Single-scan NMR spectroscopy at arbitrary dimensions

Multidimensional nuclear magnetic resonance (NMR) provides one of the foremost analytical tools available to elucidate the structure and dynamics of complex molecules in their native states. Executing this kind of experiment generally requires collecting an n-dimensional time-domain signal S, from which the spectrum arises via an appropriate Fourier analysis of its various time variables. This time-domain signal is actually measured directly only along one of the time axes, while the effects introduced by the remaining time variables are monitored via a parametric incrementation of their values throughout independent experiments. Two-dimensional (2D) NMR experiments thus usually require longer acquisition times than unidimensional experiments, 3D NMR is orders-of-magnitude more time consuming than 2D spectroscopy, etc. Very recently, we proposed and demonstrated an approach whereby data acquisition in 2D NMR can be parallelized, enabling the collection of complete 2D spectral sets within a single transient. The present paper discusses the extension of this 2D NMR methodology to an arbitrary number of dimensions. The principles of the ensuing ultrafast n-dimensional NMR approach are described, and a variety of homo- and heteronuclear 3D and 4D NMR spectra collected within a fraction of a second are presented.     

"Multi-scan single shot" quantitative 2D NMR: a valuable alternative to fast conventional quantitative 2D NMR

Quantitative Ultrafast (UF) 2D NMR is a very promising methodology enabling the acquisition of 2D spectra in a single scan. The analytical performances of UF 2D NMR have been highly increased in the last few years, however little is known about the sensitivity of ultrafast experiments versus conventional 2D NMR. A fair and relevant comparison has to consider the Signal-to-Noise Ratio (SNR) per unit of time, in order to answer the following question: for a given experiment time, should we run a conventional 2D experiment or is it preferable to accumulate ultrafast acquisitions? To answer this question, we perform here a systematic comparison between accumulated ultrafast experiments and conventional ones, for different experiment durations. Sensitivity issues and other analytical aspects are discussed for the COSY experiment in the context of quantitative analysis. The comparison is first carried out on a model sample, and then extended to model metabolic mixtures. The results highlight the high analytical performance of the "multi-scan single shot" approach versus conventional 2D NMR acquisitions. This result is attributed to the absence of t(1) noise in spatially encoded experiments. The multi-scan single shot approach is particularly interesting for quantitative applications of 2D NMR, whose occurrence in the literature has been greatly increasing in the last few years.     

Real-time monitoring of chemical transformations by ultrafast 2D NMR spectroscopy

An approach enabling the acquisition of 2D nuclear magnetic resonance (NMR) spectra within a single scan has been recently proposed. A promising application opened up by this "ultrafast" data acquisition format concerns the monitoring of chemical transformations as they happen, in real time. The present paper illustrates some of this potential with two examples: (i) following an H/D exchange process that occurs upon dissolving a protonated protein in D2O, and (ii) real-time in situ tracking of a transient Meisenheimer complex that forms upon rapidly mixing two organic reactants inside the NMR observation tube. The first of these measurements involved acquiring a train of 2D 1H-15N HSQC NMR spectra separated by ca. 4 s; following an initial dead time, this allowed us to monitor the kinetics of hydrogen exchange in ubiquitin at a site-resolved level. The second approach enabled us to observe, within ca. 2 s after the triggering of the reaction, a competition between thermodynamic and kinetic controls via changes in a series of 2D TOCSY patterns. The real-time dynamic experiments hereby introduced thus add to an increasing family of fast characterization techniques based on 2D NMR; their potential and limitations are briefly discussed.     

Sensitivity and lineshape improvement in ultrafast 2D NMR by optimized apodization in the spatially encoded dimension

Ultrafast 2D NMR allows the acquisition of a 2D spectrum in a single scan. However, even when the acquisition of ultrafast spectra is carried out under optimized conditions, the appearance and the sensitivity of 2D spectra are often not satisfactory compared with what one could expect from this promising methodology. This is due to limitations in terms of sensitivity, spectral width and resolution, and also to non-ideal lineshapes characterized by asymmetric sinc wiggles. Here, we identify the origin of these distortions by means of numerical simulations compared with experimental data. We then propose a processing approach to improve lineshapes while increasing the sensitivity of ultrafast experiments. The method consists in multiplying the Fourier transform of ultrafast echoes by an optimized apodization function. The principles of the method are described, and a variety of window functions are tested to determine optimum processing conditions. The approach is finally applied to ultrafast 2D spectra, leading to symmetric lineshapes with a sensitivity increased by a factor of 2.     

Ultrafast-based projection-reconstruction three-dimensional nuclear magnetic resonance spectroscopy

Recent years have witnessed increased efforts toward the accelerated acquisition of multidimensional nuclear magnetic resonance (nD NMR) spectra. Among the methods proposed to speed up these NMR experiments is "projection reconstruction," a scheme based on the acquisition of a reduced number of two-dimensional (2D) NMR data sets constituting cross sections of the nD time domain being sought. Another proposition involves "ultrafast" spectroscopy, capable of completing nD NMR acquisitions within a single scan. Potential limitations of these approaches include the need for a relatively slow 2D-type serial data collection procedure in the former case, and a need for at least n high-performance, linearly independent gradients and a sufficiently high sensitivity in the latter. The present study introduces a new scheme that comes to address these limitations, by combining the basic features of the projection reconstruction and the ultrafast approaches into a single, unified nD NMR experiment. In the resulting method each member within the series of 2D cross sections required by projection reconstruction to deliver the nD NMR spectrum being sought, is acquired within a single scan with the aid of the 2D ultrafast protocol. Full nD NMR spectra can thus become available by backprojecting a small number of 2D sets, collected using a minimum number of scans. Principles, opportunities, and limitations of the resulting approach, together with demonstrations of its practical advantages, are here discussed and illustrated with a series of three-dimensional homo- and heteronuclear NMR correlation experiments.     

Real-time 2D NMR identification of analytes undergoing continuous chromatographic separation

We have recently proposed and demonstrated an approach that enables the acquisition of multidimensional nuclear magnetic resonance (NMR) spectra within a single scan. A promising application opened up by this new accelerated form of data acquisition concerns the possibility of monitoring in real time the chemical nature of analytes subject to a continuous flow. The present paper illustrates such potential, with the real-time acquisition of a series of 2D 1H NMR spectra arising from a mixture of compounds subject to a continuous liquid chromatography (LC) separation. This real-time 2D NMR identification of chemicals eluted minutes apart under usual LC-NMR conditions differs from the way in which LC-2D NMR has hitherto been carried out, which relies on stopped-flow modes of operations whereby fractions are first collected and then subject to individual, aliquot-by-aliquot analyses. The real-time LC-2D NMR experiment hereby introduced can be implemented in a straightforward manner using modern commercial LC-NMR hardware, thus opening up immediate possibilities in high-throughput characterizations of complex molecules.     

Practical aspects of the simultaneous collection of COSY and TOCSY spectra

The practical aspects of some NMR experiments designed for the simultaneous acquisition of 2D COSY and 2D TOCSY spectra are presented and discussed. Several techniques involving afterglow-based, coherence transfer pathway (CTP)-based, and NMR by Ordered Acquisition using ¹H-detection (NOAH)-based strategies for the collection of different free-induction signal decays (FIDs) within the same scan are evaluated and compared. These methods offer a faster recording of these spectra in small-molecule NMR when sensitivity is not a limiting factor, with a reduction in spectrometer time about 45–60% when compared with the conventional sequential acquisition of the parent experiments. It is also shown how the optimized design of an extended three-FID approach yields one COSY and two TOCSY spectra simultaneously by combining CTP and NOAH principles in the same experiment, affording substantial sensitivity enhancements per time unit.     

UltraSOFAST HMQC NMR and the repetitive acquisition of 2D protein spectra at Hz rates

Following unidirectional biophysical events such as the folding of proteins or the equilibration of binding interactions, requires experimental methods that yield information at both atomic-level resolution and at high repetition rates. Toward this end a number of different approaches enabling the rapid acquisition of 2D NMR spectra have been recently introduced, including spatially encoded "ultrafast" 2D NMR spectroscopy and SOFAST HMQC NMR. Whereas the former accelerates acquisitions by reducing the number of scans that are necessary for completing arbitrary 2D NMR experiments, the latter operates by reducing the delay between consecutive scans while preserving sensitivity. Given the complementarities between these two approaches it seems natural to combine them into a single tool, enabling the acquisition of full 2D protein NMR spectra at high repetition rates. We demonstrate here this capability with the introduction of "ultraSOFAST" HMQC NMR, a spatially encoded and relaxation-optimized approach that can provide 2D protein correlation spectra at approximately 1 s repetition rates for samples in the approximately 2 mM concentration range. The principles, relative advantages, and current limitations of this new approach are discussed, and its application is exemplified with a study of the fast hydrogen-deuterium exchange characterizing amide sites in Ubiquitin.     

Multidimensional NMR spectroscopy for protein characterization and assignment inside cells

High-field, heteronuclear NMR spectroscopy of biological macromolecules in native cellular environments is limited by the low concentrations present and the long data acquisition times needed for the experiments. Successful 1D and 2D heteronuclear NMR data have been reported, but the 3D experiments conventionally used for protein assignment and detailed characterization are generally too long to maintain cell viability. Here we describe the successful in vivo implementation of a suite of fast 3D NMR experiments which we have used to generate the complete backbone assignment of resonances in the recombinant polypeptide GB-1 within Escherichia coli cells. The data were acquired at 600 MHz with a cold probe using the projection reconstruction experiments, (3,2)HNCA, (3,2)HNCO, and (3,2)HA(CA)NH.     

Progress in Hyperpolarized Ultrafast 2D NMR Spectroscopy

An important development in the field of NMR spectroscopy has been the advent of hyperpolarization approaches, capable of yielding nuclear spin states whose value exceeds by orders‐of‐magnitude what even the highest‐field spectrometers can afford under Boltzmann equilibrium. Included among these methods is an ex situ dynamic nuclear polarization (DNP) approach, which yields liquid‐phase samples possessing spin polarizations of up to 50 %. Although capable of providing an NMR sensitivity equivalent to the averaging of about 1 000 000 scans, this methodology is constrained to extract its “superspectrum” within a single—or at most a few—transients. This makes it a poor starting point for conventional 2D NMR acquisition experiments, which require a large number of scans that are identical to one another except for the increment of a certain t₁ delay. It has been recently suggested that by merging this ex situ DNP approach with spatially encoded “ultrafast” methods, a suitable starting point could arise for the acquisition of 2D spectra on hyperpolarized liquids. Herein, we describe the experimental principles, potential features, and current limitations of such integration between the two methodologies. For a variety of small molecules, these new hyperpolarized ultrafast experiments can, for equivalent overall durations, provide heteronuclear correlation spectra at significantly lower concentrations than those currently achievable by conventional 2D NMR acquisitions. A variety of challenges still remain to be solved before bringing the full potential of this new integrated 2D NMR approach to fruition; these outstanding issues are discussed.     

Single Scan 2D NMR Spectroscopy on a 25 T Bitter Magnet

2D NMR relies on monitoring systematic changes in the phases incurred by spin coherences as a function of an encoding time t(1), whose value changes over the course of independent experiments. The intrinsic multiscan nature of such protocols implies that resistive and/or hybrid magnets, capable of delivering the highest magnetic field strengths but possessing poor temporal stabilities, become unsuitable for 2D NMR acquisitions. It is here shown with a series of homo- and hetero-nuclear examples that such limitations can be bypassed using recently proposed 2D "ultrafast" acquisition schemes, which correlate interactions along all spectral dimensions within a single scan.     

Temperature as an Extra Dimension in Multidimensional Protein NMR Spectroscopy

NMR spectroscopy is a particularly informative method for studying protein structures and dynamics in solution; however, it is also one of the most time-consuming. Modern approaches to biomolecular NMR spectroscopy are based on lengthy multidimensional experiments, the duration of which grows exponentially with the number of dimensions. The experimental time may even be several days in the case of 3D and 4D spectra. Moreover, the experiment often has to be repeated under several different conditions, for example, to measure the temperature-dependent effects in a spectrum (temperature coefficients (TCs)). Herein, a new approach that involves joint sampling of indirect evolution times and temperature is proposed. This allows TCs to be measured through 3D spectra in even less time than that needed to acquire a single spectrum by using the conventional approach. Two signal processing methods that are complementary, in terms of sensitivity and resolution, 1) dividing data into overlapping subsets followed by compressed sensing reconstruction, and 2) treating the complete data set with a variant of the Radon transform, are proposed. The temperature-swept 3D HNCO spectra of two intrinsically disordered proteins, osteopontin and CD44 cytoplasmic tail, show that this new approach makes it possible to determine TCs and their non-linearities effectively. Non-linearities, which indicate the presence of a compact state, are particularly interesting. The complete package of data acquisition and processing software for this new approach are provided.     

(4,2)D Projection--reconstruction experiments for protein backbone assignment: application to human carbonic anhydrase II and calbindin D(28K)

Projection-reconstruction NMR experiments have been shown to significantly reduce the acquisition time required to obtain protein backbone assignment data. To date, this concept has only been applied to smaller (15)N/(13)C-labeled proteins. Here, we show that projection-reconstruction NMR techniques can be extended to larger protonated and perdeuterated proteins. We present a suite of (4,2)D triple-resonance experiments for protein backbone assignment and a Hybrid Backprojection/Lower-Value algorithm for reconstructing data with relatively weak signal-to-noise ratios. In addition, we propose a sampling theorem and discuss its implication on the choice of projection angles. We demonstrate the efficacy of this approach using the 29 kDa protein, human carbonic anhydrase II and the 30 kDa protein, calbindin D(28K).     

Fast multidimensional localized parallel NMR spectroscopy for the analysis of samples

A parallel localized spectroscopy (PALSY) method is presented to speed up the acquisition of multidimensional NMR (nD) spectra. The sample is virtually divided into a discrete number of nonoverlapping slices that relax independently during consecutive scans of the experiment, affording a substantial reduction in the interscan relaxation delay and the total experiment time. PALSY was tested for the acquisition of three experiments 2D COSY, 2D DQF‐COSY and 2D TQF‐COSY in parallel, affording a time‐saving factor of 3–4. Some unique advantages are that the achievable resolution in any dimension is not compromised in any way: it uses conventional NMR data processing, it is not prone to generate spectral artifacts, and once calibrated, it can be used routinely with these and other combinations of NMR spectra. Copyright © 2010 John Wiley & Sons, Ltd.     

The effects of molecular diffusion in ultrafast two-dimensional nuclear magnetic resonance

The so-called "ultrafast" nuclear magnetic resonance (NMR) methods enable the collection of multidimensional spectra within a single scan. These experiments operate by replacing traditional t(1) time increments, with a series of combined radiofrequency-irradiation/magnetic-field-gradient manipulations that spatially encode the effects of the indirect-domain spin interactions. Barring the presence of sizable displacements, the spatial patterns thus imparted can be read out following a mixing period with the aid of oscillating acquisition gradients, leading to a train of t(2)-modulated echoes carrying in their positions and phases the indirect- and the direct-domain spin interactions. Both the initial spatial encoding as well as the subsequent spatial decoding procedures underlying ultrafast NMR were designed under the assumption that spins remain static within the sample during their execution. Most often this is not the case, and motion-related effects can be expected to affect the outcome of these experiments. The present paper focuses on analyzing the effects of diffusion in ultrafast two-dimensional (2D) NMR. Toward this end both analytical and numerical formalisms are derived, capable of dealing with the nonuniform spin manipulations, macroscopic sample sizes, and microscopic displacements involved in this kind of sequences. After experimentally validating the correctness of these formalisms these were used to analyze the effects of diffusion for a variety of cases, including ultrafast experiments on both rapidly and slowly diffusing molecules. A series of prototypical schemes were considered including discrete and continuous encoding modes, constant- and real-time manipulations, homo- and heteronuclear acquisitions, and single versus multiple quantum modalities. The effects of molecular diffusion were also compared against typical relaxation-driven losses as they happen in these various prototypical situations; from all these situations, general guidelines for choosing the optimal ultrafast 2D NMR scheme for a particular sample and condition could be deduced.     

Fast quantitative 2D NMR for metabolomics and lipidomics: A tutorial

Nuclear magnetic resonance (NMR) is a well-known analytical technique for the analysis of complex mixtures. Its quantitative capability makes it ideally suited to metabolomics or lipidomics studies involving large sample collections of complex biological samples. To overcome the ubiquitous limitation of spectral overcrowding when recording 1D NMR spectra on such samples, the acquisition of 2D NMR spectra allows a better separation between overlapped resonances while yielding accurate quantitative data when appropriate analytical protocols are implemented. Moreover, the experiment duration can be considerably reduced by applying fast acquisition methods. Here, we describe the general workflow to acquire fast quantitative 2D NMR spectra in the “omics” context. It is illustrated on three representative and complementary experiments: UF COSY, ZF-TOCSY with nonuniform sampling, and HSQC with nonuniform sampling. After giving some details and recommendations on how to apply this protocol, its implementation in the case of targeted and untargeted metabolomics/lipidomics studies is described.     

Sub-second 2D NMR spectroscopy at sub-millimolar concentrations

A recently proposed protocol enables the acquisition of two-dimensional nuclear magnetic resonance (2D NMR) spectra within a single scan. A promising application opened up by this new data acquisition mode concerns its combination with active nuclear polarization methods, whereby spectroscopy is carried out on analytes whose spin magnetizations have been significantly enhanced over their Boltzmann thermal values. The present paper explores the potential of such combination, with the acquisition of peptide and protein 2D NMR 1H correlation spectra recorded after the samples had been subject to laser-driven chemically induced dynamic nuclear polarization (CIDNP). It is demonstrated that the speed and sensitivity enhancement afforded by these combined processes enables the acquisition of quality 2D NMR data sets within a fraction of a second, at analyte concentrations that are under 1 mM.     

Mobile phase compensation to improve NMR spectral properties during solvent gradients

A solvent compensation method based on flow injection analysis is used to obtain high quality nuclear magnetic resonance (NMR) spectra during solvent gradients. Using a binary solvent system containing D2O and CD3OD, NMR line broadening and chemical shift changes are observed with a 10% methanol per min solvent composition gradient. However, by creating a second equal but reverse gradient and combining the two solvent gradients before the NMR detector, the composition of solvent reaching the NMR flow cell is kept constant. We demonstrate a system using flow injection analysis of combining solvent gradients and show constant NMR spectral performance as a function of time as the combined flow has a constant solvent composition irrespective of the initial solvent gradient. Using this approach, methods can be developed to measure high quality NMR spectra during on-flow gradient LC-NMR experiments. The ultimate ability of this approach depends on the ability to compensate for the disturbance of the solvent gradient and reverse gradient by a pair of LC columns (the analytical and reverse gradient columns).     

Targeted projection NMR spectroscopy for unambiguous metabolic profiling of complex mixtures

Unambiguous identification of individual metabolites present in complex mixtures such as biofluids constitutes a crucial prerequisite for quantitative metabolomics, toward better understanding of biochemical processes in living systems. Increasing the dimensionality of a given NMR correlation experiment is the natural solution for resolving spectral overlap. However, in the context of metabolites, natural abundance acquisition of ¹H and ¹³C NMR data virtually excludes the use of higher dimensional NMR experiments (3D, 4D, etc.) that would require unrealistically long acquisition times. Here, we introduce projection NMR techniques for studies of complex mixtures, and we show how discrete sets of projection spectra from higher dimensional NMR experiments are obtained in a reasonable time frame, in order to capture essential information necessary to resolve assignment ambiguities caused by signal overlap in conventional 2D NMR spectra. We determine optimal projection angles where given metabolite resonances will have the least overlap, to obtain distinct metabolite assignment in complex mixtures. The method is demonstrated for a model mixture composition made of ornithine, putrescine and arginine for which acquisition of a single 2D projection of a 3D ¹H–¹³C TOCSY‐HSQC spectrum allows to disentangle the metabolite signals and to access to complete profiling of this model mixture in the targeted 2D projection plane. Copyright © 2010 John Wiley & Sons, Ltd.     

High-resolution iterative frequency identification for NMR as a general strategy for multidimensional data collection

We describe a novel approach to the rapid collection and processing of multidimensional NMR data: "high-resolution iterative frequency identification for NMR" (HIFI-NMR). As with other reduced dimensionality approaches, HIFI-NMR collects n-dimensional data as a set of two-dimensional (2D) planes. The HIFI-NMR algorithm incorporates several innovative features. (1) Following the initial collection of two orthogonal 2D planes, tilted planes are selected adaptively, one-by-one. (2) Spectral space is analyzed in a rigorous statistical manner. (3) An online algorithm maintains a model that provides a probabilistic representation of the three-dimensional (3D) peak positions, derives the optimal angle for the next plane to be collected, and stops data collection when the addition of another plane would not improve the data model. (4) A robust statistical algorithm extracts information from the plane projections and is used to drive data collection. (5) Peak lists with associated probabilities are generated directly, without total reconstruction of the 3D spectrum; these are ready for use in subsequent assignment or structure determination steps. As a proof of principle, we have tested the approach with 3D triple-resonance experiments of the kind used to assign protein backbone and side-chain resonances. Peaks extracted automatically by HIFI-NMR, for both small and larger proteins, included approximately 98% of real peaks obtained from control experiments in which data were collected by conventional 3D methods. HIFI-NMR required about one-tenth the time for data collection and avoided subsequent data processing and peak-picking. The approach can be implemented on commercial NMR spectrometers and is extensible to higher-dimensional NMR.