Rapid structural discrimination of IgG antibodies by multicharge-state collision-induced unfolding

Immunoglobulin G (IgG) antibodies are an important class of biotherapeutics that target various diseases, such as cancers, neurodegenerative disorders, and autoimmune diseases, yet rapid discrimination of IgG antibodies remains a great challenge due to heterogeneity, flexibility, and large size. Herein, we demonstrate a simplified multicharge-state collision-induced unfolding (CIU) method for rapid differentiation of four IgG isotypes that differ in terms of the numbers and patterns of disulfide bonds, bypassing tedious single charge-state selection in advance. The results presented herein reveal that gas-phase unfolding behaviors have a strong dependence on charge states outside IgG surfaces; therefore, multicharge-state CIU analysis of IgG subtypes could offer a great opportunity to gain deeper insights into their gas-phase structural differentiation. The full discrimination of IgG antibody isoforms that possess different disulfide bond numbers and even subtle disulfide bonding patterns can be achieved based on their charge-dependent gas-phase unfolding behaviors and root-mean square deviation in CIU difference spectra. Taken together, the incorporation of all charge states observed in a native ion mobility-mass spectrometry (IM-MS) experiment to CIU analysis could make this strategy sensitive to more subtle structural discrepancies, facilitating the rapid discrimination and evaluation of innovative structurally similar biotherapeutic candidates with unexplored functions.