INTRODUCTION: RNA modifications play essential roles in ribosome biogenesis, translational fidelity, and cellular stress responses. Next-generation sequencing has revolutionized our understanding of the transcriptome. However, most post-transcriptional RNA modifications remain invisible to conventional sequencing approaches due to their low abundance and chemical diversity. Here, we present a mass spectrometry-based approach for mapping RNA modifications using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), enabling targeted, site-specific detection of modified nucleosides within an RNA of interest.
METHODS: Total RNA was extracted from human K562 cells and was purified using standard approaches. Biotinylated DNA/RNA hybrid oligonucleotides were designed to anneal to regions of interest and were hybridised to the RNA by slow cooling. Annealed DNA-RNA duplexes were digested with RNase H, and the target fragments were isolated using streptavidin beads. Purified fragments were analyzed by LC-MS/MS with the aid of ion-pairing reagents on an Orbitrap Ascend Tribrid Mass Spectrometer (Thermo Scientific), desinged to characterise large and highly charged macromolecules. The resulting mass spectra were manually inspected and further annotated using BiopharmaFinder software.
RESULTS: Optimization of hybrid oligo design achieved greater than 90% RNase H-cleavage efficiency at the targeted RNA sites. Using human ribosomal RNA as a proof-of-concept substrate, we successfully enriched and isolated regions of interest from both 18S and 28S rRNA. Within these enriched fragments, we identified a methylation modification on 18S rRNA (U627), demonstrating the capacity to localize specific modifications with high confidence. We found that the workflow required only 10 µg of total RNA as starting material, with the potential for further scale-down. While RNA methylation was used here as a proof-of-concept modification, this targeted enrichment strategy is broadly applicable and can be extended to detect other RNA modification types, offering a modular platform for site-specific epitranscriptomic analysis.