運用iTRAQ化學標定分析C型肝炎病毒感染從急性到慢性不同階段之差異蛋白質體研究 Quantitative proteomics analysis from acute to chronic stages of hepatitis C virus infection by iTRAQ technology

Date
2017
Authors
林子鈺
Lin, Tze-Yu
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Abstract
肝癌屬於世界上較為普及且致命的癌症之一,約20-30%的慢性C型肝炎(HCV)患者容易病變為肝硬化或肝癌。大多數急性C型患者容易轉變成慢性C型肝炎,長期存在於肝臟中,且無明顯病徵。C型肝炎雖然有藥物治療可以醫治,但因存在較大的遺傳變異性,目前仍無疫苗可以防範。本實驗中使用Huh7.5-SEAP細胞株作為樣品,比較急性到慢性不同天數受到C型肝炎病毒感染的蛋白質和未受到感染的控制組,進行同重元素相對與絕對定量(iTRAQ)搭配串聯式質譜儀進行分析。藉由二維液相分離,等電點聚焦法(sIEF),強陽離子交換層析法(SCX)和鹼性逆相層析法(bRP)分離已被iTRAQ標記的胜肽,降低樣品的複雜性,也可藉由不同分離方法提供互補性和正交性,鑑定到更多的蛋白質。利用Gene Ontology (GO)軟體分析從急性到慢性差異蛋白質的生物路徑及功能,在本次實驗中發現的差異蛋白與RNA結合、胞外外切體、黑素體和核糖核蛋白複合物結合形成生物路徑有關,未來通過西方墨點法進行方法驗證,尋找能辨別從急性到慢性C型肝炎病毒的標記蛋白質。
Hepatocellular carcinoma (HCC) is one of the most prevalent and mortal cancer in the world, and 20% to 30% of patients with chronically hepatitis C virus (HCV) lead to liver cirrhosis and liver cancer. Most infected acute HCV people develop chronic HCV easily. Due to genetic variability of HCV, the development of antiviral drugs and vaccines becomes a real challenge. In this study, naïve Huh7.5-SEAP cells were established and infected by hepatitis C virus, then isobaric tags for relative and absolute quantitation (iTRAQ) was applied to investigate protein profiles in acute and chronic infections. The iTRAQ labeled peptides were fractionated by solution isoelectric focusing (sIEF), strong cationic exchange chromatography (SCX) and basic reverse phase chromatography (bRP) column, followed by nano-LC tandem mass spectrometric analysis. Two-dimensional liquid chromatography technique that employed on iTRAQ labeled peptides provided results with excellent complementarity and orthogonality. In the study, the differentially expressed proteins were found to be associated with RNA binding, extracellular exosome, melanosome and ribonucleoprotein complex binding. Besides, selected correlated proteins will be confirmed and validated by Western blot in the future, they could serve as novel diagnostic biomarkers for hepatitis C virus infection.
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C型肝炎病毒, 蛋白質體學, 同重元素相對與絕對定量, 串聯式液相層析質譜儀, hepatitis C virus (HCV), proteomics, iTRAQ, LC-MS/MS
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