稳定同位素标记氨基酸细胞培养联合质谱绝对定量技术在5、7、55型腺病毒鉴定中的应用  

Application of Stable Isotope Metabolic Labeling Combined with Mass Spectrometry Absolute Quantification Technology in the Identification of Adenovirus 5,7,and 55

作　　者：朱文清 李恒 张妍 徐忠伟 Zhu Wenqing;Li Heng;Zhang Yan;Xu Zhongwei(Department of Health Service,Logistics University of Chinese People’s Armed Police Force,Tianjin 300300,China)

机构地区：[1]武警部队后勤学院卫生勤务系,天津300300

出　　处：《化学世界》2023年第6期445-452,共8页Chemical World

基　　金：国家自然科学基金(No.81673651);武警部队自主科研研究(No.ZZKY20222308);武警部队后勤学院基础研究(Nos.WHJ201906,WHJ202012)资助项目。

摘　　要：利用稳定同位素标记肽段的质谱绝对定量(AQUA)技术,实现了对5、7和55型人腺病毒的准确鉴定。采用细胞培养条件下稳定同位素代谢标记(SILAC)技术培养细胞至大肠杆菌生长至对数生长期,1 mmol/L异丙基硫代-β-D-半乳糖苷(IPTG)低温诱导蛋白表达,提取并利用谷胱甘肽S转移酶(GST)纯化富集相应标记的腺病毒六邻体蛋白。通过胶消化分别消化三种不同标记策略(非标记、中度标记和重度标记)的纯化蛋白,消化肽段等量混合脱盐后经纳流液相色谱-串联质谱(Nano HPLC-MS/MS)进行定量蛋白分析。通过SILAC实现大肠杆菌的完整代谢标记,诱导表达腺病毒六邻体蛋白,通过AQUA技术实现对非标记、中度标记、重度标记六邻体蛋白标志性肽段鉴定。成功实现SILAC三种不同标记策略条件下大肠杆菌中5、7、55型人腺病毒六邻体蛋白的完整标记,为5、7、55型人腺病毒感染的快速分型检测提供了新的鉴定策略。This is to achieve the accurate identification of 5,7,and 55 types human adenovirus(HAdV)by using the technology of stable isotope labeled peptide combined with mass spectrometry absolute quantification(AQUA).Escherichia coli was grown to logarithmic phase by stable isotope metabolic labeling(SILAC)under cell culture conditions.The protein expression was induced by 1 mmol/L isoprophyl β-D-thiogalactoside(IPTG)at low temperature,and the corresponding labeled adenovirus hexon protein was extracted and purified by glutathione S-transferase(GST).The purified proteins with three different labeling strategies(native,moderately and heavily labeled)were digested by gel digestion,and the digested peptides were mixed and desalted in equal quantities.The quantitative protein analysis was carried out by nano high performance liquid chromatography mass spectrum(HPLC-MS/MS).The complete metabolic labeling of E.coli was achieved by SILAC method.and the expression of adenovirus hexon protein was induced.The signature peptides of non-standard,biddable and rescaled hexon protein were identified by AQUA.The complete labeling of human adenovirus 5,7,and 55 types human adenovirus hexon proteins in E.coli under three SILAC conditions has been successfully achieved,which provides a new identification strategy to detect 5,7,and 55 types human adenovirus infections.

关 键 词：人腺病毒 质谱绝对定量技术 稳定同位素标记氨基酸细胞培养技术 病原体检测 

分 类 号：Q816[生物学—生物工程]