Biology Reference
In-Depth Information
global metabolic pro
le of the mouse urine in the
study. It should be noted that full-resolution 2D
spectra are required primarily at the discovery
stage. Well-established NMR strategies already
exists for selective excitation if the metabolites,
and therefore the peaks of interests, are known
ahead of time. Ludwig et al. used Hadamard
encoding
94
to acquire pseudo-2D TOCSY spectra
in 10 to 20 minutes on human serum samples for
a colorectal cancer study.
95
Thirty-one frequen-
cies were selected to be used in the Hadamard
encoding scheme.
quantitative analysis of HSQC spectral data.
HSQC spectral data, even with an internal stan-
dard, cannot be used directly for the determina-
tion of molar concentration because signal
intensities are not linearly proportional to
concentration, due to factors such as differential
magnetization transfer for different
1
J
CH
values,
nonuniform RF pulse excitation, and nonuniform
relaxation. Analogous to how calibration curves
are used in mass spectroscopy, quantitation in
2D HSQC can be performed using separate cali-
bration curves for peaks of interest.
99,100
Rai
et al. proposed a mathematical solution in which
concentration is determined by multiplying peak
volume with a theoretically derived correction
factor based on J-coupling, relaxation, and
experimental parameters.
101
The most recent
development is a scheme for absolute concentra-
tion determination of individual metabolite in
a mixture using three HSQC experiments consec-
utively acquired with different repetition
times.
102,103
Data analysis was performed using
the semiautomated Newton-FMLR software
package to extrapolate a time-zero HSQC
spectrum whose peak volumes are linearly
proportional to concentration.
104
The software
Heteronuclear 2D:
1
H-
13
C HSQC
The most active area of development in multi-
dimensional experiments for metabolic pro
ling
is the
1
H-X heteronuclear single quantum coher-
ence (HSQC) experiment in which X can be
13
C,
15
N,
31
P, or other NMR active nuclei.
96
In the
experiment, proton magnetization is transferred
to the X nucleus and then back again to proton
for data acquisition to improve sensitivity.
Therefore, only X nuclei with attached protons
are detected.
The chemical shift range of protonated
carbons is about 150 ppm, making the
1
H-
13
C
HSQC the most attractive choice to resolve peaks
in the second dimension. To reduce data acquisi-
tion time, Hyberts et al. developed a forward
maximum-entropy reconstruction algorithm to
reconstruct HSQC data acquired in 12.7 hours
with nonlinear data sampling to resemble
ultrahigh-resolution HSQC collected in 3.7 days
on cell extracts.
97
To speed up data analysis,
McKenzie et al. developed an automatic peak
s
spectral deconvolution routine is applicable to
other 1D and 2D spectra.
'
TARGETED METABOLIC
PROFILING
Targeted Analysis: Stable Isotope
Tagging
Chemical derivatization with
13
C,
15
N, and
31
P
isotopic tags for select metabolite classes to
increase detection sensitivity has been explored
by Raftery and coworkers.
105
e
107
The identi
fitting routine based on a reference spectrum
created to contain all peaks seen in the HSQC
spectra for the entire sample set.
98
The mathe-
matical model for the peaks is then used to
cluster them into groups (bins) and then for-
warded to multivariate analysis. This nontar-
geted approach allows for an initial nonbiased,
qualitative analysis of the HSQC data. In
contrast, other groups are working on more
ca-
tion of the derivatized metabolites is aided by
standards derivatized under identical conditions.
The chemical shifts for 62
15
N and 32
13
C tagged
metabolites are available at
http://www.chem
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