Antigen capture and identification by mass spectrometry of complexes

Abstract

Disclosed herein is a method for proteoform-specific antigen identification. The method may comprise infusing a sample comprising an antigen and an antigen-binding protein into a mass spectrometer; isolating a complex comprising the antigen bound to the antigen-binding protein; ejecting the bound antigen from the complex; and identifying the ejected antigen and / or identifying the antigen-binding protein.

Description

[0001] ANTIGEN CAPTURE AND IDENTIFICATION BY MASS SPECTROMETRY OF COMPLEXES CROSS REFERENCE TO RELATED APPLICATIONS This application claims benefit of priority to U.S. Patent Application Ser. No.63 / 580,682, filed September 5, 2023. The contents of which is incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under grant numbers GM108569, AG069456, and CA060553 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (702581.02553.xml; Size: 15,200 bytes; and Date of Creation: August 30, 2024) is herein incorporated by reference in its entirety. FIELD OF THE INVENTION The disclosed technology is generally directed to mass spectrometry. More particularly the technology is directed to mass spectrometry of antibody-antigen complexes. BACKGROUND OF THE INVENTION Proteoforms are the many protein end products originating from a single gene, considering their modifications, sequence permutations, and isoform variants. As such sources of variation can alter both protein function and their antibody binding sites, understanding the unique composition of proteoforms is crucial in understanding function and molecular recognition in biology. To this end, top-down mass spectrometry (TDMS) can systematically discover intact proteoforms by asserting the coexistence of modifications on distinct isoforms as they exist in biomolecular complexes. Characterization starts by isolating the ionized target based on its mass-to-charge ratio (m / z). The covalent bonds of isolates are then fragmented, usually through collisions with neutral gas, electron capture, or a combination of these activation methods. Comparisons of observed and theoretical fragment ion masses then identify the species and localize the placement of various modifications within the full length protein sequence. When TDMS is coupled to immunoprecipitation enrichment (immunoprecipitation mass spectrometry, or IP-MS), this workflow identifies proteoforms after elution off the antibody. However, IP elution typically denatures noncovalent complexes and dissociates bound metals and cofactors, which means that neither the immunocomplex nor any multiproteoform complexes enriched as antigens can be directly identified. Alternatively, “native” MS preserves endogenous noncovalent interactions for characterization, simply by using buffer solutions at neutral pH containing little or no organic solvents. While there is precedent in the literature for native MS characterization of noncovalent immunocomplexes, there is limited literature on antigen ejection as a consequence of activation in the gas phase (“antigen ejection”). Furthermore, the cited example of antigen ejection was conducted at low resolution and stopped short of characterizing and confirming the antigen’s identity through fragmentation. Another work, by Zhang et al., showcases direct fragmentation of an immunocomplex (using only the fragment antigen binding region as opposed to the entire antibody), introducing ambiguity in cases where multiple antigens are bound. Accordingly, there is a need for methods for precisely and thoroughly characterizing immunocomplexes that can relate structure to function and enable identifying of antigen proteoforms and their binding sites. BRIEF SUMMARY OF THE INVENTION Disclosed herein is a method for proteoform-specific antigen identification. The method may comprise infusing a sample comprising an antigen and an antigen-binding protein into a mass spectrometer; isolating a complex comprising the antigen bound to the antigen-binding protein; ejecting the bound antigen from the complex; and identifying the ejected antigen and / or identifying the antigen-binding protein. Suitably, the sample may comprise a multiplicity of different antigens and / or antigen-binding proteins. This allows for the characterization of complex that can relate structure to function and enable the discovery of antigens and the binding sites. The Examples demonstrate that the disclosed technology is sensitive enough to distinguish closely related isoforms. Thus the disclosed technology allows for proteoform-level resolution that can reveal how specific functions are triggered such as in the adaptive immune response, including aberrant recognition of endogenous proteins underlying a diverse set of autoimmune disorders. BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. Fig. 1 After mixing the antibody and the antigen (far left), native electrospray infuses complexes into the mass spectrometer and releases them into the gas phase. Particles not bound to the antibody can be filtered away, 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. Different antigens are indicated with nearest adjacent symbol (square, circle, star, plus-sign, and none). Fig.2 Deconvolved mass spectra of components and complexes. (a) Measuring antibody- antigen interactions using a 2-site peptide (top), a 1-site peptide (middle), and a control experiment with no peptide (bottom) (Figure 5). 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 cartoonsdenote sequence similarities to and distinctions from the 2-site region shared among all Ara h 2 isoforms as illustrated in Figures 1 and 3 (indicated by bracketed legend). The antibody is also measured in its (b) monomeric and (c) dimeric forms in complex with the natural Ara h 2 antigen (Figure 8). The dimer is depicted in both unactivated (top) and activated (bottom) schemes. Greyscale circles denote isoform identity, and multi greyscale circles denote isoform ambiguity. Here, instrument configuration prioritized high-mass transmission, leaving the distribution of ejected antigens unknown. Fig. 3 (a) Filtering out of unbound antigens in a complex mixture (top) using Voltage Rollercoaster Filtering (VRF, bottom). Multi-greyscale 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 9). Figure 4. Middle-Down LC-MS Characterization of the Utilized Antibody. (a) Total ion current chromatogram from an LC-MS / MS run of the digested and reduced anti-Ara h 2 antibody. The asterisk denotes an off-target species. (b-d) Deconvolved monoisotopic mass spectra of the antibody (b) Fc region, (c) light chain (LC), and (d) Fd region. See Tables 4-6 for annotated fragmentation maps of the antibody (b) Fc region, (SEQ ID NO: 6) (c) light chain (LC), (SEQ ID NO: 7) and (d) Fd region (SEQ ID NO: 8) , respectively. Figure 5. Peptide-Mediated Dimerization in the m / z Domain. Measuring antigen-antibody interactions using a 2-site peptide (top), a 1-site peptide (middle), and a control experiment with no peptide (bottom). The antibody is detected in its monomeric (leftmost), dimeric (center), and trimeric (rightmost) forms, which are separated in mass-to-charge (m / z) space. Insets show select immunocomplex distributions in all three antibody stoichiometries. Cartoons paired with numbers indicate stoichiometries of which type of peptide is present. Figure 6. 0-Site Peptide-Antibody Experiment. Measuring antigen-antibody interactions with a 0-site control peptide. The antibody is measured in its monomeric (left), dimeric (right), and trimeric (right) forms. Cartoons of peptides indicate how many peptides are bound to the indicated form. Figure 7. LC-MS Characterization of Ara h 2. (a) The chromatogram for an LC-MS run of the reduced Ara h 2 antigen. Asterisks denote off-target species. (b-e) Deconvolved monoisotopic mass spectra (left) of Ara h 2 (b) Isoform 1 (SEQ ID NO: 9) , (c) Isoform 2 (SEQ ID NO: 10) , (d) Isoform 3 (SEQ ID NO: 11) , and (e) Isoform 4 (SEQ ID NO: 12). See Tables 7-10 for annotated fragmentation maps of Ara h 2 (b) Isoform 1, (c) Isoform 2, (d) Isoform 3, and (e) Isoform 4, respectively. Figure 8. Antigen-Mediated Dimerization in the m / z Domain. Measuring antigen-antibody interactions using the natural Ara h 2 antigen with the antibody, depicted in the mass-to-charge (m / z) space. The antibody is measured in its monomeric (leftmost) and dimeric (rightmost) forms. The dimer is depicted in both unactivated (top) and activated (bottom) schemes. Insets show select immunocomplex distributions at specified charge states. Colored dots indicate clarity or ambiguity in the annotation of antigen stoichiometries. Figure 9. Fragmentation of Isoform 2. (a) Fragmentation spectrum and (b) deconvolved mass spectrum of natural Ara h 2 Isoform 2 in a native top-down mass spectrometry experiment of the immunocomplex. See Table 11-12 for manually annotated and ProSight Lite annotated fragmentation maps of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. DETAILED DESCRIPTION OF THE INVENTION Antibody-antigen interactions are central to the immune response. Variation of protein antigens like isoforms and post-translational modifications can alter their antibody binding sites. To directly connect the recognition of protein antigens with their molecular composition, we antibody-antigen complexes were probed using native tandem mass spectrometry. The Examples demonstrate characterization of the prominent peanut allergen Ara h 2 and a convergent IgE variable region discovered in patients allergic to peanuts. In addition to measuring antigen-induced dimerization of IgE antibodies, the Examples demonstrate how immunocomplexes can be isolated in the gas phase and activated to eject, identify, and characterize proteoforms of their bound antigens. Using tandem experiments, the ejected antigens were isolated and then fragmented to identify their chemical composition. These results establish native top-down mass spectrometry as a platform for precise and thorough characterization of immunocomplexes to relate structure to function and enable the discovery of antigen proteoforms and their binding sites. Disclosed herein are methods for proteoform-specific antigen identification. The methods include the steps of infusing a sample containing an antigen and an antigen-binding protein into a mass spectrometer, isolating a complex containing the antigen bound to the antigen-binding protein, ejecting the bound antigen from the complex, and identifying the ejected antigen and / or identifying the antigen-binding protein. The methods disclosed herein are applied to samples containing antigens. The samples may be solid, liquid, or gaseous. In some cases, the antigens may include proteins, peptides, polysaccharide, lipids, or nucleic acids. In some cases, the antigens are synthetic antigens (e.g., synthetic peptide antigens) or isolated natural antigens. In some cases, the antigen may be obtained from a subject. In some cases, the sample may contain a multiplicity of different isoforms of the antigen. In some cases, the sample may contain a multiplicity of different complexes having at least one of the different isoforms of the antigen bound to the antigen-binding protein. The methods disclosed herein are applied to samples containing an antigen-binding protein. As used herein, the term ‘antigen-binding protein’ refers to molecules that can at least temporarily bind to an antigen. Examples of antigen-binding proteins include antibodies (e.g., IgA, IgD, IgE, IgG, or IgM), an antibody fragment (e.g., Fab or F(ab’)2), single-domain antibody (e.g., sdAb), or an engineered or recombinant antibody or fragment thereof (e.g., scFv, di-scFV, or tri-scFV). In some cases, the antigen-binding protein may be obtained from a subject. The methods disclosed herein may be applied to samples which contain a complex containing the antigen bound to the antigen-binding protein. In some cases, the complex is an immunocomplex. In some cases, the sample includes a multiplicity of different antigens and / or a multiplicity of different complexes having at least one of the different antigens bound to the antigen-binding protein. In some cases, the sample contains a multiplicity of different antigen- binding proteins and / or a multiplicity of complexes having the antigen bound to different antigen- binding proteins. In some cases, the different complexes are isolated from each other prior to ejecting a bound antigen from an isolated complex and identifying the ejected antigen and / or identifying the antigen-binding protein. The methods disclosed herein include infusing a sample into a mass spectrometer. As used herein, the term ‘infusing’ refers to introducing the sample to a mass spectrometer in a form which allows the mass spectrometer to sort components of the sample by m / z. There are several suitable methods and combinations of methods for introducing samples to mass spectrometers described in the art, including the liquid chromatographic methods as described in the Examples below, or gas chromatographic methods. Infusion of the sample may further include steps of ionizing components of the sample. There are many established methods for ionizing samples for mass spectrometry analysis described elsewhere in the art. Suitable ionization method includes native electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, electrospray ionization, chemical ionization, matrix-assisted laser desorption / ionization, electron ionization, and inductively coupled plasma ionization. There are many established mass spectrometry platforms described elsewhere in the art which may be used in the methods disclosed herein. As described in the Examples below, tandem mass spectrometry (MS / MS), and in particular liquid chromatography tandem mass spectrometry (LC-MS / MS) may be used. The methods disclosed herein include isolating a complex containing the antigen bound to the antigen-binding protein. Methods of isolating complexes may include isolating components of the sample on the basis of m / z. There are many suitable methods for isolating complexes established elsewhere in the art, such as sector field mass analysis, time-of-flight mass analysis, quadrupole mass analyzers, and ion traps (e.g., three-dimensional quadrupole ion trap, cylindrical ion trap, linear quadrupole ion trap, Orbitrap, etc). In some cases, the methods disclosed herein include a step of having the different complexes isolated from each other prior to ejecting a bound isoform from an isolated complex and identifying the ejected isoform. The methods disclosed herein may include filtering low mass components from the complex prior to isolating the complex. In some cases, the methods may include filtering low mass components from the sample prior to isolating the complex. In some case, filtering low mass components may include voltage roller coaster filtering. In some cases, the low mass components may include unbound antigens. In some cases, the different isoforms are ejected from the multiplicity of different complexes and one of the different isoforms is isolated and identified. In some embodiments, the different complexes are isolated from each other prior to ejecting a bound antigen from an isolated complex and identifying the ejected antigen. In some cases, the different antigens are ejected from the multiplicity of different complexes and one of the different antigens is isolated and identified. In some cases, the different antigen-binding proteins are isolated and identified after ejection of the bound antigen. The methods disclosed herein include ejecting the bound antigen from the complex. Methods for ejecting the bound antigen from the complex include using background gas. In some cases, higher energy collision dissociation (HCD). For example, ejection may include about 40 normalized collision energy, using mode charge state 25+, for HCD. In some cases, ejecting the bound antigen includes activation of the complex containing the antigen bound to the antigen- binding protein. In some cases, activating the complex removes nonspecific adducts. The methods disclosed herein include identifying the ejected antigen and / or identifying the antigen-binding protein. Methods for identification of the ejected antigen and / or identifying the antigen-binding protein may include mass spectrometry, including fragmentation of the antigen and / or antigen-binding protein into ions, mass analysis of the fragments, and detection of the fragments on a detector. Suitable methods for fragmentation may include ionization. Suitable ionization method includes native electrospray ionization, atmospheric pressure chemical ionization, atmospheric pressure photoionization, electrospray ionization, chemical ionization, matrix-assisted laser desorption / ionization, electron ionization, and inductively coupled plasma ionization. There are many established mass spectrometry platforms described elsewhere in the art which may be used in the methods disclosed herein. As described in the Examples below, tandem mass spectrometry (MS / MS). Here we demonstrate proteoform-specific antigen identification from immunocomplexes via native TDMS. By way of example, we analyzed the interactions between the four major isoforms of peanut allergen Ara h 2 and the convergently evolved variable region of an IgE, with picomolar binding affinity for Ara h 2, found in a clonal family of six plasmablasts from two unrelated peanut-allergic patients. As depicted in Figure 1, the immunocomplex and its components were analyzed together to create a link between the immune response and the specific proteoforms of bound antigens, including their binding sites. Bound complexes are then isolated by their mass-to-charge ratio and activated to eject, partially sequence, and identify the antigen(s) recognized by the recombinant antibody. The disclosed technology can identify antigens, including “orphan” antigens like those encountered in autoimmune disorders. To probe the interactions between the antibody and antigen (Figure 2a, Table 1), we subjected synthetic peptide antigens bound to a convergent set of IgE complementarity- determining region sequences, presented using an IgG1 scaffold (Table 1, Figure 4), to native MS on a Q Exactive Ultra-High Mass Range mass spectrometer (Table 2). The basic peptide contains a sequence with two binding sites (“2-site peptides”) (SEQ ID NO: 3), known to be conserved across all four Ara h 2 isoforms. An 8.5 µM:10 µM mixture of 2-site peptides with the antibody (Figure 2a, top) primarily exhibited masses corresponding to antibody dimerization (85% of the total integrated peak area as deconvolved by UniDec, Table 3) with some trimerization (8.3%).2- site peptides bound to the antibody monomer at stoichiometries of 0 (5.0%), 1 (40%), 2 (41%), and 3 (14%), and the peptides also bound to the antibody dimer in stoichiometries of 2 (60%), 3 (33%), and 4 (7.7%). The antibody trimer exhibited two-site peptide binding stoichiometries of 3 (63%), 4 (36%), and 5 (1.1%). We hypothesized that since the antibody dimers bound to a minimum of two peptides as opposed to zero peptides in the monomeric case, the peptides may act as a mediator for antibody dimerization. Otherwise, lower peptide stoichiometries would be prominent in the antibody dimer. To probe peptide-mediated dimerization, we utilized a version of the peptide where one of the antibody binding sites was replaced by a random sequence, leaving a single binding site on a peptide of comparable length (“1-site peptides”) (SEQ ID NO: 2). A 1-site peptide experiment exhibited 88% monomeric antibody form and 12% dimerization (Figure 2a, middle). Additionally, in a control experiment with no peptide (Figure 2a, bottom), the antibody exhibited low levels of self-dimerization (18%) and self-trimerization (0.73%) at a 10 µM concentration. The stark contrast between the high level of dimerization in the 2-site peptide experiment and the low level of dimerization in both the 1-site peptide experiment and the negative control shows that dimerization is strongly dependent on the presence of two antibody binding sites. The antibody dimerization exhibited during the 2-site peptide experiment was antigen- mediated. In addition to the discrepancy in dimer abundance, the highest-abundance charge state of the antibody dimer in the 2-site peptide experiment (37+) was higher than in the 1-site peptide experiment (34+) and the control (34+) (Figure 5), demonstrating different conformations and, therefore, modes of dimerization (e.g., nonspecific versus antigen-mediated). These data also show that all four Ara h 2 isoforms may be capable of mediating antibody dimerization, as the sequence containing both binding sites in the 2-site peptide is conserved across all four isoforms. The two binding sites are separated by a single glutamine residue, so their sequence proximity does not appear to be a barrier for multiple binding events. In other words, the peptide-mediated dimerization predicts antigen-mediated dimerization for all four isoforms, given that the protein’s tertiary structure does not obstruct binding regions. As antibody dimers in the 2-site peptide experiment only exhibited peptide stoichiometries of 2, 3, and 4, antigen-mediated complexation coincides with both binding regions of at least one antibody being occupied. While some stoichiometries exceed the number of available binding sites in an antigen-mediated model, such as three peptides on the antibody monomer or four peptides on the antibody dimer, these less frequent cases are likely due to nonspecific binding as shown in a “0-site” peptide (SEQ ID NO: 1) experiment (Figure 6). Therefore, most antibody complexation is binding site-directed, reinforcing the unique dimerization of the two-site experiment. While the synthetic peptide data predict that all four Ara h 2 isoforms can mediate antibody dimerization, we sought to prove this by probing immunocomplexes containing the Ara h 2 antigens (Figure 7). We mixed Ara h 2 purified from peanut flour and with the IgE construct described above (Table 1) in a 1:1 ratio; this yielded both antibody monomers (81% of total area, Figure 2b) and antibody dimers (19%; Figure 2c, top). The antigen bound to antibody monomers in stoichiometries of 0 (35%), 1 (43%), and 2 (22%). The two-antigen antibody monomer exhibited two resolved peaks corresponding to an additional light isoform (55%, isoforms 2 and 4) or an additional heavy isoform (45%, isoforms 1 and 3). Low-resolution charge deconvolution of the antigen dimer reveals that the entire antibody dimer distribution was comprised of two antibodies and two unknown antigens. To conduct the study at higher resolution (2x), the complex was isolated and activated using background gas (Higher energy Collision Dissociation, or HCD) (Figure 2c, bottom). The activation removed nonspecific adducts such that the light and heavy isoform groups could be resolved. Activation also yielded a secondary distribution (23% of total area) of antibody dimers at a higher mass-to-charge ratio (m / z) than the primary distribution (77%). This secondary distribution is comprised of two antibodies and a single antigen, primarily a light isoform (74%). Most of the 2-antigen complexes in the activated spectrum included one heavy isoform and one light isoform (53%), followed by two light isoforms (40%) and two heavy isoforms (7.8%). Despite the improved spectral clarity, the mass differences among isoforms within the same weight group were typically not resolved. The 34+ charge state, for example, did have partially resolved peaks for all four isoforms (Figure 8), but this resolution was not enough to provide satisfactory deconvolution. This experiment demonstrates that intact native MS is insufficient for deep characterization with even modest levels of antigen heterogeneity. However, the secondary distribution in the activated spectrum indicates that HCD had ejected a single antigen that carried away a disproportionate amount of charge (“asymmetric charge partitioning”). This trend validates that antigen ejection and subsequent characterization is possible, which would provide a direct link between antibody and antigen. We next conducted native TDMS on an Orbitrap Eclipse to controllably disassemble the immunocomplex (Figure 3), which was overloaded with antigen (~10:1). In this regime, we previously developed a technique called Voltage Rollercoaster Filtering (“VRF”) that markedly enhances the signals of high mass complexes while simultaneously filtering out unwanted low- mass components such as unbound antigens. With VRF, ions are accelerated into and decelerated out of a high-pressure region near the inlet of the instrument. Unlike their complexed counterparts, unbound antigens lose too much of their kinetic energy through collisions in the high-pressure region to proceed, giving rise to a filtering effect far before the excess antigens can substantially interfere with the complexes via space charge effects. Without VRF, all four isoforms were represented (25% isoform 1, 39% isoform 2, 14% isoform 3, and 23% isoform 4), and the immunocomplex was detected at <10% spectral intensity (Figure 3a, top). After applying VRF, no unbound antigen was detected, and the monomeric antibody complexes were preserved (Figure 3a, bottom). Then, we used the ion trap to isolate the immunocomplex before HCD. All four isoforms were ejected (22% isoform 1, 41% isoform 2, 15% isoform 3, 22% isoform 4), and the ejected distribution’s similarity to the distribution of the excess unbound antigens suggest that the four isoforms have comparable binding affinities (Figure 3b, including inset). Finally, after isolating a specific proteoform in the ion trap (i.e., isoform 2), the antigen was subject to further dissociation by HCD (Figure 3c). Disulfide bridges span Ara h 2, preventing complete fragmentation and sequencing; however, the dense residue-by-residue fragmentation on the C- terminus and outside the disulfide bridges was sufficient to distinguish the target. Importantly, ProSight Lite reported a P-score of 4.7x10-18for these fragmentation data (Figure 9) showing proof-of-concept for confident antigen identification. The masses of all C-terminal fragment ions support a slightly elongated terminus, consistent with the additional two residues present in isoforms 1 and 2. Furthermore, the C-terminal fragment ions localized a 14 Da loss to E130 (Figure 9), which is consistent with the E130 ^D130 substitution detected exclusively in isoforms 2 and 4 (Figure 7). The ability to distinguish isoform 2 from three other isoforms of similar mass directly from the antibody-antigen complex solidifies native TDMS as a viable method for precise characterization of the immune response. The Examples establish that native TDMS can be used to inform our understanding of the immune response. The power in this approach lies in the ability to characterize both the stoichiometry of immunocomplexes and the ejected antigens, whose subtle mass differences typically cannot be resolved by gel-based or fluorescence-based readouts. Immunoglobulin therapeutics can be evaluated against the antigens to which they were designed to target as well as disruptors of those immunocomplexes. Differences between the antigens actually bound and the bulk antigen population can reveal how and to what extent a therapeutic can bind—even in complex mixtures. Whether the antibody consists of a convergent variable region on a monoclonal IgG scaffold (as was done here) or a polyclonal population, the profile of ejected antigens reveals which of their proteoforms are recognized by immuno-capture reagent(s). The ability to directly link antigens to the immune response with proteoform-level resolution can reveal how specific functions are triggered in the adaptive immune response, including aberrant recognition of endogenous proteins underlying a diverse set of autoimmune disorders. Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. References (1) Smith, L. M.; Kelleher, N. L.; Consortium for Top Down, P. Proteoform: a single term describing protein complexity. Nat. Methods 2013, 10, 186-187. (2) Yang, X.-J. Multisite protein modification and intramolecular signaling. Oncogene 2005, 24, 1653-1662. (3) Skinner, O. S.; Haverland, N. A.; Fornelli, L.; Melani, R. D.; Do Vale, L. H. F.; Seckler, H. S.; Doubleday, P. F.; Schachner, L. 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(14) Croote, D.; Darmanis, S.; Nadeau, K. C.; Quake, S. R. High-affinity allergen-specific human antibodies cloned from single IgE B cell transcriptomes. Science 2018, 362, 1306. (15) Marty, M. T.; Baldwin, A. J.; Marklund, E. G.; Hochberg, G. K.; Benesch, J. L.; Robinson, C. V. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem.2015, 87, 4370-4376. (16) Lorenzen, K.; van Duijn, E. Native mass spectrometry as a tool in structural biology. Curr Protoc Protein Sci 2010, Chapter 17, Unit1712. (17) Laszlo, K. J.; Munger, E. B.; Bush, M. F. Folding of Protein Ions in the Gas Phase after Cation-to-Anion Proton-Transfer Reactions. Journal of the American Chemical Society 2016, 138, 9581-9588. (18) Chandler, S. A.; Benesch, J. L. P. Mass spectrometry beyond the native state. Curr. Opin. Chem. Biol.2018, 42, 130-137. (19) Light-Wahl, K. J.; Schwartz, B. L.; Smith, R. D. Observation of the Noncovalent Quaternary Associations of Proteins by Electrospray Ionization Mass Spectrometry. Journal of the American Chemical Society 1994, 116, 5271-5278. (20) Jurchen, J. C.; Williams, E. R. Origin of Asymmetric Charge Partitioning in the Dissociation of Gas-Phase Protein Homodimers. Journal of the American Chemical Society 2003, 125, 2817-2826. (21) Sciuto, S. V.; Liu, J.; Konermann, L. An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase. J. Am. Soc. Mass Spectrom.2011, 22. (22) McGee, J. P.; Melani, R. D.; Goodwin, M.; McAlister, G.; Huguet, R.; Senko, M. W.; Compton, P. D.; Kelleher, N. L. Voltage Rollercoaster Filtering of Low-Mass Contaminants During Native Protein Analysis. J. Am. Soc. Mass Spectrom.2020, 31, 763-767. (23) Gault, J.; Liko, I.; Landreh, M.; Shutin, D.; Bolla, J. R.; Jefferies, D.; Agasid, M.; Yen, H.-Y.; Ladds, M. J. G. W.; Lane, D. P.; Khalid, S.; Mullen, C.; Remes, P. M.; Huguet, R.; McAlister, G.; Goodwin, M.; Viner, R.; Syka, J. E. P.; Robinson, C. V. Combining native and ‘omics’ mass spectrometry to identify endogenous ligands bound to membrane proteins. Nat. Methods 2020, 17, 505-508. (24) Fellers, R. T.; Greer, J. B.; Early, B. P.; Yu, X.; LeDuc, R. D.; Kelleher, N. L.; Thomas, P. M. ProSight Lite: graphical software to analyze top-down mass spectrometry data. Proteomics 2015, 15, 1235-1238. (25) Melani, R. D.; Des Soye, B. J.; Kafader, J. O.; Forte, E.; Hollas, M.; Blagojevic, V.; Negrao, F.; McGee, J. P.; Drown, B.; Lloyd-Jones, C.; Seckler, H. S.; Camarillo, J. M.; Compton, P. D.; LeDuc, R. D.; Early, B.; Fellers, R. T.; Cho, B. K.; Mattamana, B. B.; Goo, Y. A.; Thomas, P. M., et al. Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire. J Proteome Res 2022, 21, 274-288. EXAMPLES Sample Preparation Three custom Ara h 2 peptides (sequence on Table 1) were produced and isolated (≥96% purity) by GenScript to test the possible antigen-binding sites. The 2-site peptide represents the isoform 1 sequence from amino acid 59 to 79 (Uniprot Q6PSU2). The sequence in isoform 1 from amino acid 61 to 76 is conserved in all four isoforms and contains two binding sites. The peptides were dissolved in ammonium acetate. Peptides and antibodies were mixed together with a final concentration of 8.5 µM peptide and 10 µM antibody. Samples were incubated on ice for at least 2 min before native TDMS analysis. For native analysis involving the antibody-antigen complex, the isolated Ara h 2 antigen was acquired from Indoor Biotechnologies (NA-AH2-1). A custom antibody previously described1containing the variable region of an IgE and expressed on an IgG scaffold (sequence on Table 1) was recombinantly produced by GenScript. Both proteins were desalted 10 times at 10,000 × g for 2-10 minutes in 3 kDa and 100 kDa Amicon Ultra centrifugal filters (Merck Millipore), respectively, with 100 mM ammonium acetate. The antibody was combined with the antigen for a final concentration of 10 µM antigen and 10 µM antibody. The mixture was incubated on ice for at least 2 min before native TDMS analysis. For middle-down analysis, 20 µg of the antibody construction was digested with IdeS protease (Promega) following the manufacturer’s protocol. After digestion, the samples were reduced and cleaned, according to Melani et al.2019 before LC-MS / MS analysis.2Intact Ara h 2 was also analyzed by LC-MS / MS. Reduction of 20 ug of protein was carried out using 8 M urea and 150 mM TCEP for 1 hour at 37°C. Following the manufacture protocol, the sample was desalted using TopTip C4 (PolyLC). The eluate was dried out using a vacuum concentrator and resuspended in buffer A (94.8% water, 5% acetonitrile, and 0.2% formic acid). Data Acquisition Middle-down antibody and intact antigen LC-MS / MS measurements were performed under denaturing conditions using an Ultimate 3000 nanoLC (Thermo Fisher Scientific) equipped with a homemade PLRP-S (5 µm particle size and 1000 Å pore size, Agilent Technologies) trap (30 mm L, 150 µm i.d.) and column (200 mm L, 75 µm i.d.) online with an Orbitrap Fusion Lumos (Thermo Fisher Scientific). The loading pump was operated at 3 μL / min, and the nano-pump gradient flow rate was set at 300 nL / min. LC separation was performed under the following gradient of buffer B (94.8% acetonitrile, 5% water, and 0.2% formic acid): 5% B from 0 to 10 min., 15% B at 13 min., 50% B at 45 min., 95% B from 47 to 49 min., 5% B from 52 to 60 min. The mass spectrometer was operated in “protein mode” with 2 m Torr of N2 pressure, and the acquisition was performed using a data-dependent top 2 MS2method. Spray voltage was set at 1,800 V, transfer capillary temperature was set at 320°C, ion funnel RF was set at 30%, and a 15 eV of source CID was applied. MS1spectra were acquired at 120,000 of resolving power (at 200 m / z), AGC target value of 5×105charges / acquisition, 100 ms. of maximum injection time, and 4 μscans. MS2used 23 of NCE for HCD and spectra were acquired at 60,000 resolving power (at 200 m / z), with target AGC values of 1×106charges / acquisition, 800 msec of maximum injection time, and 4 μscans. Precursors were quadrupole isolated used using a 3 Th isolation window, dynamic exclusion of 60 sec duration, and threshold of 2×104intensity. Samples were analyzed in technical triplicate. For native experiments, samples were sprayed via static spray using a Thermo Fisher Nanospray Flex source and medium-sized borosilicate-coated emitters. All studies concerning native antibody dimerization (using the Ara h 2 peptides or the intact antigen) were conducted on a Q Exactive Ultra-High Mass Range (UHMR) instrument from Thermo Fisher Scientific using the parameters listed in Table 2. Data was collected at the highest resolution possible while still allowing annotation: 2-4k resolution. All studies concerning native antigen-antibody ejection were conducted on an Orbitrap Eclipse (Thermo Fisher) with an HCD collision pressure of 8-20 mTorr in protein mode. Data were collected with a spray voltage of 2-2.5 kV, 2-3 microscans, 120,000 resolving power, 250- 5000 ms maximum injection time, and up to 100 averaged scans. Voltage Rollercoaster Filtering was used to filter out unbound antigen, using low levels of activation (20 V IS-CID) and optimized parameters found previously for antibodies.3After a broadband ion trap isolation of all complexes, the antigen was ejected using 40 NCE (using mode charge state 25+) for HCD, and the antigen was fragmented using 50 NCE after ion trap of solely isoform 2, charge state 8+. For these experiments, the antibody was at 5 µM, and the antigen was at 50 µM. Data was collected in full profile mode at 120k resolution with 2 microscans and an average of 100 acquisitions. Intact native (UHMR) and native top-down (Eclipse) experiments were repeated to validate the trends, challenges, and methods discussed in the main text, and these repeat experiments yielded comparable results to those reported here. Data Analysis Low-resolution (non-isotopically resolved) spectra were deconvolved using UniDec4using parameters that minimized spectral artifacts. All used parameters are listed in Table 3 according to the final spectra. Fragmentation data were manually annotated using MMass (v. 5.5.0)5and validated using ProSight Lite v.1.4 (10 ppm)6on a deconvolved spectrum using Xtract (SN 7, Qual Browser v.4.1.31.9). Annotations were made in reference to UniProt accession Q6PSU2. References in the Examples (1) Croote, D.; Darmanis, S.; Nadeau, K. C.; Quake, S. R. High-affinity allergen-specific human antibodies cloned from single IgE B cell transcriptomes. Science 2018, 362, 1306. (2) Melani, R. D.; Srzentić, K.; Gerbasi, V. R.; McGee, J. P.; Huguet, R.; Fornelli, L.; Kelleher, N. L. Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry. mAbs 2019, 11, 1351-1357. (3) McGee, J. P.; Melani, R. D.; Goodwin, M.; McAlister, G.; Huguet, R.; Senko, M. W.; Compton, P. D.; Kelleher, N. L. Voltage Rollercoaster Filtering of Low-Mass Contaminants During Native Protein Analysis. J. Am. Soc. Mass Spectrom.2020, 31, 763-767. (4) Marty, M. T.; Baldwin, A. J.; Marklund, E. G.; Hochberg, G. K.; Benesch, J. L.; Robinson, C. V. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem.2015, 87, 4370-4376. (5) Strohalm, M.; Kavan, D.; Novák, P.; Volný, M.; Havlíček, V. mMass 3: A Cross- Platform Software Environment for Precise Analysis of Mass Spectrometric Data. Anal. Chem. 2010, 82, 4648-4651. (6) Fellers, R. T.; Greer, J. B.; Early, B. P.; Yu, X.; LeDuc, R. D.; Kelleher, N. L.; Thomas, P. M. ProSight Lite: graphical software to analyze top-down mass spectrometry data. Proteomics 2015, 15, 1235-1238.

[0002] Table 1. Custom Analyte Sequences. Analyte  Sequence  0‐Site Peptide  SYGRRELRNLQRELRNLQDPD (SEQ ID NO: 1)  1‐Site Peptide  SYGRRELRNLQDPYSPSQDPD (SEQ ID NO: 2)  2‐Site Peptide  SYGRDPYSPSQDPYSPSQDPD (SEQ ID NO: 3)  EIVLTQSPGTLSLSPGGRGTLSCRTSQTINNAHLAWYQHKPGQAPR LLIYGSSERATGVPDRFSGSGSGSDFTLTISSLEAEDFAVYYCQHY Antibody Light  GRSPPYTFGPGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL Chain  NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 4)  QVQLVNSGGGVVQPGRSLRLSCVASGFTFSTFGIHWVRQAPGKGLE WVAVISNDGEKSESADSVKGRFTPSRDNSKNTVYLQMNNLRVEDTA VYYCAKVLDYSNYYYYYGMDVWGQGTTVIVSSASTKGPSVFPLAPS SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT Antibody Heavy  HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE Chain  DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID  NO: 5)

[0003] Table 2. Settings of Interest on the Q Exactive UHMR Instrument. Antige rameter Peptn‐Antibody  Paide‐Antibody Complex  Complex  Nominal Resolution  2188  2188 ‐ 4375  S‐lens RF Level  200  200  Ion Transfer Target m / z  High  High  Extended Trapping (V)  100  N / A  Normalized Collisional  Energy N / A  0 ‐ 4 Normalization Factor  N / A  1  Ultra High Vacuum  Pressure (mbar) 2.21e‐10 ‐ 2.25e‐10  1.90e‐10 ‐ 3.25e‐10 Injection Time (ms)  100  400  Detector m / z  High  High  Averaging  20  20‐26  Microscans  10  20‐30  UHMR Mode  On  On

[0004] Table 3. UniDec Parameters. 0-Site 1-Site 2-Site Complex Complex Unbound Ejected Parameter Control Peptide Peptide Peptide MS1MS2Antigen Antigen 10- 20- 10- 10- 20- 20- 1- 1- Charge Range 50 50 50 50 50 50 20 20 Mass Range 100- 100- 310- 16.2- 16.2- 100-500 100-500 100-500 (kDa) 500 350 340 18.5 18.5 Sample Mass 10 10 10 10 200 200 50 50 Interval (Da) Smooth Charge States True True True True True True True True Distribution Use Automatic False False False False False False False False m / z Peak Width Smooth Nearby Some Some Some Some Lots None Some None Points Suppress Some Some Some Some Some Some Some Some Artifacts Peak Detection 1200 1200 1200 1200 300 300 150 150 Range (Da) Peak Detection 0.001 0.001 0.001 0.001 0.1 0.1 0.01 0.25 Threshold

[0005] Table 4. Annotated antibody Fc region corresponding to Figure 4b. Bold on the fragmentation map denotes G0F glycosylation. SEQ ID NO: 6NGPSVFLFPPRPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGC Table 5. Annotated antibody light chain (LC) corresponding to Figure 4c. SEQ ID NO: 7NEIVLTQSPGTLSLSPGGRGTLSCRTSQTINNAHLAWYQHKPGQAPRLLIYGSSERATGVPDRFSGSGSGSDFTLTISSLEAEDFAVYYCQHYGRSPPYTF GPGTKLDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGECC Table 6. Annotated antibody Fd region, corresponding to Figure 4d. Italics denotes the presence of pyroglutamic acid. SEQ ID NO: 8NQVQLVNSGGGVVQPGRSLRLSCVASGFTFSTFGIHWVRQAPGKGLEWVAVISNDGEKSESADSVKGRFTPSRDNSKNTVYLQMNNLRVEDTAVYYCAKVL DYSNYYYYYGMDVWGQGTTVIVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGC Table 7. Annotated Ara h 2 Isoform 1, corresponding to Figure 7b. Bold on the fragmentation maps denote hydroxyprolines. SEQ ID NO: 9NRQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDPYSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEAL QQIMENQSDRLQGRQQEQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDR YC Table 8. Annotated Ara h 2 Isoform 2, corresponding to Figure 7c. Bold on the fragmentation maps denote hydroxyprolines, and italics on the fragmentation maps denote substitutions G40 ^E40 and E130 ^D130. SEQ ID NO: NRQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDP 10 YSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQ GRQQEQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDRYC Table 9. Annotated Ara h 2 Isoform 3, corresponding to Figure 7d. Bold on the fragmentation maps denote hydroxyprolines. SEQ ID NO: NRQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDP 11 YSPSQDPDRRDPYSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEAL QQIMENQSDRLQGRQQEQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDC Table 10. Annotated Ara h 2 Isoform 4, corresponding to Figure 7e. Bold on the fragmentation maps denote hydroxyprolines, and italics on the fragmentation maps denote substitutions G40 ^E40 and E130 ^D130. SEQ ID NO: NRQQWELQGDRRCQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDP 12 YSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQ GRQQEQQFKRELRNLPQQCGLRAPQRCDLEVESGGRDC Table 11. Manually annotated fragmentation map of Isoform 2 corresponding to FIG.9C. Each parentheses indicates a detected and annotated fragment ion that contains the region extending from the parentheses to the terminus towards which the parentheses encloses. Italics indicate the location of one-half of a disulfide bridge. Bold indicates the presence of hydroxyprolines. Underline indicate the presence of substitutions G40 ^E40 and E130 ^D130, present on isoforms 2 and 4. The double-underline indicates a region conserved in isoforms 1 and 2 of the antigen. SEQ ID NRQQWELQ)G)D)R)R)CQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDP NO: 13 YSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQ GRQQEQQFKRELRNLPQQCGLRAPQRC(D(L(E(VE(SGGRDRYC Table 12. ProSight Lite annotated fragmentation map of Isoform 2, corresponding to FIG. 9D. Each parentheses indicates a detected and annotated fragment ion that contains the region extending from the parentheses to the terminus towards which the parentheses encloses. Italics indicate the location of one-half of a disulfide bridge. Bold indicates the presence of hydroxyprolines. Underline indicate the presence of substitutions G40 ^E40 and E130 ^D130, present on isoforms 2 and 4. The double-underline indicates a region conserved in isoforms 1 and 2 of the antigen. SEQ ID NRQQWELQG)D)R)R)CQSQLERANLRPCEQHLMQKIQRDEDSYGRDPYSPSQDP NO: 14 YSPSPYDRRGAGSSQHQERCCNELNEFENNQRCMCEALQQIMENQSDRLQ GRQQEQQFKRELRNLPQQCGLRAPQRC(D(LE(VESGGRDRYC

Claims

CLAIMS 1. A method for proteoform-specific antigen identification, the method comprising: infusing a sample comprising an antigen and an antigen-binding protein into a mass spectrometer; isolating a complex comprising the antigen bound to the antigen-binding protein; ejecting the bound antigen from the complex; and identifying the ejected antigen and / or identifying the antigen-binding protein.

2. The method of claim 1, wherein the sample comprises a multiplicity of different isoforms of the antigen and / or a multiplicity of different complexes comprising at least one of the different isoforms of the antigen bound to the antigen-binding protein.

3. The method of claim 2, wherein the different complexes are isolated from each other prior to ejecting a bound isoform from an isolated complex and identifying the ejected isoform.

4. The method of claim 2, wherein the different isoforms are ejected from the multiplicity of different complexes and one of the different isoforms is isolated and identified.

5. The method of claim 1, wherein the sample comprises a multiplicity of different antigens and / or a multiplicity of different complexes comprising at least one of the different antigens bound to the antigen-binding protein.

6. The method of claim 5, wherein the different complexes are isolated from each other prior to ejecting a bound antigen from an isolated complex and identifying the ejected antigen.

7. The method of claim 5, wherein the different antigens are ejected from the multiplicity of different complexes and one of the different antigens is isolated and identified.

8. The method of claim 1, wherein the sample comprises a multiplicity of different antigen-binding proteins and / or a multiplicity of complexes comprising the antigen bound to different antigen-binding proteins.

9. The method of claim 8, wherein the different complexes are isolated from each other prior to ejecting a bound antigen from an isolated complex and identifying the ejected antigen and / or identifying the antigen-binding protein.

10. The method of claim 8, wherein the different antigen-binding proteins are isolated and identified after ejection of the bound antigen.

11. The method of any one of claims 1-10 further comprising filtering low mass components from the sample prior to isolating the complex.

12. The method of claim 11, wherein unbound antigen is filtered from the complex.

13. The method of claim 11 or 12, wherein filtering low mass components comprises voltage rollercoaster filtering.

14. The method of any one of claims 1-13 further comprising activating the complex.

15. The method of claim 14, wherein activating the complex ejects the antigen from the complex.

16. The method of claim 14-15, wherein activating the complex comprises higher energy collision dissociation.

17. The method of any one of claims 1-16, wherein the antigen-binding protein is an antibody, an antibody fragment, single-domain antibody, or an engineered or recombinant antibody or fragment thereof.

18. The method of any one of claims 1-17, wherein the antigen-binding protein is obtained from a subject.

19. The method of any one of claims 1-17, wherein the antigen is obtained from a subject.

20. The method of any one of claims 1-19, wherein the complex is an immunocomplex.

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