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Miniaturisation de la préparation d'échantillon en protéomique bottom-up pour la quantification de l'oxydation des cystéines

Abstract : Bottom-up proteomics is the most commonly used approach for protein analysis by mass spectrometry. In this approach, enzymatic digestion is one step of a long and tedious sample preparation protocol. Specific care is needed to study post-translational changes, such as cysteine oxidation, with additional steps which makes the protocol even more complex. The OcSILAC protocol, developed in our laboratory for quantifying cysteine oxidation, is a perfect illustration of this. The miniaturization of the experimental setup is an opportunity for the OcSILAC protocol to be less time and sample consuming. It is the objective of my thesis project.A microfluidic device, inspired by the Filter Aided Sample Preparation (FASP) method, was developed during this project. It is a PDMS device, incorporating a regenerated cellulose molecular filtration membrane and manufactured by gentle photolithography. In the literature, the majority of microfluidic devices dedicated to proteomics stops at proof of concept on standard reference proteins. The microchip developed during this thesis allowed the analysis of biological complex protein samples: with ten times less sample and an eight-times shorter protocol than conventional procedures. Our ChipFilter Proteolysis protocol can identify more proteins and more peptides than the previously reported FASP method. For the miniaturization of the OcSILAC protocol, the development of a biotinylated peptide enrichment unit was started using avidin magnetic beads
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Submitted on : Friday, November 13, 2020 - 11:34:12 AM
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Massamba Ndiaye. Miniaturisation de la préparation d'échantillon en protéomique bottom-up pour la quantification de l'oxydation des cystéines. Chimie analytique. Université Paris sciences et lettres, 2019. Français. ⟨NNT : 2019PSLET064⟩. ⟨tel-03003473⟩

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