title: Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved protein.
abstract: The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0359, USA.
authors.forename: Abel
authors.initials: A
authors.lastname: Baerga-Ortiz
authors.forename: Carrie A
authors.initials: CA
authors.lastname: Hughes
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Mandell
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Komives
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D003903:Deuterium; D018604:Epitope Mapping / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D006859:Hydrogen; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D011486:Protein C; D006859:Hydrogen; D003903:Deuterium; D013917:Thrombin