Ion-Pair–Free Nanoflow HILIC–MS With RNase Benchmarking for Native RNA

RNA modifications play crucial roles in regulating cellular processes, but comprehensive mapping of the human RNome still remains limited by technological challenges. Mass spectrometry (MS) is a valuable tool to analyse RNA modifications complementing sequencing-based analysis. Current MS-based oligonucleotide workflows have limited sensitivity, requiring micrograms of RNA inputs and thus hindering studies on native RNAs. Additionally, environmentally toxic ion-pairing reagents are often required. Here, we report a highly sensitive, broadly applicable oligonucleotide-MS workflow that enables analysis of nanogram-scale RNA hydrolysates and we benchmark the substrate specificity of three nucleases: RNase T1, RNase 4, and colicin E5. We developed a nano-flow hydrophilic interaction liquid chromatography (HILIC) setup compatible with common MS buffers and coupled this with high-resolution MS. Using modified NucleicAcidSearchEngine (NASE), we confidently assigned RNA hydrolysates with diverse 3’-end chemistries. Furthermore, we demonstrate that RNase 4 and colicin E5 efficiently cleave modified RNAs including pseudouridine-containing transcripts, enabling high sequence coverages. Using this workflow, we successfully mapped modifications in 25 ng of native yeast tRNA ^Phe and verified the sequence of 250 ng of a synthetic mRNA. Overall, our method provides a sensitive, high-resolution platform for oligonucleotide mass spectrometry, facilitating comprehensive analysis of RNA modifications and advancing efforts toward complete epitranscriptomic mapping.