TY - JOUR
T1 - Fused-core silica column ultra-performance liquid chromatography-ion trap tandem mass spectrometry for determination of global DNA methylation status
AU - Yang, Ill
AU - Fortin, Marie C.
AU - Richardson, Jason R.
AU - Buckley, Brian
N1 - Funding Information:
This work was supported in part by the National Institute of Environmental Health Sciences (NIEHS, Grant P30ES005022 ) and the National Institutes of Health (NIH, Grant R01ES015991 ). The authors also wish to thank Siew C. Tee for providing technical support.
PY - 2011/2/1
Y1 - 2011/2/1
N2 - 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.
AB - 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.
KW - Electrospray ionization ion trap mass spectrometry (ESI-ITMS)
KW - Global DNA methylation
KW - Ultra-performance liquid chromatography (UPLC)
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U2 - 10.1016/j.ab.2010.10.012
DO - 10.1016/j.ab.2010.10.012
M3 - Article
C2 - 20950581
AN - SCOPUS:78650291213
VL - 409
SP - 138
EP - 143
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -