title: A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples.
abstract: 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.
authors.affiliation: Department of Human Genetics, Broad Stem Cell Research Center, David Geffen School of Medicine, University of California - Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Thuc
authors.initials: T
authors.lastname: Le
authors.forename: Kee-Pyo
authors.initials: KP
authors.lastname: Kim
authors.forename: Guoping
authors.initials: G
authors.lastname: Fan
authors.forename: Kym F
authors.initials: KF
authors.lastname: Faull
mesh_terms: D044503:5-Methylcytosine / Q000032:analysis* / Q000031:analogs & derivatives; D000818:Animals; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D019175:DNA Methylation / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006900:Hydroxylation; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D006899:Mixed Function Oxygenases; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D015203:Reproducibility of Results
chemical_list: D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human