Multiplet-Assisted Peak Alignment for 1H NMR-Based Metabolomics.

NMR-based metabolomics aims at recovering biological information by comparing spectral data from samples of biological interest and appropriate controls. Any statistical analysis performed on the data matrix relies on the proper peak alignment to produce meaningful results. Through the last decades, several peak alignment algorithms have been proposed, as well as alternatives like spectral binning or strategies for annotation and quantification, the latter depending on reference databases. Most of the alignment algorithms, mainly based on segmentation of the spectra, present limitations for regions with peak overlap or cases of frequency order exchange. Here, we present our multiplet-assisted peak alignment algorithm, a new methodology that consists of aligning peaks by matching multiplet profiles of f1 traces from J-resolved spectra. A correspondence matrix with the linked f1 traces is built, and multivariate data analysis can be performed on it to obtain useful information from the data, overcoming the issues of peak overlap and frequency crossovers. Statistical total correlation spectroscopy can be applied on the matrix as well, toward a better identification of molecules of interest. The results can be queried on one-dimensional (1D) 1H databases or can be directly coupled to our previously published Chemical Shift Multiplet Database.

Short-chain fatty acids reprogram metabolic profiles with the induction of reactive oxygen species production in human colorectal adenocarcinoma cells.

Short-chain fatty acids (SCFAs) exhibit anticancer activity in cellular and animal models of colon cancer. Acetate, propionate, and butyrate are the three major SCFAs produced from dietary fiber by gut microbiota fermentation and have beneficial effects on human health. Most previous studies on the antitumor mechanisms of SCFAs have focused on specific metabolites or genes involved in antitumor pathways, such as reactive oxygen species (ROS) biosynthesis. In this study, we performed a systematic and unbiased analysis of the effects of acetate, propionate, and butyrate on ROS levels and metabolic and transcriptomic signatures at physiological concentrations in human colorectal adenocarcinoma cells. We observed significantly elevated levels of ROS in the treated cells. Furthermore, significantly regulated signatures were involved in overlapping pathways at metabolic and transcriptomic levels, including ROS response and metabolism, fatty acid transport and metabolism, glucose response and metabolism, mitochondrial transport and respiratory chain complex, one-carbon metabolism, amino acid transport and metabolism, and glutaminolysis, which are directly or indirectly linked to ROS production. Additionally, metabolic and transcriptomic regulation occurred in a SCFAs types-dependent manner, with an increasing degree from acetate to propionate and then to butyrate. This study provides a comprehensive analysis of how SCFAs induce ROS production and modulate metabolic and transcriptomic levels in colon cancer cells, which is vital for understanding the mechanisms of the effects of SCFAs on antitumor activity in colon cancer.

13C NMR metabolomics: J-resolved STOCSY meets INADEQUATE.

Robust annotation of metabolites is a challenging task in metabolomics. Among available applications, 13C NMR experiment INADEQUATE determines direct 13C-13C connectivity unambiguously, offering indispensable information on molecular structure. Despite its great utility, it is not always practical to collect INADEQUATE data on every sample in a large metabolomics study because of its relatively long experiment time. Here, we propose an alternative approach that maintains the quality of information but saves experiment time. In this approach, individual samples in a study are first screened by 13C homonuclear J-resolved experiment (JRES). Next, JRES data are processed by statistical total correlation spectroscopy (STOCSY) to extract peaks that behave similarly among samples. Finally, INADEQUATE is collected on one internal pooled sample to select STOCSY peaks that originate from the same compound. We tested this concept using the 13C-labeled endometabolome of a model marine diatom strain incubated under various settings, intending to cover a range of metabolites produced under different external conditions. This scheme was able to extract known diatom metabolites proline, 2,3-dihydroxypropane-1-sulfonate (DHPS), ß-1,3-glucan, choline, and glutamate. This pipeline also detected unknown compounds with structural information, which is valuable in metabolomics where a priori knowledge of metabolites is not always available. The ability of this scheme was seen even in sugar regions, which are usually challenging in 1H NMR due to severe peak overlap. JRES and INADEQUATE were highly complementary; INADEQUATE provided directly-bonded 13C networks, whereas JRES linked INADEQUATE networks within the same compound but broken by nitrogen or sulfur atoms, highlighting the advantage of this integrated approach.

Slice selective absorption-mode J-resolved NMR spectroscopy.

Limited chemical shift dispersion and broad multiplet patterns limit resolution in 1H NMR spectra. J-Resolved spectroscopy overcomes this problem to a great extent. However, the phase-twist line shape in J-Resolved spectroscopy allows only the magnitude mode of the experiment to be practical, which degrades resolution. Recently, various pure shift or broadband homonuclear decoupling approaches have been integrated with J-Resolved spectroscopy to eliminate the broad dispersive contribution. In the present work, we demonstrate a broadband 1H-1H J-Resolved spectrum with a greatly reduced dispersive contribution using the concept of slice selection. We show that slice selective excitation, t1 encoding, storage, and detection of the in-phase absorptive signals can be executed, while a gradient-based suppression of the dispersive antiphase signals can be performed during the storage period. In more than two spin systems, a small part of the doubly antiphase absorptive signal may also contribute to the spectrum in addition to the inphase absorptive signals. The overall effect is a reduced multiplet pattern similar to a regular J-Resolved case as the passive spins remain unflipped due to slice selective pulses. However, the effect is broadband for a fraction of the spins when all slices are considered analogous to Zangger-Sterk (ZS) broadband homo-decoupling. Further, the fresh magnetization from neighboring slices can be accessed in different scans by frequency shifting of the slice selective pulses without a recycle delay-an elegant aspect of the ZS pulse element. This allows faster signal averaging, improving sensitivity which depends on the T1 relaxation time of the signals. This method displays sensitivity up to 4-20 percent of the regular J-RES 1H signals.

Dictionary learning compressed sensing reconstruction: pilot validation of accelerated echo planar J-resolved spectroscopic imaging in prostate cancer.

OBJECTIVES: This study aimed at developing dictionary learning (DL) based compressed sensing (CS) reconstruction for randomly undersampled five-dimensional (5D) MR Spectroscopic Imaging (3D spatial + 2D spectral) data acquired in prostate cancer patients and healthy controls, and test its feasibility at 8x and 12x undersampling factors. MATERIALS AND METHODS: Prospectively undersampled 5D echo-planar J-resolved spectroscopic imaging (EP-JRESI) data were acquired in nine prostate cancer (PCa) patients and three healthy males. The 5D EP-JRESI data were reconstructed using DL and compared with gradient sparsity-based Total Variation (TV) and Perona-Malik (PM) methods. A hybrid reconstruction technique, Dictionary Learning-Total Variation (DLTV), was also designed to further improve the quality of reconstructed spectra. RESULTS: The CS reconstruction of prospectively undersampled (8x and 12x) 5D EP-JRESI data acquired in prostate cancer and healthy subjects were performed using DL, DLTV, TV and PM. It is evident that the hybrid DLTV method can unambiguously resolve 2D J-resolved peaks including myo-inositol, citrate, creatine, spermine and choline. CONCLUSION: Improved reconstruction of the accelerated 5D EP-JRESI data was observed using the hybrid DLTV. Accelerated acquisition of in vivo 5D data with as low as 8.33% samples (12x) corresponds to a total scan time of 14 min as opposed to a fully sampled scan that needs a total duration of 2.4 h (TR = 1.2 s, 32 [Formula: see text]×16 [Formula: see text]×8 [Formula: see text], 512 [Formula: see text] and 64 [Formula: see text]).

Automated metabolic assignment: Semi-supervised learning in metabolic analysis employing two dimensional Nuclear Magnetic Resonance (NMR).

Metabolomics is an expanding field of medical diagnostics since many diseases cause metabolic reprogramming alteration. Additionally, the metabolic point of view offers an insight into the molecular mechanisms of diseases. Due to the complexity of metabolic assignment dependent on the 1D NMR spectral analysis, 2D NMR techniques are preferred because of spectral resolution issues. Thus, in this work, we introduce an automated metabolite identification and assignment from 1H-1H TOCSY (total correlation spectroscopy) using real breast cancer tissue. The new approach is based on customized and extended semi-supervised classifiers: KNFST, SVM, third (PC3) and fourth (PC4) degree polynomial. In our approach, metabolic assignment is based only on the vertical and horizontal frequencies of the metabolites in the 1H-1H TOCSY. KNFST and SVM show high performance (high accuracy and low mislabeling rate) in relatively low size of initially labeled training data. PC3 and PC4 classifiers showed lower accuracy and high mislabeling rates, and both classifiers fail to provide an acceptable accuracy at extremely low size (≤9% of the entire dataset) of initial training data. Additionally, semi-supervised classifiers were implemented to obtain a fully automatic procedure for signal assignment and deconvolution of TOCSY, which is a big step forward in NMR metabolic profiling. A set of 27 metabolites were deduced from the TOCSY, and their assignments agreed with the metabolites deduced from a 1D NMR spectrum of the same sample analyzed by conventional human-based methodology.

Low Volume in Vitro Diagnostic Proton NMR Spectroscopy of Human Blood Plasma for Lipoprotein and Metabolite Analysis: Application to SARS-CoV-2 Biomarkers.

The utility of low sample volume in vitro diagnostic (IVDr) proton nuclear magnetic resonance (1H NMR) spectroscopic experiments on blood plasma for information recovery from limited availability or high value samples was exemplified using plasma from patients with SARS-CoV-2 infection and normal controls. 1H NMR spectra were obtained using solvent-suppressed 1D, spin-echo (CPMG), and 2-dimensional J-resolved (JRES) spectroscopy using both 3 mm outer diameter SampleJet NMR tubes (100 µL plasma) and 5 mm SampleJet NMR tubes (300 µL plasma) under in vitro diagnostic conditions. We noted near identical diagnostic models in both standard and low volume IVDr lipoprotein analysis (measuring 112 lipoprotein parameters) with a comparison of the two tubes yielding R2 values ranging between 0.82 and 0.99 for the 40 paired lipoprotein parameters samples. Lipoprotein measurements for the 3 mm tubes were achieved without time penalty over the 5 mm tubes as defined by biomarker recovery for SARS-CoV-2. Overall, biomarker pattern recovery for the lipoproteins was extremely similar, but there were some small positive offsets in the linear equations for several variables due to small shimming artifacts, but there was minimal degradation of the biological information. For the standard untargeted 1D, CPMG, and JRES NMR experiments on the same samples, the reduced signal-to-noise was more constraining and required greater scanning times to achieve similar differential diagnostic performance (15 min per sample per experiment for 3 mm 1D and CPMG, compared to 4 min for the 5 mm tubes). We conclude that the 3 mm IVDr method is fit-for-purpose for quantitative lipoprotein measurements, allowing the preparation of smaller volumes for high value or limited volume samples that is common in clinical studies. If there are no analytical time constraints, the lower volume experiments are equally informative for untargeted profiling.

Accelerated J-resolved 1 H-MRSI with limited and sparse sampling of ( k,t1,t2 -space.

PURPOSE: To accelerate the acquisition of J-resolved proton magnetic resonance spectroscopic imaging (1 H-MRSI) data for high-resolution mapping of brain metabolites and neurotransmitters. METHODS: The proposed method used a subspace model to represent multidimensional spatiospectral functions, which significantly reduced the number of parameters to be determined from J-resolved 1 H-MRSI data. A semi-LASER-based (Localization by Adiabatic SElective Refocusing) echo-planar spectroscopic imaging (EPSI) sequence was used for data acquisition. The proposed data acquisition scheme sampled k,t1,t2 -space in variable density, where t1 and t2 specify the J-coupling and chemical-shift encoding times, respectively. Selection of the J-coupling encoding times (or, echo time values) was based on a Cramer-Rao lower bound analysis, which were optimized for gamma-aminobutyric acid (GABA) detection. In image reconstruction, parameters of the subspace-based spatiospectral model were determined by solving a constrained optimization problem. RESULTS: Feasibility of the proposed method was evaluated using both simulated and experimental data from a spectroscopic phantom. The phantom experimental results showed that the proposed method, with a factor of 12 acceleration in data acquisition, could determine the distribution of J-coupled molecules with expected accuracy. In vivo study with healthy human subjects also showed that 3D maps of brain metabolites and neurotransmitters can be obtained with a nominal spatial resolution of 3.0 × 3.0 × 4.8 mm3 from J-resolved 1 H-MRSI data acquired in 19.4 min. CONCLUSIONS: This work demonstrated the feasibility of highly accelerated J-resolved 1 H-MRSI using limited and sparse sampling of k,t1,t2 -space and subspace modeling. With further development, the proposed method may enable high-resolution mapping of brain metabolites and neurotransmitters in clinical applications.

Revealing weak histidine 15N homonuclear scalar couplings using Solid-State Magic-Angle-Spinning NMR spectroscopy.

The tautomeric structure and chemistry of the histidine imidazole ring play active roles in many structurally and functionally important proteins and polypeptides. While in NMR spectroscopy histidine chemical shifts (e.g. 15N, 13C, and 1H) have been commonly used to characterize the tautomeric structure, hydrogen bonding, and torsion angles, homonuclear 15N scalar couplings in histidine have rarely been reported. Here, we propose double spin-echo sequences to compare the observed signals with and without a 90° pulse between the two spin-echo periods, such that their signal ratio as a function of the echo time solely depends on homonuclear scalar couplings, allowing for measuring weak homonuclear scalar couplings without influence from transverse dephasing effects, thus capable of revealing hydrogen-bond mediated 15N-15N J-couplings that can provide direct and definitive evidence for the formation of N…H…N hydrogen-bonding associated with the imidazole ring. We used two 13C,15N labeled histidine samples recrystallized from solutions at pH 6.3 and pH 11.0 to demonstrate the feasibility of this method and reveal the existence of a weak two-bond scalar coupling between the Nδ1 and Nε2 sites in the histidine imidazole ring in three tautomeric states and the presence of a hydrogen-bond mediated scalar coupling between the Nδ1 site in the imidazole ring and the backbone Nα site in the histidine neutral τ and π states. Our results demonstrate that weak 15N homonuclear scalar couplings can be measured even when their values are less than their corresponding intrinsic natural linewidths, thus providing direct and definitive evidence for the formation of N…H…N hydrogen bonding that is associated with the histidine imidazole ring.

Consecutive Queries to Assess Biological Correlation in NMR Metabolomics: Performance of Comprehensive Search of Multiplets over Typical 1D 1H NMR Database Search.

NMR-based metabolomics requires proper identification of metabolites to draw conclusions from the system under study. Normally, multivariate data analysis is performed using 1D 1H NMR spectra, and identification of peaks (and then compounds) relevant to the classification is accomplished using database queries as a first step. 1D 1H NMR spectra of complex mixtures often suffer from peak overlap. To overcome this issue, several studies employed the projections of the (tilted and symmetrized) 2D 1H J-resolved (JRES) spectra, p-JRES, which are similar to 1D 1H decoupled spectra. Nonetheless, there are no public databases available that allow searching for chemical shift spectral data for multiplets. We present the Chemical Shift Multiplet Database (CSMDB), built utilizing JRES spectra obtained from the Birmingham Metabolite Library. The CSMDB provides scoring accounting for both matched and unmatched peaks from a query list and the database hits. This input list is generated from a projection of a 2D statistical correlation analysis on the JRES spectra, p-(JRES-STOCSY), being able to compare the multiplets for the matched peaks, in essence, the f1 traces from the JRES-STOCSY spectrum and from the database hit. The inspection of the unmatched peaks for the database hit allows the retrieval of peaks in the query list that have a decreased correlation coefficient due to low intensities. The CSMDB is coupled to "ConQuer ABC", which permits the assessment of biological correlation by means of consecutive queries with the unmatched peaks in the first and subsequent queries.

PE-SERF: A sensitivity-improved experiment to measure JHH in crowded spectra.

Aiming at facilitating the analysis of molecular structure, the gradient-encoded selective refocusing methods (G-SERF) and a great number of its variants for measuring proton-proton coupling constants have been proposed. However, the sensitivity is an issue in the 2D gradient-encoded experiments, because the signal intensity is determined by the slice thickness of the sample that depends on encoding gradient and the bandwidth of selective pulses which is limited by the smallest chemical shift difference of any two coupled protons. Here, we present a method dubbed PE-SERF (perfect echo selective refocusing) which can determine all JHH values involving a selected proton with improved sensitivity compared to original G-SERF experiment. The modules of perfect echo involving selective pulses and gradient-encoded selective refocusing are combined in the method, so that the unwanted J couplings arising from coupled spin pairs in the same sample slice would be nullified. In this way, instead of single proton, a pair of coupled protons is allowed to share a sample slice, and thus the slice thickness can be increased and the spectral sensitivity can be improved. The performance of the method is demonstrated by experiments on quinine and strychnine.

Tackling the Peak Overlap Issue in NMR Metabolomics Studies: 1D Projected Correlation Traces from Statistical Correlation Analysis on Nontilted 2D 1H NMR J-Resolved Spectra.

The identification of metabolites in complex biological matrices is a challenging task in 1D 1H-NMR-based metabolomics studies. Statistical total correlation spectroscopy (STOCSY) has emerged for aiding the structural elucidation by revealing the peaks that present a high correlation to a driver peak of interest (which would likely belong to the same molecule). However, in these studies, the signals from metabolites are normally present as a mixture of overlapping resonances, limiting the performance of STOCSY. As an alternative to avoid the overlap issue, 2D 1H homonuclear J-resolved (JRES) spectra were projected, in their usual tilted and symmetrized processed form, and STOCSY was applied on these 1D projections (p-JRES-STOCSY). Nonetheless, this approach suffers in cases where the signals are very close. In addition, STOCSY was applied to the whole JRES spectra (also tilted) to identify correlated multiplets, although the overlap issue in itself was not addressed directly and the subsequent search in databases is complicated in cases of higher order coupling. With these limitations in mind, in the present work, we propose a new methodology based on the application of STOCSY on a set of nontilted JRES spectra, detecting peaks that would overlap in 1D spectra of the same sample set. Correlation comparison analysis for peak overlap detection (COCOA-POD) is able to reconstruct projected 1D STOCSY traces that result in more suitable database queries, as all peaks are summed at their f2 resonances instead of the resonance corresponding to the multiplet center in the tilted JRES spectra. (The peak dispersion and resolution enhancement gained are not sacrificed by the projection.) Besides improving database queries with better peak lists obtained from the projections of the 2D STOCSY analysis, the overlap region is examined, and the multiplet itself is analyzed from the correlation trace at 45° to obtain a cleaner multiplet profile, free from contributions from uncorrelated neighboring peaks.

DQF J-RES NMR: Suppressing the singlet signals for improving the J-RES spectra from complex mixtures.

Two-dimensional J-RESolved spectroscopy (J-RES) finds routine use in metabolomics for reducing signal overlap as it separates chemical shift and multiplet information along two frequency axes. However, only magnitude mode of the experiment is practical which prevents exploitation of its full resolving power. Tailing from high-intensity metabolite peaks often obscure nearby low-intensity metabolite peaks which leads to ambiguity in assignment of metabolites. Absorptive mode J-RES spectroscopy offers better-resolving power but comes at the cost of either sensitivity or complicated post-processing. Quite often for certain complex mixtures such as bio-fluids some components of the mixture display intense singlet signals which dominate the whole spectrum resulting in less reliable detection of weaker metabolite signals. Multi-frequency presaturation could suppress these intense singlets but will also remove the useful weaker multiplet peaks which are either totally eclipsed with the intense singlets or very close in frequency. We show that by using a double quantum filter (DQF) in magnitude mode J-RES technique, the intensity of the strong singlet metabolite peaks can be reduced relative to the intensity of the sparsely present multiplet metabolite signals. This approach leads to the identification of many weak intensity multiplet peaks which are otherwise undetected due to their overlap with intense singlet peaks in regular J-RES as well as 1D 1H spectra. Although the improved intensity of most of the weaker peaks relative to the strong singlet peaks is observed, some multiplets can disappear due to the delay-dependent modulation of the signals by the DQF. A few DQF J-RES spectra recorded with different DQF delays, therefore, produce better assignment when analyzed together. The technique is demonstrated on a mixture of eight compounds, human urine, and plant extract samples.

High-resolution two-dimensional 1H J-resolved MRS measurements on in vivo samples.

Magnetic resonance spectroscopy (MRS) provides a noninvasive tool for metabolite characterization of in vivo biological samples. Conventional MRS measurements on biological samples generally suffer from field inhomogeneity caused by intrinsic magnetic susceptibility variations inside samples. Compared to one-dimensional MRS, two-dimensional (2D) J-resolved spectroscopy enables resolving J couplings along one of the spectral dimension and benefits to metabolite identification and analyses. Intermolecular double-quantum coherences (iDQC) has been proven to be insensitive to magnetic field inhomogeneity, herein we propose a MRS approach based on iDQC evolution and optimal echo sampling scheme to achieve high-resolution 2D J-resolved measurements on biological samples. The applicability of the proposed method is evaluated with experiments on an ex vivo pig brain tissue and an in vivo rat brain tissue. Compared to conventional MRS method which is sensitive to field inhomogeneity inside investigated biological tissues, the proposed method holds immunity to this field inhomogeneity and the quality of resulting spectra may not be influenced by localized voxel size variation. The signal to noise ratio enhancement of the proposed method benefitting from the optimal echo signal sampling is verified with a solution experiment. The new method provides a promising way for high-resolution MRS measurements on biological samples. In combination with fast acquisition strategy, it may find some promising biomedical applications.

Highly Resolved Pure-Shift Spectra on a Compact NMR Spectrometer.

Benchtop NMR spectrometers experience a great success for a wide range of applications. However, their performance is highly limited by peak overlaps. Emerging "pure-shift NMR" (PS NMR) methods have been intensively used at high field to enhance the resolution by homodecoupling strategies. Here, different PS methods have been implemented on a compact NMR spectrometer operating at 43 MHz. Among the PS methods, the recent PSYCHE scheme appears more sensitive than Zangger-Sterk (ZS) experiments and offers a substantial resolution improvement as compared to 1D 1 H. On the other hand, despite their slightly lower sensitivity, ZS methods are more efficient to reduce broad signals and are more immune to strong couplings. Finally, the classical J-resolved pulse sequence is more efficient to reduce larger signals for bigger-sized molecules. The three approaches appear relevant for benchtop NMR and their combination forms an efficient toolbox to analyze a great diversity of samples.

Broadband homodecoupled time-shared 1H-13C and 1H-15N HSQC experiments.

The concepts of pure-shift NMR and time-shared NMR are merged in a single experiment. A 13C/15N time-shared version of the real-time BIRD-based broadband homodecoupled HSQC experiment is described. This time-efficient approach affords simultaneously 1H-13C and 1H-15N pure-shift HSQC spectra in a single acquisition, while achieving substantial gains in both sensitivity and spectral resolution. We also present a related 13C/15N-F2-coupled homodecoupled version of the CLIP-HSQC experiment for the simultaneous measurement of 1JCH and 1JNH from the simplified doublets observed along the direct dimension. Finally, a novel J-resolved HSQC experiment has been designed for the simple and automated determination of both 1JCH/1JNH from a 2D J-resolved spectrum.

DPFGSE-MDEC-J-resolved COSY: An efficient method for selectively measuring proton-proton spin coupling constants of the multiplet signals.

Natural products such as polyketides often possess various spin systems, consisting of a methine group directly bonded to a methyl group (e.g., ─CHA ─CHB (CH3 )─CHC ─). The methine proton HB splits into a broadened multiplet by coupling with several vicinal protons, rendering analysis difficult of n JH-H with respect to HB in double-quantum filtered COSY or exclusive COSY. For the purpose of measuring n JH-H in the aforesaid spin system, we have developed new techniques, named multifrequency homo-decoupling (MDEC)-J-resolved COSY and DPFGSE-MDEC-J-resolved COSY. This method incorporates MDEC pulse scheme into J-resolved COSY for the selective decouple of individual methyl groups, avoiding decoupling of the target protons resonating in methyl region. Determinations of n JH-H of the multiplet signals can easily be performed using the proposed pulse sequence.

Homonuclear decoupling by projection reconstruction.

Similar to J-resolved spectroscopy, also, heteronuclear multiple bond correlation (HMBC), heteronuclear single bond correlation (HSBC), and heteronuclear multiple quantum coherence (HMQC) types of correlation experiments result in homonuclear tilted multiplet patterns. On the example of the high-resolution heteronuclear single bond correlation (HR-HSBC) pulse sequence, it is shown how the tilt angle can be varied within a wide range of positive and negative values. Projection along the tilt angles in all cases results in homonuclear decoupling. Using well-known projection reconstruction techniques, the different tilt angles can be used to reconstruct a homonuclear decoupled two-dimensional correlation spectrum. The concept is proven and further refined by segmental projection reconstruction and the use of a clean in-phase heteronuclear single quantum correlation (CLIP-HSQC) spectrum with an effective zero tilt angle for further filtering. The proof of principle, its application to one-bond coupling measurement, as well as a basic HMBC, and a detailed discussion with comparison to other homodecoupling techniques are given.

Small angle double quantum spectroscopy (SAQS NMR).

A new experiment for recording double quantum spectra is introduced. The 2D DQ NMR experiment yields phase sensitive spectra with double quantum frequencies in F1. The appearance of remote peaks is vastly suppressed by using a small flip angle double quantum excitation and reconversion. Pulse sequences and phase sensitive processing are discussed. The complexity of the SQ antiphase magnetization given in larger proton spin networks could be reduced by using the option of band selective decoupling during the preparation period. In addition, an ACCORDION element is applied by incrementing the J evolution delay in concert with the t1 period. With this the excitation of double quantum coherence over a wider range of J values is achieved. A broadband homodecoupled version of the DQ experiment is proposed, where correlation peaks with singlet response at F2 chemical shifts and double quantum frequencies in F1 are obtained. We call this experiment Small Angle double Quantum Spectroscopy SAQS NMR.

Current developments in homonuclear and heteronuclear J-resolved NMR experiments.

Two-dimensional J-resolved (Jres) NMR experiments offer a simple, user-friendly spectral representation where the information of coupling constants and chemical shifts are separated into two orthogonal frequency axis. Since its initial proposal 40 years ago, Jres has been the focus of considerable interest both in improving the basic pulse sequence and in its successful application to a wide range of studies. Here, the latest developments in the design of novel Jres pulse schemes are reviewed, mainly focusing on obtaining pure absorption lineshapes, minimizing strong coupling artifacts, and also optimizing sensitivity and experimental measurements. A discussion of several Jres versions for the accurate measurement of a different number of homonuclear (JHH ) and heteronuclear (JCH ) coupling constants is presented, accompanied by some illustrative examples.