Fused-core silica column ultra-performance liquid chromatography-ion trap tandem mass spectrometry for determination of global DNA methylation status.
Epigenetic modifications, such as DNA methylation, play key roles in transcriptional regulation of gene expression. More recently, global DNA methylation levels have been documented to be altered in several diseases, including cancer, and as the result of exposure to environmental toxicants. Based on the potential use of global DNA methylation status as a biomarker of disease status and exposure to environmental toxicants, we sought to develop a rapid, sensitive, and precise analytical method for the quantitative measurement of global DNA methylation status using ultra-performance liquid chromatography with detection by ion trap tandem mass spectrometry. Using a fused-core silica column, 2'-deoxyguanosine (2dG) and 5-methyl-2'-deoxycytidine (5mdC) were resolved in less than 1 min with detection limits of 0.54 and 1.47 fmol for 5mdC and 2dG, respectively. The accuracy of detection was 95% or higher, and the day-to-day coefficient of variation was found to be 3.8%. The method was validated by quantification of global DNA methylation status following treatment of cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which reduced DNA methylation from 3.1% in control cells to 1.1% in treated cells. The sensitivity and high throughput of this method rend it suitable for large-scale analysis of epidemiological and clinical DNA samples.
Environmental and Occupational Health Sciences Institute, A Joint Institute of Rutgers University and the University of Medicine and Dentistry of New Jersey (UMDNJ), Piscataway, NJ 08854, USA.
Ill
I
Yang
Marie C
MC
Fortin
Jason R
JR
Richardson
Brian
B
Buckley
D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019175:DNA Methylation; D000077209:Decitabine; D003841:Deoxycytidine / Q000737:chemistry / Q000302:isolation & purification; D003849:Deoxyguanosine / Q000737:chemistry / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D012822:Silicon Dioxide / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004791:Enzyme Inhibitors; D003841:Deoxycytidine; D012822:Silicon Dioxide; D000077209:Decitabine; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D003849:Deoxyguanosine; D001374:Azacitidine