Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs

Abstract

Mapping, sequencing, and quantifying individual noncoding ribonucleic acids (ncRNAs), including post-transcriptionally modified nucleosides, by mass spectrometry is a challenge that often requires rigorous sample preparation prior to analysis. Previously, we have described a simplified method for the comparative analysis of RNA digests (CARD) that is applicable to relatively complex mixtures of ncRNAs. In the CARD approach for transfer RNA (tRNA) analysis, two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. This approach allows one to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging because of convoluted mass spectra arising from the natural 13C and 15 N isotopes present in the ribonuclease-digested tRNA samples. Here, we demonstrate that culturing in 12C-enriched/13C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality yield a 35 % improvement in detection of tRNA digestion products that can be uniquely assigned to particular tRNAs. These mass spectral improvements lead to a significant reduction in data processing attributable to the ease of spectral identification of labeled digestion products and will enable improvements in the relative quantification of modified RNAs by the 16O/18O differential labeling approach.

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Acknowledgments

Financial support of this work was provided by the National Science Foundation (CHE1212625) and a University of Cincinnati Department of Chemistry Doctoral Enhancement Award to C.W.

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Correspondence to Patrick A. Limbach.

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Wetzel, C., Li, S. & Limbach, P.A. Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs. J. Am. Soc. Mass Spectrom. 25, 1114–1123 (2014). https://doi.org/10.1007/s13361-014-0889-9

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Key words

  • Post-transcriptional modifications
  • tRNA
  • ncRNA
  • Comparative analysis
  • RNA sequencing
  • RNase mass mapping
  • Liquid chromatography-mass spectrometry
  • Quantitative analysis