篇名: LC-MS/MS法测定药用植物基因组DNA甲基化水平
摘要: 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=0.999 5)、0.2~100 ng/ml(r=0.999 6),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。
ABSTRACT: OBJECTIVE: To develop a detection LC-MS/MS method for global DNA methylation in medicinal plants. METHODS: 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 conducted 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.
期刊: 2016年第27卷第16期
作者: 黄琼林,文娟,李兆锦,蔡春
AUTHORS: HUANG Qionglin,WEN Juan,LI Zhaojin,CAI Chun
关键字: DNA甲基化;液相色谱-质谱联用法;胞嘧啶;5-甲基胞嘧啶;药用植物
KEYWORDS: DNA methylation; LC-MS/MS; Cytosine; 5-methylcytosine; Medicinal plants
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