Hyperpolarized NMR Metabolomics at Natural 13C Abundance

Abstract

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 ¹H 1D spectroscopy, severely limited by peak overlap. ¹³C 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 ¹³C 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 ¹³C metabolomics. Statistical analysis of resulting hyperpolarized ¹³C 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.

Figure 1

Stacked plot of d-DNP-enhanced single-scan ¹³C NMR spectra of 16 tomato samples. At right, labels “a” to “h” refer to spectra of red–ripe fruit extracts, and “i” to “p” refer to mature–green fruit extracts. Selections from 76–82, 94–110, and 174–190 ppm are shown to focus on spectrally populated regions (full spectrum in Figure S7). The sample for the blank control spectrum (top) has signals only from Na-TSP-d₄ (188.6 ppm) and EDTA·Na₂·2H₂O (182.9 ppm).

Figure 2

(a) Scores plot and (b) loadings plot of the principal component analysis (PCA) obtained from 40 spectral buckets from hyperpolarized ¹³C spectra of 16 tomato fruit extracts. Integrals were normalized to Na-TSP-d₄ as an internal reference and to the weight of the sample used for extraction. Mean centering and unit variance scaling were used in PCA. Correspondence of bucket numbers to spectral regions is given in Figure S3.

Figure 3

Coefficient of variation (CV (%)) of d-DNP enhanced ¹³C signal integrals (based on five successive experiments) for a standard metabolite mixture (pyruvate, acetate, alanine) at natural abundance using three experimental protocols (A, B, C). Solution-state spectra were detected with a 90° flip angle, after a postdissolution delay that ensured an optimum signal-to-noise ratio (S/N) for each case A, B, and C. CV at “A” was obtained using ethanol-washed NMR tubes, yielding optimum S/N at 22.8 s after dissolution. CV at “B” was obtained using NMR tubes additionally treated with Hellmanex (procedure in SI), yielding S/N optimum at 12.8 s after dissolution. CV at “C” was achieved under similar conditions as “B” but normalizing all signal integrals using the Na-TSP-d₄ internal reference as described in the text.