title: Linear epitope mapping by native mass spectrometry.
abstract: The identification of antigenic epitopes is important for the optimization of monoclonal antibodies (mAbs) intended as therapeutic agents. MS has proven to be a powerful tool for the study of noncovalent molecular interactions such as those involved in antibody-antigen (Ab-Ag) binding. In this work, we described a novel methodology for mapping a linear epitope based on direct mass spectrometric measurement of Ab-Ag complexes. To demonstrate the utility of our methodology, we employed two approaches, epitope excision and epitope extraction, to study a model system consisting of a Fab antibody fragment with specificity toward the peptide abeta(1-40). In epitope excision, the Fab and abeta(1-40) complex was treated with proteolytic enzymes and the digested complexes were directly monitored by MS under native conditions. Mass differences between the Fab-abeta complex and the Fab control revealed the size of epitope peptides that were bound to the Fab. Using the epitope extraction approach, abeta(1-40) was first digested by Lys-C, and the fragment containing the epitope was selected by Fab binding. Data analysis allowed mapping of the epitope to abeta(16-27) which is in good agreement with previously unpublished data. The utility of the methodology was demonstrated by elucidating the binding epitopes for two full-length anti-abeta(1-40) mAbs.
authors.affiliation: Lilly Corporate Center, Indianapolis, IN 46285, USA.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Lu
authors.forename: Michael R
authors.initials: MR
authors.lastname: DeFelippis
authors.forename: Lihua
authors.initials: L
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000276:immunology; D053000:Analytic Sample Preparation Methods / Q000379:methods*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D018604:Epitope Mapping / Q000379:methods*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D010447:Peptide Hydrolases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D016229:Amyloid beta-Peptides; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); D010447:Peptide Hydrolases