Current limitations of hydrogen/deuterium exchange mass spectrometry and possible ways to overcome them

Description

Hydrogen/deuterium exchange mass spectrometry (HX-MS) has been proven to be a very sensitive technique to study some aspects of protein structure and dynamics. It can be applied to study protein folding, unfolding, and stability, to measure folding or unfolding rates, to map interaction surfaces, and to determine binding constants. As with every analytical technique, there are several limitations. Whereas some of them are inherent to the method, others are related to technical issues or incomplete knowledge and, thus, can be overcome.

One of those problems is that the currently achievable spatial resolution, in terms of the amount of individual amide hydrogen exchange rates that can be measured, is limited by the number and sizes of peptides generated by pepsin. The most straightforward way to increase the resolution, namely to subject the peptides to gas-phase fragmentation in the mass spectrometer, has given inconclusive results so far. The reason for that is the occurrence of intramolecular migration of amide hydrogens upon vibrational excitation of protonated peptide ions, which destroys the information encoded by the different deuterium levels on the individual backbone amides. I will briefly discuss two strategies, which we and other groups are currently focusing on, to overcome this problem.

Another limitation is that we are far away from being able to interpret all the information obtained by HX-MS experiments. This is most obvious in the case of biomolecular interaction studies, where proteins often undergo conformational changes during binding. Since it is not possible to deduce details about the conformational changes solely from the observed differences in the hydrogen exchange rates, I would like to discuss the possibility of using this data as constraints for computational macromolecular docking prediction methods.