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. 2020 May 27;48(12):6906–6918. doi: 10.1093/nar/gkaa411

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — LC/ESI-MS characterization of uridine nucleosides from N. equitans and I. hospitalis tRNAs. (A) Analyses of uridines modified with one methyl group and registered at m/z 259.09 in positive ion mode. On left, retention times during liquid chromatography compared to known methyluridine standards. On right, fragmentation of N. equitans compound #4 confirming its structure from comparison with the m⁵U standard. The identities of compound #3 (m¹Ψ) and compound #5 (Um) were verified by the same methods (Supplementary Figure S3). Minor amounts of non-nucleoside compounds can be seen migrating at retention times close to that of m¹Ψ. (B) Uridines modified with a methyl plus a thiol group registered at m/z 275.07 in positive ion mode. Compound #7 is specific to N. equitans and matches the LC profile of the m⁵s²U standard as well as its MS/MS fragmentation pattern (right). No standards with the same LC migration were available for compounds #6 and #8, and these were identified respectively as m¹s⁴Ψ and s²Um by comparing their fragmentation patterns to a series of nucleosides containing these individual modifications (Figures 4 and 5). Small quantities of these compounds were observed in some preparations of the enriched N. equitans cells (as seen here) and are contaminants resulting from incomplete removal of all the I. hospitalis cells.