Permethylation of Ribonucleosides Provides Enhanced Mass Spectrometry Quantification of Post-Transcriptional RNA Modifications

Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, and degradation, as well as human disease states, such as cancer. However, the analysis of ribonucleoside modifications is hampered by the hydrophilicity of the ribonucleoside molecules. In this work, we used solid-phase permethylation to first efficiently derivatize the ribonucleosides and quantitatively analyze them by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. We identified and quantified more than 60 RNA modifications simultaneously by ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. The increased hydrophobicity of permethylated ribonucleosides significantly enhanced their retention, separation, and ionization efficiency, leading to improved detection and quantification. We further demonstrate that this novel approach is capable of quantifying cytosine methylation and hydroxymethylation in complex RNA samples obtained from mouse embryonic stem cells with genetic deficiencies in the ten-eleven translocation (TET) enzymes. The results match previously performed analyses and highlight the improved sensitivity, efficacy, and robustness of the new method. Our protocol is quantitative and robust and thus provides an augmented approach for comprehensive analysis of RNA modifications in biological samples.