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Supplemental Data and Information for:
Mass Spectrometry Sequencing of Transfer Ribonucleic Acids by the Comparative Analysis
of RNA Digests (CARD) Approach
Siwei Li and Patrick A. Limbach*
Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, PO Box 210172,
University of Cincinnati, Cincinnati, OH 45221-0172
*To whom correspondence should be addressed.
Phone (513) 556-1871
Fax (513) 556-9239
email [email protected]
Supplemental Table S1.
Basic Local Alignment Search Tool (BLAST) results for searching 14
randomly chosen E. coli tRNA genes against the C. koseri genome. The value reported is the
%sequence homology between the E. coli gene and that found in C. koseri.
E. coli query sequence:
tRNA: Alanine 1 (Ala1) [A]
tRNA: Cysteine (Cys) [C]
tRNA: Glutamic Acid 1 (Glu1) [E]
tRNA: Phenylalanine (Phe) [F]
tRNA: Glycine 1 (Gly1) [G]
tRNA: Histidine (His) [H]
tRNA: Isoleucine 1 (Ile1) [I]
tRNA: Leucine 1 (Leu1) [L]
tRNA: Methionine (Met) [M]
tRNA: Glutamine 1 (Gln1) [Q]
tRNA: Arginine 1 (Arg1) [R]
tRNA: Serine 1 (Ser1) [S]
tRNA: Threonine 1 (Thr1) [T]
tRNA: Valine 1 (Val1) [V]
tRNA ID
DA1660
DC1660
DE1660
DF1660
DG1660
DH1660
DI1660
DL1660
DM1660
DQ1660
DR1660
DS1664
DT1660
DV1662
E. coli
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
100%
C. koseri
100%
98%
100%
100%
100%
100%
100%
100%
100%
100%
98%
100%
97%
100%
Supplemental Table S2.
Excel file containing tRNA sequences for E. coli obtained from the
tRNAdb 2009 database (http://trnadb.bioinf.uni-leipzig.de/)1 aligned with the appropriate C. koseri
tRNA genes obtained from the Genomic tRNA Database (http://gtrnadb.ucsc.edu/)2 or from
BLAST searches against the C. koseri genome (NC_009792.1).3 Aligned sequences are then
compared as described in the text to yield anticipated singlets and doublets during comparative
sequencing.
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Supplemental Table S3.
Excel file containing tRNA sequences for E. coli obtained from the
tRNAdb 2009 database (http://trnadb.bioinf.uni-leipzig.de/)1 aligned with the appropriate S. enterica
tRNA genes obtained from the Genomic tRNA Database (http://gtrnadb.ucsc.edu/).2 Aligned
sequences are then compared as described in the text to yield anticipated singlets and doublets
during comparative sequencing.
Supplemental Figure S1.
Extracted ion chromatograms corresponding to the anticipated
doubly- and triply-charged ions of AC[s2C]U[mnm5U]CU[t6A]AGp, a singlet from tRNA-Arg(UCU),
at 5 µg and 20 µg of E. coli total tRNA after RNase T1 digestion loaded on column. The XIC
response at 31.5 min corresponds to a doubly-charged ion consistent with a base composition of
C2U2A2Gp + methyl, which is not the anticipated singlet.
Supplemental Figure S2.
Mass spectrum corresponding to the anticipated singlet [DUGp]
from C. koseri tRNA-Asp(GUC). A number of interfering ions, likely originating from the ubiquitous
digestion products CCGp (m/z 972.1), (CU)Gp (m/z 973.1), and UUGp (m/z 974.1), are present
and can interfere with detection of the DUGp singlet.
Supplemental Figure S3.
Mass spectra corresponding to the anticipated singlet CAAAGp (m/z
827.5, 2- charge) when (a) C. koseri is labeled with 18O and (b) E. coli is labeled with 18O. Although
the isotopic envelope shifts by -1 m/z unit when C. koseri is labeled with 16O, interfering RNase T1
digestion products hinder unequivocal assignment of this singlet.
Supplemental Figure S4.
(a) Collision-induced dissociation mass spectrum of the singlet
4
U[s U]AACAAAGp from E. coli total tRNAs. (b) Collision-induced dissociation mass spectrum of
the doublet [m7G]UCCCCAGp from tRNA-Thr(GGU).
Supplemental Figure S5.
Calculated codon usage frequencies for E. coli, C. koseri and T.
thermophilus. Comparative sequencing is more effective when the codon usage frequencies of the
reference and candidate organisms are similar.
References
1.
2.
3.
F. Juhling, M. Morl, R. K. Hartmann, M. Sprinzl, P. F. Stadler and J. Putz, Nucleic Acids Res,
2009, 37, D159-162.
P. P. Chan and T. M. Lowe, Nucleic Acids Res, 2009, 37, D93-97.
S. F. Altschul, W. Gish, W. Miller, E. W. Myers and D. J. Lipman, J Mol Biol, 1990, 215, 403410.
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TIC
5 µg tRNA Digest
XIC m/z 1692.7
XIC m/z 1128.1
TIC
20 µg tRNA Digest
XIC m/z 1692.7
XIC m/z 1128.1
Mass spectrum at 31.6 min
Supplemental Figure S1. Extracted ion chromatograms corresponding to the anticipated
doubly- and triply-charged ions of AC[s2C]U[mnm5U]CU[t6A]AGp, a singlet from tRNAArg(UCU), at 5 µg and 20 µg of E. coli total tRNA after RNase T1 digestion loaded on
column. The XIC response at 31.5 min corresponds to a doubly-charged ion consistent with
a base composition of C2U2A2Gp + methyl, which is not the anticipated singlet.
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CCGp - DDGp
Supplemental Figure S2. Mass spectrum corresponding to the anticipated singlet [DUGp]
from C. koseri tRNA-Asp(GUC). A number of interfering ions, likely originating from the
ubiquitous digestion products CCGp (m/z 972.1), (CU)Gp (m/z 973.1), and UUGp (m/z
974.1), are present and can interfere with detection of the DUGp singlet.
Electronic Supplementary Material (ESI) for Analyst
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a)
b)
E. coli (16O)/
C. koseri (18O)
C. koseri (16O)/
E. coli (18O)
Supplemental Figure S3. Mass spectra corresponding to the anticipated singlet CAAAGp
(m/z 827.5, 2- charge) when (a) C. koseri is labeled with 18O and (b) E. coli is labeled with
18O. Although the isotopic envelope shifts by -1 m/z unit when C. koseri is labeled with 16O,
interfering RNase T1 digestion products hinder unequivocal assignment of this singlet.
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c82-­‐
[M-­‐Ade]2-­‐
c2 c3 c4 c5 c6 c7 c8
(a)
U [s4U] A A C A A A Gp
y7 y6 y5 y4 y3 y2
y8
y82-­‐
c4
c2
y2
c3
y3
(b)
c72-­‐
y72-­‐
c6
c5
y6
y5
y4
y72-­‐
[M-­‐B]2-­‐
c2 c3 c4 c5 c6 c7
[m7G] U C C C C A Gp
y7 y6 y5 y4 y3 y2
c4-­‐BH
B=m7G y2
c2-­‐BH
[c7-­‐BH]2-­‐
y3
c3-­‐BH
y5
y4 c -­‐BH
5
c6-­‐BH
y6
Supplemental Figure S4. (a) Collision-­‐induced dissocia.on mass spectrum of the singlet U[s4U]AACAAAGp from E. coli total tRNAs. (b) Collision-­‐induced dissocia.on mass spectrum of the doublet [m7G]UCCCCAGp. Electronic Supplementary Material (ESI) for Analyst
This journal is © The Royal Society of Chemistry 2013
Supplemental Figure S5. Calculated codon usage frequencies for E. coli, C. koseri and T.
thermophilus. Comparative sequencing is more effective when the codon usage frequencies
of the reference and candidate organisms are similar.