Spectrométrie de masse des modifications induites ou post-traductionnelles de protéines : méthodologie et application à des protéines d’intérêt thérapeutique

Abstract : Protein modifications, whether post-translational (PTMs) or chemically induced, play a crucial role on the activity of proteins. Mass spectrometry (MS) techniques such as HRMS, CID/ETD MS/MS, and biochemistrybased methods for structural and kinetic characterization of protein-ligand complexes and PTMs have been developed. MS combined with several biochemical tools has been used to sequence the proteinase inhibitor gregline and to detect a novel PTM. A similar approach shows that the transposase MOS1, a model for the design of HIV integrase inhibitors, is both phosphorylated and acetylated. For the lyase Abf2, a strategy of trapping, purification, proteolysis, and DNA hydrolysis of the Abf2-DNA covalent complex, coupled to MS analysis, has been developed. Finally, the interaction between the metastasis suppressor hPEBP1 and locostatin was dissected. Upon binding to hPEBP1, locostatin undergoes hydrolysis. To identify the site targeted by locostatin, the conditions of reaction and proteolysis were optimized. The qualitative approach reveals the presence of non-specific reactions, leading to the development of 1) a mathematical model to determine the optimum bound fraction for discriminating the specific site from non-specific sites, and 2) a method for the parallel and exhaustive quantification of the degree of modification of all modified sites of a protein. These tools are widely applicable to covalent protein ligands and/or PTMs.
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Guillaume Gabant. Spectrométrie de masse des modifications induites ou post-traductionnelles de protéines : méthodologie et application à des protéines d’intérêt thérapeutique. Biochimie, Biologie Moléculaire. Université d'Orléans, 2014. Français. ⟨NNT : 2014ORLE2061⟩. ⟨tel-01246054⟩

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