[1]李盛建,王 慧,吕 磊,等.液相色谱串联质谱法检测肝癌细胞N6- 甲基腺嘌呤核苷甲基化水平[J].现代检验医学杂志,2020,35(01):20-24.[doi:10.3969/j.issn.1671-7414.2020.01.006]
　LI Sheng-jian,WANG Hui,L Lei,et al.Determiniation of N6-Methyladenosine Level inHepatoma Cell Line by LC-MS/MS[J].Journal of Modern Laboratory Medicine,2020,35(01):20-24.[doi:10.3969/j.issn.1671-7414.2020.01.006]
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液相色谱串联质谱法检测肝癌细胞N6- 甲基腺嘌呤核苷甲基化水平()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第35卷
期数:
2020年01期
页码:
20-24
栏目:
论　　著
出版日期:
2020-02-29

文章信息/Info

Title:
Determiniation of N6-Methyladenosine Level in Hepatoma Cell Line by LC-MS/MS
文章编号:
1671-7414(2020)01-020-05
作者:
李盛建1; 王 慧2; 吕 磊2; 李群英1; 须秋萍1; 周艳卿1; 张国庆2; 赵 亮1
(1. 上海宝山区罗店医院药剂科,上海 201908; 2. 上海东方肝胆外科医院药剂科,上海 200438)
Author(s):
LI Sheng-jian1;  WANG Hui2;  LÜ Lei2;  LI Qun-ying1;  XU Qiu-ping1;  ZHOU Yang-qing1;  ZHANG Guo-qing2;  ZHAO Liang1
(1.Department of Pharmacy, Shanghai Baoshan Luodian Hospital, Shanghai 201908,China; 2.Department of Pharmacy, Shanghai Eastern Hepatobiliary Surgery Hospital, Shanghai 200438,China)
关键词:
液相色谱-串联质谱;  N6-甲基腺嘌呤核苷甲基化;  肝癌细胞
分类号:
R735.7; R730.43
DOI:
10.3969/j.issn.1671-7414.2020.01.006
文献标志码:
A
摘要:
目的 建立同时测定N6-甲基腺嘌呤核苷(m6A)和腺嘌呤核苷(A)的液相色谱-串联质谱法(LC-MS/MS),检测肝癌细胞m6A甲基化水平。方法 HepG2和L02细胞mRNA经分离、消化为核苷,再经含对乙酰氨基酚为内标的甲醇沉淀处理。色谱柱为Agilent Proshell 120 EC-C18柱,流动相为0.1 g/dl甲酸水-甲醇(82:18),流速0.4 ml/min,柱温为30 ℃。质谱检测模式为多反应监测(DMRM)模式,测定m6A和A的浓度,计算细胞m6A甲基化水平。 结果 建立的LC-MS/MS法检测m6A和A的浓度分别在0.15～50.00 ng/ml和1.50～500.00 ng/ml浓度范围内线性关系良好(r > 0.999),日内、日间精密度均小于15.00 %,准确度为93.67 %～101.10 %,回收率为91.46 %～97.60 %,基质效应为90.26 %～99.27 %,样品稳定性良好。HepG2和L02细胞m6A甲基化水平分别为(0.73 ± 0.11)%和(1.26 ± 0.22)%。结论 该方法准确、快速、稳定和灵敏,可用于检测肝癌细胞m6A甲基化水平。
Abstract:
Objective To establish a LC-MS/MS method for simultaneous determination of N6-methyladenine nucleoside(m6A)and adenosine(A)for the analysis of m6A level in hepatocellular carcinoma cells.Methods mRNA of HepG2 and L02 cell lines were separated and digested into nucleosides, precipitated by methanol including acetaminophen as internal standard.The separation was achieved on a Agilent Poroshell 120 EC-C18 column using a mobile phase system of 0.1% formic acid -methanol(82:18)at a flow rate of 0.4 ml/min. The temperature of column was 30℃ and the MS detection was DMRM mode for quantifying adenine and N6-methyladenine to calculate N6-methylation level to calculated the of level m6A methylation. Results Both of m6A and A possessed good linear relationship in the linear range of 0.15～50.00 ng/ml and 1.50～500.00 ng/ml, respectively(r > 0.999). Intra- and inter-day precisions were less than 15%, the accuracy were ranged from 93.67 %～101.10 %, and the recovery were ranged from 91.46 %～101.10 %. The matrix effect were about 90.26 %～99.27 % and samples remained stable during analysis. The percentages of N6-methylation in HepG2 and L02 cells were(0.73 ± 0.11)% and(1.26 ± 0.22)% respectively. Conclusion The method is accurate, fast, stable and sensitive. It is suitable for determination of N6-methylation level in cell.

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备注/Memo

备注/Memo:
作者简介:李盛建(1988-),男,药理学硕士,主管药师,研究方向:体内药物分析,E-mail:punk8@163.com。 通讯作者:赵亮,副主任药师,研究方向:体内药物分析,E-mail:zhaoliangphar@163.com。收稿日期:2019-05-15 修回日期:2019-08-07
更新日期/Last Update: 2020-03-30