液相色谱-串联质谱法分析大鼠血液中基因组DNA羟甲基化水平

发布时间：2018-06-04 00:21

本文选题：-羟甲基胞嘧啶(hm + C)　；参考：《质谱学报》2015年03期

【摘要】：5-羟甲基胞嘧啶(5-hydroxymethylcytosine,5hm C)是由TET蛋白家族催化氧化5-甲基胞嘧啶(5-methylcytosine,5m C)生成的,对哺乳动物的基因转录和表达发挥着重要的调控作用。本研究建立了血液样品中5hm C的液相色谱-串联质谱(LC-MS/MS)测定方法,采用试剂盒法提取血液基因组DNA,在甲酸环境下加热裂解,采用HILIC色谱柱分离,电喷雾电离和多反应监测模式定量。结果表明:5hm C在0.1~50μg/L范围内线性关系良好,相关系数为0.999 4;高、中、低3个加标水平的平均回收率为95.87%,日内和日间的相对标准偏差分别为3.42%和4.12%;5hm C的检出限(LOD,S/N=3)为0.050μg/L,定量限(LOQ,S/N=10)为0.100μg/L。该方法准确、快速、稳定、灵敏,可用于血液样品全基因组中5-羟甲基胞嘧啶的检测分析。
[Abstract]:5-hydroxycytosine 5-hydroxycytosine (5HMC) is produced by the TET protein family catalyzing the oxidation of 5-methylcytosine (5-methylcytosine) 5Hc, which plays an important role in the regulation of mammalian gene transcription and expression. A method for the determination of 5hm C in blood samples by liquid chromatography-tandem mass spectrometry (LC-MS / MS) was established in this paper. The genomic DNA was extracted by kit method, then heated and cracked in formic acid environment, and separated by HILIC chromatographic column. Quantitative analysis of electrospray ionization and multi-reaction monitoring model. The results showed that the linear relationship was good in the range of 0.1 渭 g / L, the correlation coefficient was 0.999 4, and the average recovery rate of high, middle and low standard addition levels was 95.87. The relative standard deviations between day and day were 3.42% and 4.12 渭 g / L, respectively. The detection limit of LOQS / N3 was 0.050 渭 g / L, and the quantitative limit was 0.100 渭 g / L. The method is accurate, rapid, stable and sensitive. It can be used for the detection and analysis of 5-hydroxymethyl cytosine in the whole genome of blood samples.
【作者单位】：广东医学院分析中心;四川大学华西药学院;
【基金】：国家自然科学基金(21375029) 广东省学科建设基金(2013CXZDA018)资助
【分类号】：O657.63;R927.2

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