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

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

Research output: Contribution to journalArticle

89 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)7129-7136
Number of pages8
JournalAnalytical Chemistry
Volume83
Issue number18
DOIs
StatePublished - Sep 15 2011

ASJC Scopus subject areas

  • Analytical Chemistry

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