NMRTEC

Illkirch-Graffenstaden, France
Illkirch-Graffenstaden, France

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Vieville J.M.P.,French National Center for Scientific Research | Charbonnier S.,CNRS Biotechnology and Cell Signaling Laboratory | Eberling P.,French National Center for Scientific Research | Starck J.-P.,NMRTEC | Delsuc M.-A.,French National Center for Scientific Research
Journal of Pharmaceutical and Biomedical Analysis | Year: 2014

Non covalent grafting of proteins on affinity phases is a very common approach for isolation, purification and re-concentration of tagged proteins. Many biophysical studies are conducted on these grafted proteins (surface plasmon resonance, quartz crystal microbalance, etc.) showing that the integrity and function of the protein is usually maintained. However, NMR studies of such samples were not undertaken so far, due to the broadening observed on this kind of heterogeneous samples.We present here the use of the HR-MAS technology to obtain 2D NMR spectra of the MAGI-1 PDZ2/6 protein domain, C13-labeled, tagged with a His-tag and grafted on a Nickel affinity resin. We optimized the C13 Methyl SOFAST HMQC experiment allowing important gains in terms of signal-to-noise. The gain comes from the gathering of proton magnetization from the resin material to the protein under study.Several methyl signals from the unstructured C-terminal tail, which is involved in the binding of the PDZ domain to C-terminal peptides of its partners, were observed and measured. The interaction of the bound PDZ domain with cognate peptides was monitored using <500μg of protein sample. A response proportional to the peptide Kd is obtained, indicating that the method can be used to rapidly and efficiently monitor protein-ligand interactions. © 2013 Elsevier B.V.


PubMed | French National Center for Scientific Research, CNRS Biotechnology and Cell Signaling Laboratory and NMRTEC
Type: | Journal: Journal of pharmaceutical and biomedical analysis | Year: 2013

Non covalent grafting of proteins on affinity phases is a very common approach for isolation, purification and re-concentration of tagged proteins. Many biophysical studies are conducted on these grafted proteins (surface plasmon resonance, quartz crystal microbalance, etc.) showing that the integrity and function of the protein is usually maintained. However, NMR studies of such samples were not undertaken so far, due to the broadening observed on this kind of heterogeneous samples. We present here the use of the HR-MAS technology to obtain 2D NMR spectra of the MAGI-1 PDZ2/6 protein domain, C13-labeled, tagged with a His-tag and grafted on a Nickel affinity resin. We optimized the C13 Methyl SOFAST HMQC experiment allowing important gains in terms of signal-to-noise. The gain comes from the gathering of proton magnetization from the resin material to the protein under study. Several methyl signals from the unstructured C-terminal tail, which is involved in the binding of the PDZ domain to C-terminal peptides of its partners, were observed and measured. The interaction of the bound PDZ domain with cognate peptides was monitored using <500g of protein sample. A response proportional to the peptide Kd is obtained, indicating that the method can be used to rapidly and efficiently monitor protein-ligand interactions.


PubMed | French National Center for Scientific Research, CASC4DE, University of Warwick and NMRTEC
Type: Comparative Study | Journal: Analytical chemistry | Year: 2016

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) allows the correlation between precursor and fragment ions in tandem mass spectrometry without the need to isolate the precursor ion beforehand. 2D FT-ICR MS has been optimized as a data-independent method for the structural analysis of compounds in complex samples. Data processing methods and denoising algorithms have been developed to use it as an analytical tool. In the present study, the capabilities of 2D FT-ICR MS are explored with a tryptic digest of cytochrome c with both ECD and IRMPD as fragmentation modes. The 2D mass spectra showed useful fragmentation patterns of peptides over a dynamic range of almost 400. By using a quadratic calibration, fragment ion peaks could be successfully assigned. The correlation between precursor and fragment ions in the 2D mass spectra was more accurate than in MS/MS spectra after quadrupole isolation, due to the limitations of quadrupole isolation. The use of the second dimension allowed for successful fragment assignment from precursors that were separated by only m/z 0.0156. The resulting cleavage coverage of cytochrome c almost matched data provided by high-resolution FT-ICR MS/MS analysis, but the 2D FT-ICR MS method required only one experimental scan.


PubMed | Institute Of Genetique Et Of Biologie Moleculaire Et Cellulaire, CNRS Institute of Chemistry, Institute Of Recherches Servier and NMRTEC
Type: | Journal: Analytical biochemistry | Year: 2015

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R2 relaxation of the (19)F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the (19)F signal intensity after an 80-ms spin echo correlates nicely with the EC50, opening a route for NMR screening of GK activators.


Van Agthoven M.A.,Lille University of Science and Technology | Chiron L.,University of Strasbourg | Coutouly M.-A.,NMRTEC | Delsuc M.-A.,University of Strasbourg | Rolando C.,Lille University of Science and Technology
Analytical Chemistry | Year: 2012

2D FT-ICR MS allows the correlation between precursor and fragment ions by modulating ion cyclotron radii for fragmentation modes with radius-dependent efficiency in the ICR cell without the need for prior ion isolation. This technique has been successfully applied to ion-molecule reactions, Collision-induced dissociation and infrared multiphoton dissociation. In this study, we used electron capture dissociation for 2D FT-ICR MS for the first time, and we recorded two-dimensional mass spectra of peptides and a mixture of glycopeptides that showed fragments that are characteristic of ECD for each of the precursor ions in the sample. We compare the sequence coverage obtained with 2D ECD FT-ICR MS with the sequence coverage obtained with ECD MS/MS and compare the sensitivities of both techniques. We demonstrate how 2D ECD FT-ICR MS can be implemented to identify peptides and glycopeptides for proteomics analysis. © 2012 American Chemical Society.


Quinternet M.,University of Strasbourg | Quinternet M.,University of Monastir | Starck J.-P.,NMRtec | Delsuc M.-A.,University of Strasbourg | Kieffer B.,University of Strasbourg
Journal of Pharmaceutical and Biomedical Analysis | Year: 2013

New generations of drugs are using more and more often therapeutic proteins as the active ingredient, prompting the regulation agencies to adapt their analytical methods. Fast and unambiguous information on the secondary, tertiary and quaternary structure of the protein should be provided to assess the quality of these biodrugs. Recent developments of heteronuclear NMR methods, enabling their use on pharmaceutical formulated unlabeled proteins, provide an efficient way to perform such analysis, a feature that is illustrated here using various commercial formulations of insulins. © 2013 Elsevier B.V.


Van Agthoven M.A.,French National Center for Scientific Research | Coutouly M.-A.,NMRTEC | Rolando C.,French National Center for Scientific Research | Delsuc M.-A.,NMRTEC | Delsuc M.-A.,University of Strasbourg
Rapid Communications in Mass Spectrometry | Year: 2011

In two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FTICR-MS), scintillation noise, caused mostly by fluctuations in the number of ions in the ICR cell, is the leading cause for errors in spectrum interpretation. In this study, we adapted an algorithm based on singular value decomposition and first introduced by Cadzow et al. (IEE Proceedings Pt. F 1987, 134, 69) to 2D FTICR-MS and we measured its performance in terms of noise reduction without losing signal information in the 2D mass spectrum. © 2011 John Wiley & Sons, Ltd.


Quinternet M.,University of Strasbourg | Quinternet M.,French National Center for Scientific Research | Starck J.-P.,NMRtec | Delsuc M.-A.,University of Strasbourg | Kieffer B.,University of Strasbourg
Chemistry - A European Journal | Year: 2012

Heteronuclear NMR spectroscopy provides a unique way to obtain site-specific information about protein-ligand interactions. Usually, such studies rely on the availability of isotopically labeled proteins, thereby allowing both editing of the spectra and ligand signals to be filtered out. Herein, we report that the use of the methyl SOFAST correlation experiment enables the determination of site-specific equilibrium binding constants by using unlabeled proteins. By using the binding of L- and D-tryptophan to serum albumin as a test case, we determined very accurate dissociation constants for both the high- and low-affinity sites present at the protein surface. The values of site-specific dissociation constants were closer to those obtained by isothermal titration calorimetry than those obtained from ligand-observed methods, such as saturation transfer difference. The possibility of measuring ligand binding to serum albumin at physiological concentrations with unlabeled proteins may open up new perspectives in the field of drug discovery. So fast so furious: Methyl SOFAST correlation enabled the determination of site-specific equilibrium binding constants with unlabeled proteins. Very accurate dissociation constants were determined in the binding of L- and D-tryptophan to serum albumin (see figure). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Assemat O.,NMRTEC | Coutouly M.-A.,NMRTEC | Hajjar R.,NMRTEC | Hajjar R.,Institute Lavoisier | Delsuc M.-A.,Institute Of Genetique Et Of Biologie Moleculaire Et Cellulaire
Comptes Rendus Chimie | Year: 2010

The validation of molecular mass measurements by using DOSY spectrocopy is presented. A mixture of oligosaccharides has been studied and an extended model has been used. A method has been proposed and applied to correct the imperfections due either to the lock system or the room temperature regulator. © 2009 Académie des sciences.


The invention relates to a method for the comparative analysis and control of the quality of a protein preparation by means of nuclear magnetic resonance (NMR) spectrometry. This method can be used to compare three-dimensional protein conformations in different protein preparations without requiring the samples to undergo any particular preparation. In particular, the method can be used to determine if a selected protein is in the same three-dimensional conformation in different protein preparations, if it is degraded in the formulation or if it is interacting with some of the excipients present. Specifically, the method can be used for the analysis and control of the quality of therapeutic compounds, particularly biodrugs or biosimilars, in different samples, without altering said samples.

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