The Proteomics platform aim is mainly to provide a powerful technology based on 2-D gel electrophoresis and mass spectrometry for the protein identifications and characterisations. Our scientific activity involves in many collaborative programs beside service activities.

The Proteomics platform was created in December 2000 within the framework of the Genopole Institut Pasteur. Our aim is to provide a powerful technology based on 2-D gel electrophoresis and mass spectrometry for the achievement of highly competitive research programs. So far, among available procedures dealing with protein separation, 2-D gel electrophoresis is the technique that provides the highest resolution. Within the platform, the ampholines procedure is standardised and optimised for every protein extract. Furthermore, we are about to set up the immobilines procedure to optimise protein separation in the range of basic pH and to enhance the resolution of protein separation within very narrow pH ranges. With regard to mass spectrometry, we have two complementary devices, the maldi-tof (Voyager DE-STR) and an electrospray triple quadrupole (API 365 Sciex), which allow us to identify proteins from databases following the analysis of tryptic digests from gels electrophoresis.

During the year 2001, in collaboration with other research groups in the campus, we have set up two proteomic approaches to identify proteins: a global approach and a more restricted or targeted one.

Concerning the global approach, in collaboration with P. Bertin (Genetics of bacterial genomes), we performed a study on the effects of low pH on the protein patterns from Vibrio cholerae which has shown that the majority of the variant proteins we identified were part of a functional class which comprises envelope proteins as well as proteins involved in cellular processes. This analysis also allowed us to initiate the first 2-DE map of this proteome. With the same group, we went on to study the mechanisms by which toxins and proteins of Photorhabdus luminescens regulate the infectious process in the insect host. This global approach has also been conducted to identify proteins involved in the processes of virulence among various pathogens, such as Mycobacterium tuberculosis (collaboration with J.M. Reyrat, Mycobacterial genetics) and Staphylococcus aureus (collaboration with B. Fournier, Microbial biochemistry).

The restricted or targeted approach implies that prior to the proteomic analysis, the proteins must be selectively enriched by various methodologies. For example:

• Although underrepresented among the total cellular protein content, the protein fraction from a sub-cellular compartment could be visualised and mapped by appropriate procedures. In collaboration with S. Cole (Bacterial molecular genetics), we continue to identify membrane proteins from Mycobaterium tuberculosis.

• Functional analysis of protein fractions localised in particular sub-cellular compartments is more selective. With S. Mécheri (Immunoallergology), a collaborative work is under way to identify proteins with immunostimulatory potential associated with exosomes stored in murine mast cell granules. These proteins seem to be selectively induced by mast cell treatment with IL-3 or IL-4.

• Using the same approach, in collaboration with N. Guillen (Cell biology of parasitism), the analysis of phagosomes from Entamoeba histolytica, a human parasite, was undertaken to identify proteins involved in phagocytic processes.

• Understanding the complex networks of protein interactions known to be implicated in various regulatory mechanisms is essential. In collaboration with A. Jacquier (Macromolecular interaction genetics), using the TAP (tandem affinity Purification) method, we are currently investigating and analysing the formation of yeast protein-protein complexes.

• Another facet of our activity is dealing with the structural characterisation of bio-molecules. With G. Marchal (Mycobacterial reference laboratory), as part of the PTR program he is in charge of, we set up a collaborative project to characterise the Apa protein which is exported by mycobacteria. While this work is in progress, we have shown that Apa is a protein, which undergoes post-translational modifications due to the presence of mannose residues. After Apa protein has been subjected to proteolysis by subtilisine, the analysis of the resulting digest by on-line liquid chromatography mass spectrometry led to the identification of the potential sites of glycosylation.

In parallel to the scientific activity, which involves the Proteomics platform in many collaborative works, we are also dedicated to service activities.

Rousselle, Jean-Claude, jroussel@pasteur.fr