A Novel Mass Spectrometric Epitope Mapping Approach without Immobilization of the Antibody

Abstract

The development and application of a mass spectrometry-based epitope mapping procedure in solution and without immobilization of the antibody is described. Antigens were digested with proteases. Then size exclusion micro-column chromatography (SEC) was carried out prior to and upon exposure of the peptide mixtures to monoclonal antibodies. The epitope-containing peptide, affinity bound to the antibody and, thus, forming a stable complex eluted early as did all high-mass components, whereas all unbound low-mass peptides eluted late. Comparison of elution profiles in the presence and absence of the antibody showed a shift only for epitope-bearing peptides, enabling direct identification of the epitope. His-tag containing recombinant antigens Fibrillarin and RA33 were used in combination with an anti-His-tag monoclonal antibody in order to develop the method. Application of this method for the determination of an epitope on RA33 against which a monoclonal antibody was directed identified the epitope sequence (85 IDGRVVEPKRA 95) using MS/MS peptide sequencing. Advantages of this approach include low sample consumption, few handling steps, and short duration of analysis. With our method we are ultimately aiming at developing a screening procedure to identify major epitopes in patients that may be suitable in the future for stratification of patients, needed for personalized therapies.

Native and compactly folded in-solution conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of gas-phase pepstatin A – pepsin complex binding strength differences

“…30–32 These epitope mapping methods have over the years reached fairly matured stages and have remarkably advanced either by automation of solution handling procedures 33 or via reducing in-solution handling steps to mixing of antigens and antibodies in ESI-compatible buffer solutions. 34,35 Because direct transition of immune complexes from the condensed phase into the gas phase, which is routinely realized by ESI-MS, 1,10 has been recognized as the key process for successful mass spectrometric epitope mapping, this approach has been termed “intact transition epitope mapping (ITEM).” 35 ITEM-ONE identifies epitopes by determining the mass of the extracted epitope peptide upon dissociation of immune complex ions in the gas phase. 8 Determining kinetic and thermodynamic properties, that is, quantitative traits of immune complexes is possible using the ITEM-TWO method.…”