Top-Down Tandem Mass Spectrometry of tRNA Via Ion Trap Collision-Induced Dissociation

Abstract

Transfer RNA is a class of highly modified and structured non-coding RNA molecules generally comprised of 74-95 nucleotides. In this study, tandem mass spectrometry of intact multiply charged tRNA anions of roughly 25 kDa in mass has been demonstrated using a quadrupole/time-of-flight tandem mass spectrometer adapted for ion/ion reaction studies. The sample proved to be a mixture of tRNA molecules. The mass of the most abundant component of the mixture was not consistent with that of the nominal identity of the tRNA from the supplier, viz., tRNA(Phe); rather, the mass was consistent with tRNA(Phe) bearing an incomplete 3'-terminus. Multiply-charged anions from the major components were isolated in the gas phase and subjected to ion trap collision-induced dissociation without subsequent ion/ion reactions. Abundant fragments from the 5'- and 3'-termini of the molecule could be used to identify the major component as tRNA(Phe)-3'adenosine (without 3'-phosphorylation). Roughly 15% of the primary sequence of the intact tRNA was unambiguously reflected in the product ion spectrum. The existence of a possible tRNA(Phe) variant and the intact tRNA(Phe) was also supported by ion trap CID data. The multiply-charged fragment ions derived from tRNA(Phe)-3'adenosine were further charge-reduced to mostly singly- and doubly-charged species via proton transfer ion/ion reactions with benzoquinoline cations. The resulting reduction in spectral overlap and charge state ambiguity simplified interpretation of the product ion spectrum and allowed for the identification of product ions from roughly 60% of the sequence.