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. 2015 Dec 17;44(2):509–523. doi: 10.1093/nar/gkv1470

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© The Author(s) 2015. 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-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 2. — Mass spectrometric analysis of individual tRNAs isolated from stationary-phase E. coli. (A) Mass chromatograms of RNase T₁-digested fragments containing cmo⁵U and its derivatives from tRNA^Ala1 (left panels) and tRNA^Ser1 (right panels) isolated from stationary-phase E. coli. Top and middle panels: extracted-ion chromatograms (XIC) for doubly-charged negative ions of cmo⁵U34-containing fragments (CUUcmo⁵UGp of tRNA^Ala1, MW 1660.18, m/z 829.08; UCmUcmo⁵UGp of tRNA^Ser1, MW 1674.19, m/z 836.09) and mcmo⁵U34-containing fragments (CUUmcmo⁵UGp of tRNA^Ala1, MW 1674.19, m/z 836.09; UCmUmcmo⁵UGp of tRNA^Ser1, MW 1688.21, m/z 843.10), respectively. Bottom left and bottom right panels: XICs for doubly-charged negative ions of Um32/mcmo⁵U34-containing fragment of tRNA^Ala1 (CUmUmcmo⁵UGp, MW 1688.21, m/z 843.10) and mcmo⁵Um34-containing fragment of tRNA^Ser1 (UCmUmcmo⁵UmGp, MW 1702.22, m/z 850.10), respectively. The peaks marked with asterisks represent Um32/cmo⁵U-containing fragment (CUmUcmo⁵UGp, MW 1674.19, m/z 836.09) in tRNA^Ala1 and cmo⁵Um-containing fragment (UCmUcmo⁵UmGp, MW 1688.21, m/z 843.10) in tRNA^Ser1. The RNA fragments containing unmodified C32 are also present in tRNA^Ser1, but they are not described here due to high frequency of Cm32 in tRNA^Ser1 isolated from stationary-phase E. coli. (B) Modification frequencies of cmo⁵U and its derivatives at position 34 in six species of tRNAs isolated from stationary-phase E. coli. Relative composition of each modification was calculated from the peak area ratio of mass chromatograms of RNase T₁-digested fragments containing mcmo⁵Um34 (red), mcmo⁵U34 (green), cmo⁵U34 (blue) or U (gray). (C) Collision-induced dissociation (CID) spectrum of a fragment of E. coli tRNA^Ser1 containing mcmo⁵Um34. The doubly-charged negative ion of the RNase T₁-digested fragment containing mcmo⁵Um34 (m/z 850.10) was used as the precursor ion for CID. The product ions were assigned as described previously (66). Sequences of parent ion and assigned product ions are described on the upper left side of this panel. (D) Nucleoside analysis of E. coli tRNA^Ser1. Top panel: UV chromatogram at 254 nm of the four major nucleosides (C, U, G and A). Second panel: mass chromatograms of the protonated nucleosides (MH⁺) on the base peak chromatogram (BPC). Third to bottom panels: XICs for cmo⁵U (m/z 319.08), mcmo⁵U (m/z 333.09) and mcmo⁵Um (m/z 347.11), respectively. The peak marked with an asterisk represents unspecific peak. (E) CID spectrum of mcmo⁵Um nucleoside. The protonated mcmo⁵Um (MH⁺, m/z 347.11) was used as the precursor ion for CID. The N-glycoside bond cleaved to generate the base-related ion (BH₂⁺) and other product ions are assigned on the chemical structure.