Characterization by LC-MS/MS analysis of KLH vaccine conjugated with a tick antigen peptide

Keyhole limpet haemocyanins (KLH1 and KLH2) from Megathura crenulata, are multi-subunit oxygen-carrying metalloproteins of approximately 3900 amino acids, that are widely used as carrier proteins in conjugate vaccines and in immunotherapy. KLHs and their derived conjugate vaccines are poorly characterized by LC-MS/MS due to their very stable supramolecular structures with megadalton molecular mass, and their resistance to efficient digestion with standard protocols. KLH1 and KLH2 proteins were conjugated to the conserved P0 peptide (pP0), derived from the P0 acidic ribosomal protein of Rhipicephalus sp. ticks using maleimide–thiol chemistry to obtain a broad-spectrum anti-tick vaccine. The resulting KLH1– and KLH2–Cys¹pP0 conjugate vaccines were efficiently digested using the Multiple-Enzymatic Digestion Filter Aided Sample Preparation and analyzed by LC-MS/MS, enabling a sequence coverage of approximately 85% of both conjugates. Seventy-three and sixty-five percent of all lysine residues in KLH1 and KLH2, respectively, were partially conjugated to Cys¹pP0. In the quaternary structures, we found no bias toward conjugation of lysine residues exposed to either the outer surface or the inner channel. The latter may not contribute to a protective humoral response because B cell entry into the inner channel is incompatible with the entrance hole diameter. The Cys-His thioether bonds in both KLHs were determined by identifying type 1 cross-linked peptides. New post-translational modifications undescribed for the KLH such as oxidized species, were identified. This is the first report of the identification of conjugation sites of two KLH-based vaccines. These results will help translate the KLH-based conjugates into well-characterized biotechnology products.