Abstract

BACKGROUND Proteoform-level analysis of large, highly modified proteins (≥50 kDa) such as immunoglobulin E (IgE) remains challenging due to their size and molecular heterogeneity. While traditional bottom-up proteomics fails to retain proteoform-level information, top-down approaches often lack the resolution required for effective proteoform discrimination. Middle-down proteomics (MDP) overcomes these limitations by generating highly redundant peptides in the 3-15 kDa range, which retain critical combinatorial modification patterns, thereby enabling more comprehensive characterization of complex proteoforms. RESULTS To advance heterogeneity analysis of large proteins, we developed an online, tunable pepsin digestion platform that facilitates rapid and in-depth middle-down analysis of large proteins. When applied to IgE-a key allergy-related antibody with a large molecular weight (170-190 kDa) and bearing seven potential N-glycosylation sites-this platform produces high-redundancy peptides, enabling de novo sequencing of the variable region, which is critical for antigen recognition. Furthermore, analysis of middle-sized peptides allowed us to detect combined glycosylation patterns on the constant region 3 of the epsilon heavy chain (Cε3), revealing 39 Cε3 domain proteoforms. These proteoforms are important for Fcε receptor I interaction, therapeutic antibody binding, and downstream signaling. The platform also provided comprehensive glycosylation insights, including composition, occupancy, and microheterogeneity, thereby supporting detailed annotation of intact IgE proteoforms with varying glycan numbers. SIGNIFICANCE