Brief early-life non-specific incorporation of deuterium extends mean life span in Drosophila melanogaster without affecting fecundity.

We have investigated the effects of brief, non-specific deuteration of Drosophila melanogaster by including varying percentages of ²H (D) in the H2O used in the food mix consumed during initial development. Up to 22.5% deuterium oxide (D2O) in H2O was administered, with the result that a low percentage of D2O in the water increased mean life span, whereas the highest percentage used (22.5%) reduced life span. After the one-time treatment period, adult flies were maintained ad libitum with food of normal isotopic distribution. At low deuterium levels, where life span extension was observed, there was no observed change in fecundity. Dead flies were assayed for deuterium incorporation by complete hydrolysis in hot 12 N HCl solution followed by subsequent high-performance liquid chromatography/mass spectrometry (HPLC/MS). Isoleucine and leucine residues showed a small, linear dose-dependent incorporation of deuterium at non-exchangeable sites. Although high levels of D2O itself are toxic for other reasons, higher levels of deuterium incorporation, which can be achieved without toxicity by strategies that avoid direct use of D2O, are clearly worth exploring.

Improving the double quantum filtered COSY experiment by "Moving Tube" NMR.

Most 2D NMR spectra show artifacts that become increasingly more prominent as the relaxation delay between transients is decreased. Additionally, "rushing" a 2D experiment may lead to reduced sensitivity. It is shown here how to collect a DQF-COSY spectrum in less time, without artifacts, and with improved sensitivity, by a hardware solution we call Moving Tube NMR (MT NMR): the sample volume is physically moved out of the receiver coil after each transient and replaced by a fresh aliquot that is nearer to the equilibrium magnetization M(0). MT NMR was implemented with an automated mechanism that gave accurate and reproducible vertical tube movement, and a very long 5mm outer diameter (OD) NMR tube to hold a larger sample volume. Comparison of conventional and MT NMR DQF-COSY showed increased sensitivity and far reduced artifacts in the latter. The so-called t(1)-noise in the MT spectrum was no worse than in the conventional spectrum, pointing to the excellent specifications of the long 5mm OD tube, and the good mechanical handling of the automated drive. Thus, MT NMR could improve throughput for routine 2D NMR experiments without reducing sensitivity or adding artifacts, if sufficient sample is available. MT NMR could also be useful in cases of limited solubility, or for nuclei with long T(1) relaxation times.

Phase-sensitive spectral estimation by the hybrid filter diagonalization method.

A more robust way to obtain a high-resolution multidimensional NMR spectrum from limited data sets is described. The Filter Diagonalization Method (FDM) is used to analyze phase-modulated data and cast the spectrum in terms of phase-sensitive Lorentzian "phase-twist" peaks. These spectra are then used to obtain absorption-mode phase-sensitive spectra. In contrast to earlier implementations of multidimensional FDM, the absolute phase of the data need not be known beforehand, and linear phase corrections in each frequency dimension are possible, if they are required. Regularization is employed to improve the conditioning of the linear algebra problems that must be solved to obtain the spectral estimate. While regularization smoothes away noise and small peaks, a hybrid method allows the true noise floor to be correctly represented in the final result. Line shape transformation to a Gaussian-like shape improves the clarity of the spectra, and is achieved by a conventional Lorentzian-to-Gaussian transformation in the time-domain, after inverse Fourier transformation of the FDM spectra. The results obtained highlight the danger of not using proper phase-sensitive line shapes in the spectral estimate. The advantages of the new method for the spectral estimate are the following: (i) the spectrum can be phased by conventional means after it is obtained; (ii) there is a true and accurate noise floor; and (iii) there is some indication of the quality of fit in each local region of the spectrum. The method is illustrated with 2D NMR data for the first time, but is applicable to n-dimensional data without any restriction on the number of time/frequency dimensions.

Filter diagonalization using a "sensitivity-enhanced basis": improved performance for noisy NMR spectra.

The Filter Diagonalization Method (FDM) has been used to process NMR data in liquids and can be advantageous when the spectrum is sparse enough, the lines are sharp and Lorentzian, raw sensitivity is adequate, and the measured time-domain data is short, so that the Fourier Transform spectrum exhibits distorted line shapes. Noise can adversely impact resolution and/or frequency accuracy in FDM spectral estimates. Paradoxically, more complete data can lead to worse FDM spectra if there is appreciable noise. However, by modifying the numerical method, the FDM noise performance improves significantly, without apparently losing any of the existing advantages. The two key modifications are to adjust the FDM basis functions so that matrix elements computed from them have less noise contribution on average, and to regularize each dimension of a multidimensional spectrum independently. The modifications can be recommended for general-purpose use in the case of somewhat noisy, incomplete data.

Sensitivity analysis of solutions of the harmonic inversion problem: are all data points created equal?

We consider the harmonic inversion problem, and the associated spectral estimation problem, both of which are key numerical problems in NMR data analysis. Under certain conditions (in particular, in exact arithmetic) these problems have unique solutions. Therefore, these solutions must not depend on the inversion algorithm, as long as it is exact in principle. In this paper, we are not concerned with the algorithmic aspects of harmonic inversion, but rather with the sensitivity of the solutions of the problem to perturbations of the time-domain data. A sensitivity analysis was performed and the counterintuitive results call into question the common assumption made in "super-resolution" methods using non-uniform data sampling, namely, that the data should be sampled more often where the time signal has the largest signal-to-noise ratio. The numerical analysis herein demonstrates that the spectral parameters (such as the peak positions and amplitudes) resulting from the solution of the harmonic inversion problem are least susceptible to the perturbations in the values of data points at the edges of the time interval and most susceptible to the perturbations in the values at intermediate times.

Selective heteronuclear Hartmann-Hahn: a multiple-pulse sequence for selective magnetization transfer in the structural elucidation of "isotagged" oligosaccharides.

A new selective heteronuclear Hartmann-Hahn (SHEHAHA) multiple-pulse mixing sequence is proposed for the solution structure elucidation of milligram amounts of peracetylated oligosaccharides in which the acetyl groups are enriched in carbon-13, so-called "isotags". SHEHAHA accomplishes exclusive in-phase magnetization transfer between the isotag carbonyl (13)C and the proximal proton on the sugar ring. Relayed transfer around the sugar rings by proton-proton TOCSY is suppressed, while the heteronuclear transfer from the labeled carbonyl carbon to the proximal ring proton is maintained. The sequence is broadband in the sense that all acetyl groups simultaneously give good signal transfer to their respective nearest proton neighbors. The (1)H-detected spectra have decent sensitivity and excellent resolution, giving patterns that unambiguously identify common structural subunits in human glycans. Peracetylated maltitol is shown as a test case of the method. Lineshapes are pure absorption, allowing facile measurement of vicinal proton-proton couplings. Linkage points can be deduced, and the 2D correlation spectra may be useful for more ambitious prediction algorithms and machine identification by a spectral database.

"Ersatz" and "hybrid" NMR spectral estimates using the filter diagonalization method.

The filter diagonalization method (FDM) is an efficient and elegant way to make a spectral estimate purely in terms of Lorentzian peaks. As NMR spectral peaks of liquids conform quite well to this model, the FDM spectral estimate can be accurate with far fewer time domain points than conventional discrete Fourier transform (DFT) processing. However, noise is not efficiently characterized by a finite number of Lorentzian peaks, or by any other analytical form, for that matter. As a result, noise can affect the FDM spectrum in different ways than it does the DFT spectrum, and the effect depends on the dimensionality of the spectrum. Regularization to suppress (or control) the influence of noise to give an "ersatz", or EFDM, spectrum is shown to sometimes miss weak features, prompting a more conservative implementation of filter diagonalization. The spectra obtained, called "hybrid" or HFDM spectra, are acquired by using regularized FDM to obtain an "infinite time" spectral estimate and then adding to it the difference between the DFT of the data and the finite time FDM estimate, over the same time interval. HFDM has a number of advantages compared to the EFDM spectra, where all features must be Lorentzian. They also show better resolution than DFT spectra. The HFDM spectrum is a reliable and robust way to try to extract more information from noisy, truncated data records and is less sensitive to the choice of regularization parameter. In multidimensional NMR of liquids, HFDM is a conservative way to handle the problems of noise, truncation, and spectral peaks that depart significantly from the model of a multidimensional Lorentzian peak.

Sensitive, quantitative carbon-13 NMR spectra by mechanical sample translation.

Collecting a truly quantitative carbon-13 spectrum is a time-consuming chore. Very long relaxation delays, required between transients to allow the z-magnetization, M(z), of the spin with the longest T(1) to return to the equilibrium value, M(0), must precede each transient. These long delays also reduce sensitivity, as fewer transients per unit time can be acquired. In addition, sometimes T(1) is not known to within even a factor of two: a conservative guess for the relaxation delay then leads to very low sensitivity. We demonstrate a fresh method to bypass these problems and collect quantitative carbon-13 spectra by swapping the sample volume after each acquisition with a different portion where the magnetization is already equilibrated to M(0). Loading larger sample volumes of 10-20 mL into an unusually long (1520 mm) 5 mm OD. NMR tube and vertically sliding the tube between acquisitions accomplishes the swap. The relaxation delay can then be skipped altogether. The spectra are thus both quantitative, and far more sensitive. We demonstrate the moving tube technique on two small molecules (thymol and butylhydroxytoluene) and show good carbon-13 quantification. The gain in sensitivity can be as much as 10-fold for slowly-relaxing (13)C resonances. These experiments show that quantitative, sensitive carbon-13 spectra are possible whenever sufficient sample volumes are available. The method is applicable to any slow-relaxing nuclear spin species, such as (29)Si, (15)N and other low-gamma nuclei.

Enhanced spectral resolution by high-dimensional NMR using the filter diagonalization method and "hidden" dimensions.

High-dimensional (HD) NMR spectra have poorer digital resolution than low-dimensional (LD) spectra, for a fixed amount of experiment time. This has led to "reduced-dimensionality" strategies, in which several LD projections of the HD NMR spectrum are acquired, each with higher digital resolution; an approximate HD spectrum is then inferred by some means. We propose a strategy that moves in the opposite direction, by adding more time dimensions to increase the information content of the data set, even if only a very sparse time grid is used in each dimension. The full HD time-domain data can be analyzed by the filter diagonalization method (FDM), yielding very narrow resonances along all of the frequency axes, even those with sparse sampling. Integrating over the added dimensions of HD FDM NMR spectra reconstitutes LD spectra with enhanced resolution, often more quickly than direct acquisition of the LD spectrum with a larger number of grid points in each of the fewer dimensions. If the extra-dimensions do not appear in the final spectrum, and are used solely to boost information content, we propose the moniker hidden-dimension NMR. This work shows that HD peaks have unmistakable frequency signatures that can be detected as single HD objects by an appropriate algorithm, even though their patterns would be tricky for a human operator to visualize or recognize, and even if digital resolution in an HD FT spectrum is very coarse compared with natural line widths.

Minimizing the overlap problem in protein NMR: a computational framework for precision amino acid labeling.

MOTIVATION: Recent advances in cell-free protein expression systems allow specific labeling of proteins with amino acids containing stable isotopes ((15)N, (13) C and (2)H), an important feature for protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. Given this labeling ability, we present a mathematical optimization framework for designing a set of protein isotopomers, or labeling schedules, to reduce the congestion in the NMR spectra. The labeling schedules, which are derived by the optimization of a cost function, are tailored to a specific protein and NMR experiment. RESULTS: For 2D (15)N-(1)H HSQC experiments, we can produce an exact solution using a dynamic programming algorithm in under 2 h on a standard desktop machine. Applying the method to a standard benchmark protein, calmodulin, we are able to reduce the number of overlaps in the 500 MHz HSQC spectrum from 10 to 1 using four samples with a true cost function, and 10 to 4 if the cost function is derived from statistical estimates. On a set of 448 curated proteins from the BMRB database, we are able to reduce the relative percent congestion by 84.9% in their HSQC spectra using only four samples. Our method can be applied in a high-throughput manner on a proteomic scale using the server we developed. On a 100-node cluster, optimal schedules can be computed for every protein coded for in the human genome in less than a month. AVAILABILITY: A server for creating labeling schedules for (15)N-(1)H HSQC experiments as well as results for each of the individual 448 proteins used in the test set is available at http://nmr.proteomics.ics.uci.edu.

SOGGY: solvent-optimized double gradient spectroscopy for water suppression. A comparison with some existing techniques.

Excitation sculpting, a general method to suppress unwanted magnetization while controlling the phase of the retained signal [T.L. Hwang, A.J. Shaka, Water suppression that works. Excitation sculpting using arbitrary waveforms and pulsed field gradients, J. Magn. Reson. Ser. A 112 (1995) 275-279] is a highly effective method of water suppression for both biological and small molecule NMR spectroscopy. In excitation sculpting, a double pulsed field gradient spin echo forms the core of the sequence and pairing a low-power soft 180 degrees (-x) pulse with a high-power 180 degrees (x) all resonances except the water are flipped and retained, while the water peak is attenuated. By replacing the hard 180 degrees pulse in the double echo with a new phase-alternating composite pulse, broadband and adjustable excitation of large bandwidths with simultaneous high water suppression is obtained. This "Solvent-Optimized Gradient-Gradient Spectroscopy" (SOGGY) sequence is a reliable workhorse method for a wide range of practical situations in NMR spectroscopy, optimizing both solute sensitivity and water suppression.

From gene to HSQC in under five hours: high-throughput NMR proteomics.

A simple, rapid, in vitro cell-free protein expression system, Expressway NMR, is introduced and used to express the small ubiquitin-related modifier protein SUMO-1. This 12 kDa molecule is challenging for NMR as it has limited solubility and requires relatively high salt (200 mM) for stability in solution. Starting with the gene, the cell-free system, and milligram amounts of nitrogen-15 isotopically enriched amino acids, sufficient protein is produced in 4 h to obtain a high-resolution 2D HSQC spectrum of the protein in 40 min. This time would be closer to 10 min with the aid of a higher sensitivity salt-tolerant cryogenic NMR probe. With all protein purification steps included, and aggressive data processing using the filter diagonalization method (FDM), it is but 6 h from gene to heteronuclear single quantum coherence (HSQC). As the cell-free system is nearly background-free, it is also possible to work with the crude reaction mixture, in which case only a total of 5 h is required. Sample stability over time, whether crude extract or purified, was notable, with no significant change in the 15N-1H HSQC spectrum over 6 months at 4 degrees C (300 muM, pH 6.1, capped NMR tube). The combination of a turnkey, high-yield, protease-free in vitro protein expression system, an optimized sensitivity-enhanced HSQC pulse sequence, and FDM processing makes this scheme an attractive first step to rapidly assess the suitability of proteins for complete solution structure determination.

Rapid high-resolution four-dimensional NMR spectroscopy using the filter diagonalization method and its advantages for detailed structural elucidation of oligosaccharides.

Four-dimensional nuclear magnetic resonance spectroscopy with high resolution of signals in the indirect dimensions is reported as an implementation of the filter diagonalization method (FDM). Using an oligosaccharide derivatized with 13C-labeled acetyl isotags, a four-dimensional constant-time pulse sequence was tailored for conjoint use with the FDM. Results demonstrate that high resolution in all dimensions can be achieved using a relatively short experimental time period (19 h), even though the spectrum is highly congested in the direct and all three indirect dimensions. The combined use of isotags, constant-time pulse sequences, and FDM permits rapid isolation of sugar ring proton spin systems in multiple dimensions and enables all endocyclic J-couplings to be simply measured, the key goal to assigning sugar stereochemistry and anomeric configuration. A general method for rapid, unambiguous elucidation of spin systems in oligosaccharides has been a long-sought goal of carbohydrate NMR, and isotags combined with the FDM now enable this to be easily performed. Additional general advantages of the FDM program for generating high-resolution 2D slices in any dimension from a 4D spectrum are emphasized.

Rapid 3D NMR using the filter diagonalization method: application to oligosaccharides derivatized with 13C-labeled acetyl groups.

Rapid 3D NMR spectroscopy of oligosaccharides having isotopically labeled acetyl "isotags" was made possible with high resolution in the indirect dimensions using the filter diagonalization method (FDM). A pulse sequence was designed for the optimal correlation of acetyl methyl protons, methyl carbons, and carbonyl carbons. The multi-dimensional nature of the FDM, coupled with the advantages of constant-time evolution periods, resulted in marked improvements over Fourier transform (FT) and mirror-image linear prediction (MI-LP) processing methods. The three methods were directly compared using identical data sets. A highly resolved 3D spectrum was achieved with the FDM using a very short experimental time (28 min).

Ultra-high resolution 3D NMR spectra from limited-size data sets.

The advantage of the filter diagonalization method (FDM) for analysis of triple-resonance NMR experiments is demonstrated by application to a 3D constant time (CT) HNCO experiment. With a 15N-,13C-labeled human ubiquitin sample (1.0 mM), high spectral resolution was obtained at 500 MHz in 25 min with only 6-8 increments in each of the CT dimensions. This data set size is about a factor of 50-100 smaller than typically required, yet FDM analysis results in a fully resolved spectrum with a sharp peak for each HNCO resonance. Unlike Fourier transform (FT) processing, in which spectral resolution in each dimension is inversely proportional to the acquisition time in this dimension, FDM is a true multi-dimensional method; the resolution in all dimensions is determined by the total information content of the entire signal. As the CT dimensions of the 3D HNCO signal have approximate time-reversal symmetry, they can each be doubled by combining the usual four hyper-complex data sets. This apparent quadrupling of the data is important to the success of the method. Thus, whenever raw sensitivity is not limiting, well-resolved n-dimensional spectra can now be obtained in a small fraction of the usual time. Alternatively, to maximize sensitivity, evolution periods of faster relaxing nuclei may be radically shortened, the total required resolution being obtained through chemical shift encoding of other, more slowly relaxing, spins. Improvements similar to those illustrated with a 3D HNCO spectrum are expected for other triple-resonance spectra, where CT evolution in the indirect dimensions is implemented.

Cascaded z-filters for efficient single-scan suppression of zero-quantum coherence.

A simple and robust method to suppress zero-quantum coherence (ZQC) in NMR experiments, in a single scan and with very high suppression ratio, is described. It is an appreciable improvement on a previous technique by Thrippleton and Keeler [Angew. Chem. Int. Ed. 42 (2003) 3938]. The method, called a z-filter cascade, preserves longitudinal, or z-magnetization, with high efficiency. Losses depend mostly on T1 relaxation but not T2 relaxation mechanisms. At the same time, suppression of ZQC can be essentially complete in a single scan. The time duration of the z-filter cascade scales inversely to representative chemical shift differences between the coupled spins, and is typically a few tens of milliseconds. The high efficiency of the zero-quantum suppression and excellent retention of the desired z-magnetization, in a single scan without resort to phase cycling or difference spectroscopy, makes the z-filter cascade a useful new pulse sequence building block for a whole range of NMR experiments. In cases where unwanted residual ZQC may have previously contributed to baseline " t1-noise" in two-dimensional NMR spectra, the z-filter cascade can deliver a noteworthy improvement in spectral quality.

Regularized resolvent transform for direct calculation of 45 degrees projections of 2D J spectra.

The regularized resolvent transform (RRT) has been applied in a novel way to J-resolved spectra. This involves the direct calculation of the 45 degrees projection without constructing the 2D spectrum. The results show a significant resolution enhancement over that obtained by the 45 degrees projection of a 2D Fourier spectrum, even for much larger signals. In particular, RRT is able to resolve peaks that belong to different overlapping multiplets in a very crowded spectral region, where the conventional technique fails for any signal size. The resolving power of this method along with the significantly shorter signals required, make this method a powerful tool in spectral assignment.

Processing DOSY spectra using the regularized resolvent transform.

A new method for processing diffusion ordered spectroscopy (DOSY) data is presented. This method, the regularized resolvent transform (iRRT-the i denoting the adaptation of the method to evaluate the inverse Laplace transform), is better than conventional processing techniques for generating 2D DOSY spectra using data that has poor chemical shift resolution. From the same data, it is possible to use the iRRT to generate 1D subspectra corresponding to different components of the sample mixture; these subspectra compare favorably to 1D spectra of the pure substances. Both the 2D spectra and the 1D subspectra offer a vast improvement over results generated using a conventional processing technique (non-linear least-squares fitting). Consequently, we present the iRRT as a stable and reliable tool for solving the inverse Laplace transform problem present in experiments such as DOSY.

Progress on the two-dimensional filter diagonalization method. An efficient doubling scheme for two-dimensional constant-time NMR.

An efficient way to treat two-dimensional (2D) constant-time (CT) NMR data using the filter diagonalization method (FDM) is presented. In this scheme a pair of N- and P-type data sets from a 2D CT NMR experiment are processed jointly by FDM as a single data set, twice as large, in which the signal effectively evolves in time for twice as long. This scheme is related to "mirror-image" linear prediction, but with the distinction that the data are directly used, without any preprocessing such as Fourier transformation along one dimension, or point-by-point reflection. As the signal has nearly perfect Lorentzian line shape in the CT dimension, it can be efficiently handled by the FDM approach. Applied to model and experimental signals, the scheme shows significant resolution improvement, and appears to tolerate noise reasonably well. Other complex aspects of multidimensional FDM are discussed and illustrated.

Adjustable, broadband, selective excitation with uniform phase.

An advance in the problem of achieving broadband, selective, and uniform-phase excitation in NMR spectroscopy of liquids is outlined. Broadband means that, neglecting relaxation, any frequency bandwidth may be excited even when the available radiofrequency (RF) field strength is strictly limited. Selective means that sharp transition edges can be created between pure-phase excitation and no excitation at all. Uniform phase means that, neglecting spin-spin coupling, all resonance lines have nearly the same phase. Conventional uniform-phase excitation pulses (e.g., E-BURP), mostly based on amplitude modulation of the RF field, are not broadband: they have an achievable bandwidth that is strictly limited by the peak power available. Other compensated pulses based on adiabatic half-passage, like BIR-4, are not selective. By contrast, inversion pulses based on adiabatic fast passage can be broadband (and selective) in the sense above. The advance outlined is a way to reformulate these frequency modulated (FM) pulses for excitation, rather than just inversion.