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研究生: 林怡青
Lin, Yi-Ching
論文名稱: 2-氨基-3-甲基-3H-咪唑並[4,5-F]喹啉誘導HepG2細胞之差異蛋白質體學分析
Differential proteomic analysis of HepG2 cell induced by 2-Amino-3-methyl-3H-imidazo[4,5-F]quinoline
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 118
中文關鍵詞: 雜環胺類差異蛋白質體學2-氨基-3-甲基-3H-咪唑並[4,5-F]喹啉
英文關鍵詞: heterocyclic amine, differential proteomics, 2-Amino-3-methyl-3H-imidazo[4,5-F]quinoline
DOI URL: http://doi.org/10.6345/NTNU201900711
論文種類: 學術論文
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  • IQ (2-amino-3-methyl-3H-imidazo[4,5-F]quinoline),為一種常見於食品中的雜環胺類,並被國際癌症組織列為2A級致癌物。目前已有研究證實,IQ對於動物具有致癌性,特別是在肝臟部分尤其顯著,但關於雜環胺類導致癌症的途徑仍尚未研究完畢。本實驗使用iTRAQ (同重位素相對及絕對定量)方法針對以IQ刺激不同時間長度的HepG2 cell樣品進行分析,為了增加鑑定到的蛋白質數目,搭配了不同的三種分餾方法,包含等電聚焦分級分離、鹼性逆相層析法、強陽離子交換層析法,且比較其正交性與互補性。再經由質譜進行分析,結果為三種分餾方法共鑑定到3057個蛋白質及10712個不重複胜肽,並以鹼性逆相層析法所鑑定到的數目最多。且在其中挑選出了391個差異蛋白質,並以Gene Ontology進行分類比較;並在其中又選出較具趨勢性的蛋白質分為上下調進行KEGG pathway分析,主要與ribosome, proteasome, spliceosome, Parkinson’s disease 有較大的相關性,並挑選出其中蛋白質表現下調較明顯的幾個基因(Ribosome- RPS26、RPS4X、RPL37A、RPS24,Parkinson’s disease-SLC25A4、NDUFA8)以qPCR實驗驗證,其中以RPS26、RPL37A趨勢與蛋白質定量結果最為相同。

    IQ (2-amino-3-methyl-3H-imidazo[4,5-F]quinoline), belonged to heterocyclic amine (HCA) family, is classified as a probable human carcinogen (class 2A) by the International Agency for Research on Cancer (IARC). Studies to date indicate that IQ is carcinogenic to animals, especially in liver. Still, the mechanism of HCA-induced cancer in protein level is not well-understood. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was applied for the investigation of the HepG2 protein profiles with different IQ-treat times. In order to obtain higher complementarity, orthogonality and more protein identifications, two-dimensional separation techniques were used as fractionation strategies for the iTRAQ labeled peptides, including solution isoelectric focusing (sIEF), basic reverse phase chromatography (bRP), and strong cationic exchange chromatography (SCX). Protein identification and quantitation was then accomplished by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. A total of 10712 unique peptides and 3057 proteins was successfully identified. Among these three fractionation strategies, bRP fractionation gave the most protein and unique peptide identifications. Moreover, 271 differentially expressed proteins were selected and found to be highly associated with ribosome, proteasome, spliceosome, Parkinson’s disease. Some of these differential genes (Ribosome- RPS26、RPS4X、RPL37A、RPS24,Parkinson’s disease-SLC25A4、NDUFA8) were confirmed and validated using qRT-PCR.

    目錄 i 圖目錄 v 表目錄 viii Abstract ix 中文摘要 xi 縮寫 xii 第一章 緒論 1 第一節 雜環胺類化合物 1 第二節 IQ (2-Amino-3-methyl- 3H-imidazo[4,5-F]quinoline) 4 第三節 質譜儀技術 5 第四節 蛋白質身份鑑定 10 第五節 差異蛋白質體學 (Differential proteomics) 12 第六節 液相層析分離技術 16 第七節 定量反轉錄聚合酶連鎖反應(Quantitative reverse-transcription polymerase chain reaction, qRT-PCR) 21 第八節 研究動機 23 第二章 實驗材料與方法 25 第一節 細胞存活率測定 25 第二節 定量即時聚合酶連鎖反應 26 第三節 蛋白質樣品製備 29 第四節 樣品純化濃縮 30 第五節 蛋白質濃度測定 31 第六節 蛋白質水解和化學標定iTRAQ試劑 32 第七節 第一維分餾策略 33 第八節 自製C18離心管柱去鹽 39 第九節 奈米級超高效能液相層析電噴灑串聯式質譜儀 (nanoLC ESI tandom mass spectrometry) 40 第十節 資料分析 50 第三章 實驗結果與討論 52 第一節 細胞存活率測定 52 第二節 定量即時聚合酶連鎖反應 54 第三節 蛋白質濃度測定 56 第四節 以等電聚焦分級分離儀分餾樣品之質譜鑑定結果 58 第五節 以鹼性逆向層析法分餾樣品之質譜鑑定結果 63 第六節 以強陽離子交換層析法分餾樣品之質譜鑑定結果 66 第七節 三種不同分餾方法之正交性與互補性 69 第八節 蛋白質相對定量 71 第九節 蛋白質相關生物作用途徑 74 第十節 確校 83 第四章 結論與未來展望 91 參考文獻 92 附表 98

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