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Illkirch-Graffenstaden, France

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. Source

Van Agthoven M.A.,Lille University of Science and Technology | Van Agthoven M.A.,University of Warwick | Chiron L.,University of Strasbourg | Coutouly M.-A.,NMRTEC | And 4 more authors.
International Journal of Mass Spectrometry | Year: 2014

2D FT-ICR MS, introduced by Pfändler et al. (Chem. Phys. Lett. 138 (1987) 195), allows one to correlate precursor and fragment ions in complex samples without requiring ion isolation. Recent advances in electronics, computer capacities, and gas-free in-cell fragmentation techniques open up new perspectives for 2D FT-ICR MS as an analytical technique. The pulse sequence consists of two encoding pulses separated by an incremental delay, followed by an observe pulse. In our previous 2D FT-ICR MS work we used three pulses of equal duration and amplitude. However, signal intensity was low because it was distributed over a series of intense harmonics. Using a simple theoretical model to analytically express ion fragmentation and 2D FT-ICR MS ion trajectories, we obtained a nearly pure signal when the maximum radius of the ions during the encoding pulses is within the laser beam. By adjusting the experimental parameters of the encoding pulses according to the calculation on the same cyclotron radius, we strongly decrease the intensity of harmonic peaks. We also discuss the effect of increasing the amplitude of the observe pulse, which affects precursor and fragment ion peaks differently in terms of signal-to-noise ratio. The 2D mass spectra obtained with the optimized pulse sequence show a much higher signal-to-noise ratio, even without using denoising algorithms. © 2014 Elsevier B.V. Source

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. Source

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. Source

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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