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研究生：	韓嘉莉
論文名稱：	開發可針對多重樣品進行膜蛋白體定量分析之策略 A Multiplexed Quantitative Strategy for Membrane Proteomics: Opportunities for Mining Therapeutic Targets for Disease
指導教授：	陳玉如
學位類別：	博士 Doctor
系所名稱：	化學系 Department of Chemistry
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	94
中文關鍵詞：	蛋白質體學、質譜儀、膜蛋白質
論文種類：	學術論文
相關次數：	點閱：169 下載：0
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膜蛋白質調控許多細胞內的重要功能，許多膜蛋白質的異常表現被指出與疾病相關，很多膜蛋白質為藥物治療的標的蛋白質，因此，全面性地定量膜蛋白質可增進我們對膜蛋白質調控疾病的機制及其訊息傳遞的了解。為了可以針對多重樣品做全面性及有效的膜蛋白質體分析，我們發展一個新的定量技術平台，結合新開發的膠體輔助蛋白質水解法、iTRAQ定量試劑以及二維液相層析串聯質譜法等技術。本論文的第一部分著重在定量策略的開發以及此策略的效能評估，新開發的膠體輔助蛋白質水解法可容許高濃度的溶劑及介面活性劑，並且可以藉由反覆沖洗過程將其移除，有效地提高了膜蛋白質的溶解度、蛋白質水解及質譜鑑定率。此新開發的蛋白質水解方法已應用在插管病人肺沖洗液的蛋白質體研究上，成功建立現今最完整的肺沖洗液蛋白質體。另外結合膠體輔助蛋白質水解法與iTRAQ標定技術可增加蛋白質定性及定量的準確度，從四個由HeLa細胞獨立製備的膜蛋白質樣品的定量分析測試，結果顯示此一新平台具有高準確性及再現性（相對標準誤差 < 12%，錯誤率低於 8%）；在嚴謹的蛋白質鑑定條件下，此策略有效地定量了520個膜蛋白質（91%），並且每個具有transmembrane helix（TMH）的高疏水性蛋白質，皆能以平均高達14.1個胜肽片段來定量。除此之外，大多數蛋白質的訊號強度皆有明顯增加，經過蛋白質結構預測分析後，顯示膠體輔助蛋白質水解法可以更有效地溶解具有多個TMH的高疏水性膜蛋白質，增加了穿透膜胜肽片段的偵測率，在結果中，高達19個TMH的膜蛋白質可被成功的定量，據我們所知，此結果為現今分析膜蛋白質體數量及涵蓋率最高的記錄，在定量的精確度及精準度也超越了現今其他定量方法！
本論文的第二部份，我們將此一技術平台應用在兩個不同的生物系統的膜蛋白質體上，第一個生物系統為具有多發性腎囊腫疾病（autosomal-dominant polycystic kidney disease , ADPKD）的老鼠，雖然多發性腎囊腫是腎臟疾病中最常見且最容易致命的遺傳疾病，然而其疾病的蛋白質表現與病理機制仍有待釐清，我們希望能藉由發展之新方法找出和老鼠腎臟病變有關的膜蛋白質。經過分析正常鼠及病鼠的細胞膜蛋白體後，我們成功定量了791個蛋白質，其中67個及37個蛋白質為大於兩倍增加及減少的變異蛋白質，有部分的變異蛋白質在過去曾被報導與ADPKD相關，這些蛋白質參予了包括細胞增生及死亡、細胞與細胞之間或者細胞與介質間的溝通、溶質運輸和膜蛋白質極化等功能，在這些變異蛋白質中，有部分蛋白質已被證實為藥物標的運輸及受體蛋白質，例如EGFR已被證實可有效治療ADPKD；另外有部份蛋白質則被使用於其他疾病的治療上。此新的定量平台不僅可幫助我們了解ADPKD的制病機制，也找到數個可能作為藥物標的之膜蛋白質。
第二個生物系統為人類的大腸直腸癌組織，根據行政院衛生署公布的國人十大癌症排行榜中，大腸直腸癌在男性及女性比例都佔第三位，藉由不同癌症期數（Dukes’ A、B、C和D）病人的正常及腫瘤組織之比較分析，將有利於找出可作為初期診斷的生物標的蛋白質或作為手術前後診察的判斷依據。在初步分析八個病人的結果中，我們定量了304個蛋白質，其中39個蛋白質是四期八個病人中皆有出現且具有兩倍以上變化的變異蛋白質，Carcinoembryonic antigen-related cell adhesion molecule (CEA)在B期及C期的病人組織中大量表現，是目前臨床使用為診斷大腸癌的生物標的蛋白質，其他還包括細胞分泌以及位於細胞膜上的蛋白質，有機會成為疾病診斷及藥物治療的標的分子。
綜合以上兩個應用研究，我們相信此一新的技術平台可以有效且全面性地分析及定量膜蛋白質體，更可應用於各種不同刺激或病理的生物系統及細胞、組織及體液等各種樣品，幫助瞭解疾病的致病原因、找出有效診斷及治療的生物標的蛋白質！

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 multiplexed, comprehensive and robust quantitation of the membrane proteome, we developed a strategy combining gel-assisted digestion, iTRAQ labeling, and LC-MS/MS. The first part of thesis focus on the methodology development and performance evaluation of the new strategy. The gel-assisted digestion offers advantages of diverse compatibility with high concentrations of detergent and salts for efficient solubilization, denaturation and digestion of membrane proteins. This new digestion method was applied to the proteomic profile of bronchoalveolar lavarge fluid (BALF) from patients with ventilator-associated pneumonia (VAP). The data obtained from the cohort of VAP patients demonstrated that our approach provides in-depth description of the BALF proteome. The gel-assisted digestion also improved identification and quantitation accuracy by peptide-level isotopic tagging of amino groups. Quantitation of four independently purified membrane fractions from HeLa cells gave high accuracy (< 8% error) and precision (< 12% RSD), demonstrating a high degree of consistency and reproducibility of this quantitation platform. Under stringent identification criteria (false discovery rate = 0%), the strategy efficiently quantified membrane proteins; as many as 520 proteins (91%) were membrane protein—each quantified based on average of 14.1 peptides per integral membrane protein. In addition to significant improvements in signal intensity for most quantified proteins, most remarkably, topological analysis revealed that the biggest improvement was achieved in detection of transmembrane peptides from integral membrane proteins with up to 19 transmembrane helices. To the best of our knowledge, this level of coverage exceeds that previously achieved using MS and provides superior quantitation accuracy compared with other methods.
In the second part of thesis, the new strategy was applied to two biology systems. The first example presents the first proteomic delineation of phenotypic expression in a mouse model of autosomal-dominant polycystic kidney disease (ADPKD) tissues. Although ADPKD is the most prevalent and potentially lethal inherited human renal disease, systematic mapping of phenotypic expression of the disease and uncovering perturbed cellular networks remain to be further investigated. By characterizing kidney cell plasma membrane from wild-type versus PKD1 knockout mice, 791 proteins were quantified and 67 and 37 proteins showed ≥ 2-fold up-regulation and down-regulation, respectively. Some of these differentially expressed membrane proteins are involved in the mechanisms underlying major abnormalities in ADPKD, including epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, ion and fluid secretion, and membrane protein polarity. Among these proteins, targeting therapeutics to certain transporters/receptors, such as epidermal growth factor receptor, has proven effective in preclinical studies of ADPKD; others are known drug targets in various diseases. Our method demonstrates how comparative membrane proteomics can provide insight into the molecular mechanisms underlying ADPKD and the identification of potential drug targets, which may lead to new therapeutic opportunities to prevent or retard the disease.
The second example focuses on the human colorectal cancer (CRC). Colorectal cancer is a worldwide disease and it is equally common in both men and women. Quantitative analysis of differentially expressed membrane proteins in the paired normal and cancerous human colorectal cancer (CRC) tissues will provide opportunity for discovery of biomarker candidates. The preliminary data shows that 304 proteins were quantified from 8 patients in Dukes’ A, B, C, and D stages; 39 proteins show ≥ 2-fold differentially expression in all the eight patients. The identified up-regulation of carcinoembryonic antigen-related cell adhesion molecule 6 and 5 (CEA) in Dukes’ B and C patients were consisted with previous literatures on their roles as biomarker of colorectal cancer. Several secreted proteins and plasma membrane proteins, that might be potential diagnostic biomarkers or drug targets, were also quantified in this study.
Taken together, this multiplexed quantitation platform provides a generic and powerful complement to membrane proteomics. We expect that the approach can be a generic strategy to investigate differential expression of membrane proteins in many sample types including cells, tissues and body fluids under different environmental and pathophysiological conditions to discover membrane protein markers or to delineate the pathogenesis of certain diseases.

中文摘要..................................................1
ABSTRACT……………………………………………………………..3
CONTENT………………………………………………………………6
LIST OF FIGURES………………………………………………...….11
LIST OF TABLES……………………………………..………………17
ABBREVIATIONS.................................................................................18
CHAPTER 1 INTRODUCTION……………………………..…….…20
1    The significance of membrane proteins……………..………………...……20
2    Challenges for Mass Spectrometry-Based Identification of membrane proteins…………………………….….……………………………………21
2.1    Solubilization and digestion of membrane proteins………..…..…22
2.2    Protein/peptide separation methods for membrane proteomic study…………………………………………………….…………22
3    Methodologies for Quantitative Membrane Proteomics……………………24
3.1 Two dimentional gel electrophoresis and differential gel electrophoresis………………………………...……………………………24
3.2 Chemical labeling for quantitative membrane proteomics…………...26
4    Specific aims in this study………………………………………………….31
CHAPTER 2 METHODOLOGY DEVELOPMENT:
A MULTIPLEXED QUANTITATIVE STRATEGY FOR MEMBRANE PROTEOMICS……...……………………………….. 33
1 Rational of a multiplexed, comprehensive, and quantitative strategy for membrane proteomics…………………..……………………………………………33
2 Experimental procedures…………..…………..…………………...….….. 33
        2.2.1 Materials………………………………………………………….…33
        2.2.2 Isolation of membrane proteins from HeLa cell…………………….34
2.3 Digestion of membrane proteins………………………………….…34
2.4 iTRAQ labeling and fractionation by strong cation exchange (SCX) chromatography……………………………………………………………36
2.5 LC-ESI MS/MS analysis………………………………………….…37
2.6 Data processing and analysis………………………………………...38
2.7 iTRAQ quantitation software………………………………………..39
2.8 Annotations…………………………………………………………..40
3 Results and discussion……………………………..…..…..……………….41
        2.3.1 Selection and optimization of digestion methods…………...………41
3.2 Assessment of reproducibility and compatibility with iTRAQ labeling by quantitative analysis of four replicate preparations…………………….43
3.3 Large-scale quantitative membrane proteomics by gel-assisted digestion, iTRAQ labeling, and SCX fractionation with comparison to SDS-assisted in-solution digestion………………………………………...44
3.3.1 Workflow and experimental design…….……………………44
3.3.2 Comprehensive identification and quantification of membrane proteins………………………………………………………………45
3.3.3 Improved peptide recovery and quantification accuracy by gel-assisted digestion and iTRAQ labeling………………………….48
3.3.4 Improvement of quantification of transmembrane-spanning domains………………………………………………………………49
4 Summary……………………………………………………………………50
CHAPTER 3 APPLICATION I:
PROTEOMIC PROFILES OF BRONCHOALVEOLAR LAVARGE FLUID FROM PATIENTS WITH VENTILATOR-ASSOCIATED PNEUMONIA…………………………………………………….……52
1 Introduction………………………………………………………………...52
2 Experimental procedures…………………………………………………...54
2.1 Materials………………………………………………………….…54
2.2 Preparation of BALF…………………………..……………………55
2.3 Enzymatic digestion of BALF proteins……………………………..56
2.4 Fractionation of tryptic peptides by SCX chromatography…………56
2.5 Protein identification by RPLC-ESI-MS/MS……………………….57
2.6 Data processing and database search………………..………………57
2.7 Western blot and statistical analysis………………………………...58
3 Results and discussion…………………………………………………...…59
3.1 Patient characteristics……………………………………………….59
3.2 Proteomic profiles of BALF from VAP and non-VAP groups………59
3.3 Semi-quantitative comparison between VAP and non-VAP groups...62
3.4 Western blot analysis among VAP patients and non-VAP controls…62
3.5 Differentially expressed proteins in the VAP group are functionally involved in the pathogenesis of the disease………………………………..63
3.5.1 Cell structure and motility in response to mechanotransduction…………………………………..……….……63
3.5.2 Innate and adaptive immune responses……………………...64
3.5.3 Antimicrobial function……………………………………….65
3.5.4 Glucose metabolism…………………………………………66
4 Summary……………………………………………………………………66
CHAPTER 4 APPLICATION II:
PROTEOMIC PROFILE OF AUTOSOMAL-DOMINANT POLYCYSTIC KIDNEY DISEASE IN MICE...…………………….68
1 Introduction………………………………………………………………...68
2 Experimental procedures…………………………………………………...68
2.1 Materials…………………………………………………………….69
2.2 Animals and preparation of kidney membrane fraction…………….69
2.3 Digestion of membrane proteins……………………………………70
2.4 iTRAQ labeling and fractionation by strong cation exchange (SCX) chromatography……………………………………………………………71
2.5 LC-ESI MS/MS analysis………………………….…………………71
2.6 Data processing and analysis………………………………………...72
2.7 Annotations…………………………………………………………..74
2.8 Western blot analysis………………………………………………...74
3 Results and Discussion……………………………………………………...75
3.1 Quantitative analysis of membrane proteins from the ADPKD mouse………………………………………………………………………75
3.2 Systemic manifestation of the altered membrane proteome provides insight into the molecular mechanism and opportunities for effective pathophysiology-based therapy for ADPKD………………………………77
4 Summary……………………………………………………………………80
CHAPTER 5 APPLICATION III:
PROTEOMIC PROFILE OF HUMAN COLORECTAL CANCER TISSUES………………………………………………………………..82
1 Introduction………………………………………………………………...82
2 Experimental procedures…………………………………………………...83
2.1 Extraction of membrane proteins from human colorectal cancer tissues………………………………………………………………………83
2.2 Digestion of membrane proteins……………………………………..84
2.3 iTRAQ labeling and LC-ESI MS/MS analysis………………………84
2.4 Data processing and analysis………………………………………...85
2.5 Annotations…………………………………………………………..87
3 Results and discussion………………………………………………………87
3.1 Quantitative comparison of membrane proteins in tumor and adjacent non-tumor tissues from patient with human colorectal cancer……….……87
3.2 Differentially expressed membrane proteins in human colorectal cancer may be utilized as biomarker candidates for early diagnostics and drug-based therapy…………………………………………………………88
4 Summary……………………………………………………………………90
CHPATER 6 CONCLUSION AND FUTURE PERSPECTIVE…...91
REFERENCE………………………………………………………….94
                                

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