Protein epitope mapping by mass spectrometry.
A mass spectrometric method is described for the rapid mapping of linear epitopes in proteins that are bound by monoclonal antibodies. The method consists of three steps. In the first step, an antigen protein is digested by a proteolytic enzyme to produce an appropriate set of peptide fragments. In the second step, peptide fragments containing the linear epitope are selected and separated from the pool of peptide fragments by immunoprecipitation with the monoclonal antibody. In the final step, the immunoprecipitated peptides are identified by matrix-assisted laser desorption mass spectrometry. The method allows the rapid determination of antigenic sites without tedious peptide synthesis or protein mutagenesis. The approach is demonstrated through the mapping of epitopes in two peptides (melittin and glucagon-like peptide-1 7-37) against which monoclonal antibodies were raised. In addition to epitope mapping, the successful coupling between matrix-assisted laser desorption mass spectrometry and immunoprecipitation provides a potentially powerful tool for determining binding sites between proteins.
Zhao
Y
Y
Rockefeller University, New York, New York 10021.
Chalt
B T
BT
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis / Q000737:chemistry; D005934:Glucagon / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D052216:Glucagon-Like Peptide 1; D013058:Mass Spectrometry; D008555:Melitten / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry* / Q000276:immunology; D011233:Precipitin Tests; D011498:Protein Precursors / Q000032:analysis / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D011498:Protein Precursors; D008555:Melitten; D052216:Glucagon-Like Peptide 1; D005934:Glucagon