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

Abstract

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 theInternational 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.