Use of High-Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) to Quantify Modified Nucleosides

Rebecca Rodell, Ning Tsao, Adit Ganguly, Nima Mosammaparast

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Physiological and chemically induced modifications to nucleosides are common in both DNA and RNA. Physiological forms of these modifications play critical roles in gene expression, yet aberrant marks, if left unrepaired, may be associated with increased genome instability. Due to the low prevalence of these marks in most samples of interest, a highly sensitive method is needed for their detection and quantitation. High-performance liquid chromatography, coupled to mass spectrometry (HPLC-MS), provides this high degree of sensitivity while also being adaptable to nearly any modified nucleoside of interest and still maintaining exquisite specificity. In this chapter, we demonstrate how to use HPLC-MS to analyze the catalytic activity of a nucleic acid demethylase, to quantify the prevalence of N6-methyladenosine from RNA, and to determine the kinetics of alkylation damage repair.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages125-140
Number of pages16
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2444
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • AlkB
  • Epitranscriptomics
  • Mass spectrometry
  • Methylation
  • Nucleoside

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