title: [Analysis of global deoxyribonucleic acid 5-hydroxymethylcytosine in tissue by liquid chromatography-tandem mass spectrometry].
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 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.
authors.affiliation: Guangdong Medical College, Zhanjiang 524023, China.
authors.forename: Liyu
authors.initials: L
authors.lastname: Chen
authors.forename: Lijian
authors.initials: L
authors.lastname: Zhang
authors.forename: Liangtao
authors.initials: L
authors.lastname: Zhang
authors.forename: Chun
authors.initials: C
authors.lastname: Cai
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry / Q000031:analogs & derivatives; D000818:Animals; D001923:Brain Chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D004247:DNA / Q000032:analysis*; D019175:DNA Methylation; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA