Chemical Amination/Imination of Carbonothiolated Nucleosides During RNA Hydrolysis

Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has become the gold‐standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state‐of‐the‐art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non‐natural mcm5isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher‐energy collisional dissociation (HCD)‐based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium‐buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications. Discovery of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium‐buffered mild basic conditions is described. The general approach for discovery uses higher‐energy collisional dissociation‐mass spectrometry (HCD‐MS) and isotope labeling of RNA.