Characterization by LC-MS/MS of two conjugate vaccines using KLH as carrier protein

Keyhole limpet haemocyanins (KLH1 and KLH2) from Megathura crenulata, are large ( 3900 amino acids) multi-subunit oxygen-carrying metalloproteins, that are widely used as carrier proteins in conjugate vaccines and in immunotherapy. KLHs and their derived conjugate vaccines are poorly characterized by mass spectrometry due to the very stable supramolecular structures with megadalton molecular mass, and the resistance to be efficiently digested with standard protocols using specific proteases. KLH1 and KLH2 proteins were conjugated to the conserved twenty-one amino acids pP0 peptide, derived from the P0 acidic ribosomal protein of Rhipicephalus sp. ticks by using the maleimide-thiol chemistry. The resultant KLH1- and KLH2-Cys1pP0 conjugate vaccines were efficiently digested using the MED-FASP procedure, and analyzed by LC-MS/MS allowing approximately 85% sequence coverage of both conjugates. New PTMs not described for the KLH such as oxidized species were identified. The conjugation sites and Cys-His thioether bonds were determined by identifying cross-linked peptides using software developed for cross-linking mass spectrometry experiments. Conjugation sites were validated by combining sequence coverage, the presence of diagnostic and linker fragment ions in the MS/MS spectra, and the multi-peak pattern in the extracted ion chromatograms. In summary, 124 out of 170 Lys (75%) and 99 out 150 Lys (66%) were found conjugated to Cys1pP0 in KLH1 and KLH2, respectively. In total, four and seven Cys-His thioether bonds were experimentally determined in KLH1 and KLH2, respectively. This is the first report of the conjugation sites identification of two KLH-based vaccines determined by mass spectrometry