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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-Cys 1 pP0 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 Cys 1 pP0. 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.

Rationale: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable., Methods: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18 O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30., Results: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18 O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker., Conclusions: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates., (© 2024 John Wiley & Sons Ltd.)