Pytheas: A Software to Map RNA Modifications via Tandem Mass Spectrometry

Abstract

Ribonucleic acids (RNA) such as ribosomal RNA (rRNA) and messenger RNA (mRNA) are decorated at the post-transcriptional level by more than 150 characterized chemical modifications. These modifications range from positional isomerization to the addition of complex chemical groups to nucleosides. In the last decades, RNA modifications have been shown to enhance RNAs structural diversity and their intramolecular interactions, as well as promoting RNA diverse functions within complex regulatory networks involved in cellular metabolism. To characterize and quantify RNA modifications, tandem Mass Spectrometry (MS/MS) has been proven to be a powerful and flexible analytical tool. We present here a novel software pipeline, Pytheas, to assist in the interpretation of MS/MS data with the goal to characterize modified RNAs. Through an interactive multi-step process, Pytheas generates a theoretical database from arbitrary RNA sequences and the user-defined parameters such as digestion enzymes, nucleotide chemical compositions and isotopic labeling. Then it uses an empirical scoring function derived from the SEQUEST proteomics platform to score measured MS/MS spectra vs the theoretical library, adopting the target-decoy approach for the false discovery rate (FDR) estimation. The final steps allow visualization of the scored spectra and map the modifications on the RNA sequence. Pytheas is freely available, easy to use and highly customizable; at any step the user can set parameters relevant for their experiments, such as type and number of modifications or instrument-based uncertainty windows on m/z measurements. The applicability of Pytheas has been tested using the Liquid Chromatography MS/MS datasets acquired on different instruments, and was successful in mapping known and discover novel modifications present in tRNAs and B. subtilis 16S and 23S RNA.