Hydrogen deuterium exchange and other mass spectrometry- based approaches for epitope mapping

Abstract

Antigen-antibody interactions are a fundamental subset of protein-protein interactions responsible for the “survival of the fittest.” Determining the interacting interface of the antigen, called an epitope, and that on the antibody, called a paratope, is crucial to antibody development. Because each antigen presents multiple epitopes (unique footprints), sophisticated approaches are required to determine the target region for a given antibody. Although X-ray crystallography, Cryo-EM, and nuclear magnetic resonance can provide atomic details of an epitope, they are often laborious, poor in throughput, and insensitive. Mass spectrometry-based approaches offer rapid turnaround, intermediate structural resolution, and virtually no size limit for the antigen, making them a vital approach for epitope mapping. In this review, we describe in detail the principles of hydrogen deuterium exchange mass spectrometry in application to epitope mapping. We also show that a combination of MS-based approaches can assist or complement epitope mapping and push the limit of structural resolution to the residue level. We describe in detail the MS methods used in epitope mapping, provide our perspective about the approaches, and focus on elucidating the role that HDX-MS is playing now and in the future by organizing a discussion centered around several improvements in prototype instrument/applications used for epitope mapping. At the end, we provide a tabular summary of the current literature on HDX-MS-based epitope mapping.

Integrating Dynamic Network Analysis with AI for Enhanced Epitope Prediction in PD-L1:Affibody Interactions

“…For epitope mapping of antibodies and non-antibody sca↵olds, a variety of experimental methods have been employed, including X-ray crystallography, NMR, mass spectrometry, peptide arrays and deep mutational scanning-based approaches. [14][15][16][17][18][19][20][21][22] Computational methods have potential to be faster and less costly, however, even advanced artificial intelligence (AI)-based AlphaFold and RoseTTAFold [23][24][25][26] have faced challenges in accurately predicting protein-protein interactions. [27][28][29][30] While success cases exist, such as the model of a PD-L1/CD80 complex, 31 large screening studies have shown a low number of highly-accurate antibody-antigen complex prediction.…”

Single-domain antibodies reveal unique borreliacidal epitopes on the Lyme disease vaccine antigen, Outer surface protein A (OspA)

“…To better resolve antibody-OspA interactions within Bin 1, five V H Hs were subjected to epitope mapping by hydrogen exchange (HX)-mass spectrometry (MS) analysis using protocols optimized for OspA (33, 44). HX-MS is an increasingly powerful tool for epitope mapping in which the HX reaction is conducted in solution and captures antibody-induced changes in antigen backbone flexibility; strong reductions in HX are interpreted as points of antigen-antibody contact (45). As predicted, HX-MS indicated that all five V H Hs in bin 1 protected regions within OspA’s central β-sheet (33).…”

Hydrophilic Interaction Liquid Chromatography at Subzero Temperature for Hydrogen–Deuterium Exchange Mass Spectrometry

“…Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a widely used tool for investigating the dynamics of proteins and their interactions in vitro − and promises to become a valuable tool for studies in vivo . For the biopharmaceutical discovery and development sector, HDX-MS data have become important for understanding interactions of proteins with covalently bound small-molecule drugs and also with large hydrogen-bonded protein and glycoprotein ligands, resulting in better understandings of their mechanism of action. − HDX-MS data are used to substantiate and protect intellectual property, to support biologics license applications, and to evaluate the physicochemical similarity between a biosimilar candidate and the originator product. − Most similarity comparisons are qualitative; however, scientists have made significant progress toward quantitative evaluations of HDX-MS data, including free energy determinations of biophysical processes. − As HDX-MS measurement precision improves, quantitative evaluations may become ubiquitous.…”