Epitope mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex by proteolysis followed by matrix-assisted laser desorption ionization mass spectrometry.
We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDI/TOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDI/TOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDI/TOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.
Papac
D I
DI
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Hoyes
J
J
Tomer
K B
KB
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000378:metabolism; D001839:Bombesin / Q000276:immunology*; D002918:Chymotrypsin / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology; D019886:Gastrin-Releasing Peptide; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D010455:Peptides; D019886:Gastrin-Releasing Peptide; D000626:Aminopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D013820:Thermolysin; D001839:Bombesin