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Claire Ramus

New insight methodologies for the differential analysis of membrane proteins

Published on 1 April 2005
Thesis presented April 01, 2005

Abstract:
Study of the dynamic protein expression, which is essential for the understanding of cellular functions and mechanisms, requires the development of differential proteomic strategies. The involvement of membrane proteins in numerous essential cellular processes makes their targeted analysis particularly interesting.
In order to specifically study membrane proteins, over the last few years, proteomics approaches have been developed and are now routinely used in our laboratory. Relying on this experience we undertook the development of different strategies for the quantitative analysis of these previously enriched membrane proteins.
• Firstly, differential isotopic labeling of proteins using chemical reagents such as ICAT was developed to the study of a model membrane protein: the bovine ADP/ATP mitochondrial translocator. The optimized​ strategy, involving labeling proteins in the presence of high concentrations of SDS and urea, was then applied to the quantitative analysis of thylakoid membrane preparations (from Arabidopsis thaliana planta) or of membrane fractions obtained from murine embryonic stem cells. The identification of a membrane protein specifically expressed in totipotent stem cells fully validates our strategy.
• During the course of the extensive study of the Arabidosis thaliana chloroplast envelope membrane, a quantitative protein analysis was also carried out using synthetic trypsic peptides. This strategy was applied to compare the protein expression patterns of envelope proteins from wild type and mutant plants, the latter being characterized by the deletion of the gene coding for the hypothetical IE18 translocator.
The development of these different methods also contributed to the evaluation of new software modules that are currently being developed in the laboratory for quantitative proteomic analyses.

Keywords:
Mass spectrometry, Proteomics, Membrane proteins, Isotope labeling, Protein profiling