Development of Universal Mass Exclusion List (UMEL) for RNA modification mapping

Understanding the location of modified nucleosides in RNA sequences is crucial to understanding their biochemical significance. Mapping the sequence location of modified nucleosides from low-abundance RNAs is challenging. Here, we report the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS) exclusion list strategy that enhances sequence information from modified oligonucleotides. This approach, compatible with standard RNA modification mapping methods that utilize LC-MS/MS, enables the exclusion of any unmodified oligonucleotide from fragmentation during MS/MS, thereby enabling enhanced dissociation of modified oligonucleotides. This universal exclusion list is applicable to natural RNAs of any type from any organism. We find that this approach generates at least 10% more mapped RNase T1 digestion products than using DDA alone. To demonstrate the broad utility of this approach for discovery-based analyses, RNA modification mapping of total tRNAs from four distinct organisms spanning both prokaryotic and eukaryotic domains was conducted.