Quantification of Metabolites from Two-Dimensional Nuclear Magnetic Resonance Spectroscopy: Application to Human Urine Samples

Abstract

We present a general scheme for metabolite quantification from a two-dimensional (2D) (1)H-(13)C heteronuclear single quantum correlation (HSQC) nuclear magnetic resonance (NMR) experiment of body fluids observed in natural abundance. The scheme of quantification from 2D HSQC spectra consists of measurement of relaxation parameters of proton resonances, such as T(1) and T(2) of the metabolites and (1)H-(13)C heteronuclear J-coupling for accurate quantification. The measured cross-peak volume from 2D HSQC NMR spectra is multiplied by a calculated correction factor (which depends upon two-dimensional NMR experimental parameters and relaxation parameters) to measure accurate quantification of the metabolite. The correction factor is theoretically derived from the solution of the Bloch equation and product operator formalism. The accuracy of the scheme is tested on a solution containing a mixture of amino acids of known concentration. For human urine samples, the accuracy of the method for measuring the concentration of various metabolites was tested with spike-in experiments. The scheme is general in nature and can be applied to any other body fluid samples for metabonomic studies. We also test the measured cross-peak volume of various metabolites from 2D (1)H-(13)C HSQC NMR spectra of human urine samples for clustering analysis with scatter plots, making the scheme complete for metabolic profiling.

Intrinsically disordered chromatin protein NUPR1 binds to the C-terminal region of Polycomb RING1B

“…We think that those variations are due to error of the measurements in the spectra, even though we corrected the measured values by the figure obtained when setting the receiving gain of the spectrometer. It is also well known that the measurement of intensities in 2D HSQC NMR spectra is plagued with several difficulties (42), and even the viscosity of the medium (which is altered by the presence of other molecules) can affect the value measured (43), even though at the total protein concentrations used in this work (∼500 μM) we have not observed broadening in the spectra of isolated NUPR1 (34) or C-RING1B (17). The facts that (i) broadening is only observed when titration is carried out (i.e., when the corresponding protein partner is added) and (ii) broadening of the signals of C-RING1B in the HSQC spectra follows a different pattern when the mutants are titrating (with molecular weight similar to that of the wild type, discussed below) suggest that the binding between the two proteins is mainly responsible for the broadening of signals.…”

Quantitative NMR Studies of Multiple Compound Mixtures

“…Though the signal attenuation factor can be theoretically calculated [15], it strictly requires that all related resonance-specific parameters are known, for example, chemical shift, which could affect the off-resonance effect, J-couplings, which could affect the INEPT transfer efficiency, related bond lengths and interatom space distance together with CSA tensor, which affect the relaxation rate by dipole-dipole interaction or by the CSA. More demanding are the sample conditions, such as the viscosity and temperature of the sample, and knowing how they affect the molecular rotational tumbling time and thus also the relaxation rate.…”

“…(e) Alternatively, chemometric analytic approach can be used to resolve multiple compound complex mixtures through decomposition of trilinear DOSY diffusion data [13]. (f ) 2D NMR spectra of mixtures could contain higher proportion of resolved peaks, but the signals are usually more difficult to quantify because of resonance-specific signal attenuation during the coherence transfer periods as the result of relaxation, imperfect pulses, and mismatch of the insensitive nuclei enhanced by polarization transfer (INEPT) delay with specific J-couplings [14,15]. The signal attenuation factor can be theoretically calculated provided all related parameters are known for each specific compound [15], or be experimentally determined [16].…”

“…(f ) 2D NMR spectra of mixtures could contain higher proportion of resolved peaks, but the signals are usually more difficult to quantify because of resonance-specific signal attenuation during the coherence transfer periods as the result of relaxation, imperfect pulses, and mismatch of the insensitive nuclei enhanced by polarization transfer (INEPT) delay with specific J-couplings [14,15]. The signal attenuation factor can be theoretically calculated provided all related parameters are known for each specific compound [15], or be experimentally determined [16]. The signal intensities in 2D NMR spectra can also be related to the quantity of the compound of interest in the mixtures by reference to a standard curve constructed using concentration reference mixtures of synthetic compounds [3].…”

Metabolic Profiling for the Detection of Bladder Cancer

“…At the initial discovery stage, investigators increasingly have started to invest in more time to collect either twodimensional (2D) homonuclear ( 1 H-1 H) or heteronuclear ( 1 H-13 C) NMR experiments for better metabolome coverage [40,41]. Once a panel of potential metabolites has been identified, validation on a larger sample set can be performed in a much faster manner using Hadamard encoding schemes for the 2D NMR experiments, which would reduce the experimental time to that comparable to a 1D 1 H experiment [42•].…”