MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies
CE Parker, KB Tomer - Molecular biotechnology, 2002 - Springer
CE Parker, KB Tomer
Molecular biotechnology, 2002•SpringerProteolytic digestion of proteins bound to immobilized antibodies, combined with matrix
assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound
peptides, can be a powerful technique for epitope determination. Binding of the protein to
the antibody is done while the protein is in its native, folded state. A purified protein is not
required for this procedure, because only proteins containing the antigenic determinant will
bind to the antibody in the initial step. The method makes use of the resistance of the …
assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound
peptides, can be a powerful technique for epitope determination. Binding of the protein to
the antibody is done while the protein is in its native, folded state. A purified protein is not
required for this procedure, because only proteins containing the antigenic determinant will
bind to the antibody in the initial step. The method makes use of the resistance of the …
Abstract
Proteolytic digestion of proteins bound to immobilized antibodies, combined with matrix assisted laser desorption (MALDI) mass spectrometric identification of the affinity-bound peptides, can be a powerful technique for epitope determination. Binding of the protein to the antibody is done while the protein is in its native, folded state. A purified protein is not required for this procedure, because only proteins containing the antigenic determinant will bind to the antibody in the initial step.
The method makes use of the resistance of the antibody to enzymatic digestion. Enzymatic cleavage products of the antigenic protein not containing the epitope are washed off the beads, leaving the epitope-containing fragments affinity bound to the immobilized antibody. Dissociation of the antigen-antibody complex prior to mass spectrometric analysis is unnecessary because the affinity-bound peptides are released by the MALDI matrix crystallization process, although the antibody remains covalently attached to the sepharose beads.
This epitope-mapping protocol has been used in the determination of both continuous and discontinuous epitopes on both glycosylated and unglycosylated proteins.
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