All NMR experiments were performed
at 298 K on a Bruker Avance III 800 MHz spectrometer equipped with
a cryogenically cooled probe and Z-gradients suitable for inverse
detection. A few spiking experiments were performed on a Bruker 700
MHz spectrometer equipped with a room temperature probe and Z-gradients
suitable for inverse detection. The one-pulse or NOESY pulse sequence
along with the CPMG (Carr–Purcell–Meiboom–Gill)
pulse sequence, all with water suppression using presaturation, were
used for 1H 1D NMR experiments. To confirm unknown metabolite
identification, spectra were obtained after each addition of 5–10
μL of stock solution (1 mM) of the authentic compounds to the
methanol-precipitated (2:1 v/v) serum samples (seeSI Table S1); in the case of volatile compounds, such as acetone,
ethanol, 2-butanol, dimethylamine, and urea, spiking experiments were
performed using ultrafiltered serum samples. To enable comparison
of metabolite concentrations from various protein precipitation methods
(SI Table S2), the CPMG experiments were
performed with 128 transients and a sufficiently long recycle delay
(D1 = 15 s). To aid unknown metabolite identification, homonuclear
two-dimensional (2D) experiments, such as 1H–1H double quantum filtered correlation spectroscopy (DQF-COSY)
and 1H–1H total correlation spectroscopy
(TOCSY) experiments, were performed on serum samples after protein
precipitation using methanol (2:1 v/v). The 2D experiments were performed
with suppression of the residual water signal by presaturation during
the relaxation delay. For DQF-COSY and TOCSY experiments, sweep widths
of 9600 Hz were used in both dimensions; 512 or 400 FIDs were obtained
with t1 increments for DQF-COSY or TOCSY,
respectively, each with 2048 complex data points. The number of transients
used was 16, and the relaxation delay was 2.0 s for DQF-COSY and 1.5
s for TOCSY. The resulting 2D data were zero-filled to 1024 points
in the t1 dimension. A 90° shifted
squared sine-bell window function was applied to both dimensions before
Fourier transformation. Chemical shifts were referenced to the internal
TSP signal for 1H 1D or 2D spectra. Bruker Topspin versions
3.0 or 3.1 software packages were used for NMR data acquisition, processing,
and analyses.
at 298 K on a Bruker Avance III 800 MHz spectrometer equipped with
a cryogenically cooled probe and Z-gradients suitable for inverse
detection. A few spiking experiments were performed on a Bruker 700
MHz spectrometer equipped with a room temperature probe and Z-gradients
suitable for inverse detection. The one-pulse or NOESY pulse sequence
along with the CPMG (Carr–Purcell–Meiboom–Gill)
pulse sequence, all with water suppression using presaturation, were
used for 1H 1D NMR experiments. To confirm unknown metabolite
identification, spectra were obtained after each addition of 5–10
μL of stock solution (1 mM) of the authentic compounds to the
methanol-precipitated (2:1 v/v) serum samples (see
ethanol, 2-butanol, dimethylamine, and urea, spiking experiments were
performed using ultrafiltered serum samples. To enable comparison
of metabolite concentrations from various protein precipitation methods
(
performed with 128 transients and a sufficiently long recycle delay
(D1 = 15 s). To aid unknown metabolite identification, homonuclear
two-dimensional (2D) experiments, such as 1H–1H double quantum filtered correlation spectroscopy (DQF-COSY)
and 1H–1H total correlation spectroscopy
(TOCSY) experiments, were performed on serum samples after protein
precipitation using methanol (2:1 v/v). The 2D experiments were performed
with suppression of the residual water signal by presaturation during
the relaxation delay. For DQF-COSY and TOCSY experiments, sweep widths
of 9600 Hz were used in both dimensions; 512 or 400 FIDs were obtained
with t1 increments for DQF-COSY or TOCSY,
respectively, each with 2048 complex data points. The number of transients
used was 16, and the relaxation delay was 2.0 s for DQF-COSY and 1.5
s for TOCSY. The resulting 2D data were zero-filled to 1024 points
in the t1 dimension. A 90° shifted
squared sine-bell window function was applied to both dimensions before
Fourier transformation. Chemical shifts were referenced to the internal
TSP signal for 1H 1D or 2D spectra. Bruker Topspin versions
3.0 or 3.1 software packages were used for NMR data acquisition, processing,
and analyses.