A method for direct mass spectrometry–based identification of mono-methylated RNA nucleoside positional isomers and its application to the analysis of Leishmania ribosomal RNA

RNA post-transcriptional modifications are common in all kingdoms of life, and are predominantly affiliated with methylations at various nucleobase positions. Methylations occur frequently at specific sites on the RNA nucleobases and appear to regulate site-specific intermolecular/intramolecular interactions. Herein, we present a method that utilizes liquid chromatography-mass spectrometry (LC-MS) to identify positional mono-methylated RNA nucleoside isomers. The method produces profiles of in-source fragmentation and subsequent MS2 (pseudo-MS3) of RNase-digested fragments of an RNA, and distinguishes between positional methylated nucleobase isomers by comparing their intra-nucleobase fragment ion profiles with signature profiles derived from authentic isomers. For method validation, we independently determined the positions of all known mono-methylated nucleoside isomers in the Escherichia coli 18S/23S rRNAs. As proof of concept, we further applied this technology to fully characterize the base-modified ...