臺灣博碩士論文加值系統

大腸直腸癌在全世界為主要發生及造成死亡的原因。在全球的癌症致死率中排名第三位。在近期的癌症研究當中，有許多證據證實了在惡性腫瘤的發展過程中，酪胺酸磷酸化蛋白質參與腫瘤細胞浸潤及轉移的調控。根據以上的論點，此篇研究的目的即是比較SW480大腸直腸原位腺癌細胞和具淋巴轉移能力的SW620細胞株中具有差異性表現量的酪胺酸磷酸化蛋白質，藉此更加了解在大腸直腸癌轉移相關之訊息傳遞路徑中所扮演的角色。藉由細胞貼附試驗及FAK西方墨點法可驗證到SW620細胞株的轉移能力較SW480強。由實驗室發展出分析策略中先篩選出可能為酪胺酸磷酸化胜肽的訊號；在365 m/z訊號值中可鑑定到280酪胺酸磷酸化胜肽含有287酪胺酸磷酸化修飾位置相對應於261蛋白質。最後，利用非標定定量法可以從這兩株細胞找到103具有差異性表現量的酪胺酸磷酸化蛋白質(P〈0.05)。從這些修飾位置中，我們可以找到2個新穎序列及4個已知的激酶及磷酸激酶，包括EGFR, Src, ALK和SHP1。而且，10個篩選出的酪胺酸磷酸蛋白質在SW480細胞具高表現量，另3個在SW620細胞具高表現量。之後利用Metacore來進行蛋白質的交互作用分析比對，發現其中10個酪胺酸磷酸蛋白質與SOX2,FKHR,E2F1等與轉移與細胞週期基因相關。由以上上述資訊可以提供我們未來進一步去了解大腸直腸癌轉移的新方向外，從本研究找到出與大腸直腸癌相關的標的酪胺酸磷酸化蛋白質，也可提供做為在轉移上的研究，或是用來發展或設計藥物標靶來阻止癌症轉移的發生。

Colorectal cancer (CRC) is the third most common cause of cancer death in the world and also is a major cause of worldwide morbidity and mortality. In recent studies, plenty of evidences indicated that tyrosine-phosphorylated (pTyr) proteins were involved in malignant tumor cell invasion and metastatic regulation. In this study, the goal is to get a better understanding of metastasis-associated signaling pathway underlying human colorectal cancer. We identified the differentially expressed pTyr proteins from the same patient of primary colorectal adenocarcinoma SW480 cell and lymph node metastasis SW620 cell are compared. By performing adhesion assay and Western blotting of FAK, the data showed that SW620 metastatic ability were better than SW480. Using our analytical strategy for filtering out the potential pTyr peptide signals, we found the signals of 365 m/z values to be identified 280 pTyr peptides containing 287 pTyr sites from 261 proteins. Finally, the 103 differentially expressed pTyr proteins (p〈0.05) were found in the two cells using the label-free quantitative analysis. From this list of site, we extracted two novel consensus sequences and four known motifs for specific kinases and phosphatases including EGFR, Src, ALK, and SHP1. Moreover, 10 target pTyr proteins were with higher level in SW480 cells while 3 with higher levels in SW620 cells. After interactome analysis by Metacore software, 10 of 13 target pTyr proteins are interacted with SOX2,FKHR,E2F1 metastasis or cell-cycle genes. Above this information may provide us a direction to understand CRC metastasis and those pTyr protein targets identified in this study may provide novel information to not only mechanistic aspects of colorectal cancer metastasis but also drug targets for hampering the cancer metastatic processes.

摘要 I
Abstract II
致謝 III
List of Table VII
List of Figure VIII
Abbreviations IX
Chapter 1 Overview of the Research 1
1.1 Colorectal Cancer(CRC) 1
1.1.1 Introduction of CRC 1
1.1.2 CRC Metastasis 1
1.2 Protein Phosphorylation 2
1.2.1 Post-translational Modification(PTM) 2
1.2.2 The Role of Tyrosine-phosphorylated Protein in Cancer and Clinical Application 2
1.3 The Application of Phosphoproteomic in Cancer Research 2
Chapter 2 Objective 4
Chapter 3 Material and Methods 5
3.1 Experimental Workflow 5
3.2 pTyr Proteins Collection 6
3.2.1 Cell Line Background 6
3.2.2 Cell Culture 6
3.3 Check Cell Lines’ Metastatic Ability 6
3.3.1 Cell Adhesion Assay 6
3.4 Sample Preparation for Mass Spectrometry Analysis 7
3.4.1 Cell Lysis Collection 7
3.4.2 Protein Concentration Measurement(Protein Assay) 8
3.4.3 Immunoprecipitation by pTyr-specific Antibodies 8
3.4.4 Stacking Gel 9
3.4.5 In-gel Digestion 11
3.4.6 TiO2 Micro-columns Enrichment and Alkaline Phosphatase Treatment 12
3.5 Mass Spectrometry Analysis 13
3.5.1 Liquid Chromatography Mass Spectrometry(LC-MS) Analysis 13
3.5.2 Identification of Potential Phosphopeptide Signals by iPhos Software 13
3.5.3 Targeted Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis 14
3.5.4 pTyr Protein Identification by In-house Mascot Search Engine 15
3.5.5 Label-free Quantitative Analysis 15
3.6 Western Blotting 16
Chapter 4 Result and Discussion 18
4.1 pTyr Protein Collection 18
4.2 Check cell lines’ Metastatic Ability 20
4.2.1 Adhesive Ability of SW480 and SW620 20
4.2.2 Metastatic Marker of Focal Adhesion Kinase (FAK) Protein 22
4.3 Sample Preparation for Mass Spectrometer Analysis 24
4.4 pTyr Proteins Identification and Quantitative 26
4.4.1 Liquid Chromatography Mass Spectrometry(LC-MS) Analysis 26
4.4.2 Selection of Potential pTyr Signals by iPhos Software 26
4.4.3 Phosphotyrosine Proteins Present at Different Levels in SW480 and SW620 Cells 28
4.4.4 PhosphoSite Plus (PSP) and PHOSIDA Database Search 41
4.4.5 Motif Analysis and Kinase Prediction 42
4.4.6 Filtration of the Candidates as the Targets in SW480 ＆ SW620 Cell lines 43
4.4.7 Metacore Analysis 48
4.4.8 Western Blotting Validation 49
Chapter 5 Conclusions 50
Chapter 6 Reference 51
Chapter 7 Appendix 56

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