液相色谱-串联质谱分析组织中基因组脱氧核糖核酸羟甲基胞嘧啶水平

5-羟甲基胞嘧啶通过阻止脱氧核糖核酸(DNA)甲基化转移酶1(DMNT1)甲基化胞嘧啶来影响DNA甲基化的程度。本文建立了液相色谱-串联质谱(LC-MS/MS)测定组织中全基因组5-羟甲基胞嘧啶水平的方法。采用苯酚-氯仿提取组织DNA,提取的DNA用88%甲酸在140 ℃下裂解,DNA裂解液加入同位素胞嘧啶作内标,经N2吹干后,加乙腈-水(9:1, v/v)溶解,用LC-MS/MS检测5-羟甲基胞嘧啶的含量,并计算全基因组中5-羟甲基胞嘧啶的水平。结果表明,5-羟甲基胞嘧啶的线性范围为0.1～30 ng/mL,相关系数为0.9969,检出限(信噪比为3计)和定量限(信噪比为10计)分别为0.057 ng/mL和0.090 ng/mL;日内相对标准偏差和日间相对标准偏差分别为5.13%和6.24%;加标回收率为90.24%～97.53%。用该方法检测了大鼠大脑组织DNA羟甲基化水平,平均结果为0.66%。该方法简便,重现性好,灵敏度较高,能满足全基因组5-羟甲基胞嘧啶定量检测的要求。

Analysis of global deoxyribonucleic acid 5-hydroxymethylcytosine in tissue by liquid chromatography-tandem mass spectrometry

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.