Bottom-up hydrogen deuterium exchange mass spectrometry: data analysis and interpretation

Abstract

Hydrogen Deuterium Exchange (HDX) Mass Spectrometry (MS) is a sensitive analytical technique that provides information on protein conformation and dynamics in solution. It is commonly used in the study of protein-ligand and protein-protein interactions and more recently in the pharmaceutical industry for epitope mapping, screening drug candidates and in the comparison of biopharmaceuticals to biosimilars. HDX-MS monitors the exchange of protein backbone hydrogen atoms with deuterium in solution. Recent advancements in HDX automation and data analysis, have taken the emphasis off developing a fundamental understanding of HDX, which is still lacking. This tutorial review will cover the different mechanisms of exchange and how the exchange reaction is affected by various factors. We also explore the basis of data analysis and the difficulties that often arise in the interpretation of site-specific and segment-averaged HDX data, such as overlapping isotopic distributions and correct identification of peptides. Initial data analysis generates a list of peptides and the deuterium incorporation of each peptide at each labeling time point, i.e., a set of deuterium uptake profiles. Data interpretation and error analysis is subsequently required to ensure that deuterium uptake profiles accurately reflect conformational dynamics in solution. Finally, this review will also discuss the different ways in which HDX data can be represented and how the data can be interpreted.

Rapid characterization of structural and functional similarity for a candidate bevacizumab (Avastin) biosimilar using a multipronged mass‐spectrometry‐based approach

“…Arguably less well‐recognized is the rapidly developing capability of MS instruments to analyze large, intact biomolecules under native‐like conditions . This, combined with coupled techniques like ion mobility spectrometry (IMS) and hydrogen/deuterium exchange (HDX) allows MS to support higher order structure analysis, in some cases at single residue resolution …”

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

“…To refine these ambiguous antibody binding sites, we subjected V1B11 and JNM-D1 to epitope mapping by hydrogendeuterium exchange mass spectrometry (HX-MS), as described recently (31). HX-MS affords peptide-level resolution of antibody binding sites and is therefore an extremely useful methodology for epitope localization (33). As part of this analysis, we also included JNM-C12 (bin 1) since it also demonstrated a degree of cross-cluster competition.…”

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

“…HX-MS is increasingly being used for the purpose of B cell epitope identification (47,49,50,52,53). The general principles of HX-MS and how the technique relates to other epitope mapping strategies have been covered in several recent reviews (45,54). In a particularly informative study, Malito and colleagues subjected a single MAb directed against factor H binding protein from Neisseria meningitidis to epitope mapping by peptide array, HX-MS, and X-ray crystallography (50).…”

“…2A and B, the rate of HX by peptide 1 (residues 0 to 11) was unaffected by PB10, while HX of peptide 38 (residues 93 to 107) was considerably slower in the presence of PB10. A reduction in HX across a peptide is interpreted as protection due to protein-protein contacts and, therefore, diagnostic of epitopic regions (45). The magnitude of this protection was quantified using the deuteration difference normalized by maximal deuteration as follows: , and were applied successfully to V H H-RTA interactions, where we compared the HX-MS output to a known X-ray crystal structure (51).…”

“…Hydrogen exchange-mass spectrometry (HX-MS) was the method of choice based on recent success in a wide variety of pathogens, including HIV, the malaria parasite, Neisseria meningitidis, and Staphylococcus aureus (45)(46)(47)(48)(49)(50). In a recent study, we successfully adapted HX-MS for the purpose of identifying epitopes recognized by a family of RTA-specific single-domain alpaca-derived antibodies (V H Hs) (51).…”