Journal
ANALYTICAL BIOCHEMISTRY
Volume 412, Issue 2, Pages 203-209Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.01.026
Keywords
Mass spectrometry; Multiple reaction monitoring (MRM); DNA methylation; DNA hydroxymethylation; 5-Methyl-cytosine (5mC); 5-Hydroxymethyl-cytosine (5hmC); Induced pluripotent stem cells (iPSCs)
Funding
- CIRM [RC1-0111]
- NIH [RO1 NS 051411]
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The recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC in the same sample. We have developed a method using liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (LC-ESI-MS/MS-MRM) to simultaneously measure levels of 5mC and 5hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5hmC quantitation methods such as end labeling with thin layer chromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required to measure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming. (C) 2011 Elsevier Inc. All rights reserved.
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