Matrix-assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody.
In this study we have applied epitope excision and epitope extraction strategies, combined with matrix assisted laser desorption/ionization mass spectrometry, to determine the fine structure of epitopes recognized by a polyclonal antibody to human immunodeficiency virus envelope glycoprotein gp120. This is the first application of this approach to epitope mapping on a large, heavily glycosylated protein. In the epitope excision method, gp120 in the native form is first bound to the antibody immobilized on sepharose beads and cleaved with endoproteinase enzymes. In the epitope extraction method, the gp120 was first proteolytically cleaved and then allowed to react with the immobilized antibody. The fragments that remain bound to the antibody, after repeated washing to remove the unbound peptides, contain the antigenic region that is recognized by the antibody, and the bound peptides in both methods can be characterized by direct analysis of the immobilized antibody by matrix assisted laser desorption ionization/mass spectrometry. In this study we have carried out epitope excision and extraction experiments with three different enzymes and have identified residues 472-478 as a major epitope. In addition, antigenic regions containing minor epitopes have also been identified.
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.
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S
Jeyarajah
C E
CE
Parker
M T
MT
Summer
K B
KB
Tomer
D000595:Amino Acid Sequence; D002268:Carboxypeptidases; D018983:DNA Footprinting; D000939:Epitopes / Q000032:analysis*; D015699:HIV Envelope Protein gp120 / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D007120:Immunochemistry; D007931:Leucyl Aminopeptidase; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000939:Epitopes; D015699:HIV Envelope Protein gp120; D002268:Carboxypeptidases; D007931:Leucyl Aminopeptidase