Biology Reference
In-Depth Information
of TSP.
59
Here, TSP exhibited chemical shift vari-
ability due to differences in magnetic suscepti-
bility across the tissue section.
application and general robustness.
63
The
increase in sensitivity of cryogenic probes has
the unwanted consequence of an increase in the
already very large water signal, making water
suppression on a cryogenic probe, especially at
higher magnetic
External Standard
D
2
O (needed for lock) and the reference stan-
dard can be kept separated from precious metab-
olomics samples using two coaxial tubes. The
same coaxial tube can be used for all samples.
In this case, TSP or DSS can be used with no
problems at all. The NMR supply vendor New
Era has a microtube capillary system for limited
volume samples. Baratin et al. introduced elec-
tronic referencing as an alternative chemical shift
referencing technique in which an arti
cult due to
a phenomenon called radiation damping. Radia-
tion damping has been known since the 1950s
and is discussed in detail by Mao and Ye.
64,65
The most widely used 1D experiment is the
on-resonance saturation of the water peak with
a weak RF
field, more dif
rst
increment of the 2D NOESY (nuclear Over-
hauser effect spectroscopy) pulse sequence.
66,67
A spectrum of human urine acquired using this
method is shown in
Figure 1
A with identi
field in conjunction with the
cially
generated signal is employed for referencing
and assessing metabolite concentrations in vivo.
This procedure has been applied to 1D and 2D
liquid-state NMR spectroscopy.
60,61
A modi
ca-
tion of some endogenous metabolites. The water
signal is selectively irradiated twice during the
experiment, once during the equilibrium period
and again during the mixing time. Protons
undergoing rapid intermolecular exchange
with water, for example NH
2
, OH, and COOH
groups, are also attenuated in the process, which
is particularly advantageous for suppressing the
large broad signal from the NH
2
group of urea in
urine samples. The phase cycling of the RF
pulses in the NOESY pulse sequence also remove
contributions from water that are outside the
detector coil region which usually appear as
a broad hump. The 90
pulse width is dependent
on salt concentration. There are three 90
RF
pulses in the NOESY sequence; therefore, care
should be taken to optimize the 90
ed
version of the technique to account for changes
in the dielectric properties in different samples
was proposed by Mehr et al.
62
The hardware
and software for this technique has been incor-
porated into current NMR spectrometer systems.
ONE-DIMENSIONAL NMR
METHODS:
1
H,
13
C,
31
P
1
H
NMR active nuclei commonly used to study
biological samples include
1
H,
13
C,
15
N, and
31
P. One-dimensional (1D)
1
H NMR spectros-
copy is the most popular choice due to its high
prevalence in metabolites, high natural abun-
dance (99.985%), and high sensitivity (i.e., high
gyromagnetic ratio). The two main obstacles in
1D
1
H data acquisition of biological
flip angle.
Three other recent variations of the water pre-
saturation technique are (1) FLIPSY (FLIP-angle
adjustable one-dimensional NOESY) experi-
ment,
68,69
(2) pre-SAT180,
70
and (3) incorpora-
tion of the Carr-Purcell-Meiboom-Gill (CPMG)
spin-echo pulse train into the NOESY-presat
experiment.
36
Biological samples containing macromole-
cules can be studied directly without the need
for their physical removal. The signal contribu-
tion from protein and other high molecular
weight components are removed by two spectral
fluids are
(1) suppression of the very large water signal
(
55 M) and (2) the presence of protein signals
if not already removed at the sample preparation
stage. Numerous water suppression techniques
have been proposed, but only a few are widely
used in metabolic pro
w
ling due to their ease of
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