Probing molecular interactions in intact antibody: antigen complexes, an electrospray time-of-flight mass spectrometry approach.
Using a combination of nanoflow-electrospray ionization and time-of-flight mass spectrometry we have analyzed the oligomeric state of the recombinant V antigen from Yersinia pestis, the causative agent of plague. The mass spectrometry results show that at pH 6.8 the V antigen in solution exists predominantly as a dimer and a weakly associated tetramer. A monoclonal antibody 7.3, raised against the V antigen, gave rise to mass spectra containing a series of well-resolved charge states at m/z 6000. After addition of aliquots of solution containing V antigen in substoichiometric and molar equivalents, the spectra revealed that two molecules of the V antigen bind to the antibody. Collision-induced dissociation of the antibody-antigen complex results in the selective release of the dimer from the complex supporting the proposed 1:2 antibody:antigen stoichiometry. Control experiments with the recombinant F1 antigen, also from Yersinia pestis, establish that the antibody is specific for the V antigen because no complex with F1 was detected even in the presence of a 10-fold molar excess of F1 antigen. More generally this work demonstrates a rapid means of assessing antigen subunit interactions as well as the stoichiometry and specificity of binding in antibody-antigen complexes.
Tito
M A
MA
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, Oxford OX1 3QT, United Kingdom.
Miller
J
J
Walker
N
N
Griffin
K F
KF
Williamson
E D
ED
Despeyroux-Hill
D
D
Titball
R W
RW
Robinson
C V
CV
D000465:Algorithms; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015010:Yersinia pestis / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011994:Recombinant Proteins