(HDX-MS) strategies currently being used for protein structure and dynamics measurements include bottom-up and top-down approaches. The peptide-based bottom-up approach has no limit in protein size, but the small size of peptic peptides can lead to significant back exchange and incomplete sequence coverage, and the spatial resolution is often limited to peptide level. The top-down approach tackles these issues by analyzing intact proteins, but its success decreases rapidly as the protein size increases. In this presentation, we will discuss our development of a new ‘middle-down’ approach and its application in antibody characterization with ca. 100% sequence coverage.

Disulfide linkages in the antibody limit the ETD cleavage in top-down experiments and thus need to be. By performing ECD on the light chain and heavy chain separately after reduction and HPLC separation, we achieved complete sequence coverage for the light chain, but only 50% for the heavy chain (45 kDa). To expand the applicability of top-down HDX to larger proteins, we attempted to digest antibodies under HDX quench conditions into a limited number of specific fragments, We found three very large protein fragments after a brief proteolysis (1 min) of HER with a molecular weight of 12-25 kDa, that were well separated by HPLC within a 12 min run. The specific fragments obtained this way covered 100% of the light chain, and 95.3% of the heavy chain, representing a total coverage of 96.8% for the whole antibody. ETD fragmentation on these reached a spatial resolution of less than two residues and complete coverage for every fragment. The effect of deglycosylation on the HER antibody structure as revealed by HDX with top-down ETD and the structural characterization of intact antibodies will be presented, and the advantages and disadvantages of different HDX-MS strategies will be discussed.