Metabolomics
plays a pivotal role in systems biology, and NMR is
a central tool with high precision and exceptional resolution of chemical
information. Most NMR metabolomic studies are based on
1
H 1D spectroscopy, severely limited by peak overlap.
13
C NMR benefits from a larger signal dispersion but is barely used
in metabolomics due to ca. 6000-fold lower sensitivity. We introduce
a new approach, based on hyperpolarized
13
C NMR at natural
abundance, that circumvents this limitation. A new untargeted NMR-based
metabolomic workflow based on dissolution dynamic nuclear polarization
(d-DNP) for the first time enabled hyperpolarized natural abundance
13
C metabolomics. Statistical analysis of resulting hyperpolarized
13
C data distinguishes two groups of plant (tomato) extracts
and highlights biomarkers, in full agreement with previous results
on the same biological model. We also optimize parameters of the semiautomated
d-DNP system suitable for high-throughput studies.
Various micelle parameters viz., critical micelle concentration (CMC), counter-ion binding (b), aggregation number (N), hydrodynamic radius (R h ), micelle zeta potential (f) and energetic parameters, free energy of micellization (DG 0 m ), enthalpy of micellization (DH 0 m ) and entropy of micelle formation (DS 0 m ) were determined for sodium dodecylsulfate, and dodecyltrimethylammonium bromide in the presence of NaCl for the former and NaBr for the latter. Conductometry and calorimetry methods were used for the measurements of CMC and energetic parameters. The fluorimetric (static quenching) method was employed to determine N and dynamic light scattering to estimate R h and f. The conductometrically determined b was verified from the CMC values by calorimetry using the Corrin-Harkins equation. The results found for the two surfactants of identical tails but different head groups have been presented and discussed. A detailed report on the salt effect using salts containing counter-ions the same as those in the surfactant is found only limitedly in the literature.
Abstract. NMR-based analysis of metabolite mixtures provides crucial
information on biological systems but mostly relies on 1D 1H
experiments for maximizing sensitivity. However, strong peak overlap of
1H spectra often is a limitation for the analysis of inherently complex biological mixtures. Dissolution dynamic nuclear polarization (d-DNP) improves NMR sensitivity by several orders of magnitude, which enables 13C NMR-based analysis of metabolites at natural abundance. We have recently demonstrated the successful introduction of d-DNP into a full
untargeted metabolomics workflow applied to the study of plant metabolism.
Here we describe the systematic optimization of d-DNP experimental settings
for experiments at natural 13C abundance and show how the resolution,
sensitivity, and ultimately the number of detectable signals improve as a
result. We have systematically optimized the parameters involved (in a
semi-automated prototype d-DNP system, from sample preparation to signal
detection, aiming at providing an optimization guide for potential users of
such a system, who may not be experts in instrumental development). The
optimization procedure makes it possible to detect previously inaccessible
protonated 13C signals of metabolites at natural abundance with at
least 4 times improved line shape and a high repeatability compared to a
previously reported d-DNP-enhanced untargeted metabolomic study. This
extends the application scope of hyperpolarized 13C NMR at natural
abundance and paves the way to a more general use of DNP-hyperpolarized NMR
in metabolomics studies.
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