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. 2014 May 15;42(11):7346–7357. doi: 10.1093/nar/gku390

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© The Author(s) 2014. 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 License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — Mass spectrometric analysis of bovine mt tRNA^Ala for assignment of post-transcriptional modifications. (A) Secondary structure of bovine mitochondrial tRNA^Ala with post-transcriptional modifications determined in this study. The position numbers of the modifications are displayed according to the nucleotide numbering system from the tRNA compilation (40). Symbols for modified nucleosides are as follows: m¹A, 1-methyladenosine; m²G, N²-methylguanosine and Ψ, pseudouridine. Watson–Crick base pairs are indicated by solid lines, whereas G–U pairs are indicated by asterisks. (B) Nucleoside analysis of bovine mitochondrial tRNA^Ala. Left, top panel: UV chromatogram at 254 nm of the four major nucleosides (C, U, G and A). Left, lower panels: extracted-ion chromatograms (XIC) for the protonated ion of m¹A nucleoside (m/z 282, black line) with its base ion (m/z 150, gray line) (second panel), m²G nucleoside (m/z 298, black line) with its base ion (m/z 166, gray line) (third panel) and Ψ nucleoside (m/z 245, black line) (bottom panel). The XIC for the base ion (20% upper offset) is overlaid on the XIC for the nucleoside ion. Right: mass spectra of m¹A and m²G. Cleavage positions for the base-related ions are indicated on the chemical structures. (C) RNA fragment analysis of RNase T₁ digests of bovine mitochondrial tRNA^Ala. Assigned fragments are indicated on the base peak chromatogram (BPC) in the first panel. ‘p’ stands for the terminal phosphate group. The XIC for the doubly charged negative ion of a modification-containing fragment (AUUUm¹Am²Gp, m/z 982.6) is indicated in the second panel. Because m²G at position 10 is a partial modification, both AUUUm¹Am²Gp and AUUUm¹AGp were detected. (D) RNA fragment analysis of RNase A digests of bovine mitochondrial tRNA^Ala. Assigned fragments are indicated on the BPC. The XIC for the doubly charged negative ion of the 5′-terminal fragment (pGAGGAUp, m/z 1047.6) is indicated in the second panel. (E) A CID spectrum of a cyanoethylated RNA fragment to determine the location of a Ψ site. The doubly charged negative ion of the RNA fragment (m/z 1617.7) shown in the inset was used as the precursor ion for CID. The product ions were assigned according to McLuckey et al. (41). The asterisks in the spectrum denote product ions containing ce¹Ψ. (F) Whole mass analysis of intact bovine mt tRNA^Ala. A series of multiply charged negative ions is shown in the mass spectrum. The charge values are indicated in parentheses. The observed mass obtained by deconvoluting the mass spectra is shown in the inset.

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