title: 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.
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.
authors.affiliation: Department Chemistry & Biochemistry, Florida State University, Tallahassee, Florida 32306, United States.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhang
authors.forename: LeAnna N
authors.initials: LN
authors.lastname: Willison
authors.forename: Pallavi
authors.initials: P
authors.lastname: Tripathi
authors.forename: Shridhar K
authors.initials: SK
authors.lastname: Sathe
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Roux
authors.forename: Mark R
authors.initials: MR
authors.lastname: Emmett
authors.forename: Greg T
authors.initials: GT
authors.lastname: Blakney
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Zhang
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
mesh_terms: D000577:Amides / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000737:chemistry* / Q000276:immunology; D052179:Antigens, Plant / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017357:Cyclotrons; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D005583:Fourier Analysis; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D000577:Amides; C478880:Ana o 2 allergen, cashew; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D052179:Antigens, Plant; D011994:Recombinant Proteins; D006859:Hydrogen; D003903:Deuterium