Permethylation of ribonucleosides provides enhanced mass spectrometry quantification of post-transcriptional modifications

Abstract

Abstract Chemical modifications of RNA are associated with fundamental biological processes such as RNA splicing, export, translation, degradation, as well as human disease states such as cancer. However, the analysis of ribonucleoside modifications is impeded due to the hydrophilicity of the ribonucleoside molecules. In this research, we used solid-phase permethylation to derivatize the ribonucleosides, and the permethylated ribonucleosides, which were then quantitively analyzed using a liquid chromatography−tandem mass spectrometry (LC−MS/MS)-based method. The solid-phase permethylation efficiently derivatized the ribonucleosides, and more than 60 RNA modifications were simultaneously monitored using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) performed in the dynamic multiple reaction monitoring (dMRM) mode. Because of the increased hydrophobicity of permethylated ribonucleosides, this method enhanced retention, separation, and ionization efficiency, resulting in improved detection and quantification when compared to existing analytical strategies of RNA modifications. We applied this new approach to measure the extent of cytosine methylation and hydroxymethylation in RNA obtained from mouse embryonic stem cells with genetic deficiencies in ten-eleven translocation (TET) enzymes. The results matched previously performed analyses and highlighted the sensitivity, efficacy, and robustness of the new method. The advantage of this method enables comprehensive analysis of RNA modifications in biological samples. Abstract Figure