Characterizing Intact IgA2 Antibody and Its Size Variants Using Native Mass Spectrometry and a Tandem MS-based Charge Reduction Approach

Characterization of protein structures at multiple levels plays an essential role in study on protein functions. Structural analysis at the intact protein or intact complex level allows access of information on aspects including level of post-translational modification (PTM), distribution of variants and stoichiometry of binding. Glycosylation is the most common and complex type of PTM, and most natural and therapeutic proteins are glycoproteins. With the increasing level of glycosylation, the heterogeneity of glycoprotein systems increases, the difference in biochemical properties between glycans and polypeptide chains compromises the accuracy of characterization using conventional tools. Mass spectrometry (MS) measures the mass-to-charge ratios of ionized protein species regardless of such difference, while the native MS strategy allows preservation of non-covalent interactions and direct analysis of intact protein and their complexes. However, the accuracy and even feasibility of native MS measurement of intact glycoprotein species are frequently hindered by signal overlapping caused by the wide mass distribution of the heterogeneous proteoforms, and inconsistency of mass profiles between proteoform populations in different charge states due to their differing ionization responses. Although several approaches based on charge reduction were developed to solve these problems, their applicability is limited by the efficiency of charge resolution or availability of electron-based or proton-based gas-phase reaction modules in commercial models of mass spectrometers. In this work, a tandem MS-based approach was used to characterize the intact size variants of an IgA2 monoclonal antibody (mAb) that incorporated non-covalent subunits. This approach utilized both charge reduction effect and the mass balance rule of the dissociation reactions, and employed collision induced dissociation (CID) or higher energy collisional dissociation (HCD) as the fragmentation reaction. Through parallel measurement of this IgA2 system using limited charge reduction approaches for both ensemble and subpopulations of proteoforms, as well as this tandem MS-based approach and cross-validation, the accuracy provided by these approaches was evaluated. The results suggested that when fragments can be released with high specificity and a defined mass, the tandem-MS based approach can provide sufficient accuracy for both charge and mass determination of heterogeneously glycosylated protein complexes at the intact level. This approach may be used as a universal solution to characterization of a larger variety of heterogeneously glycosylated protein systems.