期刊
ANALYTICAL CHEMISTRY
卷 83, 期 18, 页码 7129-7136出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac201501z
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资金
- NIH [R01 GM78359]
- NSF Division of Materials Research [DMR-06-54118]
- USDA [NIFA-AFRI 2010-04213]
- Almond Board of California
- State of Florida
The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and IFS, 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.
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