome from the roots of wild-type Arabidopsis and pho2 mutant. Recent advances indicate that PHO2, a ubiquitin-conjugating E2 enzyme (UBC24), plays a crucial role in regulating phosphate (Pi) uptake and translocation in Arabidopsis thaliana; however, the molecular mechanism downstream of the PHO2 signaling pathway remain unknown. Because membrane proteins subjected to ubiquitin-mediated proteolysis are very likely participated in the PHO2 signaling pathway, the membrane proteomics was employed to reveal the potential targets of PHO2 in this study. Based on our recently developed multiplexed quantitation strategy combining gel-assisted digestion, iTRAQ labeling and 2D-LC/MS/MS, total of 2795 proteins were quantified, of which 245 proteins showed statistically significant variation between wild-type and pho2 plants grown under two different conditions. To our knowledge, this dataset presents the largest Arabidopsis membrane proteome reported so far; as many as 1519 membrane proteins were ananotated as membrane proteins or containing at least one potential transmembrane helice. Of note, 65 proteins of differentially expressed proteins in the pho2 roots were annotated with transporter-related activity. Among them, the Pi transporter traffic facilitator 1 (PHF1) and many Pi transporters in the PHT1 family (PHT1;1, PHT1;2, PHT1;3 and PHT1;4) were upregulated in the pho2 roots, which suggests they could be responsible for the enhanced activities of Pi uptake and translocation in the pho2. Moreover, western analyses of these proteins and genetic study on the phf1 mutant supported the role of PHT1 family and PHF1 in the downstream pathway of PHO2. This study has provided insights into the molecular mechanism regulating Pi acquisition in plants.
The second example focuses on the membrane proteomics profiling of human embryonic stem cell. The SEMI-based label-free and iTRAQ-based quantitation methods were both employed for comparing the membrane proteome between undifferentiated and differentiated human embryonic stem cell. Using label-free approach, 981 proteins were identified with 95% confidence and 867 proteins were quantified. Among these proteins, significantly altered proteins between groups were compared with the results of iTRAQ labeling (3369 identified proteins) and showed consistent conclusions. Our method not only demonstrated its power on discovery of specific hESC markers for the corresponding ES cell state but may help to shed light on the mechanisms for self-renewal and differentiation.

誌謝.......................................................i
中文摘要..................................................ii
Abstract..................................................iv
Table of Contents........................................vii
List of Figures..........................................xii
List of Tables...........................................xvi
Abbreviations..........................................xviii
CHAPTER 1 INTRODUCTION.....................................1
1.1 Significance of Membrane Proteins......................1
1.2 Methods for Quantitative Membrane Proteomics...........2
1.2.1 Two-dimensional Gel Electrophoresis (2DE)............2
1.2.2 Stable Isotope Labeling Quantitation.................3
1.2.2.1 Metabolic Labeling in Cell Culture.................4
1.2.2.2 Enzymatic Labeling.................................5
1.2.2.3 Absolute Quantitation (AQUA).......................5
1.2.2.4 Chemical Labeling..................................6
1.2.3 Label-free Quantitation.............................10
1.3 Introduction of Phosphate Uptake in Arabidopsis thaliana and the Molecular Mechanism of PHO2 Signaling Pathway...................................................14
1.4 Membrane Proteome of Human Embryonic Stem Cell........16
1.4.1 Introduction of Human Embryonic Stem Cells..........16
1.4.2 Significance of Membrane Proteome in Stem Cell Biology...................................................17
1.5 Objective of this Study...............................19
CHAPTER 2 MATERILS AND METHODS............................21
2.1 Materials.............................................21
2.2 Sample Preparation....................................21
2.2.1 Plant Growth and Preparation of Total Root Membrane Fraction..................................................21
2.2.2 Culture and Differentiation of Stem Cells...........22
2.2.2.1 Feeder-Free Culture of Undifferentiated Human Embryonic Stem Cells (hES5)...............................22
2.2.2.2 Differentiation of Human Embryonic Stem Cells (hES5) from Embryonic Bodies to Embryoid Body (EB) Outgrowth.................................................23
2.2.3 Cell Culture of HeLa Cells..........................23
2.2.4 Membrane Protein Preparation........................24
2.2.4.1 Membrane Protein Purification from Cell Lines.....24
2.2.4.2 Membrane Protein Purification from Mouse Liver Tissues...................................................25
2.3 Gel-assisted Digestion for Membrane Proteins..........25
2.4 Protein and Peptide Assays............................26
2.4.1 Coomassie (Bradford) Protein Assay Kit (Pierce, Rockford, IL).............................................26
2.4.2 BCATM Protein Assay Kit (Pierce, Rockford, IL)......27
2.5 Desalting and Concentration...........................27
2.6 Quantitative Proteomic Analysis by iTRAQ Labeling.....28
2.6.1 iTRAQ Labeling Reaction with Four Stages of Peptides..................................................28
2.6.2 Peptide Fractionation by Strong Cation Exchange Chromatography............................................28
2.6.3 LC-MS/MS Analysis by Q-TOF Premier..................29
2.6.4 Data Analysis.......................................29
2.6.4.1 Database Search and Protein Identification........29
2.6.4.2 Quantification by Multi-Q.........................30
2.7 Quantitative Proteomic Analysis by Label-free Quantitation..............................................31
2.7.1 LC-MS/MS Analysis by Q-TOF Premier..................31
2.7.2 Database Search and Protein Identification..........32
2.7.3 Quantification by IDEAL-Q...........................33
2.8 Prediction the Transmembrane Helices and Gene Ontology Classification............................................33
2.9 Western Blot Analysis.................................34
2.10 Immunostaining and Fluorescence Microscopy...........34
CHAPTER 3 RESULTS.........................................36
3.1 Highly Sensitive and Multiplexed Quantitation Method for Membrane Proteomics Using Label-free and iTRAQ Labeling Strategies................................................36
3.2 Recovery of Membrane Protein Purification from Cells and Tissues...............................................37
3.2.1 Purification Recovery of Cell Model.................37
3.2.2 Starting Materials in Tissue Model..................38
3.3 Optimized Protocol for Label-free Quantitation........38
3.3.1 Optimization of LC Gradient and Acquisition Mode of LC-MS/MS..................................................38
3.3.2 Quantitation Accuracy and Reproducibility in Different LC Gradient.....................................40
3.3.3 Quantitation Accuracy and Reproducibility Obtained from Different Amount of Starting Materials...............41
3.4 Optimization for iTRAQ Strategy: Quantitation Accuracy and Reproducibility in Different Amount of Starting Materials.................................................42
3.5 Comparison of Quantitation Performance between Label-free and iTRAQ Labeling Strategies by Replicate Experiments...............................................44
3.6 Multiplexed Quantitation of Membrane Proteome of Wild-type Plants and pho2 Mutant..........................45
3.6.1 Reproducibility for Purification and Quantitation of Membrane Proteome.........................................45
3.6.2 Comparatively Quantitative Analysis of Membrane Proteins from Roots of Wild-type Plants and pho2 Mutant...46
3.7 Membrane Proteomics Analysis of Human Embryonic Stem Cells during Differentiation..............................48
3.7.1 Large-Scale Quantitative Membrane Proteomics of Stem Cells by iTRAQ-based Quantitation Method..................49
3.7.1.1 Optimized LC Gradient for iTRAQ Labeled Peptides in Different Fractions from SCX Separation...................49
3.7.1.2 Reproducibility for Purification and Quantitation.50
3.7.1.3 Quantitative Membrane Proteomics between Human Embryonic Stem Cell and Embryoid Body.....................51
3.7.2 Large-Scale Quantitative Membrane Proteomics of Stem Cells by Label-free Quantitation Method...................53
3.7.2.1 Reproducibility of Purification and Quantitation of Membrane Proteins on Stem Cell............................53
3.7.2.2 Quantitative Analysis of Membrane Proteomics of Human Embryonic Stem Cells and Embryoid Bodies............54
CHAPTER 4 DISCUSSION......................................56
4.1 Highly Sensitive Quantitation Strategy Provides Large-scale Quantification and Accuracy for Membrane Proteome..................................................56
4.2 Multiplexed Quantitation of Membrane Proteome of Wild-type Plants and pho2 Mutant..........................58
4.2.1 Pathway Mapping of Differentially Expressed Proteins from Roots of Wild-type Arabidopsis and pho2 Mutant.......59
4.3 Membrane Proteomics Analysis of Human Embryonic Stem Cells during Differentiation..............................61
4.3.1 Comparison of the Label-free and iTRAQ Quantitation
Methods via Membrane Proteome of Human ESC................61
4.3.2 Comparative Membrane Proteome of Stem Cells for the Discovery of Potential Biomarkers.........................62
4.3.2 Pathway Mapping of Differentially Expressed Proteins
from hESC and EB during Cell Differentiation..............67
CHAPTER 5 CONCLUSION......................................69
Referances................................................72
Figures...................................................89
Tables...................................................121
Appendix.................................................163

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