Proteomics - CNRS URA 2185  


  HEADDr NAMANE Abdelkader / abdelkader.namane@pasteur.fr
  MEMBERS Dr HEM Sonia / HOURDEL Véronique / LAURENT Christine / LENORMAND Pascal / Dr ROUSSELLE Jean-Claude / ETIENNE Annie


  Annual Report

Proteomics is the study of the subsets of protein present in different parts of an organism and how they change with time and varying conditions.

Two-dimensional electrophoresis (2DE) currently remains the most popular separation technique for complex protein mixtures, and allows proteome mapping and differential protein expression profiling here performed with SameSpots software. Universal or specific staining of proteins is achieved with fluorescent dyes. Among them, we are now using LavaPurple total protein stain since it shows betterperformance in sensitivity, dynamic range, and MS compatibility.

Gel-free proteomics techniques based on mono- or multidimensional liquid chromatography coupled with tandem mass spectrometry (MDLC-MSMS) complement our 2DE techniques. In 2009, a “LTQ Orbitrap velos ETD” mass spectrometer coupled with a nano HPLC was acquired to improve this approach.

Numerous proteomics approaches were carried out in the laboratory. We mainly focused on comparative analyses of changes in relative protein abundance in response to a specific biological condition (growth, mutation, infection). Stable isotope labeling (iTRAQ) followed by LC-ESI-MSMS or LC-MALDI-MSMS and differential 2D analyses were used for this aim.

2DE can visualize protein isoforms, variants and posttranslational modifications. To this end, the combination of narrow range pH gradient with specific immuno-detection by western blotting was applied to the study of phosphorylated isoforms of NRP in various physiological contexts (collaboration with R. Weil and A. Israël, Cell Signaling and Activation) as well as for the detection of phosphorylated isoforms of protein kinase IKKε (collaboration with E.Meurs, Hepacivirus and Innate Immunity).

"Targeted proteomics” involves initial protein enrichment by various methodologies. This approach was widely used for different projects, especially for protein-protein interactions and phosphoproteome analyses.For instance, after several enrichment steps at the peptide level (IMAC, TiO2), LC-MS/MS analyses led to the identification of Leishmaniadonovaniphosphoproteome (collaboration with G Spaeth, Parasite Virulence).

Proteomics can also be used to make a protein inventory in an organism. Using LC-MSMS, we have identified cytosolic and membrane proteins from the flagellum ofTrypanosoma bruceiusing several techniques for sample preparation (collaboration with P Bastin,Trypanosome Cell Biology).

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

Keywords: Proteomics, 2D-electrophoresis, mass spectrometry, LC-MSMS, protein isoforms, signaling, malaria

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  Publications

Kisseleva-Romanova E, Lopreiato R, Baudin-Baillieu A, Rousselle JC, Ilan L, Hofmann K, Namane A, Mann C, Libri D. (2006) Yeast homolog of a cancer-testis antigen defines a new transcription complex. EMBO Journal 25(15):3576-85.

Choumet V, Carmi-Leroy A, Laurent C, Lenormand P, Rousselle JC, Namane A, Roth C, Brey PT. (2007) The salivary glands and saliva of Anopheles gambiae as an essential step in the Plasmodium life cycle: a global proteomic study. Proteomics 7(18):3384-94

Morales MA, Watanabe R, Laurent C, Lenormand P, Rousselle JC, Namane A, Späth GF. (2008) Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages. Proteomics 8(2): 350-63

Stingl K, Schauer K, Ecobichon C, Labigne A, Lenormand P, Rousselle JC, Namane A, De Reuse H. (2008) In Vivo interactome of Helicobacter pylori urease revealed by tandem affinity purification. Molecular and Cellular Proteomics,7(12):2429-41

Tafelmeyer P, Laurent C, Lenormand P, Rousselle JC, Marsollier L, Reysset G, Zhang R, Sickmann A, Stinear TP, Namane A, Cole ST. (2008) Comprehensive Proteome Analysis of Mycobacterium ulcerans and Quantitative Comparison of Mycolactone Biosynthesis. Proteomics, 8(15):3124-38



  Web Site

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Activity Reports 2009 - Institut Pasteur
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