Analysis of the epitranscriptome with ion-pairing reagent free oligonucleotide mass spectrometry

Methods Enzymol. 2021:658:111-135. doi: 10.1016/bs.mie.2021.06.024. Epub 2021 Jul 27.


RNA modifications gain growing attention as a new frontier in the life sciences but with the rise of RNA vaccines also in biomedical drug development. Impeccable characterization of RNA modifications within their sequence context remains an analytical challenge. Oligonucleotide mass spectrometry (ON-MS), an approach similar to bottom-up proteome analysis, is capable of defining a short 5-15 nucleotide sequence context of an RNA modification while delivering information on the chemical character of the modified nucleotide. Commonly, ON-MS requires the use of ion pairing reagents for ON separation which is not compatible with most other MS-based applications and only few laboratories run dedicated MS instruments for the task. Here, we present an ON-MS technique which is independent of ion pairing reagents and can be used on any available mass spectrometer without risking its sensitivity for other analytes. In this chapter, we describe the experiments necessary for ON-MS method development, ON-MS application to native and synthetic RNAs and finally a guideline for data analysis.

Keywords: AlkB; Epitranscriptome; In vitro transcription; LC-MS/MS; Mass spectrometry; Oligonucleotides; Quantification; RNA modification; RNA, Stable isotope labeling, Ion-pairing reagent free, Low resolution MS, SEC, RNase T1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Indicators and Reagents
  • Mass Spectrometry
  • Nucleotides*
  • Oligonucleotides*
  • RNA


  • Indicators and Reagents
  • Nucleotides
  • Oligonucleotides
  • RNA