Determination of primary protein structure has long been manageable and increasingly automated by the hyphenated technique
of liquid chromatography and mass spectrometry (LC–MS). Higher order structure (HOS) can now also be mapped with the same
tools, such as disulfide bond mapping, due to the increased power and capabilities of LC–MS (1). Most recently, the commercialization
of ion mobility mass spectrometry and hydrogen deuterium exchange by mass spectrometry (HDX–MS also referred to as H–D exchange
or HXMS) has changed the shape of analytical technology (2). Researchers in the field are now confident that numerous technical
challenges of this powerful analytical approach have been overcome (3, 4).
HDX–MS uses a simple principle, first exploited decades ago by Lindestrom-Lang, that the solvent accessibility of amide protons
in proteins varies dynamically. By examining the changes over the course of an experiment, different protein states can be
compared with each other to understand protein binding, to map epitopes, to understand the effect of modifications or small
molecule binding, or even to manage the effects of formulation (5). Smaller sample sizes (e.g., picomoles) may be used compared
with other techniques, and HDX–MS can be used to study proteins that are hard to purify. Typically, pepsin digestion, which
cleaves the protein into small peptides, is used. The labeling is quenched chemically, and deuterium incorporation can be
localized to short stretches (e.g., 5 to 10 amino acids) of the primary structure by measuring the resultant mass change in
that peptide (6).
However, deuteration is a dynamic process, and deuterons and protons will exchange back and forth. To help control this process
after the initial uptake, the analytical separation is cooled to 0 °C to manage the "back" exchange. The cold pathway and
chemical quenching dramatically slow the reversal of deuteration and provide a window for chromatographic separation as close
to the MS detector as possible (1, 7). In one instrument, built with guidance from an academic and industry partnership, sub-2-μm
ultra-performance LC (UPLC) separations take place within a cooled environment that is paired with robotics-enabled injection
(Waters, nanoACQUITY UPLC System with HDX Technology). This technique works for large molecules such as antibodies, the characterization
of protein-ligand interactions, mutation effects, and epitope mapping (8–12).
Commercialization of HDX–MS
Commercializing a complete solution for HDX–MS—from robotic process handling to data handling—was an important leap for industry,
in that it adopted a hitherto academic technique into the real world (13). The underlying factors that were addressed to ensure
successful adoption by industry were that mass spectrometry detection be robust and the instrument be engineered for simple
start-up by scientists. In research departments, companies that have adopted HDX–MS use MS in several different ways, principally
to fill the therapeutic pipeline. For example, in new medicines laboratories, HDX–MS can rapidly perform epitope mapping to
tie up intellectual property in the paratope and epitope (14).
Incorporation of brute capabilities, such as UPLC and ion mobility mass spectrometry (IMS), greatly facilitates the studies.
As more complex proteins are analyzed, it also becomes increasingly useful to include ion mobility separations to disentangle
the data (2). IMS provides an orthogonal differentiation to mass differences and chromatographic separation. Untangling the
puzzle of data is therefore easier because there are more angles to look at (i.e., better specificity). Researchers can also
begin to look at intermediates in the binding process and understand more clearly why certain domains are active and others
are less active. This technique has recently been demonstrated in studies of kinase binding and the detailed understanding
of allosteric effects (15).
Historically, curating HDX–MS data manually was inefficient and required expert interpretation. Because interpretation is
a repetitive process that requires counting and measuring spectra, the process can be automated. Building software specifically
designed to systematically select spectra with predetermined criteria and measure the mass change of the deuterated forms
was one of the criteria to make HDX–MS acceptable to industry (13). No longer do users need to be experts in instrumentation
and interpretation; HDX–MS has now become accessible to biologists and biochemists.
Tools appropriate to biologists and biochemists are not enough; the data display needs to be more intuitive. More recent iterations,
therefore, have concentrated on showing conformation in relation to a protein map or to differences between two states that
relate to the protein sequence, rather than the spectral readout from the instrument themselves. This display requires transposition,
overlaying of primary sequence, and color schemes that are more appropriate to human visual interpretation. Recent developments
have allowed the superposition of the MS readouts onto three-dimensional crystal structures of proteins or as "heat maps"
of change related to primary sequence (13). These intuitive plotting schemes provide a rapid, visual comparison of different
states, such as bound and free forms of the proteins of interest (13). A "butterfly" chart provides a rapid visual overview
of the average relative fractional exchange for all peptides in each labeling time-point. The "inflection" points of the chart
highlight the total amount of exchange as well as the rate of exchange, thus highlighting the active regions of the protein
at a glance. The entire polypeptide backbone of each form can now be compared for both spatial and temporal information.