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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
Author(s) -
BaergaOrtiz Abel,
Hughes Carrie A.,
Mandell Jeffrey G.,
Komives Elizabeth A.
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.4670102
Subject(s) - epitope , monoclonal antibody , epitope mapping , chemistry , thrombin , hydrogen–deuterium exchange , thrombomodulin , linear epitope , antibody , microbiology and biotechnology , mass spectrometry , biochemistry , biology , chromatography , platelet , immunology
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.