Epitope fine mapping by mass spectrometry: Investigations of immune complexes consisting of monoclonal anti-HpTGEKP antibody and zinc finger protein linker phospho-hexapeptides.

We describe the mass spectrometric analysis of the binding strength of a monoclonal mouse antibody (clone 18E9.D9, BioLegend, London, England) which was raised against the predominantly occuring phosphorylated linker sequence, HpTGEKP, of C2H2 zinc finger proteins (ZNF proteins) using a synthetic phospho-hexapeptide as epitope peptide. The epitope peptide resembles the sequence motive which occurs with highest frequency in all C2H2 zinc finger proteins. As this anti-HpTGEKP antibody is assumed to bind to various related phosphorylated C2H2 ZNF protein linker sequences as well, related phospho-peptide sequences were investigated for binding strength differences. To select the top ten phosphorylated linker sequence motives of all human C2H2 ZNF proteins, all potential C2H2 ZNF gene linker sequences were extracted from the Human Genome Reference Consortium database (Build 38, patch release 13). DNA sequence stretches with stop-codons were removed and the DNA codons from the remaining sequences were translated. The resulting amino acid sequences of all C2H2 ZNF protein linkers were ranked according to their frequencies of occurrence. From these top ten C2H2 ZNF protein linker sequences the respective phospho-hexapeptides were synthesized and their binding behaviors towards the anti-HpTGEKP phospho-peptide antibody were investigated by "Intact Transmission Epitope Mapping – Thermodynamic Weak-force Order (ITEM-TWO)".