Biology Reference
In-Depth Information
that it can be used as a reference. A major
improvement to peak identi
are spread along two dimensions to enable both
unambiguous identi
cation and accu-
rate quantitative analysis of metabolites using
1D NMR spectra is provided by Chenomx
Inc.
101,102
Metabolite identi
cation and accurate quan-
titation of an enhanced pool of metabolites.
Various 2D NMR methods that provide comple-
mentary information on metabolite identity
and metabolite concentrations are used. 2D
J-resolved spectroscopy, correlation spectros-
copy (COSY), TOCSY, heteronuclear single
quantum-coherence (HSQC) spectroscopy,
and heteronuclear multiple-bond correlation
(HMQC) spectroscopy experiments are the
most commonly used. Projections of 2D
J-resolved spectra are much simpler as each
multiplet that arises due to spin
e
spin couplings
(J-coupling) is collapsed to a single peak.
104,105
However, care should be exercised in quanti-
tating metabolites using this experiment since,
as for the 1D CPMG experiment, peak integrals
are affected by transverse relaxation during the
long evolution period. The 2D HSQC experi-
ment is particularly attractive due to its much
higher resolving power, which arises from the
wider chemical shift ranges of heteronuclei
such as
13
C and
15
N. Lower sensitivity due to
the low gyromagnetic ratios of
13
C and
15
N,
combined with their low natural abundances
is, however, a major challenge that limits wider
utility of such 2D experiments. Nevertheless,
the latest advances in NMR instrumentation,
including the development of
cation is facilitated
by modeling of the spectral features using
spectra of pure compounds from pH and spec-
trometer frequency
e
dependent chemical shift
databases. Accurate metabolite concentrations
are then obtained by comparing modeled
metabolite peak integrals with that of an
internal reference compound such as DSS. This
method allows identi
cation of peaks, even if
they are shifted due to altered conditions, such
as pH, and provides accurate concentration
values that can then be inputted for further
multivariate statistical analysis. The approach
is semiautomated and needs manual selection
of appropriate metabolites from the library
and manual feedback in the
fitting of peaks.
More recently, a method that performs auto-
mated spectral deconvolution of 1D NMR
spectra was presented.
80
It uses an algorithm,
AutoFit, to reconstruct experimental spectra using
a reference compound library. The algorithmopti-
mizes a number of parameters, including a recali-
bration of reference spectral library based on
position, intensity, and the peak line width from
a reference compound such as DSS, from the
experimental spectrum. An alternative approach,
based on a Bayesian
eld
magnets, cryogenic and microcoil probes, and
isotope labeling approaches, are contributing
to the increased utility of these experiments
for metabolomics applications.
A protocol for the determination of metabo-
lite concentrations was evaluated using the
1
H-
13
C HSQC spectrum.
106
Here, the 2D peak
integrals are compared with calibration curves
obtained from 2D spectra of three equimolar
mixtures of standard compounds to predict
the metabolite concentrations. Improving on
this quantitation strategy, an algorithm to
deconvolute 2D NMR peaks was developed.
107
It constructs simple time domain models with
the fewest number of peaks, whose frequency
strong
fitting algorithm, was
recently proposed.
103
Although this approach is
currently limited to small spectral regions due to
its computational requirements,
it operates in
a fully automated mode.
Metabolite Quantitation Using 2D NMR
In 1D
1
H NMR, the relatively narrow spectral
region in which all metabolite signals are
observed results in peak overlap and limits the
number of metabolites that can be identi
ed
and quanti
ed accurately. This problem can be
greatly alleviated by making use of two-
dimensional (2D) NMR spectra, in which peaks
Search WWH ::
Custom Search