Top-Down Characterization of Yeast tRNA by Gas-Phase Fractionation and Collisionally Activated Dissociation Informed by Electron Photodetachment Charge Reduction Mass Spectrometry

Many transfer RNAs (tRNAs) are heavily modified and exhibit high sequence heterogeneity, making them challenging targets for characterization. Tandem mass spectrometry can interrogate multiple covalent modifications present on a single type of tRNA 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 offer a compelling strategy. Here we report a gas-phase fractionation MS/MS method in which narrow segments of a mass spectrum are isolated and subjected to either ultraviolet (UV) photoactivation or collisionally activated dissociation (CAD). UV photoactivation causes charge reduction via electron photodetachment, dispersing the precursor ions over a broader <i>m</i>/<i>z</i> range and alleviating overlap of isotope distributions, streamlining their identification from a database of tRNA sequences. In a complementary experiment, CAD generates diagnostic fragmentation patterns, allowing confident identification of tRNAs preidentified based on the electron photodetachment 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.