biostudies-literatureUnknown44(6)Yamauchi YPseudouridine (5-ribosyluracil, Ψ) is the only 'mass-silent' nucleoside produced by post-transcriptional RNA modification. We describe here a novel mass spectrometry (MS)-based method for direct determination of Ψ in RNA. The method assigns a Ψ-containing nucleolytic RNA fragment by an accurate measurement of a signature doubly dehydrated nucleoside anion ([C9H7N2O4](1-),m/z207.04) produced by collision-induced dissociation MS, and it determines the Ψ-containing nucleotide sequence by pseudo-MS(3), i.e. in-source fragmentation followed by MS(2) By applying this method, we identified all of the known Ψs in the canonical human spliceosomal snRNAs and, unexpectedly, found two previously unknown Ψs in the U5 and U6 snRNAs. Because the method allows direct determination of Ψ in a subpicomole quNucleic acids researche59https://www.ebi.ac.uk/biostudies/studies/S-EPMC4824092biostudies-literatureA mass spectrometry-based method for direct determination of pseudouridine in RNA.PMC4824092Yamagishi YNakayama HIsobe THigo DTakahashi NYamauchi YNobe YIzumikawa KTaoka MfalseA mass spectrometry-based method for direct determination of pseudouridine in RNA.Pseudouridine (5-ribosyluracil, Ψ) is the only 'mass-silent' nucleoside produced by post-transcriptional RNA modification. We describe here a novel mass spectrometry (MS)-based method for direct determination of Ψ in RNA. The method assigns a Ψ-containing nucleolytic RNA fragment by an accurate measurement of a signature doubly dehydrated nucleoside anion ([C9H7N2O4](1-),m/z207.04) produced by collision-induced dissociation MS, and it determines the Ψ-containing nucleotide sequence by pseudo-MS(3), i.e. in-source fragmentation followed by MS(2) By applying this method, we identified all of the known Ψs in the canonical human spliceosomal snRNAs and, unexpectedly, found two previously unknown Ψs in the U5 and U6 snRNAs. Because the method allows direct determination of Ψ in a subpicomole qu2016-01-01T00:00:00Z2016 Apr2025-04-04T10:28:22.857Z2019-03-27T03:11:25ZS-EPMC48240922667372510.1093/nar/gkv1462