Capturing Conformational Changes in Biotherapeutics by Hydrogen Deuterium Exchange and UHPLC–MS - By providing information on the relative accessibility of locations within a protein, HDX by


Capturing Conformational Changes in Biotherapeutics by Hydrogen Deuterium Exchange and UHPLC–MS
By providing information on the relative accessibility of locations within a protein, HDX by mass spectrometry opens new windows into the higher order structure of biomolecules.

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Figure 5. A part of peptide map coverage of interferon alpha-2b by pepsin online digestion using data-independent acquisition MS. The high coverage with many overlapping peptides is helps locate regions of uptake more accurately on the sequence map.6
A peptide map generated by non-specific proteases can be highly complex and rapid chromatography may lead to many coeluting peptides. In the past, mass spectrometers were programmed to use the ion signal intensity to switch to MS–MS mode, known as data-dependant acquisition (DDA). Despite ever more sophisticated techniques to cope with switching back and forth, low abundance peptides still can be missed. Even worse, targeted DDA techniques are poor at quantitation and sometimes the analysis has to be repeated because of lack of reproducibility. So a robust strategy that simultaneously provides quantitation and identification has productivity advantages for a biotherapeutics developer.

Figure 6. UHPLC–MS is a method of unbiased data acquisition that comprehensively catalogs complex samples in a single analysis.
By using data-independent acquisition MS, a comprehensive dataset can be generated without prior knowledge of the sample. This dataset is achieved by acquiring two parallel channels, one at low collision energy (CE) and the other at high CE. The resulting raw data files contain parallel data channels with the molecular ions in one, and fragment ions in the other (Figure 6). The time link between these channels is used to automatically relate the molecular ions and their fragments by a number of criteria such as retention time and peak characteristics. From a single injection, LC–MS can provide comprehensive structural information. The data generated are quantitative, and the simplicity of the technique means that non-experts can use it. The comprehensive nature of the technique means that all of the peptides are measured, without bias being introduced because of peptide signal intensity.7 This is an important feature for HDX studies where key information might be found in low intensity peptides.

More Information in Less Time

The incorporation of robotics is an aspect that helps make such complex experiments more routine and less prone to error. The labeling, quenching, and injection can be fully automated to better handle laborious HDX experiments. The variability involved in time-sensitive labeling experiments is minimized by robotics and leaves scientists free to do higher level tasks instead. Biotherapeutics developers thereby obtain greater levels of information in less time, and make better use of their capital, both human and hardware.

Possible Future Trends

Ion mobility mass spectrometry (IMS) has been commercialized for a few years, and early publications already showed that HDX studies benefitted by helping to reduce spectral complexity.8 This additional dimension of separation is likely to speed up adoption of HDX with MS, although the lack of automated data processing will remain a challenge in the short term. Only now that HDX is moving into the mainstream is there greater impetus to rely on software rather than specialized human interpretation.

In prototype work with a Synapt HDMS System (Waters Corporation), gas-phase reactions have been carried out to simplify the tasks and reduce the steps needed to provide useful information. One example is recent work on gas-phase HDX, where instead of performing the exchange in solution outside the instrument, it was instead performed inside the mass spectrometer,9 demonstrating that it is possible to perform rapid and efficient gas-phase HDX. The advances in instrument architecture have made this type of exploratory study possible, re-using commercially available instrumentation with a minimum of modification.

Because different parts of the protein are being labeled, greater understanding of the dynamic behavior of the proteins in the gas phase is possible. Naturally, much work remains to be done to relate this information to biological conditions, but it is already clear that the speed of this technique has positive implications for comparing different samples. Furthermore, the simplicity of the experiment makes it appealing for routine use in environments where a quick view of conformation would be beneficial.


HDX by mass spectrometry opens new windows into the dynamics of biomolecules by providing information on the relative accessibility of different conformations of a protein, or locations within a protein. As instruments have developed, a number of important advances have made HDX with mass spectrometry a more accessible tool to study the HOS of biotherapeutics. Industry and regulatory trends have very clearly moved toward an emphasis on conformation and the relationships between biomolecules. Therefore HDX has received a welcome impetus as one of the tools that is likely to be adopted rapidly in more areas of biotherapeutic analysis.

JOOMI AHN is senior research chemist and ST JOHN SKILTON, PHD, is a senior marketing manager, both at Waters Corporation, Milford, MA, 508.478.2000,

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