色谱2012，Vol.30Issue(5)：533-537,5.DOI:10.3724/SP.J.1123.2011.12015
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
Analysis of global deoxyribonucleic acid 5-hydroxymethylcytosine in tissue by liquid chromatography-tandem mass spectrometry
摘要
Abstract
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℃, dried under nitrogen, followed by spiking with cyto-sine-13C15N2 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 x2.1 mm, 1. 7ΜM) by gradient elution with 10 mmol/L ammonium acetate and ace-tonitrile 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.关键词
液相色谱-串联质谱/5-羟甲基胞嘧啶/脱氧核糖核酸羟甲基化/组织Key words
liquid chromatography-tandem mass spectrometry (LC-MS/MS)/5-hydroxymethylcytosine (5-hmCyt)/ deoxyribonucleic acid (DNA) hydroxymethylation/ tissue分类
化学化工引用本文复制引用
陈丽宇,张立坚,张良滔,蔡春..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[J].色谱,2012,30(5):533-537,5.基金项目
广东医学院团队基金项目. （）
