Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry

John P. McGee; Rafael D. Melani; Ben Des Soye; Derek Croote; Valerie Winton; Stephen R. Quake; Jared O. Kafader; Neil L. Kelleher

doi:10.1021/jasms.3c00235

Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry

John P. McGee, Rafael D. Melani, Ben Des Soye, Derek Croote, Valerie Winton, Stephen R. Quake, Jared O. Kafader, Neil L. Kelleher^*

^*Corresponding author for this work

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

Abstract

Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.

Original language	English (US)
Pages (from-to)	2093-2097
Number of pages	5
Journal	Journal of the American Society for Mass Spectrometry
Volume	34
Issue number	10
DOIs	https://doi.org/10.1021/jasms.3c00235
State	Published - Oct 4 2023

Funding

This study was funded by the National Institute of Health under a grant from the National Institute of General Medical Sciences P41 GM108569 (NLK); National Institute on Aging F31 AG069456 (JPM); the Northwestern Medicine Dr. Michael M. Abecassis Transplant Innovation Endowment Grant; NCI CCSG P30 CA060553 (awarded to the Robert H. Lurie Comprehensive Cancer Center). The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Keywords

Orbitrap
antibody
antigen
complex-up
native
top-down

ASJC Scopus subject areas

Structural Biology
Spectroscopy

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10.1021/jasms.3c00235

Cite this

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title = "Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry",

abstract = "Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.",

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note = "Funding Information: This study was funded by the National Institute of Health under a grant from the National Institute of General Medical Sciences P41 GM108569 (NLK); National Institute on Aging F31 AG069456 (JPM); the Northwestern Medicine Dr. Michael M. Abecassis Transplant Innovation Endowment Grant; NCI CCSG P30 CA060553 (awarded to the Robert H. Lurie Comprehensive Cancer Center). The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: {\textcopyright} 2023 American Society for Mass Spectrometry. Published by American Chemical Society. All rights reserved.",

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N2 - Antibody-antigen interactions are central to the immune response. Variation of protein antigens such as isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we probed antibody-antigen complexes by using native tandem mass spectrometry. Specifically, we characterized the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients who are allergic to peanuts. In addition to measuring the antigen-induced dimerization of IgE antibodies, we demonstrated how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, we isolated the ejected antigens and then fragmented them to identify their chemical composition. These results establish native top-down mass spectrometry as a viable platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites.

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Immunocomplexed Antigen Capture and Identification by Native Top-Down Mass Spectrometry

Abstract

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Keywords

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