Speaker
Description
Antibodies show tremendous structural diversity shaping their biological functions in immunity and immune pathologies. While IgG1 Fc domains have been extensively characterized by MS approaches, more complex antibody structures (including Fab glycosylated IgGs or isotypes such as IgA or IgM) are less well understood. This presentation showcases the integration of miniaturized bottom-up, middle-up and intact mode workflows for dissecting the Fab and Fc hetereogeneity of endogenous antibodies, with a particular focus on defining glycoform profiles. By employing specific capturing approaches in combination with selective hinge-region cleavage strategies, specific antibody populations and fragments are isolated and characterized revealing intrinsic differences. Native-mode affinity-capillary electrophoresis with mass spectrometry allows to assess how antibody proteoforms including Fc and Fab glycosylation define Fc-receptor interactions. This work provides insights into molecular details of antibody functionalities in health and disease.
We applied the middle-up approach to characterize IgG Fc portions of anti‐citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) from both synovial fluid and plasma samples, and compared them to the proteoform profiles of total or bulk IgG from the same samples. We observed differences in isotype and allotype usage as well as Fc glycosylation between the different antibody populations. Remarkably, both IgG1 agalactosylation and IgG4 subclass usage appeared to associate with disease activity as well as erythrocyte sedimentation rate, providing indications of a possible contribution of these antibody variants to RA etiology.
References
Blöchl et al. 2025 Fc Proteoforms of ACPA IgG Discriminate Autoimmune Responses in Plasma and Synovial Fluid of Rheumatoid Arthritis Patients and Associate with Disease Activity. Advanced Sciences. e2408769. doi: 10.1002/advs.202408769.
Gstöttner et al. 2024 Benchmarking glycoform-resolved affinity separation - mass spectrometry assays for studying FcγRIIIa binding. Front Immunol. 15:1347871. doi:10.3389/fimmu.2024.1347871.
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