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系統識別號 U0026-0706201511380100
論文名稱(中文) iPhos: 使用鹼性磷酸酶輔助液相層析質譜技術研究磷酸化蛋白質體之網頁工具
論文名稱(英文) iPhos: a web tool to streamline the alkaline phosphatase-assisted LC-MS phosphoproteome investigation
校院名稱 成功大學
系所名稱(中) 電機工程學系
系所名稱(英) Department of Electrical Engineering
學年度 103
學期 2
出版年 104
研究生(中文) 張宏存
研究生(英文) Hong-Tsun Chang
學號 N26021157
學位類別 碩士
語文別 英文
論文頁數 54頁
口試委員 指導教授-吳謂勝
口試委員-曾大千
口試委員-鄭宏祺
口試委員-陳健生
口試委員-廖寶琦
中文關鍵字 蛋白質磷酸化  針對性的液相層析質譜  鹼性磷酸  非標記定量 
英文關鍵字 protein phosphorylation  targeted LC-MS/MS  alkaline phosphatase  label-free quantification 
學科別分類
中文摘要 iPhos 為一網站軟體工具組用以促進及有效率的進行蛋白質體學分析之研究,並且藉著鹼性磷酸酶與磷酸化蛋白質之間的作用,協助蛋白質體特性的分析。全面性的蛋白質體學分析對於細胞之間的訊息傳導研究極為重要,但是細胞中的磷酸化胜的數量十分稀少,因此使用質譜技術來分析蛋白質體仍是一項挑戰。為了克服這些障礙,與我們共同研究的廖寶琦老師實驗室過去提出一個全面性分析的策略,藉由使用鹼性磷酸酶的特性並搭配高解析度之質譜儀設備的流程來實現,此策略可更準確的從樣本中鑑定出磷酸化胜肽。儘管此流程在應用上都是可行的,但完整流程大部分都需要繁雜的步驟及耗費大量的人力,因此為了讓以上流程更有效率,iPhos 提供人性化操作介面及協助整個實驗流程的數據探勘進行,以得到更加全面性的研究結果。我們利用 iPhos 軟體工具組做了進一步的分析,從人類轉移肺癌細胞 (CL1-5) 的分析結果中證實了 iPhos 工具組的效用,並在 CL1-5 細胞之控制組與施以藥物 dasatinib 細胞之實驗組的比較研究中,我們更證實 iPhos 工具組的實用性,而且也提出基於使用 iPhos 促進液相層析質譜技術的磷酸化蛋白質體研究的實驗報告結果。除此以外,我們也比較了以單純的數據依賴型數據採集為基礎的 (DDA-based) 質譜分析與針對性型的質譜分析之差別,從這兩種蛋白質體策略的研究中發現,iPhos 促進的針對性型質譜分析比單純數據依賴型數據採集為基礎的質譜分析得到更全面且可靠的磷酸化胜肽定性與定量之結果。iPhos 軟體工具組及範例資料樣本皆可在下列網址取 http://cosbi3.ee.ncku.edu.tw/iPhos/.
英文摘要 iPhos is a web based software toolkit to facilitate and streamline the work-flow of the alkaline phosphatase-assisted phosphoproteome characterization. Comprehensive characterization of the phosphoproteome in living cells is critical in signal transduction research. But the low abundance of phosphopeptides among the total proteome in cells remains a challenge in mass spectrometry-based proteomic analysis. To overcome the obstacles, an alternative analytic strategy to confidently identify phosphorylated peptides by using the alkaline phosphatase (AP) treatment in combination with high-resolution mass spectrometry was provided by Liao’s group. While the process is applicable, the key integration along the pipeline was mostly done by tedious manual work. In order to be effective and convenient, iPhos not only has a user friendly web interface but also assists the experiment work-flow in data analysis. We further demonstrated the utility of the iPhos toolkit on the data of human metastatic lung cancer cells (CL1-5). In the comparison study of the control group of CL1-5 cell lysates and the treatment group of dasatinib-treated CL1-5 cell lysates, we demonstrated the applicability of the iPhos toolkit and reported the experimental results based on the iPhos-facilitated phosphoproteome investigation. Additionally, we also compared the strategy with pure DDAbased LC-MS/MS phosphoproteome investigation. The results of iPhos-facilitated targeted LC-MS/MS analysis convey more thorough and confident phosphopeptide identification than the results of pure DDA-based analysis. The iPhos software toolkit and sample tutorial data are available online at http://cosbi3.ee.ncku.edu.tw/iPhos/.
論文目次 中文摘要I
Abstract III
Acknowledgement IV
List of Tables VII
List of Figures VIII
List of Abbreviations IX
Chapter 1 Introduction 1
1.1 Motivation and Literature Review ........1
1.1.1 Technique for detecting protein phosphorylation ....2
1.1.2 Systematic ways of finding pTyr-phosphorylated peptides ...4
1.2 Contribution of this study .........4
1.3 Thesis organization ..........6
Chapter 2 Methods and Material 7
2.1 iPhos toolkit implementation .........7
2.1.1 Raw data conversion .........7
2.1.2 Peak Extraction Assister .........9
2.1.3 Module-1: phosphopeptide signal extraction .....10
2.1.4 Module-2: inclusion list generation for targeted LC-MS/MS ..11
2.1.5 Module-3: integration of phosphopeptide identification and patternbased
label-free quantitative analysis ......11
2.2 The demonstration dataset ..........15
2.2.1 Cell lysate preparation ........16
2.2.2 Immunoprecipitation and in-gel digestion ......16
2.2.3 Phosphopeptide enrichment by TiO2 pipette tip and AP treatment .17
2.2.4 NanoLC-MS analysis .........18
2.2.5 Targeted LC-MS/MS analysis and database search ...19
Chapter 3 Results and Discussion 22
3.1 iPhos user interface and user guides .......22
3.1.1 Raw data preprocessing .........23
3.1.2 iPhos Peak Extraction Assister ........23
3.1.3 iPhos Module-1 ..........24
3.1.4 iPhos Module-2 ..........29
3.1.5 iPhos Module-3 ..........31
3.2 CL1-5 cell lysate phosphoproteome investigation assisted by iPhos ...35
3.2.1 A demonstration example ........38
3.2.2 Comparison with DDA-based phosphoproteome investigation .39
3.2.3 Confidence evaluation ........41
Chapter 4 Conclusion 46
References 48
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