title: A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking.
abstract: The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best with highly purified antibodies. Here we report the use of capture antibodies to reduce the need for purification of the antibody in the mass spectrometric determination of the epitope. In this new method, a secondary Fc-specific antibody, covalently bound to Sepharose beads, is used to capture the primary antibody (the antibody of interest). After capture, the two antibodies are cross-linked. The antigen is then bound to the immobilized antibodies and subjected to proteolysis using several successive proteinases. In this study, this strategy is demonstrated with a crude mouse anti-ACTH IgG solution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new method (1) results in a higher binding capacity of the bound antibody to ACTH, (2) does not require purification of the antibody of interest, and (3) dramatically reduces the chemical background in the MALDI mass spectra.
authors.affiliation: National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Peter
authors.forename: K B
authors.initials: KB
authors.lastname: Tomer
mesh_terms: D000324:Adrenocorticotropic Hormone / Q000737:chemistry / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D018604:Epitope Mapping / Q000379:methods*; D051379:Mice; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000906:Antibodies; D000324:Adrenocorticotropic Hormone