A nanogram-sensitive workflow for oligonucleotide mass spectrometry using ion-pair-free nanoflow HILIC and RNase benchmarking.

RNA modifications regulate diverse cellular processes, yet comprehensive characterization of modified RNA sequences remains technically challenging. Mass spectrometry provides direct chemical information on RNA, but current oligonucleotide-based workflows typically require micrograms of RNA input and often rely on ion-pairing reagents for chromatographic separation, limiting their applicability to scarce or native RNA samples. Here, we establish a sensitive oligonucleotide mass spectrometry workflow that combines ion-pair-free nanoflow hydrophilic interaction liquid chromatography with systematic benchmarking of controlled RNA cleavage strategies. We compared RNase T1, RNase 4, and colicin E5 and evaluated how reaction conditions influence cleavage specificity, fragment length distribution, and terminal chemistries of RNA hydrolysates. The resulting workflow enables robust LC-MS/MS analysis using standard MS-compatible buffers and supports confident oligonucleotide identification through NucleicAcidSearchEngine (NASE) database searching. Using this approach, we achieved high sequence coverage from nanogram-scale RNA inputs, enabling modification analysis of 25-50 ng native yeast tRNAPhe and sequence verification of 250 ng synthetic mRNA. Together, this work establishes a sensitive and broadly applicable platform for oligonucleotide mass spectrometry and provides practical guidance for RNase selection and digestion strategies. The method expands the applicability of RNA MS to low-input samples and supports future studies of RNA sequence and modification landscapes.