Nucleoside Analysis by Hydrophilic Interaction Liquid Chromatography Coupled with Mass Spectrometry

RNA molecules contain a wide variety of chemical modifications that cannot be deduced from the genomic sequence. RNA modifications confer a chemical diversity to simple RNA molecules, enabling a greater variety of biological functions. To detect RNA modifications, highly sensitive analytical tools are required. Liquid chromatography/mass spectrometry (LC/MS) has been playing a vital role in analyzing minor modified nucleosides in RNA specimens from various sources. Reverse-phase chromatography (RPC) has been used for LC/MS for a long time because RPC is compatible with electrospray ionization (ESI) MS. However, RPC is not always suitable for detecting hydrophilic or polar nucleosides. We here describe a different mode of LC/MS for detecting RNA modifications using hydrophilic interaction liquid chromatography (HILIC). HILIC/ESI-MS is a valuable alternative for profiling modified nucleosides.