Intact Transition Epitope Mapping-Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)

ITEM-THREE is a novel mass spectrometry-based epitope mapping method. To perform ITEM-THREE, antibody-epitope peptide immune-complexes are first generated in electrospray-compatible solutions in which the antibodies maintain their in-solution functionality, by adding an antibody of interest to a mixture of peptides/ digest of antigen from which at least one of the peptides holds the antibody's epitope. This mixture is directly nano-electrosprayed without purification. Identification of the epitope peptide is executed within a mass spectrometer which provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a step-wise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope containing peptide fragment ions are recorded and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen.