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Updated: Mar 28, 2026
2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
Published on: July 10, 2020
A mass spectrometry-based method for direct determination of pseudouridine in RNA
Yoshio Yamauchi1, Yuko Nobe1, Keiichi Izumikawa2
1Department of Chemistry, Graduate School of Science and Engineering, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji-shi, Tokyo 192-0397, Japan Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan.
Abstract:
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 quantity of RNA, it will serve as a useful tool for the structure/function studies of a wide variety of non-coding RNAs.
