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

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。

Determiniation of N6-Methyladenosine Level in Hepatoma Cell Line by LC-MS/MS

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.