A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samplesMaterials and methodsCell cultureDerivation of human iPSCsDNA extraction and hydrolysisDNA standardsMRM quantitationTET1 cloning, virus production and infectionImmunocytochemistrySouthern blot analysisBisulfite sequencingResultsMS characterization of 5hmC, 5mC, and CValidation of the MRM methodMeasuring 5hmC and 5mC in somatic and iPSCsDiscussionAcknowledgmentsSupplementary dataReferencesA sensitive mass spectrometry method for simultaneous quantification of DNAmethylation and hydroxymethylation levels in biological samplesThuc Lea,b,1, Kee-Pyo Kima,1,2, Guoping Fana,⇑, Kym F. FaullbaDepartment of Human Genetics, Broad Stem Cell Research Center, David Geffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095, USAbPasarow Mass Spectrometry Laboratory, Department of Psychiatry and Biobehavioral Sciences and Semel Institute for Neuroscience and Human Behavior, DavidGeffen School of Medicine, University of California – Los Angeles, Los Angeles, CA 90095, USAarticle infoArticle history:Received 17 November 2010Received in revised form 13 January 2011Accepted 19 January 2011Available online 24 January 2011Keywords:Mass spectrometryMultiple reaction monitoring (MRM)DNA methylationDNA hydroxymethylation5-Methyl-cytosine (5mC)5-Hydroxymethyl-cytosine (5hmC)Induced pluripotent stem cells (iPSCs)abstractThe recent discovery of 5-hydroxymethyl-cytosine (5hmC) in embryonic stem cells and postmitotic neu-rons has triggered the need for quantitative measurements of both 5-methyl-cytosine (5mC) and 5hmC inthe same sample. We have developed a method using liquid chromatography electrospray ionization tan-dem mass spectrometry with multiple reaction monitoring (LC–ESI–MS/MS–MRM) to simultaneouslymeasure 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 layerchromatography and radiolabeling by glycosylation. Only 50 ng of digested genomic DNA is required tomeasure the presence of 0.1% 5hmC in DNA from mouse embryonic stem cells. Using this procedure, weshow that human induced pluripotent stem cells exhibit a dramatic increase in 5mC and 5hmC levelscompared with parental fibroblast cells, suggesting a dynamic regulation of DNA methylation andhydroxymethylation during cellular reprogramming.Ó 2011 Elsevier Inc. All rights reserved.The pattern of methylated cytosine residues in DNA provides aninheritable epigenetic code that regulates gene expression duringdevelopment. The covalent addition of a methyl group at the 5-po-sition of cytosine primarily occurs in the CpG dinucleotide and iscatalyzed by a family of DNA methyltransferases (Dnmts),3includ-ing maintenance Dnmt1 and de novo Dnmt3a and Dnmt3b. DNAmethylation is involved in various biological processes such as geno-mic imprinting, silencing of retroviral transposons, X chromosomeinactivation, and cellular differentiation. Mechanistically, promotermethylation can lead to transcriptional repression directly by inhib-iting transcriptional binding or indirectly by recruiting various pro-teins, including methyl CpG binding domain proteins (MBDs),corepressors, and histone modification enzymes involved in chroma-tin remodeling [1–6]. Importantly, many studies have shown thatDNA methylation is a dynamic process in cellular proliferation anddifferentiation and is tightly regulated in normal development. Aber-rant DNA methylation patterns and mechanisms are deleterious tothe developing central nervous system (CNS) [6–9].Recently, there has been renewed interest in another relatedmammalian DNA modification, 5-hydroxymethyl-cytosine(5hmC). Significant levels of 5hmC are found in the developedmurine CNS and in embryonic stem cells [10–13]. The in vivo addi-tion of a hydroxyl group onto 5-methyl-cytosine (5mC) is catalyzedby 2-oxoglutarate oxygenase Tet1, Tet2, and Tet3 [10,13]. There arealso reports that 5hmC can be formed by other mechanismsbesides the Tet pathway, including ultraviolet (UV) irradiation of5mC in aerated aqueous solution [14] and DNA methyltransferasereaction of cytosine with formaldehyde [15]. To date, only the Tetpathway has been demonstrated to produce 5hmC in mammaliangenomic DNA.Speculation that 5hmC is involved in the DNA demethylationpathway comes from the two reported mechanisms of converting5hmC into cytosine (C). Bacterial DNA methyltranferases catalyzethe removal of formaldehyde from 5hmC, thereby converting0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2011.01.026⇑Corresponding author. Fax: +1 310 794 5446.E-mail address: [email protected] (G. Fan).1These authors contributed equally to this work.2Present address: Center of Cell and Gene Therapy, Baylor College of Medicine,Houston, TX 77030, USA.3Abbreviations used: Dnmt, DNA methyltransferase; CNS, central nervous system;5hmC, 5-hydroxymethyl-cytosine; 5mC, 5-methyl-cytosine; UV, ultraviolet; C, cyto-sine; LC–ESI–MS/MS–MRM, liquid chromatography electrospray ionization tandemmass spectrometry with multiple reaction monitoring; mESC, mouse embryonic stemcell; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; LIF,leukemia inhibitory factor; hESC, human embryonic stem cell; MEF, mouse embry-onic fibroblast; iPSC, induced pluripotent stem cell; PVDF, polyvinylidene difluoride;PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; SDS, sodiumdodecyl sulfate; PCR, polymerase chain reaction; UPLC, ultra-performance liquidchromatography; dC, deoxycytidine; cDNA, complementary DNA; BSA, bovine serumalbumin; DAPI, 40,6-diamidino-2-phenylindole; SSC, salt sodium citrate; RT, reversetranscription; 5mdC, 5-methyl-20-deoxycytidine; 5hmdC, 5-hydroxym ethyl-20-deoxycytidine.Analytical Biochemistry 412 (2011) 203–209Contents lists available at ScienceDirectAnalytical Biochemistryjournal homepage: www.elsevier.com/locate/yabio5hmC to C [15]. Another deformylation mechanism involves thephotochemical hydration of 5hmC in basic solution [14]. However,these two possible DNA demethylation mechanisms have yet to beconfirmed in mammalian models.Some of the most commonly used methods for profiling andquantification of DNA methylation, such as bisulfite sequencingand