Biomedical Engineering Reference
In-Depth Information
condition,
one
should
take
great
care
of
large
one-bond
homonuclear
couplings.
Although carbonyl carbons (C9) suffer from fast relaxation due to their large
CSA, carbonyl direct detection has been the preferred choice as this nucleus is
coupled only to the alpha carbon (C
a
)(
1
J
C9Ca
5 55 Hz),
25-29
and C9 and C
a
have discrete chemical shifts. Several methods have been proposed to remove
1
J
C9Ca
from the direct C9-detection experiments by acquisition methods, and/or
while processing spectra. After acquisition, the spectral complexity canbe
'virtually' removed by computational deconvolution of the spectra, usinga
maximum-entropy algorithm.
25,26
Server et al. compared 3D HCACO
experiments with and without deconvolution and reported an increase in the
S/N of almost the full theoretical factor of 2. The post-acquisition super-
position of the negative and positive components of the 2C
a
z
C9
y
antiphase
doublet is another option.
29
In this method, a linear combination of the
original spectrum with the absolute value representation of the same spectrum
is calculated and, in theory, a factor of
2
p
gain in the S/N can be expected. In
addition, by omitting the refocusing period that converts the 2C
a
z
C9
y
anti-
phase into in-phase, unwanted signal losses during this period can be
avoided.
29
Indeed, a two-fold improvement in the sensitivity of the shorter
anti-phase pulse sequence compared to the longer refocused in-phase
experiment was observed for a 25 kDa protein, and the improvement is
expected to increase with increasing molecular weight.
39
In the crowded region
of the spectrum, however, loss of information by signal overlap will lead toa
smaller gain in sensitivity in the deconvoluted spectrum. There is also an
experiment called COCAINE (CO-CA in- and anti-phase spectra with
sensitivity enhancement), which selects one of the two doublet components
in a way similar to
1
H
15
N TROSY HSQC.
65
Selecting a single component within the split peaks using in-phase/anti-phase
(IPAP) or Spin-State-Selective Excitation (S
3
E) schemes are other elegant
solutions for removing the
1
J
C9Ca
coupling and are widely used.
66,67
In the
IPAP scheme, the removal of the splitting is accomplished by recording anti-
phase as well as in-phase components for each increment.
27
The sum and
difference of these two components is calculated, shifted to the centre of the
original multiplet, and summed up again to obtain a singlet with two-fold
intensity. However, the IPAP scheme costs at least a duration of 1/(2
1
J
C9Ca
)
y9 ms, to ensure that anti-phase coherences are completely be converted into
in-phase. The S
3
E scheme, on the other hand, yields the same sensitivity gain as
IPAP but using a shorter building block with a duration of 1/(4
1
J
C9Ca
) y4.5
ms. Therefore, it is easy to expect that the S
3
E scheme may have better
sensitivity than IPAP in a large system and/or in high magnetic fields. One
should mention that, in cases where the coupling to a
15
N nucleus needs to be
refocused, the S
3
E element will not shorten the refocusing delay, since the
smaller J
CN
couplings dominate the total delay length. A suite of pulse
sequences that use IPAP and/or S
3
E building blocks were established mainly
by the Bertini and Bermel groups.
31,43
This proton-less series of experiments
