Unit: Proteomics

Director: NAMANE Abdelkader

The most important 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, as well as service activities.

The Proteomics platform, component of the Pasteur-Génopole®, is part of the Department of Structural Biology and Chemistry. Its objective is to provide a powerful technology for two-dimensional electrophoresis and mass spectrometry for the scientific community. The two-dimensional gel electrophoresis is still the most popular technique for soluble protein separation. Since 2002, we have implemented the "Immobiline" procedure in order to optimize the separation of proteins in narrow pH ranges or at basic pH values. It complemented the "Ampholine" method standardized and optimized for proteins originating from various bacterial or eukaryote cells.

With regard to mass spectrometry, we are equipped with three instruments: 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 enabled us to carry out structural characterisations of biomolecules and protein identifications in databases after in-gel digestion. The last acquired QqTOF instrument will give us access to the up-to-date proteomics approaches using the mono or multidimensional liquid chromatography coupled with tandem mass spectrometry.

Two proteomics approaches were followed during the year 2003. The "global" analysis allows visualization and identification of the variations in the whole proteome. In Photorhabdus luminescens we explored the regulation of metabolism with the control of the infectious process, and we identified proteins involved in these mechanisms (J.F. Charles, Genetics of Bacterial Genomes). The same approach was followed to identify proteins implied in the regulator expression PhoP/PhoR from Mycobacterium tuberculosis (M. Jackson and B. Gicquel, Mycobacterial Genetics). Using 2D electrophoresis, comparative analyses of the protein patterns from various mutants of the Notch1a factor were carried out to identify phosphorylated residues in the PEST C-terminus domain (N. Gupta, Molecular Biology of the Gene).

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. After HPLC purification, we carried out structural studies of peptidoglycan fragments from Neisseria meningitidis, a human pathogen. About 28 muropeptides have been characterised (I Boneca, Pathogenesis of Mucosal Bacteria). We have also been interested in the proteolysis sites of the Urokinase receptor (CD87/uPAR) by proteinases of physiopathological interest in order to better understand the expression of the associated CD87 chimiotactic activity (D Pidard, Innate Host Defense and Inflammation). We began the nuclear and mitochondrial partner identification of the pro-aptotic protein AIF (Apoptosis Inducing Factor), isolated by immunoprecipitation (S. Susin, Apoptosis and Immune System). Otherwise, using conditional mutants in yeast followed by selective purifications with the TAP (tandem Affinity Purification) approach, we identified proteins involved in the pre-60s maturation and we began to understand their assembly order. We have identified about 50 partners. Mass spectrometry quantitative approaches are going to be applied using stable isotopes: the ICAT (Isotope-Coded Affinity Tag) and the SILAC (Stable-Isotopes Labelling with Amino acids in Cell culture) (A. Jacquier, Genetics of the Macromolecular Interactions).

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, protein identifications

Activity Reports 2003 - Institut Pasteur

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