heteronuclear single quantum coherence spectra

HETERONUCLEAR SINGLE QUANTUM COHERENCE SPECTRA FREE

Recent technical advances with higher magnetic fields and the introduction of cryogenic probes have drastically increased the sensitivity and thereby reduced experimental times for inverse detection experiments of other nuclei such as 13C and 31P. Ī vast majority of NMR-based metabolomics studies have been based on 1D 1H NMR experiments because of the high sensitivity of the 1H nucleus. The drawback of NMR spectroscopy is the inherent low sensitivity compared to MS-based methods, but it has nevertheless become a cornerstone in metabolomic studies. Most important, NMR spectroscopy is highly reproducible, does not require any sample derivatization and gives detailed structural information about the components of a mixture. NMR (nuclear magnetic resonance) spectroscopy is well suited for analysis of complex mixtures of organic compounds and has some distinct advantages compared to other analytical techniques such as GC-MS (gas chromatography–mass spectrometry) and LC-MS (liquid chromatography-mass spectrometry). The correlation plots highlight cross-peaks belonging to each individual compound, not limited by long-range magnetization transfer as conventional NMR experiments. ConclusionsĬorrelation plots prepared by statistically correlating 1H- 13C Heteronuclear Single Quantum Correlation spectra from human biofluids provide unambiguous identification of metabolites. The identities of these metabolites were confirmed by comparing the correlation plots with reported NMR data, mostly from the Human Metabolome Database. Resultsįrom 50 1H- 13C Heteronuclear Single Quantum Correlation spectra, 23 correlation plots resembling pure metabolites were constructed. In this paper, we demonstrate how a similar approach can be used to identify metabolites in human biofluids (post-prostatic palpation urine). Similar correlation of 2D 1H- 13C Heteronuclear Single Quantum Correlation spectra was recently applied to investigate the structure of heparine. For the identification of individual metabolites in metabolomics, correlation or covariance between peaks in 1H NMR spectra has previously been successfully employed. NMR spectroscopy is an excellent technique for analysis of mixtures of organic compounds and gives a detailed chemical fingerprint of most individual components above the detection limit. All rights reserved.Identification of individual components in complex mixtures is an important and sometimes daunting task in several research areas like metabolomics and natural product studies. Our results agree with those from studies using different techniques, providing additional evidence for the utility of 19F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.Ĭopyright © 2020 Biophysical Society. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. We also measured binding as a function of temperature.

HETERONUCLEAR SINGLE QUANTUM COHERENCE SPECTRA FREE

The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. Their agreement demonstrates that one-dimensional 19F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. We then compare the results from lineshape analysis of one-dimensional 19F spectra to those from two-dimensional 1H- 15N heteronuclear single quantum coherence spectra. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. Here, we use one-dimensional 19F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common.

Heteronuclear single quantum coherence spectra