1. 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.
- Author
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Zhang Q, Willison LN, Tripathi P, Sathe SK, Roux KH, Emmett MR, Blakney GT, Zhang HM, and Marshall AG
- Subjects
- Antigen-Antibody Complex immunology, Antigens, Plant genetics, Antigens, Plant immunology, Cyclotrons, Deuterium chemistry, Deuterium Exchange Measurement methods, Fourier Analysis, Hydrogen chemistry, Recombinant Proteins chemistry, Recombinant Proteins immunology, Recombinant Proteins metabolism, Amides chemistry, Antibodies, Monoclonal immunology, Antigen-Antibody Complex chemistry, Antigens, Plant chemistry, Epitope Mapping methods, Mass Spectrometry methods
- Abstract
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.
- Published
- 2011
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