Initial peak assignments relied on established
literature values, specifically, the human metabolome database,17 (link) the biological magnetic resonance data bank,18 (link) and publications from our laboratory on the
serum metabolome.7 (link),19 (link) Unknown metabolite identification
involved a combination of literature/database searches,17 (link) chemical shift, peak multiplicity, and J couplings measurements, and comprehensive 2D DQF-COSY
and TOCSY spectral analyses. The putative new compounds were finally
confirmed by spiking with authentic compounds (seeSI Table S1). Chenomx NMR Suite Professional Software package
(version 5.1; Chenomx Inc., Edmonton, Alberta, Canada) was used to
quantitate the metabolites. This software allows fitting spectral
lines using the standard metabolite library for 800 MHz 1H NMR spectra and, in particular, the determination of concentrations
in complicated, overlapped spectral regions. One complication that
arises is that the proximity of chemical shift values for multiple
metabolite signals often result in the software providing multiple
library hits for the same metabolite peak; the correct metabolite
identification therefore relied on the newly established metabolite
identification as annotated for a typical 1H NMR spectrum
(vide infra). Peak-fitting with reference to the internal TSP signal
enabled the determination of absolute concentrations for identified
metabolites in protein-precipitated serum except for 2-oxoisovaleric,
which was absent in the Chenomx library and was therefore quantitated
by manual integration using the Bruker Topspin versions 3.0 or 3.1
software package.
literature values, specifically, the human metabolome database,17 (link) the biological magnetic resonance data bank,18 (link) and publications from our laboratory on the
serum metabolome.7 (link),19 (link) Unknown metabolite identification
involved a combination of literature/database searches,17 (link) chemical shift, peak multiplicity, and J couplings measurements, and comprehensive 2D DQF-COSY
and TOCSY spectral analyses. The putative new compounds were finally
confirmed by spiking with authentic compounds (see
(version 5.1; Chenomx Inc., Edmonton, Alberta, Canada) was used to
quantitate the metabolites. This software allows fitting spectral
lines using the standard metabolite library for 800 MHz 1H NMR spectra and, in particular, the determination of concentrations
in complicated, overlapped spectral regions. One complication that
arises is that the proximity of chemical shift values for multiple
metabolite signals often result in the software providing multiple
library hits for the same metabolite peak; the correct metabolite
identification therefore relied on the newly established metabolite
identification as annotated for a typical 1H NMR spectrum
(vide infra). Peak-fitting with reference to the internal TSP signal
enabled the determination of absolute concentrations for identified
metabolites in protein-precipitated serum except for 2-oxoisovaleric,
which was absent in the Chenomx library and was therefore quantitated
by manual integration using the Bruker Topspin versions 3.0 or 3.1
software package.
