Mass spectrometry remains an important method for analysis of modified nucleosides ubiquitously present in cellular RNAs, in particular for ribosomal and transfer RNAs that play crucial roles in mRNA translation and decoding. Furthermore, modifications have effect on the lifetimes of nucleic acids in plasma and cells and are consequently incorporated into RNA therapeutics. To provide an analytical tool for sequence characterization of modified RNAs, we developed Pytheas, an open-source software package for automated analysis of tandem MS data for RNA. This dataset contains the analysis of 14N and 15N-labeled 16S RNA from E. coli, including all the known RNA modifications (excluding pseudouridines). The analysis has been performed using three different protocols and instruments: Agilent Q-TOF, Waters Synapt G2-S, and Thermo Scientific Orbitrap Fusion Lumos.