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.

Keywords: Orbitrap; antibody; antigen; complex-up; native; top-down.

Figure 1

After the antibody and the antigen are mixed (far left), native electrospray infuses complexes into the mass spectrometer. Particles not bound to the antibody can be filtered away on the basis of m/z, and the complex can be activated to eject the bound antigen. Free antigens can then be targeted for profiling and deep characterization, revealing the presence of isoforms and location of modifications.

Figure 2

Deconvolved mass spectra of components and complexes (see the detailed experimental methods in the Supporting Information). (a) Measuring antibody–antigen interactions using a two-site peptide (top), a one-site peptide (middle), and a control experiment with no peptide (bottom) (Figure S2). The antibody is measured quantitatively in its monomeric (left), dimeric (center), and trimeric (right) forms. Cartoons indicate how many peptides are bound to the indicated form. Asterisks denote deconvolution artifacts. Peptide colors denote sequence similarities to and distinctions from the two-site region shared among all Ara h 2 isoforms as illustrated in Figures 1 and 3. The antibody is also measured in its (b) monomeric and (c) dimeric forms in complex with the natural Ara h 2 antigen (Figure S5). The dimer is depicted in both unactivated (top) and activated (bottom) schemes. Colored circles denote isoform identity, and multicolored circles denoate isoform ambiguity. Here, the instrument configuration prioritized high-mass transmission, leaving the distribution of ejected antigens unknown.

Figure 3

(a) Filtering out of unbound antigens in a complex mixture (top) using voltage rollercoaster filtering (VRF, bottom). Multicolored circles denote isoform ambiguity, and an asterisk denotes an unknown feature that is removed in the subsequent ion trap isolation step. (b) The filtered immunocomplex is activated to eject all proteoforms of bound antigens. The inset shows the deconvolved mass spectrum of the ejected antigen in comparison to that of the antigen that was filtered out using VRF. (c) Depictions of the four Ara h 2 isoforms as well as what regions are supported through the detected fragment ions of isoform 2 (Figure S6).