Unit: Proteomics (Platform)
Director: Namane Abdelkader
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 platform, component of the "Pasteur-Génopole® Ile-de-France", is part of the Department of Structural Biology and Chemistry. Its objective is to provide a powerful technology in two-dimensional electrophoresis and mass spectrometry to the scientific community.
The two-dimensional gel electrophoresis is still the most popular technique separation in the soluble protein field. This year, notable improvements have been made in sample preparations (extraction buffers, enrichment processes, cleaning kits), and new focusing programs were standardized. Implementation of protein fractionation before electrophoresis using the Zoom Fractionator (Invitrogen) of protein staining by fluorescent dyes (Sypro Ruby and ProQ Diamond) were carried out.
With regard to mass spectrometry, we are equipped with three instruments, which are, in the acquisition rank, an electrospray triple quadrupole instrument, API 365 (ABI-MDS-SCIEX), a Maldi-TOF instrument, Voyager DE-STR (ABI-PerSeptive Biosystems), and a QqTOF instrument, QSTAR XL (ABI-MDS-SCIEX). The two first instruments allow us structural characterisations of biomolecules and protein identifications in databases after in-gel digestion. The hybrid instrument, QSTAR XL enables us to reach the proteomic approach using the mono or multidimensional liquid chromatography coupled with tandem mass spectrometry (MDLC-MSMS) or "shotgun proteomics").
Three types of proteomics approaches using all technologies available on the platform have been used. The global proteomics approach allows carrying out an inventory of the protein contents. By instance, the study of the Mycobacterium ulcerans proteome is ongoing in several steps consisted by the identification of soluble proteins, protein membranes and secreted proteins. Theses protein identifications are made by Peptide Mass Fingerprinting (PMF) using 2D-gel separations associated with Maldi-tof or by MDLC-MSMS approach (Gilles Reysset, Bacterial Molecular Genetics). Comparative proteomics approaches allow the visualization and the identification of variations in protein expression. We studied the variation of protein expression in mosquito salivary glands infected or not infected by Plasmodium falciparum, using firstly the classical 2D-gels approach and secondly the iTRAQ approach (Applied Biosystems) coupled with LC-MSMS (V Choumet, GPH Anopheles).
The "targeted" proteomics implies initial protein enrichment by various methodologies. This approach was widely used for different projects in the platform. Thus, the Candida albicans phosphoproteome study was initiated by electrophoresis and will be developed in 2006 (S Goyard, Fungal Biology and Pathogenicity). We have got on the structural study of peptidoglycan fragments from human pathogens such as Helicobacter pylori and Listeria monocytogenes. (Ivo BONECA, Pathogenesis of Mucosal Bacteria). We also identified proteins obtained by immunoprecipitation from people suffering from brain malaria (S Pied, Immunophysiopathology of Infections).
Selective purifications of protein complexes issued from yeast conditional mutants with the TAP (Tandem Affinity Purification) approach lead to the identification of protein involved in pre-60s maturation. So we began to understand the assembly order of proteins. Mass spectrometry-based quantitative proteomics approaches using stable isotopes are applied such as the iTRAQ coupled with LC-MSMS and the SILAC coupled with the Maldi-Tof. These quantitative results are very encouraging to understand the protein order assembly within the maturation process (A. Jacquier, Molecular Interaction Genetics). Moreover, an approach of protein identifications in metabolic networks by the TAP method is also applied to protein analyses from Helicobacter pylori (H of Reuse, Pathogenesis of Mucosal Bacteria).
In parallel to our scientific activity, which involves the Proteomics platform in many collaborative projects, we are also dedicated to service activities.
Keywords: proteomics, electrophoresis, mass spectrometry, LC-MSMS, proteins