液相色谱串联质谱法检测肝癌细胞N^6-甲基腺嘌呤核苷甲基化水平

Determiniation of N^6-Methyladenosine Level in Hepatoma Cell Line by LC-MS/MS

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。

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 Hep G2 and L02cell 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-C18column 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.ResultsBoth 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 Hep G2and L02cells 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.