ac201501z_si_003.pdf (603.62 kB)
Epitope Mapping of a 95 kDa Antigen in Complex with Antibody by Solution-Phase Amide Backbone Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
journal contributionposted on 2011-09-15, 00:00 authored by Qian Zhang, LeAnna N. Willison, Pallavi Tripathi, Shridhar K. Sathe, Kenneth H. Roux, Mark R. Emmett, Greg T. Blakney, Hui-Min Zhang, Alan G. Marshall
The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and 1F5, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and the results were compared to previous mapping by immunological and mutational analyses. Antibody 2B5 defines a conformational epitope, and 1F5 defines a linear epitope. Intact murine IgG antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen–monoclonal antibody (Ag–mAb) complexes. mAb-complexed and uncomplexed (free) rAna o 2 were then subjected to HDX. HDX instrumentation and automation were optimized to achieve high sequence coverage by protease XIII digestion. The regions protected from H/D exchange upon antibody binding overlap and thus confirm the previously identified epitope-bearing segments: the first extension of HDX monitored by mass spectrometry to a full-length antigen–antibody complex in solution.
homohexameric food allergen proteinAntibody 2 B 5ion cyclotron resonance mass spectrometry95 kDa Antigenantigenmass spectrometryFTICR MScashew AnaFourier Transform Ion Cyclotron Resonance Mass SpectrometryThe epitopesrAna1 F 52 B 5protease XIII digestionIntact murine IgG antibodiesEpitope Mappingantibody binding overlapsequence coverageHDX instrumentation