Poster abstracts

Poster number 136 submitted by Collin Wetzel

Improving Comparative Analysis of tRNAs by Mass Spectrometry Using Isotopic Enrichment

Collin Wetzel (Department of Chemistry, University of Cincinnati), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)

Abstract:
Mapping and sequencing of individual transfer ribonucleic acids (tRNAs) can prove to be very difficult, requiring rigorous sample preparation prior to analysis. Previously our group has presented a simplified method for the comparative analysis of RNA digests (CARD).1,2 In the CARD approach two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. With this approach we are able 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 due to convoluted mass spectra arising from the natural 13C and 15N isotopes present in 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 enable one to use existing software analysis tools to reduce data processing steps, and these improvements enable one to obtain more biologically relevant information from the analysis.

References:
1. Siwei Li and Patrick A. Limbach (2012). Method for comparative analysis of ribonucleic acids using isotope labeling and mass spectrometry. Analytical chemistry, 84 8607–8613.
2. Siwei Li and Patrick A. Limbach (2013). Mass spectrometry sequencing of transfer ribonucleic acids by the comparative analysis of RNA digests (CARD) approach. The Analyst, 138 1386–1394.

Keywords: Mass spectrometry, Isotope enrichment depletion, Modified RNA