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 for the scientific community, a powerful technology in two-dimensional electrophoresis and mass spectrometry.

The two-dimensional gel electrophoresis is still the most popular technique in soluble protein separation. Since 2002, we have implemented the "immobilines" procedure in order to optimize the separation of proteins in narrow pH ranges or at basic pH values. It complemented the "ampholines" method standardized and optimized for proteins originating from various bacterial or eukaryote cells. Improvements in sample preparations (fractionation, cleaning kits) were initiated during this year.

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), acquired in 2003. The two first instruments allowed us structural characterisations of biomolecules and protein identifications in databases after in-gel digestion. The most recently acquired QqTOF instrument will give us access to the up-to-date proteomics approaches, mainly the shotgun proteomics approach, using single or multidimensional liquid chromatography coupled with the tandem mass spectrometry, nD LC-MSMS.

Three proteomics approaches were applied during the year 2004. Comparative approaches allow the visualization and the identification of variations in protein expression in the whole proteome. In bacteria-host interactions between Photorhabdus luminescens and Bombyx mori, we identified proteins involved in the control of the infectious process in insects (JF Charles, Genetics of Bacterial Genomes). With global proteomics approaches we can establish a protein inventory from a microorganism. A Proteome study of Mycobacterium ulcerans is in progress including identifications of soluble proteins, membrane proteins and exported proteins. This study uses Maldi-tof-MS experiments for protein identifications by peptide mass fingerprinting and nD LC-MSMS experiments for shogun proteomics (Gilles Reysset, Bacterial Molecular Genetics).The "targeted" approach implies an initial 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. After purification by CLHP, we have continued structural studies of peptidoglycan fragments from human pathogen such as Helicobacter pylori and Listeria monocytogenes (Ivo Boneca, Pathogenesis of Mucosal Bacteria). We have also continued the study of proteolysis sites of the Urokinase receptor (CD87/uPAR) by proteinases of physiopathological interest for better understanding the expression of the associated CD87 chimiotactic activity (D. Pidard, Innate Host Defense and Inflammation). Besides this, using conditional mutants in yeast followed by selective purifications of protein complexes with the TAP (Tandem Affinity Purification) approach, we identified proteins involved in the pre-60s maturation and we began to understand their order of assembly. We have identified about 50 partners. Mass spectrometry-based quantitative proteomics approaches are applied using stable isotopes: the ICAT (Isotope-Coded Affinity Tag) and the SILAC (Stable Isotopes Labelling with Amino acids in Cell culture). The preliminary results are very encouraging (A. Jacquier, Genetics of the Macromolecular Interactions).

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

Activity Reports 2004 - Institut Pasteur

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