Biology Reference
In-Depth Information
editing methods: (1) relaxation-based editing
based on the CPMG spin-echo pulse train to
selectively retain signals from small molecules,
as in
Figure 1
B (middle), and (2) diffusion-
based gradient
for the effects of B
1
inhomogeneity, pulse miscali-
bration, and frequency offsets.
77
The use of
composite adiabatic pulses in the pulse sequence
ensures uniform RF excitation and makes the
experiment highly amendable for high
filter editing experiments, which
can selectively retain either small or large mole-
cules based on experimental parameter settings,
as in
Figure 1
B (bottom).
71
e
73
The CPMG spin-
echo pulse train, (
eld
work.
78
Another 1D carbon pulse sequence, the
Z-restored spin-echo, developed by Xia et al.,
corrects the often seen severe baseline distortions
in carbon spectra collected on cryogenic probes.
79
The spectral distortions are caused by the long
preacquisition delay (
-180
-
)
n
, exploits the differ-
ential T
2
relaxation rate of large molecules such
as proteins and small molecule metabolites.
Note that signals from metabolites bound to
protein will also be lost. A third strategy to
remove NMR peaks from macromolecules after
NMR data collection is by a mathematical trans-
formation developed by Maher et al. called
virtual relaxation edited spectroscopy (RESY).
74
This method may be useful for the analyses of
archived NMR spectra of whole serum and
plasma.
s
s
20
m
s) to allow time for
RF pulse ring-down in the coil. These two pulse
sequence improvements will allow researchers
to better exploit 1D
13
C spectra at natural abun-
dance for metabolite analysis.
w
31
P
Phosphorous-31 is 100% abundant and is very
useful for studying phospholipids and metabo-
lites involved in energy metabolism. Examples
of 1D
31
P spectroscopy include studies using
cell and tissue extracts and biological
13
C
The carbon chemical shifts of small molecules
is spread over 200 ppm. However, the high spec-
tral dispersion is offset by
13
C low natural abun-
dance (1.11%) and low gyromagnetic ratio
resulting in poor sensitivity overall. To over-
come these shortcomings, Keun et al. used a cryo-
genic probe speci
uids.
80
e
82
Compared to
13
C,
31
P is not as widely used, and
many studies focus on choline phospholipid
metabolism in cancer cells. Gabellieri et al. used
31
P to monitor choline kinase inhibition in cancer
cells by following the phosphorylation rates of
phosphocholine, ATP, ADP, AMP, and Pi.
83
cally optimized for direct
carbon detection to show the feasibility of col-
lecting 1D
13
C spectra of rat urine in 30 minutes
per sample with results comparable to 17 hours
on a conventional room temperature probe opti-
mized for proton detection.
75
Other dif
2D METHODS
ling studies use
2DNMR experiments collected on select samples
exclusively for metabolite con
The majority of metabolic pro
rmation or identi-
culties with carbon detection
include themuchwider range of T
1
(longitudinal)
and T
2
(transverse) relaxation times, which
require a long equilibrium delay to allow the
carbon magnetization to return towards equilib-
rium. Piotto et al. improved the original DEFT
(driven equilibrium Fourier transform)
76
pulse
sequence to reduce data acquisition time by
returning the carbon magnetization back to equi-
librium after each scan while also compensating
fication of statistically signi
cant peaks. Global
metabolic pro
ling of biological samples using
multidimensional NMR experiments suffers
from the lack of fast data acquisition techniques
like those developed for protein NMR spectros-
copy. These methods are not easily translated to
metabolomics due to the high dynamic range of
peak intensities and lack of stable isotope enrich-
ment. Similarly, advancements in post
e
data
Search WWH ::
Custom Search