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    Proteomics: 3P5

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    Leaders

     

    The 3P5 proteomics facility is a Paris Descartes University–equipped platform housed both in the Cochin Institute and in the Necker research center. 3P5 was granted the IBiSA national label since 2009 and was certified ISO 9001-compliant since 2012. The team has a very strong expertise on biochemistry and sample preparation techniques dedicated to IEF and LC MS analyses. The platform develops state-of-the-art analysis methods of global protein expression and of specific post translational modifications using the most recent analytical machines. 3P5 is opened to academic and private research teams whatever their geographical localization.

    Equipments:

    The 3P5 proteomic Core facility @ Cochin is fully equipped for discovery-, targeted-, and quantitative proteomics. IEF (Iso Electro Focalisation)-based technologies with 2D-DIGE and NanoProTM nanofluidic immunodetection systems allows fine isoform and proteoform separations for both preparative and analytical analyses. An automated protein digester and 3 high resolution mass spectrometers (LTQ- Orbitrap Velos, Qexactive+, Orbitrap Fusion) hyphenated to u3000 RSLC nanoHPLC chromatographers allow high throughput routine identification and quantification of thousands of proteins out of μg- to sub ng-range of proteins from whole cell lysates to isolated molecular complexes. The informatics equipment allows both a secured storage and performant analyses of the data generated by the different analyzers.

    Provided services

    Identification of proteins is a frequent demand of the research teams. During the past 6 years around 22 000 gel pieces, most of them coming from 2D-DIGE quantitative projects or characterization of purified proteins have been processed. Identification success reaches 100%. Moreover, many projects require the identification of proteins specifically associated with a bait protein (Affinity-purification MS). Although we do not directly perform the purification of the complexes which is operated by the research teams themselves, we provide a strong biochemical expertise for the realization of these projects in addition to the final identification of the purified proteins.

    • Differential proteomics. The platform set up different methods to compare complex proteomes. Among these, 2D-DIGE technology and several gel-free methods including isotopic labeling methods (SILAC and pulsed SILAC) for relative quantification of proteins. Reporter-based quantification techniques such as ITRAQ and TMT chemical labeling methods are also available to compare whole cell proteomes but also subcellular proteomes e.g. secreted proteins and microvesicules. The platform did set up the label-free relative and absolute quantification according to methods developed by Mann’s group. 

    Post translational modification analyses are also frequently requested by the research teams. Most of these requests concern phosphorylation either on a single target protein but also frequently against the whole proteome (phospho-proteomic study). We have also realized high throughput studies of acetylation and methylation using specific antibodies to immunoprecipitate peptides carrying these modifications. A nanofluidic apparatus (ProteinSimple, NanoPro 1000) enabling detection of protein expression and post translational modifications with high throughput using extremely small samples is also available in the platform to complement the post-traductional analysis offer. This apparatus is one of the few systems allowing determination whether several post translational modifications of a target protein are carried out by the same molecule or by different molecules.