posted on 2021-09-21, 17:07authored byIvan Vuckovic, Aleksandar Denic, M. Cristine Charlesworth, Milovan Šuvakov, Shane Bobart, John C. Lieske, Fernando C. Fervenza, Slobodan Macura
We
described several postprocessing methods to measure protein
concentrations in human urine from existing 1H nuclear
magnetic resonance (NMR) metabolomic spectra: (1) direct spectral
integration, (2) integration of NCD spectra (NCD = 1D NOESY-CPMG),
(3) integration of SMolESY-filtered 1D NOESY spectra (SMolESY = Small
Molecule Enhancement SpectroscopY), (4) matching protein patterns,
and (5) TSP line integral and TSP linewidth. Postprocessing consists
of (a) removal of the metabolite signals (demetabolization) and (b)
extraction of the protein integral from the demetabolized spectra.
For demetabolization, we tested subtraction of the spin-echo 1D spectrum
(CPMG) from the regular 1D spectrum and low-pass filtering of 1D NOESY
by its derivatives (c-SMolESY). Because of imperfections in the demetabolization,
in addition to direct integration, we extracted protein integrals
by the piecewise comparison of demetabolized spectra with the reference
spectrum of albumin. We analyzed 42 urine samples with protein content
known from the bicinchoninic acid (BCA) assay. We found excellent
correlation between the BCA assay and the demetabolized NMR integrals.
We have provided conversion factors for calculating protein concentrations
in mg/mL from spectral integrals in mM. Additionally, we found the
trimethylsilyl propionate (TSP, NMR standard) spectral linewidth and
the TSP integral to be good indicators of protein concentration. The
described methods increase the information content of urine NMR metabolomics
spectra by informing clinical studies of protein concentration.