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.

Figure 1. Improvement of HDX System (Myoglobin)

Deuterium uptake for 16 segments of myoglobin after exchange for 4 and 8 hours (old vs new system). The optimization of HDX instrumentation and automation gives increased deuterium uptake for all 16 segments, due to reduced back-exchange.

Figure 2. Sequence Coverage for Common Segments for Both Antibodies

Sequence coverage for analyzed identical segments of different rAna o 2 forms. Sequence coverage for identical proteolytic segments of free rAna o 2 and rAna o 2 complexed with mAb 2B5 (top) or mAb 1F5 (bottom). The segments cover 90% (mAb 2B5) and 80% (mAb 1F5) of the sequence based on 126 (mAb 2B5) and 77 (mAb 1F5) identical segments. All segments for both complexes were identified in at least 2 of the 3 replicates for every H/D exchange period.

Figure 2. Sequence Coverage for Common Segments for Both Antibodies

Sequence coverage for analyzed identical segments of different rAna o 2 forms. Sequence coverage for identical proteolytic segments of free rAna o 2 and rAna o 2 complexed with mAb 2B5 (top) or mAb 1F5 (bottom). The segments cover 90% (mAb 2B5) and 80% (mAb 1F5) of the sequence based on 126 (mAb 2B5) and 77 (mAb 1F5) identical segments. All segments for both complexes were identified in at least 2 of the 3 replicates for every H/D exchange period.

Figure 3. Effect of Dual Enzyme Digestion

Dual enzyme digestion of the rAna o 2 antigen. The red segments are generated by 2 minute protease XIII digestion (with 4 M urea to unfold the proteins and TCEP to break disulfide bonds) and the blue segments are generated by 1 min pepsin digestion followed by 1 min protease XIII digestion. Only those segments identified in all 3 replicates are pictured. The dual enzyme digestion approach produces more segments than obtained by protease XIII digestion alone and provides better sequence resolution, especially for large proteins.

Figure 4. HDX after 4 hour for 2B5 mAb Binding

Deuterium uptake for selected segments of rAna o 2 after exchange for 4 hours (free vs. complexed with mAb 2B5). Segments with significant protection (>30%) upon 2B5 binding are bracketed in red.

Figure 5. D Uptake Profiels for Selected Segments

Deuterium incorporation vs. H/D exchange period for selected segments of free and complexed rAna o 2. Segments 29–37, 31–41, 32–41 and 41–48 exhibit less deuterium uptake for rAna o 2 complexed with mAb 2B5, thereby defining the 2B5 epitope. Deuterium uptake profiles are similar for segments 160–175 and 282–293. For the binding of mAb 1F5, only one segment (265–289) shows significant protection on complexation, thereby defining its epitope.

Figure 5. D Uptake Profiels for Selected Segments

Deuterium incorporation vs. H/D exchange period for selected segments of free and complexed rAna o 2. Segments 29–37, 31–41, 32–41 and 41–48 exhibit less deuterium uptake for rAna o 2 complexed with mAb 2B5, thereby defining the 2B5 epitope. Deuterium uptake profiles are similar for segments 160–175 and 282–293. For the binding of mAb 1F5, only one segment (265–289) shows significant protection on complexation, thereby defining its epitope.

Figure 6. HDX Epitope Mapped onto X-ray Structure

Structural models for trimeric Ana o 2. Left: The sequences of Ana o 2 and its glycinin homologue are aligned with the HDX-defined 2B5 and 1F5 epitopes boxed in purple and red, respectively. Asterisk (*) denotes aa identity, colon (:) indicates aa similarity. Right: The epitopes are colored similarly for the trimeric X-ray model (pdb ID 1od5).