While a number of approaches have been developed to analyze liquid chromatography tandem mass spectrometry (LC-MS/MS) data obtained from modified oligonucleotides, the majority of these methods require analyzing every MS/MS spectrum de novo to sequence the oligonucleotide and place the modification. Spectral matching is an alternative approach for analyzing MS/MS data that is based on creating a library of annotated MS/MS spectra against which individual MS/MS data can be searched. Here, we have adapted the existing NIST spectral matching software to enable its use in the interpretation of MS/MS data obtained from modified oligonucleotides. In particular, we demonstrate the utility of this approach to identify specific post-transcriptionally modified nucleosides in particular transfer RNAs (tRNAs) obtained through a conventional RNA modification mapping experimental protocol. Spectral matching was found to be an efficient approach for screening for known modified tRNAs by using the experimental data as the library and previously annotated RNase T1 digestion products of tRNAs as the reference spectra. The utility of spectral matching for rapid analysis of multiple LC-MS/MS analyses was demonstrated by screening mutant strains of Streptococcus mutans to identify the enzyme(s) responsible for synthesizing the tRNA position 37 modification threonylcarbamoyladenosine (t6 A).
Keywords: LC-MS/MS; RNA sequencing; modified nucleosides; spectral matching; tRNA; tandem mass spectrometry.
© 2019 John Wiley & Sons, Ltd.