Expanding the epitranscriptomic RNA sequencing and modification mapping mass spectrometry toolbox with field asymmetric waveform ion mobility and electrochemical elution liquid chromatography

Abstract

AbstractPost-transcriptional modifications of RNA strongly influence RNA structure and function. Recent advances in RNA sequencing and mass spectrometry (MS) methods have identified over 140 of these modifications on a wide variety of RNA species. Most next-generation sequencing approaches can only map one RNA modification at a time, and while MS can assign multiple modifications simultaneously in an unbiased manner, MS cannot accurately catalog and assign RNA modifications in complex biological samples due to limitations in fragment length and coverage depth. Thus, a facile method to identify novel RNA modifications while simultaneously locating them in the context of their RNA sequences is still lacking. We combined two orthogonal modes of RNA ion separation before mass-spectrometry identification: high-field asymmetric ion mobility separation (FAIMS) and electrochemically modulated liquid chromatography (EMLC). FAIMS RNA-MS increases both coverage and throughput, while the EMLC LC-MS orthogonally separates RNA of different length and charge. The combination of the two methods offers a broadly applicable platform to improve length and depth of MS-based RNA sequencing while providing contextual access to the analysis of RNA modifications.