NMR Chemical Shift Ranges of Urine Metabolites in Various Organic Solvents

Signal stability is essential for reliable multivariate data analysis. Urine samples show strong variance in signal positions due to inter patient differences. Here we study the exchange of the solvent of a defined urine matrix and how it affects signal and integral stability of the urinary metabolites by NMR spectroscopy. The exchange solvents were methanol, acetonitrile, dimethyl sulfoxide, chloroform, acetone, dichloromethane, and dimethyl formamide. Some of these solvents showed promising results with a single batch of urine. To evaluate further differences between urine samples, various acid, base, and salt solutions were added in a defined way mimicking to some extent inter human differences. Corresponding chemical shift changes were monitored

Pure shift amide detection in conventional and TROSY-type experiments of 13^{13}C,15^{15}N-labeled proteins

Large coupling networks in uniformly $^{13}$C,$^{15}$N-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on 13C-BIRDr,X. As a result the full homo- and heteronuclear coupling network can be suppressed at low power leading to real singlets at substantially improved resolution and uncompromised sensitivity. The method is tested on a small globular and an intrinsically disordered protein (IDP) where the average spectral resolution is increased by a factor of ~ 2 and higher. Equally important, the approach works without saturation of water magnetization for solvent suppression and exchanging amide protons are not affected by saturation transfer

SORDOR pulses: expansion of the Böhlen–Bodenhausen scheme for low-power broadband magnetic resonance

A novel type of efficient broadband pulse, called second-order phase dispersion by optimised rotation (SORDOR), has recently been introduced. In contrast to adiabatic excitation, SORDOR-90 pulses provide effective transverse 90∘ rotations throughout their bandwidth, with a quadratic offset dependence of the phase in the x,y plane. Together with phase-matched SORDOR-180 pulses, this enables the Böhlen–Bodenhausen broadband refocusing approach for linearly frequency-swept pulses to be extended to any type of 90$^∘$/180$^∘$ pulse–delay sequence. Example pulse shapes are characterised in theory and experiment, and an example application is given with a $^{19}$F-PROJECT experiment for measuring relaxation times with reduced distortions due to J-coupling evolution

Band-selective universal 90° and 180° rotation pulses covering the aliphatic carbon chemical shift range for triple resonance experiments on 1.2 GHz spectrometers

Biomolecular NMR spectroscopy requires large magnetic field strengths for high spectral resolution. Today’s highest fields comprise proton Larmor frequencies of 1.2 GHz and even larger field strengths are to be expected in the future. In protein triple resonance experiments, various carbon bandwidths need to be excited by selective pulses including the large aliphatic chemical shift range. When the spectrometer field strength is increased, the length of these pulses has to be decreased by the same factor, resulting in higher rf-amplitudes being necessary in order to cover the required frequency region. Currently available band-selective pulses like Q3/Q5 excite a narrow bandwidth compared to the necessary rf-amplitude. Because the maximum rf-power allowed in probeheads is limited, none of the selective universal rotation pulses reported so far is able to cover the full $^{13}$C aliphatic region on 1.2 GHz spectrometers. In this work, we present band-selective 90° and 180° universal rotation pulses (SURBOP90 and SURBOP180) that have a higher ratio of selective bandwidth to maximum rf-amplitude than standard pulses. Simulations show that these pulses perform better than standard pulses, e. g. Q3/Q5, especially when rf-inhomogeneity is taken into account. The theoretical and experimental performance is demonstrated in offset profiles and by implementing the SURBOP pulses in an HNCACB experiment at 1.2 GHz

Determination of Configuration and Conformation of a Reserpine Derivative with Seven Stereogenic Centers Using Molecular Dynamics with RDC‐Derived Tensorial Constraints

NMR-based determination of the configuration of complex molecules containing many stereocenters is often not possible using traditional NOE data and coupling patterns. Making use of residual dipolar couplings (RDCs), we were able to determine the relative configuration of a natural product containing seven stereocenters, including a chiral amine lacking direct RDC data. To identify the correct relative configuration out of 32 possible ones, experimental RDCs were used in three different approaches for data interpretation: by fitting experimental data based singular value decomposition (SVD) using a single alignment tensor and either (i) a single conformer or (ii) multiple conformers, or alternatively (iii) using molecular dynamics simulations with tensorial orientational constraints (MDOC). Even though in all three approaches one and the same configuration could be selected and clear discrimination between possible configurations was achieved, the experimental data was not fully satisfied by the methods based on single tensor approaches. While these two approaches are faster, only MDOC is able to fully reproduce experimental results, as the obtained conformational ensemble adequately covers the conformational space necessary to describe the molecule with inherent flexibility

Urinary NMR Profiling in Pediatric Acute Kidney Injury—A Pilot Study

Acute kidney injury (AKI) in critically ill children and adults is associated with significant short- and long-term morbidity and mortality. As serum creatinine- and urine output-based definitions of AKI have relevant limitations, there is a persistent need for better diagnostics of AKI. Nuclear magnetic resonance (NMR) spectroscopy allows for analysis of metabolic profiles without extensive sample manipulations. In the study reported here, we examined the diagnostic accuracy of NMR urine metabolite patterns for the diagnosis of neonatal and pediatric AKI according to the Kidney Disease: Improving Global Outcomes (KDIGO) definition. A cohort of 65 neonatal and pediatric patients (0–18 years) with established AKI of heterogeneous etiology was compared to both a group of apparently healthy children (n = 53) and a group of critically ill children without AKI (n = 31). Multivariate analysis identified a panel of four metabolites that allowed diagnosis of AKI with an area under the receiver operating characteristics curve (AUC-ROC) of 0.95 (95% confidence interval 0.86–1.00). Especially urinary citrate levels were significantly reduced whereas leucine and valine levels were elevated. Metabolomic differentiation of AKI causes appeared promising but these results need to be validated in larger studies. In conclusion, this study shows that NMR spectroscopy yields high diagnostic accuracy for AKI in pediatric patients

Selective 1Hα NMR methods to reveal functionally relevant proline cis/trans isomers in IDPs

It is important to identify proline cis/trans isomers that appear in several regulatory mechanisms of proteins, and to characterize minor species that are present due to the conformational heterogeneity in intrinsically disordered proteins (IDPs). To obtain residue level information on these mobile systems we introduce two H-1(alpha)-detected, proline selective, real-time homodecoupled NMR experiments and analyze the proline abundant transactivation domain of p53. The measurements are sensitive enough to identify minor conformers present in 4-15 % amounts; moreover, we show the consequences of CK2 phosphorylation on the cis/trans-proline equilibrium. Using our results and available literature data we perform a statistical analysis on how the amino acid type effects the cis/trans-proline distribution. The methods are applicable under physiological conditions, they can contribute to find key proline isomers in proteins, and statistical analysis results may help in amino acid sequence optimization for biotechnological purposes