Characterization of Post-Transcriptional RNA Modifications by Sheathless Capillary Electrophoresis–High Resolution Mass Spectrometry

Over the past decade there has been a growing interest in RNA modification analysis. High performance liquid chromatography–tandem mass spectrometry coupling (HPLC–MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we propose a novel and simple workflow based on capillary zone electrophoresis–tandem mass spectrometry (CE–MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. By first totally digesting the purified RNA, prior to CE–MS/MS analysis, we were able to identify the nucleoside modifications. Then, using a bottom-up approach, sequencing of the RNAs and mapping of the modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.