Knowledge of the structure and dynamics of proteins and protein
assemblies is critical both for understanding the molecular basis of physiological
and patho-physiological processes and for guiding drug design. While X-ray
crystallography and nuclear magnetic resonance spectroscopy are both excellent
techniques for this purpose, both suffer from limitations, including the requirement
for high quality crystals and large amounts of material. Recently, hydrogen/
deuterium exchange measured using mass spectrometry (HXMS) has emerged as a
powerful new tool for the study of protein structure, dynamics and interactions in
solution. HXMS exploits the fact that backbone amide hydrogens can exchange
with deuterium when a protein is incubated in D2O, and that the rate of the
exchange process is highly dependent on the local structural environment. Several
features of HXMS make it an especially attractive approach, including small
sample requirements and the ability to study extremely large protein assemblies
that are not amenable to other techniques. Here, we provide an overview of HXMS
and describe several recent applications to problems of medical interest. After
reviewing the molecular basis of the H/D exchange process, the different steps of
the HXMS experiment – labeling, rapid proteolysis, fragment separation and mass
measurement – are described, followed by a discussion of data analysis methods.
Finally, we describe recent results on the application of HXMS to 1) mapping
drug/inhibitor binding sites and detecting drug induced conformational changes, 2)
studying viral capsid structure and assembly, and 3) characterizing the structure of
pathological protein conformations, specifically amyloid fibrils.
Keywords: Hydrogen exchange, mass spectrometry, protein dynamics, amyloid, drug
screening, viral capsid.
