Comprehensive Characterization of tRNA by Ultra High-Performance Liquid Chromatography High-Resolution Accurate Mass Spectrometry

Transfer ribonucleic acid (tRNA) are the smallest RNA in the translational triune and contain the greatest density of post-transcriptional modifications than any other RNA types in the cell. Due to the size of tRNA studying these modifications usually entails enzymatic digestion followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Here we report an advancement in Intact Mass Analysis for identification of tRNA through deconvolution of high resolution accurate mass spectrometry facilitated using a secondary alkylamine as an ion pairing reagent during reverse phase chromatography. We identify in isolated and total S. cerevisiae tRNA 3’ CCA variations and show that most tRNA lack a 3’ adenosine with the lesser abundant species having the expected CCA termini. We identify a previously unknown stable demethylated Wybutosine intermediate for tRNA PHE and identify low abundant contaminating tRNAs in an isolated tRNA PHE sample. Confirmation of identities was verified through traditional mass spectrometric nucleoside and mass mapping experiments.