Laboratoire de Chimie Bactérienne UMR 7283

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8 novembre 2017: 1 événement


  • Séminaire IMM

    Mercredi 8 novembre 2017 11:00-12:30 - Martial Rey - Institut Pasteur

    Séminaire METHODS

    Résumé :

    "Cross-linking Mass Spectrometry, from discovery to structural biology."
    Structural studies of large protein complexes, in their quests of analyzing larger edifices, tend to combine multiple biophysical techniques like small angle X-ray scattering, cryo-electron microscopy (cryo-EM), cross-linking mass spectrometry (XL-MS), … In this panel, XL-MS development accelerated impressively the past decade with the development of numerous cross-linkers, several dedicated software and improved mass spectrometry-based approaches. Thanks to these improvements, XL-MS has shown numerous successes from blinded structural modelling in silico (CASP11)(1) of several complexes to the characterization of many large protein complexes like the transcribing Pol IIp-CE complex(2) or the Human Nuclear Pore Complex(3). In those integrated approaches XL-MS demonstrated a very good ability to bridge length scales from overall molecular architecture down to atomic resolution, brought by Cryo-EM or X-ray structures.

    Apart from being a structural technique giving middle spatial range constraints (1-2 nm length contacts), XL-MS can also be used to identify partners in immunoprecipitation approach coupled with MS analyses (IP-MS). But it now begins to be used in vivo, for instance it has been used to characterize the interactome of bacterial pathogens to gain insight on their antibiotic resistance mechanisms(4).

    In this world of fast technical improvements, the IP Mass Spectrometry lab focused on developing a very sensitive XL-MS method combining better cross-linking reactivity(5) and one-step enrichment approach. This new protocol allowed us to identify at least 30 times more cross-linked peptides than the classical XL-MS approach proving its extreme sensitivity(6).

    1. A. Belsom et al., Blind testing cross-linking/mass spectrometry under the auspices of the 11th critical assessment of methods of protein structure prediction (CASP11). Wellcome Open Res 1, 24 (2016).

    2. F. W. Martinez-Rucobo et al., Molecular Basis of Transcription-Coupled Pre-mRNA Capping. Mol Cell 58, 1079-1089 (2015).

    3. K. H. Bui et al., Integrated structural analysis of the human nuclear pore complex scaffold. Cell 155, 1233-1243 (2013).

    4. X. Wu et al., In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075. Nat Commun 7, 13414 (2016).

    5. C. Nury et al., A novel bio-orthogonal cross-linker for improved protein/protein interaction analysis. Anal. Chem. 87, 1853-1860 (2015).

    6. M. Rey, I. Lopes-Neira, M. Dupré, M. Duchateau, J. Chamot-Rooke, XL-FASP : A New Integrated Cross-Linking Workflow to Study Extra Large Membrane Protein Complexes. Anal. Chem., Submitted (2018).

    Lieu : Salle Jacques Senez

    En savoir plus : Séminaire IMM