Mapping discontinuous epitopes recognized by human enhancing antibodies In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein:protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in proteinprotein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. We have initiated a project in which we will 1) probe an epitope on membrane embedded HIV gp160 recognized by the human neutralizing Ab 4E10 and probe the discontinuous epitope on the HIV gp120 trimeric spike recognized by a human neutralizing Ab. We are presently having a stable gp160 trimeric spike engineered using LipoParticles. LipoParticles are a patented process to express integral membrane proteins on a non-infectious particle. MAb 2909 recognizes a discontinuous epitope on the native trimeric spike conformation of the HIV envelope protein. It does not recognize monomeric gp120 or gp160.
| Hager-Braun, Christine; Hochleitner, Elisabeth O; Gorny, Miroslaw K et al. (2010) Characterization of a discontinuous epitope of the HIV envelope protein gp120 recognized by a human monoclonal antibody using chemical modification and mass spectrometric analysis. J Am Soc Mass Spectrom 21:1687-98 |
| Dhungana, Suraj; Williams, Jason G; Fessler, Michael B et al. (2009) Epitope mapping by proteolysis of antigen-antibody complexes. Methods Mol Biol 524:87-101 |
