Chen Liyu, Zhang Lijian, Zhang Liangtao, Cai Chun
Guangdong Medical College, Zhanjiang 524023, China.
Se Pu. 2012 May;30(5):533-7. doi: 10.3724/sp.j.1123.2011.12015.
As preventing deoxyribonucleic acid (DNA) methylation transferase 1 (DNMT1) methylation of the target cytosine, 5-hydroxymethylcytosine could cause alterations in DNA methylation. A rapid analytical method for the determination of the degree of global DNA hydroxymethylation in tissues using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. After the extraction with phenol-chloroform, DNA was hydrolyzed to nucleobases by 88% formic acid at 140 degrees C, dried under nitrogen, followed by spiking with cytosine-13C15SN2 as internal standard, and reconstituted in a mixture of acetonitrile-water (9:1, v/v) for the analysis with LC-MS/MS. The LC separation was performed on a BEH HILIC column (100 mm x 2.1 mm, 1.7 microm) by gradient elution with 10 mmol/L ammonium acetate and acetonitrile as mobile phases. The analytes were detected by MS/MS with positive ion electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode to satisfy qualitative and quantitative detections. The results showed that the linear range of the calibration curve for 5-hydroxymethylcytosine was 0.1-30 ng/mL, and the correlation coefficient was higher than 0.99. The limit of detection (LOD, S/N = 3) and the limit of quantification (LOQ, S/N = 10) were 0.057 and 0.090 ng/mL, respectively. The intraday and interday precisions were 5.13% and 6.24%, respectively. The recoveries of the spiked standards varied from 90.24% to 97.53%. The method was applied to the analysis of DNA from cerebrums of rats, and the average degree of 5-hydroxymethylcytosine was 0.66%. The method is simple, reproducible, sensitive and suitable for the quantitative analysis of 5-hydroxymethylcytosine in global DNA from tissues.
由于5-羟甲基胞嘧啶可阻止靶胞嘧啶的脱氧核糖核酸（DNA）甲基转移酶1（DNMT1）甲基化，其可导致DNA甲基化改变。已开发出一种使用液相色谱-串联质谱法（LC-MS/MS）测定组织中整体DNA羟甲基化程度的快速分析方法。用苯酚-氯仿萃取后，DNA在140℃下用88%甲酸水解为核碱基，氮气吹干，然后加入胞嘧啶-13C15SN2作为内标，再用乙腈-水（9:1，v/v）混合溶液复溶，用于LC-MS/MS分析。在BEH HILIC柱（100 mm×2.1 mm，1.7 µm）上以10 mmol/L醋酸铵和乙腈为流动相进行梯度洗脱，实现LC分离。采用正离子电喷雾电离（ESI+）在多反应监测（MRM）模式下通过MS/MS检测分析物，以满足定性和定量检测。结果表明，5-羟甲基胞嘧啶校准曲线的线性范围为0.1-30 ng/mL，相关系数高于0.99。检测限（LOD，S/N = 3）和定量限（LOQ，S/N = 10）分别为0.057和0.090 ng/mL。日内和日间精密度分别为5.13%和6.24%。加标标准品的回收率在90.24%至97.53%之间。该方法应用于大鼠大脑DNA分析，5-羟甲基胞嘧啶的平均程度为0.66%。该方法简单、可重复、灵敏，适用于组织中整体DNA中5-羟甲基胞嘧啶的定量分析。
