Title: High information throughput analysis of nucleotides and their isotopically enriched
isotopologues by direct-infusion FTICR-MS.
Pawel Lorkiewicz 1, Richard M. Higashi 1,2,3, Andrew N. Lane 2,3, Teresa W-M. Fan 1,2,3.
1. Department of Chemistry, University of Louisville, 2210 S. Brook St, Rm. 348 John W.
Shumaker Research Building, Louisville, KY 40292, USA
2. Center for Regulatory Environmental Analytical Metabolomics, 2210 S. Brook St., Louisville,
KY 40292, USA
3. JG Brown Cancer Center, Clinical Translational Research Building, 505 S. Hancock St.,
Louisville, KY 40202, USA
Correspondence to: Teresa W-M. Fan
Department of Chemistry, University of Louisville,
2210 S. Brook St, Rm. 348 John W. Shumaker Research
Building, Louisville, KY 40292, USA
twmfan@gmail.com
Tel: 502-852-6448
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Fig. S1 Effect of C18 tip processing on MS peak intensity. Intensities of monoisotopic (m0)
peaks in the negative ion mode FTICR mass spectra of a 2.5 µm mixture of standards were
measured with and without processing with C18 tip. The intensities were normalized to the ATP
m0 peak. Error bars represent standard error of the mean associated with each averaged value
(n=3).
Before
Processing
After C-18
processing
140
120
% ATP
100
80
60
40
20
Fig. S2 Effect of MSA on
Hex-PP
Ac-CoA
GDP-Man
UDP-GNAc
UDP-Glc
UMP
UDP
UTP
NADPH
NADP+
NADH
NAD
GMP
GTP
CDP
CMP
CTP
cAMP
AMP
ADP
0
13
C-isotopologue distributions of nucleotides in polar extract of A549
cells grown in [U-13C]-glucose. A549 cells were grown in [U-13C]-glucose with or without MSA,
extracted, processed, and analyzed as in Figure 3. The Spectral regions shown correspond to
ATP untreated (panel A) and MSA treated (panel B); UTP/CTP untreated (panel C) and MSA
treated (panel D). Also shown is the spectral comparison of the ribose and glucose region of the
1-D 1H-{13C} HSQC NMR data for the same untreated and MSA treated extracts (E). The two
spectra were normalized by spectral parameters and cell protein weight such that the intensity
of the
C-attached 1H signals was directly comparable. Four 1’-ribosyl signals, one for adenine
13
nucleotides (13C-1’-AXP), two for NAD+, and one for the uracil-containing nucleotides were
assigned along with the assignment of two 1-glucosyl signals of UDPG (13C-G1-UDPG) and
UDP-GlcNAc (cf. (Fan and Lane, 2008))
2
80
ATP 13C2
Relative Abundance (%)
(x10)
60 ATP 12C
ATP 13C1
40
A
ATP 13C5
100
(x10)
(x10)
60
ATP 13C3
C
80
ATP 13C4
(x10)
ATP 13C6
ATP 13C8
40
(x10)
ATP 13C7
20
UTP 13C5
100
20
100
UTP 13C2
CTP 12C
CTP 13C5
(x10)
UTP 12C
UTP 13C3
(x10)
UTP
13C
UTP 13C8
6
UTP (x10)
13C
7
100
B
80
D
80
60
60
40
40
20
20
UTP 13C1
(x10)
0
0
505 506 507 508 509 510 511 512 513 514 515 516 480 481 482 483 484 485 486 487 488 489 490 491 492
m/z
m/z
E
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Table S1. The accuracy and precision for the determination of isotopologue ratios of
nucleotides at natural abundance in A549 cell extracts.
The ion counts of m0 and m0+1 peaks from three separate analyses of a crude A549 cell extract
were measured to determine the analytical mean and standard deviation (SD) (precision) of the
ratio m0+1/m0. For each measurement, three highest intensity stored and transformed spectra
(each representing 5 transients) with 18-25 ms of AGC-mediated ion sampling time were
summed. The accuracy was assessed as the % deviation of the average to the theoretical value
for a natural abundance of 1.07% (Rosman and Taylor, 1997). Except for ATP, the ratio of
m0+2/m0 was too low to measure reliably. For ATP, the measured ratio of m0+2/m0 was 0.43%
versus 0.53% for the theoretical value, and the larger error is attributed presumably to the low
peak intensity.
Nucleotide
% Intensity
SD/mean
m0+1/m0
% Deviation
from theory
ATP
10.56 ± 0.13
0.0123
2.4
GTP
10.80±0.34
0.031
0.2
CTP
9.83±0.35
0.036
1.0
UTP
9.76±0.34
0.035
0.31
References
Fan, T.W.-M. and Lane, A.N. (2008) Structure-based profiling of Metabolites and Isotopomers
by NMR. Progress in NMR Spectroscopy 52, 69-117
Rosman, K. and Taylor, P. (1997) Isotopic Compositions Of The Elements IUPAC.
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