Simultaneous acquisition of three NMR spectra in a single experiment for rapid resonance assignments in metabolomics

Abstract

NMR-based approach to metabolomics typically involves the collection of two-dimensional (2D) heteronuclear correlation spectra for identification and assignment of metabolites. In case of spectral overlap, a 3D spectrum becomes necessary, which is hampered by slow data acquisition for achieving sufficient resolution. We describe here a method to simultaneously acquire three spectra (one 3D and two 2D) in a single data set, which is based on a combination of different fast data acquisition techniques such as G-matrix Fourier transform (GFT) NMR spectroscopy, parallel data acquisition and non-uniform sampling. The following spectra are acquired simultaneously: (1) 13 C multiplicity edited GFT (3,2)D HSQC-TOCSY, (2) 2D [ 1 H-1 H] TOCSY and (3) 2D [ 13 C-1 H] HETCOR. The spectra are obtained at high resolution and provide high-dimensional spectral information for resolving ambiguities. While the GFT spectrum has been shown previously to provide good resolution, the editing of spin systems based on their CH multiplicities further resolves the ambiguities for resonance assignments. The experiment is demonstrated on a mixture of 21 metabolites commonly observed in metabolomics. The spectra were acquired at natural abundance of 13 C. This is the first application of a combination of three fast NMR methods for small molecules and opens up new avenues for high-throughput approaches for NMR-based metabolomics.

Rapid nuclear magnetic resonance data acquisition with improved resolution and sensitivity for high‐throughput metabolomic analysis

“…These approaches can be classified as experiments that use reduced sampling approaches such as nonuniform sampling (NUS), [31][32][33][34][35][36][37] projection NMR, [29,30] Hadamard NMR, [53][54][55] and phase-modulated (PM) NMR [38] and those based on fast-pulsing techniques [56][57][58][59] such as fast heteronuclear multiple quantum coherence (HMQC). In addition to these two categories, another type of sampling approach involves multiple receivers [29,30] and ultrafast single-scan NMR techniques. [33,[39][40][41][42][43][44][45][46][47][48][49][50][51][52] This is summarized in Table 1.…”

“…In metabolomics, parallel NMR data acquisition can be used for recording different 2D experiments required for resonance assignments simultaneously. [29,30,74] For instance, the most widely used 2D NMR experiments in metabolomics are as follows: 2D [ 1 H, 1 H] TOCSY, 2D [ 13 C, 1 H] HSQC, and 2D [ 13 C, 1 H] HSQC-TOCSY. These three spectra can be conceptualized as three orthogonal projections (2D planes) of 3D [ 13 C, 1 H] HSQC-TOCSY ( Figure 2).…”

“…[100,101] Two or more fast NMR methods can be combined to get better results in a short duration. [29,30,[70][71][72][102][103][104][105][106][107] Multireceiver experiments are made more efficient by combining with other fast NMR techniques such as Hadamard NMR, [72,102] NUS, [72,103] projection NMR, [29,30,72] or ultrafast NMR techniques. [71,72] Various combinations of these experiments are possible; hence, establishing the optimum combination of fast NMR technique for addressing various challenges in metabolomics and in biomolecular NMR remains an underexplored area.…”

“…After the first INEPT, the protons attached to 12 C are frequency labeled during t 1 , and then subjected to isotropic mixing for observing the 2D [ 1 H, 1 H] TOCSY correlations during t 2 . [ 29,30,74 ] The experiments are implemented in a manner such that the FIDs from each of the three different experiments are acquired simultaneously and stored separately. The spectra obtained are termed as “dual receiver (DR)” spectra.…”

An efficient combination of BEST and NUS methods in multidimensional NMR spectroscopy for high throughput analysis of proteins

“…13 In such situations, the required multidimensional NMR measurements have to be recorded within the life span of the native state of the protein under study. In this context, several strategies have been proposed: Filter Diagonalisation Method (FDM), 14 Reduced Dimensionality (GFT 15,16 and APSY 17 ), Non-Uniform Sampling (NUS), [18][19][20][21][22] projection reconstruction, 23 Shaped Arrayed data acquisition protocol (SHARC NMR), 24 Hadamard NMR, 25 Ultrafast, 26 Covariance NMR, 27 multiple-receiver techniques, [28][29][30][31][32] longitudinal relaxation optimization, 33,34 cooling overall spin temperature (COST), 35 HSQC-based multi-dimensional out-and-back experiments, 36 Band-Selective Optimized-Flip-Angle-Short-Transient (SOFAST), 37 and Band-selective Excitation Short-Transient (BEST) 38 methods, which help to reduce the nD-NMR experimental times to a signicant extent. Atreya et al 34 have demonstrated the importance of combined implementation of two fast NMR techniques (longitudinal relaxation optimization 33 and GFT 15 ) for obtaining the chemical shi information in short instrumental times.…”