摘要
目的:建立基于液质联用技术的定量检测环鸟苷酸-腺苷酸(cGAMP)的方法体系,用于评价细胞内DNA感受器环鸟苷酸-腺苷酸合成酶(cGAS)的活性。方法:在人白血病单核细胞系U937中,以脂质体转染鲱鱼精DNA(HT-DNA)的方式诱导cGAMP合成,采用甲醇-乙腈萃取法提取细胞中的cGAMP分子,真空干燥后利用液相色谱-质谱多反应监测技术(LC-MS/MRM)对cGAMP进行定量检测。同时利用蛋白质免疫印迹法和实时荧光定量PCR体系对细胞中cGAMP产生后的常规生物学效应指标进行检测,以评价该方法的可靠性。结果与结论:建立的基于液质联用技术检测方法实现了对细胞中cGAMP分子的定量分析。该方法具有特异性好、灵敏度高的特点,为深入研究cGAS的活性调控机制提供了技术支撑。
Objective:Establish a method for quantitative detection of cyclic GMP-AMP(cGAMP)based on liq uid chromatography-mass spectrometry(LC-MS)to evaluate the activity of intracellular DNA receptor cyclic GMPAMP synthase(cGAS).Methods:HT-DNA was transfected into human leukemia monocyte cell line U937 to in duce cGAMP production.Then cGAMP was extracted by methanol-acetonitrile extraction and was quantitatively de tected by LC-MS/multiple reaction monitoring(LC-MS/MRM).At the same time,the conventional biological effect indicators after cGAMP production in cells were detected by Western blotting and real-time quantitative PCR sys tem to evaluate the reliability of the method.Results&Conclusion:The established method based on LC-MS technology was successfully used inquantitative analyzing of cGAMP in cells.This method can be used in further mechanism studies of cGAS regulation.
作者
郭增林
韩秋影
毛劼
王娜
周涛
陈媛
GUO Zeng-Lin;HAN Qiu-Ying;MAO Jie;WANG Na;ZHOU Tao;CHEN Yuan(National Center of Biomedical Analysis,Beijing 100850,China)
出处
《生物技术通讯》
CAS
2020年第1期61-65,87,共6页
Letters in Biotechnology
基金
国家自然科学基金(81672734)。
