Top-Down Characterization of a Yeast tRNA Extract by Gas-Phase Fractionation Electron Photodetachment Charge Reduction Mass Spectrometry

Mass spectrometry has proven to be a highly effective tool for characterization of RNA post-transcriptional modifications. For both synthetic RNAs and ones from biological sources, mass spectrometry can interrogate multiple, co-occurring covalent modifications at the nucleotide level simultaneously. To expand the capacity of MS-based methods to determine co-occurring modifications, top-down methodologies that examine intact RNAs offering a compelling strategy. Many transfer RNAs (tRNAs) are heavily modified and exhibit high sequence heterogeneity, making them challenging targets for MS/MS analysis. Here we report an on-line desalting approach coupled to a gas-phase fractionation method in which narrow segments of a mass spectrum are isolated and subjected to ultraviolet photodissociation (UVPD) and collisional activation dissociation (CAD). UV photoactivation causes charge reduction via electron photodetachment, dispersing the precursor ions over a broader m/z range and alleviating overlap of isotope distributions, streamlining their identification from a database of tRNA sequences. CAD generates diagnostic fragmentation patterns, allowing confident identification of tRNAs pre-identified based on the UVPD charge-reduction process. In total, over 350 fragment ion searches were conducted for 50 CAD spectra, yielding thorough sequence coverage of several tRNAs including four (Tyr GPA, Asp GUC, Ser GCU, Thr IGU) which have not to date been characterized by top-down mass spectrometry.