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Epigenetic Methods in Neuroscience Research pp 87–96Cite as

Simultaneous Quantification of Global 5mC and 5hmC Levels in the Nervous System Using an HPLC/MS Method

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Part of the book series: Neuromethods ((NM,volume 105))

Abstract

Methylation of DNA at the 5′ carbon of cytosine (5mC) is a key epigenetic regulator of gene expression required for the proper development and function of the central nervous system. Observations of experience-dependent fluctuations in the patterns of 5mC at neuronal gene enhancers, promoters and intragenic regions have prompted great interest in the role of DNA methylation mechanisms in the brain. In addition, the recent discovery of 5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5mC that acts as a DNA demethylation intermediate has offered mechanistic insight into how these changes in 5mC levels are achieved. As a result, numerous techniques have now been adapted or newly developed to quantify and profile 5mC and 5hmC in the genome. Here we describe a method using high performance liquid chromatography electrospray ionization tandem mass spectrometry with multiple reaction monitoring (HPLC-ESI-MS/MS-MRM) for the simultaneous detection and quantification of global 5mC and 5hmC levels present in genomic DNA from nervous-system derived samples. HPLC-ESI-MS/MS-MRM analysis offers the highest levels of sensitivity, selectivity, and precision available for the determination of global changes in these epigenetic modifications.

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Acknowledgements

This work was supported by NIMH grant MH57014. Primary neuronal culture and brain images were provided by Mikael C. Guzman-Karlsson. We would like to thank Jeremy Day and J. David Sweatt for comments and suggestions in the editing of this manuscript.

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Authors and Affiliations

  1. Department of Pharmacology & Toxicology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    Daniel L. Ross

  2. Department of Neurobiology, University of Alabama at Birmingham, 1825 University Blvd, Shelby 1070, Birmingham, AL, 35294, USA

    Garrett A. Kaas

  3. Evelyn F. McKnight Brain Institute, University of Alabama at Birmingham, Birmingham, AL, 35294, USA

    Garrett A. Kaas

Authors
  1. Daniel L. Ross
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  2. Garrett A. Kaas
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Corresponding author

Correspondence to Garrett A. Kaas .

Editor information

Editors and Affiliations

  1. Neuroscience Center, University of Helsinki, Helsinki, Finland

    Nina Karpova

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Ross, D.L., Kaas, G.A. (2016). Simultaneous Quantification of Global 5mC and 5hmC Levels in the Nervous System Using an HPLC/MS Method. In: Karpova, N. (eds) Epigenetic Methods in Neuroscience Research. Neuromethods, vol 105. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2754-8_5

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  • DOI: https://doi.org/10.1007/978-1-4939-2754-8_5

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2753-1

  • Online ISBN: 978-1-4939-2754-8

  • eBook Packages: Springer Protocols

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