A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking

The combination of limited proteolysis and MALDI-TOF mass spectrometry has become an important tool for the determination of epitopes but works best w ith highly purified antibodies. Here we report the use of capture antibodie s to reduce the need for purification of the antibody in the mass spectrome tric determination of the epitope. In this new method, a secondary Fc-speci fic antibody, covalently bound to Sepharose beads, is used to capture the p rimary antibody (the antibody of interest). After capture, the two antibodi es are cross-linked. The antigen is then bound to the immobilized antibodie s and subjected to proteolysis using several successive proteinases. In thi s study, this strategy is demonstrated with a crude mouse anti-ACTH IgG sol ution and adrenocorticotropin (ACTH). Comparing this strategy with previous methods where the antibody is bound directly to activated beads, the new m ethod (1) results in a higher binding capacity of the bound antibody to ACM , (2) does not require purification of the antibody of interest, and (3) dr amatically reduces the chemical background in the MALDI mass spectra.