DÉVELOPPEMENT DE NOUVELLES MÉTHODOLOGIES PAR SPECTROMÉTRIE DE MASSE A TRÈS HAUTE RÉSOLUTION POUR L'ANALYSE STRUCTURALE DE COMPLEXES PROTÉIQUES

Abstract : The association of hydrogen/deuterium exchange and mass spectrometry (HDX/MS) has been emerged as a powerful analytical tool for probing structural and dynamic features of proteins. In classical bottom-up approach, the deuterated protein is digested by pepsin and the proteolytic digest is analyzed by mass spectrometry. Unfortunately, this approach suffers from two main drawbacks: (1) the resolution, which is limited by the size of the peptides after digestion and (2) the back-exchange that can take place in solution before the analysis by mass spectrometry. Classical tandem mass spectrometry cannot be used to improve resolution since Collision Induced Dissociation (CID) experiments on deuterated peptides have been shown for intramolecular deuterium migration before backbone cleavage ("scrambling"). Therefore, there is a great interest in the development of alternative HDX/MS approaches in order to improve back-exchange, scrambling and resolution problems. In this work, a complete optimized workflow for robust top-down HDX/MS has been set up. New system has been developed for introduction of the deuterated samples in the mass spectrometer, named "cryosource" and electron-based activation techniques, Electron Capture/Transfer Dissociation, were used. ECD and ETD have been shown recently to fragment, in proper experimental conditions, entire deuterated proteins with minimized scrambling and single residue resolution. The cryosource leads to negligible back-exchange during long periods of time and allows low flow rates. Optimization of all parameters was done using model peptides and protein. Our final goal was to use this methodology for the structural analysis of the complex aIF2.
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Submitted on : Monday, November 12, 2012 - 12:04:01 PM
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Ophélie Robine. DÉVELOPPEMENT DE NOUVELLES MÉTHODOLOGIES PAR SPECTROMÉTRIE DE MASSE A TRÈS HAUTE RÉSOLUTION POUR L'ANALYSE STRUCTURALE DE COMPLEXES PROTÉIQUES. Chimie analytique. Ecole Polytechnique X, 2012. Français. ⟨pastel-00750754⟩

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