posted on 2024-02-06, 15:36authored byYang Yang, Daniil G. Ivanov, Michael D. Levin, Bogdan Olenyuk, Oscar Cordova-Robles, Brittany Cederstrom, Jan E. Schnitzer, Igor A. Kaltashov
Large immune complexes formed by the cross-linking of
antibodies
with polyvalent antigens play critical roles in modulating cell-mediated
immunity. While both the size and the shape of immune complexes are
important determinants in Fc receptor-mediated signaling responsible
for phagocytosis, degranulation, and, in some instances, autoimmune
pathologies, their characterization remains extremely challenging
due to their large size and structural heterogeneity. We use native
mass spectrometry (MS) supplemented with limited charge reduction
in the gas phase to determine the stoichiometry of immune complexes
formed by a bivalent (homodimeric) antigen, a 163 kDa aminopeptidase
P2 (APP2), and a monoclonal antibody (mAb) to APP2. The observed (APP2·mAb)n complexes populate a wide range of stoichiometries
(n = 1–4) with the largest detected species
exceeding 1 MDa, although the gas-phase dissociation products are
also evident in the mass spectra. While frequently considering a nuisance
that complicates interpretation of native MS data, limited dissociation
provides an additional dimension for characterization of the immune
complex quaternary structure. APP2/mAb associations with identical
composition but slightly different elution times in size exclusion
chromatography exhibit notable differences in their spontaneous fragmentation
profiles. The latter indicates the presence of both extended linear
and cyclized (APP2·mAb)n configurations.
The unique ability of MS to distinguish between such isomeric structures
will be invaluable for a variety of applications where the biological
effects of immune complexes are determined by their ability to assemble
Fc receptor clusters of certain density on cell surfaces, such as
platelet activation by clustering the low-affinity receptors FcγRIIa
on their surface.