The Hydrogen/Deuterium eXchange Mass Spectrometry activity at Institut Pasteur
Hydrogen Deuterium eXchange followed by Mass Spectrometry (HDX-MS) is a powerful and recognized biophysical tool in structural biology capable of probing protein/ligand interactions, conformational changes, and protein folding and dynamics (Figure 1). HDX-MS measures the changes in mass imposed by the replacement of backbone amide hydrogens by deuterium added in excess in the surrounding environment. The rate of exchange relies on the folding and dynamics of proteins making backbone amide hydrogens excellent structural probes (more details about the principle of hydrogen exchange can be found here).
We are currently using an optimized version of the HDX-MS pipeline commercialized by the Waters Company composed of a Synapt G2-Si HDMS mass spectrometer with ETD and IMS capabilities, a LEAP-Pal robot for automated sample handling, a cooled HDX manager that maintains the valves, columns and associated tubings at 0°C and Class M nanoACQUITY UPLC pumps (Fundings: CACSICE Equipex, C2RT Institut Pasteur). Our pipeline has been completed with the development of a software named “MEMHDX” to aid in the rapid statistical validation and visualization of large HDX-MS datasets. A schematic overview of a classical HDX-MS experiment performed with our optimized HDX-MS workflow is presented in Figure 2.
The majority of our projects focus on soluble, membrane-associated or integral membrane proteins that are either involved in infectious disease or considered as potential drug targets. Most of these projects are carried out in collaboration with internal or external collaborators. Our expertise in HDX-MS include:
- Analysis of the conformation and dynamic of intrinsically disordered proteins (IDPs) and large proteins (> 150 kDa).
- Analysis of small molecule/protein interactions.
- Analysis of protein/protein interaction (e.g., activation of the CyaA toxin) and identification of binding sites (e.g. Epitope mapping for both the academic and pharmaceutical sector).
- Analysis of protein/DNA interactions.
- Analysis of the conformation and dynamic of integral membrane proteins (e.g., the human glutamate transporteur EAAT1).
- Optimization and improvement of the HDX-MS pipeline (e.g. MEMHDX; details about the principle of the software can be found here).
Main Projects and Collaborators
- Structural analysis of B. pertussis CyaA toxin – Collaboration with the group of Alexandre Chenal, Institut Pasteur, Paris.
- Structural analysis of viral IDPs – Collaboration with the group of Sonia Longhi, CNRS, Marseille.
- Epitope mapping – Collaboration with Michel-Robert Popoff and Alexandre Chenal, Institut Pasteur, Paris.
- Deciphering the structural changes associated with ion binding on the human glutamate transporter – Collaboration with the group of Nicolas Reyes, Institut Pasteur, Paris.
- Identification of protein/aptamer binding sites by HDX-MS – Collaboration with the groupe of Marcel Hollenstein, Institut Pasteur, Paris.
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