Abstract
Global DNA methylation was classically considered the relative percentage of 5-methylcysine (5mC) with respect to total cytosine (C). Early approaches were based on the use of high-performance separation technologies and UV detection. However, the recent development of protocols using mass spectrometry for the detection has increased sensibility and permitted the precise identification of peak compounds based on their molecular masses. This allows work to be conducted with much less genomic DNA starting material and also to quantify 5-hydroxymethyl-cytosine (5hmC), a recently identified form of methylated cytosine that could play an important role in active DNA demethylation. Here, we describe the protocol that we currently use in our laboratory to analyze 5mC and 5hmC by mass spectrometry. The protocol, which is based on the method originally developed by Le and colleagues using Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry (triple Quadrupole (QqQ)) detection, allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels starting from just 1 μg of genomic DNA, which allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels.
Similar content being viewed by others
References
Herman JG, Baylin SB (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349:2042–2054
Cokus SJ, Feng S, Zhang X et al (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215–219
Finnegan EJ, Kovac KA (2000) Plant DNA methyltransferases. Plant Mol Biol 43:189–201
Zhang X, Yazaki J, Sundaresan A et al (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126:1189–1201
Bender J (2004) DNA methylation and epigenetics. Annu Rev Plant Biol 55:41–68
Feinberg AP, Cui H, Ohlsson R (2002) DNA methylation and genomic imprinting: insights from cancer into epigenetic mechanisms. Semin Cancer Biol 5:389–398
Payer B, Lee JT (2008) X chromosome dosage compensation: how mammals keep the balance. Annu Rev Genet 42:733–772
Doerfler W (1991) Patterns of DNA methylation—evolutionary vestiges of foreign DNA inactivation as a host defense mechanism. A proposal. Biol Chem 372:557–564
Lippman Z, Gendrel AV, Black M et al (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430:471–476
Ronemus MJ, Galbiati M, Ticknor C et al (1996) Demethylation-induced developmental pleiotropy in Arabidopsis. Science 273:654–657
Esteller M (2008) Epigenetics in cancer. N Engl J Med 358:1148–1159
Jones PA, Baylin SB (2007) The epigenomics of cancer. Cell 128:683–692
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204–220
Beard C, Li E, Jaenisch R (1995) Loss of methylation activates Xist in somatic but not in embryonic cells. Genes Dev 9:2325–2334
Okano M, Bell DW, Haber DA et al (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257
Kim GD, Ni J, Kelesoglu N et al (2002) Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases. EMBO J 21:4183–4195
Rhee I, Bachman KE, Park BH et al (2002) DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 416:552–556
He YF, Li BZ, Li Z et al (2011) Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333:1303–1307
Morales-Ruiz T, Ortega-Galisteo AP, Ponferrada-Marin MI et al (2006) DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases. Proc Natl Acad Sci U S A 103:6853–6858
Wu SC, Zhang Y (2010) Active DNA demethylation: many roads lead to Rome. Nat Rev Mol Cell Biol 11:607–620
Berdyshev GD, Korotaev GK, Boiarskikh GV et al (1967) Nucleotide composition of DNA and RNA from somatic tissues of humpback and its changes during spawning. Biokhimiia 32:988–993
Bjornsson HT, Sigurdsson MI, Fallin MD et al (2008) Intra-individual change over time in DNA methylation with familial clustering. JAMA 299:2877–2883
Wilson VL, Smith RA, Ma S et al (1987) Genomic 5-methyldeoxycytidine decreases with age. J Biol Chem 262:9948–9951
Kulis M, Esteller M (2010) DNA methylation and cancer. Adv Genet 70:27–56
Moore LE, Pfeiffer RM, Poscablo C et al (2008) Genomic DNA hypomethylation as a biomarker for bladder cancer susceptibility in the Spanish Bladder Cancer Study: a case-control study. Lancet Oncol 9:359–366
Baccarelli A, Bollati V (2009) Epigenetics and environmental chemicals. Curr Opin Pediatr 21:243–251
Kim M, Bae M, Na H et al (2012) Environmental toxicants-induced epigenetic alterations and their reversers. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 30:323–367
Tajuddin SM, Amaral AF, Fernandez AF et al (2014) LINE-1 methylation in leukocyte DNA, interaction with phosphatidylethanolamine N-methyltransferase variants and bladder cancer risk. Br J Cancer 110:2123–2130
Woo HD, Kim J (2012) Global DNA hypomethylation in peripheral blood leukocytes as a biomarker for cancer risk: a meta-analysis. PLoS One 7:e34615
Feil R, Fraga MF (2012) Epigenetics and the environment: emerging patterns and implications. Nat Rev Genet 13:97–109
Valledor L, Hasbún R, Meijón M et al (2007) Involvement of DNA methylation in tree development and micropropagation. Plant Cell Tissue Organ Cult 91:75–86
Berdasco M, Fraga MF, Esteller M (2009) Quantification of global DNA methylation by capillary electrophoresis and mass spectrometry. Methods Mol Biol 507:23–34
Torano EG, Petrus S, Fernandez AF et al (2012) Global DNA hypomethylation in cancer: review of validated methods and clinical significance. Clin Chem Lab Med 50:1733–1742
Le T, Kim KP, Fan G et al (2011) A sensitive mass spectrometry method for simultaneous quantification of DNA methylation and hydroxymethylation levels in biological samples. Anal Biochem 412:203–209
Valledor L, Escandon M, Meijon M et al (2014) A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms. Plant J 79:173–180
Acknowledgments
We thank Ronnie Lendrum for editorial assistance. This work has been financially supported by the following: Fondo de Investigaciones Sanitarias FIS/FEDER (PI11/01728 to AF.F. and PI12/01080 to M.F.F.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contract: CP11/00131 to A.F.F.); the Spanish National Research Council (CSIC; 200820I172 to M.F.F.); Fundación Ramón Areces (to M.F.F). The IUOPA is supported by the Obra Social Cajastur, Spain.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Fernandez, A.F., Valledor, L., Vallejo, F., Cañal, M.J., Fraga, M.F. (2018). Quantification of Global DNA Methylation Levels by Mass Spectrometry. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7481-8_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7479-5
Online ISBN: 978-1-4939-7481-8
eBook Packages: Springer Protocols