Identification, localization, and relative quantitation of pseudouridine in RNA by tandem mass spectrometry of hydrolysis products

The constitutional isomers uridine (U) and pseudouridine (Ψ) cannot be distinguished from each other by simple mass measurements of RNA or its fragments because the conversion of U into Ψ is a “mass-silent” post-transcriptional modification. Here we propose a new mass spectrometry based method for identification, localization, and relative quantitation of Ψ in RNA consisting of ∼20 nucleotides that does not require chemical labeling. Our approach takes advantage of the different fragmentation behavior of uridine (N-glycosidic bond) and pseudouridine (C-glycosidic bond) residues in RNA upon collisionally activated dissociation.