Attomol-level quantification of chemically modified ribonucleosides enabled by capillary porous graphitic carbon columns in nano LC-MS

Abstract

Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited the development of methods for reliable detection and accurate quantification of these modifications. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials as stationary phases for reversed-phase separation of ribonucleosides and found that both PGC matrices have excellent retention and separation characteristics, as well as the capability to separate structural isomers. Using PGC matrices in nLC-MS yielded excellent signal-to-noise ratios in a detection range spanning up to six orders of magnitude, allowing for the analysis of individual ribonucleosides down to attomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary PGC columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of chemically modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.