Hydrogen/deuterium exchange with MS
St. John Skilton, PhD is senior manager, business operations, LSD, pharmaceutical life sciences, Waters Corp.
Invention, adaptation, and innovation have played a role in developing commercial hydrogen/deterium (H/D) exchange (HDX) systems.
HDX as a technique was pioneered by Lindstrom Lang in the 1940s. The basic principle is straightforward: place a molecule
in a solution of D2O and measure how many–and how quickly–hydrogen atoms exchange with deuterium atoms. From this measure, the higher order structure
of the molecule, typically a protein, could be inferred. Some regions of a protein would have faster uptake than others, and
regions can be compared to provide a detailed picture of the three-dimensional structure of the protein. This allows proteins
in different states or with mutations to be compared and correlated to biological activity. Lang used crude techniques, such
as scintillation meters, to time the different rates at which deuterated versus nondeuterated samples fell through a liquid-filled
tube under the action of gravity. The biotechnology industry has since adopted this method as a routine technique (1, 2).
Although HDX had previously been performed with high-performance liquid chromatography (HPLC), HPLC had weaker separation
power. For HDX with mass spectrometry (MS), ultra-performance liquid chromatography (UPLC) separations allow more detailed
measurements, an important consideration for use with proteins as large as antibodies. UPLC, therefore, improved the efficiency
of HDX by having a higher degree of separation concomitant with robustness One challenge in quantifying deuterium uptake was
the dynamic nature of the process. Deuteration is not a one-way street: molecules in solution will exchange back and forth
dynamically, so in order to measure the process accurately, the exchange had to be chemically quenched to a pH of 2.5 (for
proteins) and the analytical separation simultaneously cooled to 0 °C to manage the "back" exchange. Operating at such cold
temperatures was not the sort of treatment chromatographic systems were originally designed for, so innovation was needed
was to make a refrigerator unit integrated with the UPLC system. Having a cold pathway manages the reversal of the deuteration
and brings the chromatographic separation as close to the detector as possible (3).
Figure 1 (HDX/MS): A depiction of the relative deuterium uptake for interferon helps one visualize and interpret the higher
order protein structure related to conformational change. The uptake measurements are made at the peptide level for multiple
time points across the experiment. Each uptake measurement is superimposed on the 3D structure of the protein, typically obtained
from an X-ray representation. (FIGURE 1 (HDX/MS) IS COURTESY OF THE AUTHOR)
Another element in advancing HDX with UPLC/MS was the availability of a detection system that could cope with the complexity
of the analysis and be able to make reproducible, quantitative measurements. The detection technique most appropriate for
this need was an existing methodology applied in a new way: mass spectrometry with MSE (Waters Corp.). In MSE methodology, peptides are detected intact and then fragmented in rapid succession in the collision cell of the mass spectrometer,
dozens of time per second (1). This method is possible in QTof and SYNAPT spectrometers, which are hybrid, tandem mass spectrometers
(Waters Corp.) Therefore, for the same (peptide) ion, an accurate mass and an amino acid sequence is available. From the differences
in masses between deuterated and undeuterated peptides, a precise determination of uptake, as well as proof of assignment,
can simultaneously be made. This gain in information content allows the precise location of conformational changes (4, 5,
As more complex proteins are analysed, it also becomes increasingly useful to include ion-mobility separations to disentangle
the data (6, 2). Ion mobility separations are built in to SYNAPT mass spectrometers and have helped to further advance the
HDX/MS applications area (7).
In HDX studies, data are produced across multiple time points, multiple species, and with replicates. Curating this data manually
is not time-efficient and requires expert interpretation. The interpretation is a repetitive process that requires counting
and measuring spectra, so the process can be automated with some in-built intelligence. Software (DynamX, Waters Corp.) is
designed to systematically select spectra with predetermined criteria and measure the mass change of the deuterated form.
The software automation was greatly simplified by having sharper peaks and better separation with UPLC, and the comprehensive
nature of the MSE detection. This automation, along with the capability to sort and display data, has been an important advance.
1. I.A. Kaltashov et al., J. Am. Soc. Mass. Spectrom. 21 (3), 323–337 (2010).
2. R.E. Iacob, J.P. Murphy, and J.R. Engen, Rapid Commun. Mass Spectrom. 22 (18), 2898–2904 (2008).
3. T.E. Wales, Anal. Chem. 80 (17), 6815–6820 (2008).
4. J. Engen, Anal. Chem. 81 (19), 7870–7875 (2009).
5. Z. Jianming et al., Nature
463 (7280), 501–506 (2010)
6. K.D. Rand et al., "Gas-Phase Hydrogen/Deuterium Exchange in a Traveling Wave Ion Guide for the Examination of Protein
Confirmations, Anal. Chem. online, DOI 10.1021ac901897x, Nov. 18, 2009.
7. R.E. Iacob et al., Proc. Natl. Acad. Sci. USA
106 (5), 1386–1391 (2009).