Systems Biology (Methods in Molecular Biology)

3.2 Sample
Preparation for NMR
Spectroscopy

1. Dissolve each sample in 1 ml of 10 mM D 2 O phosphate-
   buffered saline solution at pH¼7.4.


2. Homogenize samples by using a vortex mixer for 1 min.


3. Centrifuge samples at 14,000gfor 10 min at 20C to obtain
   fecal water. After centrifugation, transfer 600μl of each result-
   ing supernatant into a 5 mm NMR tube.

3.3 Acquisition,
Processing,
and Postprocessing
of NMR Data

3.3.1 NMR Setup

1. Set temperature to 298 K.


2. Properly position a representative sample inside the probe and
   leave 5 min to equilibrate the sample temperature.


3. For obtaining a good signal-to-noise ratio: adjust the probe-
   head tuning and matching; lock and shim the sample on D 2 O;
   calibrate the 90pulse length; determine the power, length,
   and frequency offset for HDO signal suppression by using the
   presaturation pulse.


4. Once an optimal signal is obtained, transfer the setting para-
   meters to the other samples (seeNotes 2–4).

3.3.2 Two-Dimensional
2D^1 H J-Resolved

For the analysis of fecal water samples, J-resolved pulse sequence is

used to observe resonances better, as they are partially or

completely buried in a typical 1D spectrum. This sequence

improves the quality of the metabolic information extracted.

1. Acquire 2D^1 H J-resolved (JRES) NMR spectra using a double
   spin echo sequence [9],suppressing the residual water signal
   with the presaturation technique.


2. Use the following parameters to acquire the JRES spectra:
   transients per increment, 16; total increments, 32; dummy
   scans, 16; data points, 16k; spectral width for direct (F2 or
   chemical shift) dimension, 6 kHz; spectral width for indirect
   (F1 or J-coupling) dimension, 40 Hz; relaxation delay, 2 s.


3. Processing the NMR data carrying out the following opera-
   tions: zero-fill the F1 data to 256 data points; multiply each
   Free Induction Decay (FID) with a combined sine-bell/
   exponential function in the F2 dimension and a sine-bell func-
   tion in the F1 dimension; apply Fourier Transform to each
   dimension; tilt the spectra by 45; symmetrize the spectra
   about F1 dimension; calibrate chemical shifts to the TSP
   methyl protons at 0.00 ppm; apply a zero-order baseline cor-
   rection of spectrum.


4. Exporting the proton-decoupled skyline projections (p-JRES)
   in a suitable format (arrange the exported 1D-skyline projec-
   tions into a matrix of N samples (rows) by M variables (col-
   umns)) for subsequent postprocessing treatment.

330 Luca Casadei et al.