title: High-throughput and cost-effective global DNA methylation assay by liquid chromatography-mass spectrometry.
abstract: An affordable and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the accurate and precise determination of global DNA methylation levels in peripheral blood. Global DNA methylation extent was expressed as the ratio of methylated 2'-deoxycytidine (5MedC) to 2'-deoxyguanosine (dG), which were obtained after DNA extraction and hydrolysis and determined by positive electrospray LC-ESI-MS/MS. The cost-effective internal standards (15)N(3)-dC and (15)N(5)-dG were incorporated for the accurate quantification of 5MedC and dG, respectively. The desired nucleoside analytes were separated and eluted by LC within 2.5 min on a reverse phase column with a limit of detection of 1.4femtomole on column for 5MedC. Sample preparation in 96-well format has significantly increased the assay throughput and filtration was found to be a necessary step to assure precision. Precision was performed with repeated analysis of four DNA QC sample over 12 days, with mean intra- and inter-day CVs of 6% and 11%, respectively. Accuracy was evaluated by comparison with a previously reported method showing a mean CV of 4% for 5 subjects analyzed. Furthermore, application of the assay using a benchtop orbitrap LCMS in exact mass full scan mode showed comparable sensitivity to tandem LCMS using multiple reaction monitoring.
authors.affiliation: University of Hawaii Cancer Center, Honolulu, 96813, USA.
authors.forename: Xingnan
authors.initials: X
authors.lastname: Li
authors.forename: Adrian A
authors.initials: AA
authors.lastname: Franke
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D003362:Cost-Benefit Analysis; D004247:DNA / Q000302:isolation & purification; D019175:DNA Methylation; D003841:Deoxycytidine / Q000737:chemistry; D003849:Deoxyguanosine / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000191:economics / Q000379:methods; D006868:Hydrolysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000191:economics / Q000379:methods*
chemical_list: D003841:Deoxycytidine; D004247:DNA; D003849:Deoxyguanosine