A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples

Anal Biochem. 2011 May 15;412(2):203-9. doi: 10.1016/j.ab.2011.01.026. Epub 2011 Jan 24.


The recent discovery of 5-hydroxymethyl-cytosine (5 hmC) in embryonic stem cells and postmitotic neurons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5 mC) and 5 hmC 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 5 mC and 5 hmC in digested genomic DNA. This method is fast, robust, and accurate, and it is more sensitive than the current 5 hmC 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% 5 hmC in DNA from mouse embryonic stem cells. Using this procedure, we show that human induced pluripotent stem cells exhibit a dramatic increase in 5 mC and 5 hmC levels compared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation and hydroxymethylation during cellular reprogramming.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Methylcytosine / analysis*
  • Animals
  • Catalytic Domain
  • Chromatography, Liquid
  • Cytosine / analogs & derivatives*
  • Cytosine / analysis
  • DNA Methylation / genetics*
  • DNA-Binding Proteins / chemistry
  • Embryonic Stem Cells / metabolism*
  • Fibroblasts / metabolism
  • Humans
  • Hydroxylation
  • Induced Pluripotent Stem Cells / metabolism*
  • Mass Spectrometry / methods*
  • Mice
  • Mixed Function Oxygenases
  • Proto-Oncogene Proteins / chemistry
  • Reproducibility of Results


  • DNA-Binding Proteins
  • Proto-Oncogene Proteins
  • 5-hydroxymethylcytosine
  • 5-Methylcytosine
  • Cytosine
  • Mixed Function Oxygenases
  • TET1 protein, human