Departments of Chemistry and Molecular Biosciences and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.
Department of Bioengineering, Stanford University, Stanford, California 94305, United States.
J Am Soc Mass Spectrom. 2023 Oct 4;34(10):2093-2097. doi: 10.1021/jasms.3c00235. Epub 2023 Sep 8.
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.
抗体-抗原相互作用是免疫反应的核心。蛋白质抗原（如同工型和翻译后修饰）的变异可以改变它们的抗体结合位点。为了直接将蛋白质抗原的识别与其分子组成联系起来，我们使用天然串联质谱法探测抗体-抗原复合物。具体来说，我们对突出的花生过敏原 Ara h 2 和在对花生过敏的患者中发现的趋同 IgE 可变区进行了表征。除了测量抗原诱导的 IgE 抗体二聚体外，我们还展示了如何在气相中分离免疫复合物并将其激活以喷射、鉴定和表征其结合抗原的蛋白形式。使用串联实验，我们分离出喷射出的抗原，然后将其片段化以鉴定其化学成分。这些结果确立了天然自上而下的质谱法作为一种可行的平台，用于精确和彻底地表征免疫复合物，将结构与功能联系起来，并能够发现抗原蛋白形式及其结合位点。
