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

doi:10.1021/ac201501z

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 journal › Article › peer-review

110 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 language	English (US)
Pages (from-to)	7129-7136
Number of pages	8
Journal	Analytical Chemistry
Volume	83
Issue number	18
DOIs	https://doi.org/10.1021/ac201501z
State	Published - Sep 15 2011
Externally published	Yes

ASJC Scopus subject areas

Analytical Chemistry

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10.1021/ac201501z

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Zhang, Q., Willison, L. N., Tripathi, P., Sathe, S. K., Roux, K. H., Emmett, M. R., Blakney, G. T., Zhang, H. M., & Marshall, A. G. (2011). 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. Analytical Chemistry, 83(18), 7129-7136. https://doi.org/10.1021/ac201501z

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. / Zhang, Qian; Willison, Leanna N.; Tripathi, Pallavi et al.
In: Analytical Chemistry, Vol. 83, No. 18, 15.09.2011, p. 7129-7136.

Research output: Contribution to journal › Article › peer-review

Zhang, Q, Willison, LN, Tripathi, P, Sathe, SK, Roux, KH, Emmett, MR, Blakney, GT, Zhang, HM & Marshall, AG 2011, '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', Analytical Chemistry, vol. 83, no. 18, pp. 7129-7136. https://doi.org/10.1021/ac201501z

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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.",

author = "Qian Zhang and Willison, {Leanna N.} and Pallavi Tripathi and Sathe, {Shridhar K.} and Roux, {Kenneth H.} and Emmett, {Mark R.} and Blakney, {Greg T.} and Zhang, {Hui Min} and Marshall, {Alan G.}",

year = "2011",

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doi = "10.1021/ac201501z",

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AU - Sathe, Shridhar K.

AU - Roux, Kenneth H.

AU - Emmett, Mark R.

AU - Blakney, Greg T.

AU - Zhang, Hui Min

AU - Marshall, Alan G.

PY - 2011/9/15

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N2 - 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.

AB - 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.

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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

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