Direct identification of protein epitopes by mass spectrometry without immobilization of antibody and isolation of antibody-peptide complexes.

A rapid new approach is described that combines the selectivity and sensitivity of immunoaffinity and mass spectrometric based techniques for mapping protein epitopes. The approach alleviates the need to immobilize antibody, extract the antibody-peptide complex, and dissociate bound peptide, which are requirements of other methods. It avoids problems associated with limited proteolysis of an antigen-antibody complex particularly in the vicinity of the binding domain which can hinder identification of the epitope. Epitopic peptides are identified from a direct comparison of the matrix-assisted laser desorption ionization mass spectra of the antibody reaction mixture and an unreacted control. Samples are prepared for mass spectrometric analysis by heat-assisted and electrospray deposition to afford reproducible spectra that enable epitopic peptides to be identified in complex mixtures analyzed at the femtomole level. Indirect evidence is presented to suggest that the antibody-peptide complex is resilient to both sample deposition and the ionization event. The utility and sensitivity of the approach are illustrated for the lysozyme model. IgG-binding domains of human lysozyme are identified, and one epitope is refined to six residues that comprise part of an extended beta-loop region.