Hydrogen deuterium exchange (HDX) studies on proteins, pioneered by Linderstrom-Lang in the 1940s, provides information that
helps clarify tertiary structure. In the 1990s, linking HDX to mass spectrometry made it possible to characterize ever larger
biomolecules and to identify the location of deuterium uptake. In the past three years, another advance has taken place with
the development of cooled, fast separation chromatography combined with mass spectrometers that can separate ions in multiple
orthogonal dimensions. Further, the technique has matured to the level where it is no longer an esoteric research topic, but
a tool that can be applied over several stages in biotherapeutic development. This broad applicability facilitates faster
development for promising biotherapeutics. This article examines some of the recent advances in HDX technology and how these
advances have informed practical applications.
Hydrogen deuterium exchange (HDX) studies on labile hydrogens in proteins were pioneered by Hvidt and Linderstrom-Lang starting
in the 1940s.1 They studied the dynamic exchange of labile hydrogens with deuterium in protein structures in deuterated solvent. This same
principle is exploited today with analytical techniques that far outperform those available two generations ago.
Recent technological improvements in ultra high pressure liquid chromatography (UHPLC) and mass spectrometry (MS) have provided
separation and detection tools to delve into fine-grained detail unimagined as little as five years ago. The precision with
which modern mass spectrometers can measure changes in molecular weight means that the location of deuterium uptake can be
accurately determined. The extent of deuterium uptake depends on protein structure and conformation and thus provides insights
that are vital to biotherapeutic development. Consequently, HDX is likely to become a routine technique in the near future.
HDX with mass spectrometry complements the information obtained by crystallography, X-rays, and other analytical methods.
Furthermore, HDX by UHPLC–MS requires far less sample than those methods and sometimes can supply information when those techniques
do not work.
Most recently, fast separation by UHPLC–MS at 0 °C has allowed HDX techniques to mature into a tool that is usable by biochemists
and can be used routinely in biotherapeutic development. Such routine use facilitates a faster path for promising biotherapeutics. This article examines some of the recent advances in this technology
and how those advances have informed practical applications.
Higher Order Structure (HOS) Characterization of Biopharmaceutical Products
As the industry has developed, the concept of well-characterized biological products (WCBP) emerged.2 Detailed information about the higher order structure (HOS) of biotherapeutics provides better characterization. As a result,
regulatory agencies are now encouraging sponsors to provide more information on dynamic studies of a biopharmaceutical product,
particularly at the submission stage.3 Methods in routine use for HOS studies include calorimetry, nuclear magnetic resonance, X-ray crystallography, and advanced
fluorescence. With the advent of ultra-fast HDX with UHPLC–MS, a new level of detail is possible.4,5 HDX with mass spectrometry already is an important tool at the discovery stage for leading biotech companies, and is likely
to rapidly progress into other departments.
Basic Principles of HDX
The basic principle of HDX is that amide hydrogens on the backbone of a biomolecule are more exposed in solution and therefore
more prone to exchanging with deuterium in a solution of D2O. Over a time-course experiment, the number of hydrogens that are exchanged can be measured by MS and therefore the degree
of activity at various sites can be inferred. Certain locations are more prone to exchange because they are less protected.
These locations can be identified, sometimes to the residue level, and mapped to the sequence. Thereafter, the relationship
to biological activity can be correlated to a three-dimensional map of the protein.
Modern HDX workflows tend to favor LC combined with ESI Q-Tof MS (Figure 1), because of the level of detail required. HDX
experiments benefit from faster UHPLC separations with sub-2-µm particles: The chromatographic efficiency obtained means a
shorter run time and a better resolution than with HPLC, with a smaller amount of protein loaded. Being able to perform separations
in less than 10 min minimizes the loss of deuteration. This loss is termed "back-exchange," reflecting that the amide deuteriums
in the protein backbone can re-exchange to hydrogen in solution. Lower pH, fast separations, and a cold environment all contribute
to minimizing this effect.
Figure 1. The workflow of a typical hydrogen-deuterium exchange (HDX) experiment with mass spectrometry (MS) detection. The
digestion and chromatographic portion of the experiment is kept at 0 °C to minimize back exchange.
In the workflow outlined in Figure 1, the protein in H2O is continuously labeled with deuterium and sampled at multiple time points (from seconds to hours). Typically a 15- to 20-fold
excess of D2O is used in physiological buffer at neutral pH. The exchange is quenched at each time point by lowering the pH to 2.5 and
the temperature to 0 °C for each sample drawn.
Two pathways are open to the user. For rapid monitoring of global conformational changes, the protein is injected directly
into the MS detector. For local analysis, to determine the specific location of changes in deuteration, peptides are digested
online using an immobilized pepsin column. Pepsin is a non-specific enzyme, and has the advantage of working at a lower pH.
Figure 2. Fluidic schematic for online pepsin digestion using ultra high pressure liquid chromatography (UHPLC) with hydrogen-deuterium
exchange (HDX). The peptide separation is performed inside the HDX manager at 0 °C.
The reproducibility of digestion and chromatographic separation is an important component of a successful experiment, allowing
the comparison of the same peptide across different time points (Figure 3). The mass shifts for selected peptides can be compared.
The faster exchange occurs for a peptide, the more accessible is that site in solution relative to other locations on the
protein. Therefore, the relative folding and dynamics also can be inferred from the different rates of uptake at different
locations. Differences in uptake for different conditions can be mapped onto tertiary structures for easier visualization.
Figure 3. HDX chromatographic separation of Interferon alpha 2b peptides by online digestion and their ESI–MS spectra. The
chromatography is identical for all time points. In the illustration, the same peptide is extracted at each of the time points
and varies in mass-to-charge ratio (m/z) because of increasing uptake of deuterium. The blue bar indicates the m/z of the
12C isotope with no deuteration.
Calmodulin is a calcium-binding protein, and the loop regions with an EF-hand motif are folded when the calcium is added in
protein solution. The highlighted region in Figure 4A depicts the apo and holo versions of the protein. No calcium is bound
in the apo version; the corresponding area in the holo version of the protein is folded differently. The color scheme in Figure
4B superimposes a visual representation of the differing proportions of deuterium incorporation on the sequence (a "heat map").
In the apo form, a yellow color represents >60% of relative deuterium uptake, whereas the same location in the holo form bound
with calcium is <10% relative uptake by the end of the experiment (4 h).
Figure 4a. A model structure of calmodulin protein in apo and holo forms. Highlighted in yellow is the region responsible
for calcium binding. The different deuterium incoporation is observed under two forms of the conformational state.
Peptide Mapping for HDX
Mass spectrometry has contributed to studying tertiary structure by increasing the information available. Biomolecules in
an HDX experiment typically are digested with a non-specific enzyme. By using a data-independent acquisition MS technique
(MSE ), the many overlapping peptides can be identified with high confidence, as shown in Figure 5.7 The linear sequence coverage can be up to 100% because of the many overlapping peptides, and therefore the location of uptake
can be pinpointed more precisely.
Figure 4b. The color scheme for percent relative deuterium uptake of the calmodulin peptide, residues 46–65. This region showed
a different amount and rate of exchange because of the conformation differences between the apo and holo forms. The information
displayed in this heat map can be superimposed on the 3D model as in Figure 4a for easier visualization.