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Développement d'une approche analytique non ciblée pour l'étude des protéines dans les milieux complexes, environnementaux et biologiques

Abstract : Recent advances in proteomics have been spurred by the rapid development of hybrid and/or high-resolution mass analysers (HRMS/MS). These powerful instrumentations have led to significant improvements in « Bottom-up » approach and have enabled to deepen our knowledge on the functionality of biological systems (structure, function, metabolism, dynamic, etc).Despite their high sensibilities, the potential of such instruments could be significantly lessened by an imperfect sample pre-treatment. In this context, current sample pretreatments follow multi-steps experimental workflows, which alternatively lead to low recoveries of proteins. In this line, this study aims at developing a simple and versatile strategy in order to reduce protein losses and enhance their detection in gel-free LC-MS analysis. First, an analytical method based on liquid chromatography and tandem mass spectrometry was developed to detect and quantify complex peptides mixture. Secondly, a universal, simple and fast purification approach was designed with the aim to purify protein extracts in only one-step. For this purpose, the molecular reactivity, dynamics and conformational changes of proteins at each development step were comprehensively investigated with a set of spectroscopic techniques, in order to select the best strategy. Finally, different factors influencing extraction of proteins were investigated with the goal in the long term to propose an on-demand extraction protocol for direct analysis of proteins by LC-HRMS/MS.
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Balkis Eddhif. Développement d'une approche analytique non ciblée pour l'étude des protéines dans les milieux complexes, environnementaux et biologiques. Chimie analytique. Université de Poitiers, 2017. Français. ⟨NNT : 2017POIT2291⟩. ⟨tel-01951522⟩

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