Rapid Screening for Potential Epitopes Reactive with a Polycolonal Antibody by Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry

Abstract

The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

Epitope mapping of 7S cashew antigen in complex with antibody by solution‐phase H/D exchange monitored by FT‐ICR mass spectrometry

“…H/D exchange results for free rAna o 1 and rAna o 1 complexed with mAb 2G4. For each of the proteolytic peptides common to free rAna o 1 and rAna o 1 complexed with mAb 2G4, the relative D‐uptake increase for the antigen on binding to antibody (ARDD) is calculated as described previously . Rather than calculation based on common peptides, the ARDD calculated based on peptides, covering the segment of interest but not necessarily of the same length, are annotated by asterisks.…”

“…Other approaches to epitope mapping include X‐ray crystallography, nuclear magnetic resonance (NMR), chimeric molecule analysis, alanine scanning mutagenesis, hydrogen–deuterium exchange, electron microscopy, and phage display . The gold standard for epitope characterization is X‐ray crystallography of the antigen–antibody complex; however, it is not readily applicable for many antigens and antibodies, and imposes daunting requirements in terms of the amount of material, crystallinity, and data analysis.…”

“…Recent improvements in HDX analysis monitored by high resolution FT‐ICR MS include automation, faster chromatographic separation, more efficient protein digestion, and an enhanced data analysis package . The improved HDX‐MS methodology has achieved successful epitope mapping in various biological systems …”

Protein Crystallography in Vaccine Research and Development

“…Another area, partly introduced above, is the use of HDX-MS for the rapid characterization of protein–protein interfaces, with obvious application to antigen–antibody complexes. In contrast with crystallographic or cryo-EM approaches, HDX-MS also holds the intriguing possibility that such studies might be applicable to polyclonal antibody–antigen mixtures [ 179 ]. Further, the addition of electron transfer dissociation (ETD) technology to the HDX-MS approach is likely to enable improvements in the resolution of structural MS-based epitope mapping studies [ 180 ].…”

Mapping the contact surfaces in the Lamin A:AIMP3 complex by hydrogen/deuterium exchange FT-ICR mass spectrometry

“…Hydrogen/deuterium exchange (HDX) was performed as previously described [ 40 ]. HDX samples were prepared in 5 μL volume at 40 μM (His-Tev-AIMP3, His-Strep-TrxA-LmnA, and AIMP3:LmnA complex (AIMP3 and LmnA were mixed in 1:1 ratio to form the complex.)…”