Fine optimization of a dissolution dynamic nuclear polarization experimental setting for ¹³C NMR of metabolic samples

Abstract

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.

Quantitative NMR spectroscopy of complex mixtures

“…5% on complex metabolite mixtures), 99 and a thorough optimization of the experimental workflow has been carried out to improve its sensitivity and resolution, making it compatible with metabolomics. 100 A first application has been reported to an untargeted metabolomics study on model plant extracts at natural abundance (Fig. 6).…”

Hyperpolarized 13C NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization

“…While the performance of d‐DNP has been shown for the analysis of relatively concentrated plant extracts, the analysis of biofluids constitutes a more challenging target, both in terms of concentration and matrix complexity. Recently, we reported a multi‐parametric optimization of the d‐DNP experimental workflow [15] that paves the way towards such application, by allowing a significant increase in sensitivity and resolution. In particular, this systematic optimization focused on sample preparation ( i.e .…”

“…d‐DNP experiments were carried out with an optimized semi‐automated d‐DNP workflow [15] . Samples were polarized at 1.2 K with frequency‐modulated microwave irradiation using gated cross‐polarization [13] .…”

“…radical concentration and DNP juice composition) and dissolution parameters ( i.e . dissolution duration, choice of dissolution solvent and stabilisation delay) to ensure i) a high and robust hyperpolarization in the solid‐state and ii) intense, narrow and repeatable 13 C signals in the liquid‐state [15] …”

“…d-DNP experiments were carried out with an optimized semi-automated d-DNP workflow. [15] Samples were polarized at 1.2 K with frequency-modulated microwave irradiation using gated cross-polarization. [13] After 20 min of polarization, 5 mL of hot solvent (D 2 O or CD 3 OD) were used to rapidly dissolve the sample and bring it to the nearby liquid-state spectrometer through a transfer line equipped with a magnetic tunnel.…”