LC-MS/MS法测定药用植物基因组DNA甲基化水平Δ

摘要

OBJECTIVE:To develop a detection LC-MS/MS method for global DNA methylation in medicinal plants. METH-ODS:Genomic DNA was isolated using plant DNA extraction kit,and then hydrolyzed by 88% formic acid at 140 ℃. After dried with nitrogen,extracted DNA was dissolved again with mobile phase. LC separation was performed on HILIC column with mobile phase consisted of 7 mmol/L ammonium formate-acetonitrile (gradient elution) at flow rate of 0.3 ml/min. The analysis was con-ducted by tandem MS with positive ion electrospray ionization in multiple reaction monitoring(MRM)mode. The ratio of genomic DNA methylation in 10 commonly used medicinal plants was calculated. RESULTS:The linear ranges of Cyt and 5mC were 1-500 ng/ml(r=0.999 5)and 0.2-100 ng/ml(r=0.999 6). The relative standard deviations(RSDs)of accuracy were 1.12% and 3.68%(n=6). The RSDs of intra-day precision were 2.36% and 4.02% for Cyt and 5mC,respectively (n=5). The RSDs of inter-day precision were 1.04% and 3.54% for Cyt and 5mC,respectively (n=3). The RSDs of repeatability test were 1.53% and 3.27%for Cyt and 5mC,respectively(n=6). The recoveries of Cyt and 5mC were 98.7%-102.1% and 91.2%-103.5%. The percentages of global DNA methylation in 10 medicinal plants were ranged from 17.63% to 25.18%. CONCLUSIONS:LC-MS/MS method is simple,rapid,sensitive and precise,and can be used for the detection of global DNA methylation in medicinal plants.%目的：建立测定药用植物基因组DNA甲基化水平的液相色谱-串联质谱法（LC-MS/MS）。方法：采用试剂盒提取药用植物基因组DNA，并用88％甲酸在140℃下裂解DNA，经氮气吹干后，用流动相重新溶解。采用HILIC亲水作用色谱柱，以7 mmol/L甲酸铵水溶液-乙腈为流动相进行梯度洗脱，流速为0.3 ml/min。采用电喷雾离子源正离子模式进行监测，多反应监测（MRM）模式下进行定量分析，计算10种常见药用植物基因组的DNA甲基化率。结果：Cyt、5mC检测质量浓度线性范围分别为1～500（r＝0.9995）、0.2～100 ng/ml（r＝0.9996），精密度试验的RSD分别为1.12％和3.68％（n＝6），日内稳定性试验的RSD分别为2.36％和4.02％（n＝5），日间稳定性试验的RSD分别为1.04％和3.54％（n＝3），重复性试验的RSD分别为1.53％和3.27％（n＝6），方法回收率分别为98.7％～102.1％、91.2％～103.5％。10种药用植物基因组的DNA甲基化率在17.63％～25.18％之间。结论：LC-MS/MS法简便、快速、灵敏度高、精密度好，可用于药用植物基因组DNA甲基化水平的检测。