Analysis of protein conformation and dynamics by hydrogen/deuterium exchange MS

Abstract

Understanding as much as possible about proteins in the shortest amount of time has long been a goal of hydrogen exchange (HX) MS. Recent technological advances have led to improvements in the technique, but has this goal yet been achieved? (To listen to a podcast about this Feature, please go to the Analytical Chemistry Web site at pubs.acs.org/journal/ancham.).

Figure 1

The growth of HX MS since 1990. The number of publications (red squares) and citations (blue circles) was determined using ISI Web of Science (Thompson Reuters) with a keyword search for “hydrogen exchange mass spectrometry” over the period 1991-2008. The totals for 1991-2008 were 1374 publications and 25,249 citations. During that same period, 16 laboratories accounted for 25% of the papers in the field.

Figure 2

HX into proteins. Labile NHs, rendered here as blue balls, can become deuterated when a protein is placed in a D₂O solution. Other hydrogens that exchange too slowly or too quickly to be measured are not shown. The rate of deuteration depends on factors including solvent accessibility, hydrogen bonding, pH, and temperature. In this figure, for example, NHs that are not hydrogen bonded and reside near the surface of the protein become deuterated rapidly, but highly solvent-exposed NHs in structured elements at the surface (several α-helices in this case) are protected from exchange by hydrogen bonding.

Figure 3

Backbone amide hydrogens sorted according to secondary structure: those in α-helices are shown in green, those in β-sheets are shown in yellow, and the remainder are shown in blue. If structural elements such as helices or multistrand beta sheets (such as those indicated by white arrows) remain highly stable during protein-ligand interactions, there may be no change to deuteration at NHs. Interactions driven by side chains may not perturb backbone amide HX.

Figure 4

The general steps that make up a typical HX MS experiment. The old techniques shown are being replaced by the newer, better methods at each step during the overall experiment.

Figure 5

Deuteration of a recombinant biopharmaceutical monoclonal antibody (adapted from Houde et al., ref. 25). The relative deuterium levels for this antibody are shown after 10 minutes of labeling. Some regions are much more deuterated than others. Data such as these form the baseline against which comparisons can be made to determine if the conformation of the molecule is altered (for example, by post-translational modifications or storage conditions).