Detection of the Common RNA Nucleoside Pseudouridine in Mixtures of Oligonucleotides by Mass Spectrometry

Abstract

Pseudouridine, an isomer of uridine, is probably the most common of many posttranscriptional RNA modifications found in nature. Although mass spectrometry has become widely used in the characterization of modified nucleic acids, its application to the recognition and sequence placement of pseudouridine has not been straightforward, particularly in the case of complex mixtures such as those resulting from selective enzymatic hydrolysis of RNA into oligonucleotides. We report results of a study of the characteristic dissociation reactions of pseudouridine-containing oligonucleotides following ionization by electrospray and use of those pathways in an LC/MS-based method applicable to direct analysis of RNase digests of RNA. As a consequence of the C−C (rather than C−N) glycosidic bond of pseudouridine, the otherwise common dissociation paths involving base loss do not occur, resulting in characteristic formation of a set of low-mass negative ions containing the intact glycosidic bond (m/z 225, 207, 189, 165, 164, 139), which permit recognition of pseudouridine-containing oligonucleotides. Those components can subsequently be subjected to sequence analysis by MS/MS, in which enhancement of selective sequence-determining ions (a-, w-, y-types), and absence of a − base ions, are observed at the site of pseudouridylation. Also, selected reaction pathways can be monitored in the LC/MS/MS analysis that are indicative of pseudouridine at the 5‘ terminus (m/z 225 → 165), internal positions (m/z 207 → 164), and in the RNase T1-derived product ΨpGp (m/z 668 → 207) arising from the RNA sequence ...GΨG... These procedures can be effectively integrated into an existing suite of LC/ESI-MS-based methods designed for the analysis of posttranscriptionally modified sites in RNA.