|Director : Abdelkader NAMANE (email@example.com)|
The major aim of the Proteomics platform is to provide a powerful technology for protein identification and characterization based on 2-D gel electrophoresis, liquid chromatography and mass spectrometry. Our activity is based on joint research programs with various units of the campus and service activities.
The Proteomics Technical Platform (TP3), component of the Genopole-Institut Pasteur, is part of the Department of Structural Biology. Its objective is to provide the scientific community with a powerful technology for two-dimensional electrophoresis and mass spectrometry. The two-dimensional gel electrophoresis is still the most popular technique in protein separation. During 2002, we implemented the "Immobiline" procedure in order to optimize the separation of proteins in narrow pH ranges or at basic pH values. It complements the "Ampholine" method standardized and optimized for proteins originating from various bacterial or eukaryote cells. With regard to mass spectrometry, we are equipped with two instruments: one of the maldi-tof type (Voyager DE-STR), while the other is an electrospray triple quadrupole instrument (API 365 Sciex). They allow protein identification in databanks after in-gel trypsin digestion and structural characterization. A hybrid instrument QqTOF (quadrupole time of flight) type (QSTAR XL, Sciex) will be installed in 2003.
Two proteomics approaches were used during the year 2002. The "global" analysis allows visualization and identification of the variations in the whole proteome. In Photorhabdus luminescens we explored the control mechanisms of toxin synthesis and the proteins implicated in the infectious process in the insect host (PH Bertin and JF Charles, Genetics of Bacterial Genomes). The same approach was used to identify proteins participating in the virulence of Mycobacterium tuberculosis (J.M. Reyrat, Mycobacterial Genetics) and of Staphylococcus aureus (B.Fournier, Microbial Biochemistry).
The "targeted" approach implies the preliminary enrichment of proteins of interest by various methods. Thus, analysis of proteins from an isolated cellular compartment enables visualization of low abundance species for a sub proteome inventory. In this direction, we undertook the identification of Mycobacterium tuberculosis membrane proteins (S. Cole, Bacterial Molecular Genetics). The functional analysis of protein fractions with a defined cellular localization is very selective. In collaboration with the Immuno-Allergy Unit (S. Mecheri) we began the protein identification with immunostimulatory potential associated with exosomes stored in murine or human mast cell granules. These proteins are selectively induced by mast cell treatment with IL-3 or IL-4. We have already identified several tens of soluble and membrane proteins. Analysis of the Entamoeba histolytica phagosomes was continued in order to identify proteins implicated in the phagocytosis process of this human parasite (N Guillen, Cell Biology of Parasitism). With this same approach analysis of the cytosolic fractions of murine cells NK is carried out (C Roth, Cytokines and Lymphoid Development).
By using a selective method of purification (Tandem Affinity Purification) we identified several protein complexes in yeast, required for ribosomal RNA maturation (A. Jacquier, Macromolecular Interaction Genetics).
Another side of our activity is related to structural characterization of biomolecules. Within the framework of the PTR 10 leaded by G. Marchal (Mycobacterium Reference Laboratory), we characterized the Apa protein which is exported by mycobacteria in mannosylated form. After subtilisin digestion of Apa protein, samples were analyzed by liquid chromatography coupled with mass spectrometry. We thus highlighted the potential glycosylation sites and the number of mannosylated residues.
In parallel to our scientific activity, which involves the Proteomics platform in many collaborative works, we are also dedicated to service activities.
Keywords: proteomics, electrophoresis, mass spectrometry, analysis, identification, proteins
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