Analysis of higher-order protein structures and molecular interactions with HDX-MS – University of Copenhagen

Analysis of higher-order protein structures by HDX-MS

Proteins can be viewed as molecular machines inside a cell factory, and in order to understand how these machines function or malfunction during disease, we need to identify the parts of the machines that move or bind to other machines during work. The same is true for understanding the action of drugs at the molecular level as almost all drugs work by interacting with select parts of the cellular protein machinery and a rapidly growing proportion of drugs are themselves protein-based (biopharmaceuticals). 

The HDX-MS experiment: A sensitive analytical approach for studying the conformation, dynamics and molecular interactions of proteins in solution is to monitor the exchange of hydrogen atoms attached to the backbone linkages in the protein with those from the surrounding solvent (HDX). The amide HDX of a protein reports on the conformation of the protein because regions of stabile structure will exchange at a slower rate than unstructured or highly dynamic regions. By diluting the protein into an aqueous buffer containing deuterium (see figure 1), the exchange of hydrogen with deuterium in the protein will give rise to an increase in mass, since deuterium weights one Dalton more than hydrogen, which can be monitored as a function of time by mass spectrometry (MS). Perturbations to the local structural environment of particular amino acid residues in a protein, for example by a rearrangement or interaction with a small or large ligand molecule, will alter the HDX rate of hydrogens on those residues. The change in HDX can be detected at high sensitivity by MS and can, for instance, be used to localize regions of conformational change in the protein or map sites engaged in ligand binding.

Figure 1. Schematic overview of the hydrogen/deuterium exchange mass spectrometry (HDX-MS) experiment.

HDX-MS presents a very sensitive technique to detect and estimate the degree of difference in conformation and dynamics between two protein forms, as well as pinpoint the location of such difference (Figure 2).  The method is becoming an increasingly important technique for characterising biopharmaceuticals and drug interactions and detailed structural analysis of proteins and protein systems difficult or impossible to access with other traditional methods such as NMR spectroscopy or X-ray diffraction.

Figure 2. High-resolution HDX-MS experiments: integrating electron transfer dissociation MS/MS into the HDX-MS workflow (Rand et al. Acc. Chem. Res. 2014).

The Protein Analysis Lab has an autosampler robot (Figure 3), which provides automated preparation of protein samples for higher throughput HDX-MS analyses.

Figure 3. Automated robotic sample preparation for HDX-MS experiments by using a LEAP automsampler PAL.

Current HDX-MS projects and expertise in the lab:

  • Analysis of the conformation and dynamics of proteins of pharmaceutical interest
  • Analysis of the interactions of drugs/ligands and protein targets
  • Analysis of protein-lipid interactions and the conformation of integral membrane proteins
  • Improving the applicability and resolution of the HDX-MS method
  • Using gas-phase fragmentation (MS/MS) for HDX-MS analysis at high-spatial resolution

Figure 4. Localisation of interactions and structural changes in proteins upon ligand or complex formation. Here illustrated in the case of a monoclonal antibody and the neonatal Fc receptor (Jensen et al. Mol.Cell.Proteom. 2015)