超高效液相色谱-串联质谱法同时测定肿瘤细胞mRNA中12种正常核苷和修饰核苷  

Simultaneous Determination of Twelve Normal and Modified Nucleosides in mRNA of Cancer Cells by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry

作　　者：罗阳旭 戴开金[2] 杜娟[1,3] 肖华弟[1] 郑玲 陈训财 马安德 罗奇志 LUO Yang-xu;DAI Kai-jin;DU Juan;XIAO Hua-di;ZHENG Ling;CHEN Xun-cai;MA An-de;LUO Qi-zhi(Hygiene Detection Center,School of Public Health,Southern Medical University,Guangzhou 510515,China;Guangdong Southern Medical University Technology Development Department CO.,Ltd.,Southern Medical University,Guangzhou 510515,China;Forensic Science Center,Southern Medical University,Guangzhou 510515,China;Department of Forensic Toxicology,School of Forensic Medicine,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学公共卫生学院卫生检测中心,广东广州510515 [2]南方医科大学广东南方医大科技开发有限公司,广东广州510515 [3]南方医科大学司治鉴定中心,广东广州510515 [4]南方医科大学法医学院法医毒物学系,广东广州510515

出　　处：《分析测试学报》2020年第8期950-960,共11页Journal of Instrumental Analysis

基　　金：Project on the Integration of Industry,Education and Research of Guangdong Province(2012B091100371)。

摘　　要：建立了一种肿瘤细胞mRNA中12种正常核苷和修饰核苷的分析方法。肿瘤细胞样品经总RNA提取、mRNA纯化和mRNA水解后,加入8-溴鸟苷(8Br-G)为内标,以0.1%甲酸水溶液和0.1%甲酸甲醇溶液为流动相,采用Agilent RRHD SB-C18(2.1 mm×50 mm,1.8μm)色谱柱进行分离,电喷雾电离源正离子扫描(ESI+),多反应监测(MRM)模式检测。该方法符合方法学验证要求,12种正常核苷和修饰核苷在一定浓度范围内线性关系良好,相关系数(r)均大于0.99,定量下限(LOQ)为0.17~3.13 ng/mL,检出限(LOD)为0.08~1.56 ng/mL。将建立的方法应用于人鼻咽癌5-8F细胞系、人宫颈癌Hela细胞系和人胚肾上皮293T细胞系,成功地检测出多种修饰核苷水平。该方法细胞用量少(每样品2×106个),分析时间短(每样品6 min),灵敏度高,可为癌症研究和临床诊断等提供一种有效的分析方法。An ultra-high performance liquid chromatography-tandem mass spectrometric( UPLC-MS/MS) method was developed for the simultaneous determination of twelve normal and modified nucleosides in mRNA of cancer cells. Total RNA extraction,mRNA purification and mRNA hydrolyzation were performed using cultured cancer cell samples from commercial kits,with 8-bromoguanosine( 8 Br-G) as internal standard. Twelve normal and modified nucleosides were separated on an Agilent RRHD SB-C18( 2. 1 mm × 50 mm,1. 8 μm) column with 0. 1% formic acid methanol solution and 0. 1% formic acid solution as mobile phases,and determined by UPLC-MS/MS with positive electrospray ionization( ESI+) under multiple reaction monitoring( MRM) mode. The method could meet the requirements for method validation. There were good linear relationships for twelve normal and modified nucleosides in mRNA of cancer cells in the certain concentration ranges with their correlation coefficients( r) larger than 0. 99. The limits of quantitation and limits of detection were in the ranges of 0. 17-3. 13 ng/m L and 0. 08-1. 56 ng/m L,respectively. The method was then applied to human nasopharyngeal carcinoma 5-8 F cell line,human cervical cancer Hela cell line and human embryonic kidney epithelial 293 T cell line,with successful determination of various modified nucleosides. The method is capable of simultaneous quantitation for twelve normal and modified nucleosides in mRNA with short analysis time( 6 min per sample),low number of cells( 2 × 10~6 per sample) and high sensitivity,which could provide a powerful tool for cancer research and clinical diagnosis.

关 键 词：核苷 修饰核苷 MRNA 肿瘤细胞 超高效液相色谱-串联质谱法(UPLC-MS/MS) 

分 类 号：O657.63[理学—分析化学] Q525[理学—化学]