Quantification of DNA Methylation and Its Oxidized Derivatives Using LC-MS

Franziska R Traube; Sarah Schiffers; Thomas Carell

doi:10.1007/978-1-0716-1294-1_6

Quantification of DNA Methylation and Its Oxidized Derivatives Using LC-MS

Methods Mol Biol. 2021:2272:77-94. doi: 10.1007/978-1-0716-1294-1_6.

Authors

Franziska R Traube^#¹, Sarah Schiffers^#^{1

2}, Thomas Carell³

Affiliations

¹ Department of Chemistry, Ludwig-Maximilians-Universität München and Center for Integrated Protein Science Munich (CIPSM), Munich, Germany.
² S.S. National Institutes of Health, Bethesda, MD, USA.
³ Department of Chemistry, Ludwig-Maximilians-Universität München and Center for Integrated Protein Science Munich (CIPSM), Munich, Germany. [email protected].

^# Contributed equally.

PMID: 34009610
DOI: 10.1007/978-1-0716-1294-1_6

Abstract

The amount of 5-methyl-2'-deoxycytidine (m⁵dC) and its oxidized derivatives 5-hydroxymethyl-dC (hm⁵dC), 5-formyl-dC (f⁵dC), and 5-carboxy-dC (ca⁵dC) inside mammalian cells provides valuable information concerning cellular state and fate. LC-MS methods enable reliable quantification of these noncanonical DNA modifications in the low femtomolar range. Here, we describe a broadly applicable protocol to quantify m⁵dC, hm⁵dC, f⁵dC, and ca⁵dC in vertebrate-derived cells using ultra-HPLC triple quadrupole MS (UHPLC-QQQ-MS).

Keywords: Isotope-dilution mass spectrometry; Quantification of noncanonical DNA nucleosides; UHPLC-QQQ-MS; gDNA isolation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Chromatography, Liquid / methods*
DNA / analysis*
DNA / chemistry*
DNA / genetics
DNA Methylation*
Epigenesis, Genetic*
Humans
Oxidation-Reduction
Tandem Mass Spectrometry / methods*

Substances

DNA