MALDI-TOF and MALDI-TOF/TOF mass spectrometry as a method to test RNA as substrate of RNA modifying enzymes

摘要

First, we showed that the 29-mer RNA corresponding to the acceptor steem and the T seem-loop of P. abyssi tRNA~(asp) is substrate of both _(Pab)Trml and _(Tth)Trml, as demonstrated by the ion peaks at m/z 1326.15 and 1340.17 in the MS spectrum of the 29-mer RNA digestion, incubated with _(Pab)Trml, and by the ion peak at m/z 1326.07 in the MS spectrum of the 29-mer RNA digestion, incubated with _(Tth)Trml. MSMS experiment indicates that _(Pab)Trml methylates both adenines 57 and 58, whereas ~(Tth)Trml methylates only A58. Then, we determined that neither _(Pab)Trml nor _(Tth)Trml methylate the 2-aminopurine containing mini-RNA on the 2-aminopurine base. This knowledge will allow us to differentiate the methyltransferase step from the other catalytic events, so that we can assign the rates for the RNA binding step and base flipping event by stopped-flow kinetics.