Characterization of a discontinuous epitope of the human immunodeficiency virus (HIV) core protein p24 by epitope excision and differential chemical modification followed by mass spectrometric peptide mapping analysis

A combination of epitope excision, epitope extraction, and differential che mical modification followed by mass spectrometric peptide mapping was used for the characterization of a discontinuous epitope that is recognized by t he mouse anti-HIV-p24 monoclonal antibody 5E2.A3. In epitope excision, the protein is first bound to an immobilized antibody and then digested with pr oteolytic enzymes. In epitope extraction, the protein is first digested and subsequently allowed to react with the antibody. After epitope excision of the p24-5E2.A3 complex with endoproteinase Lys-C, a large fragment remaine d affinity bound corresponding to amino acids 1-158 of HIV-p24 (fragment 1- 158). Further digestion, however, resulted in loss of affinity. Moreover, n o affinity-bound fragments were observed after an epitope extraction experi ment. These data from the epitope excision and extraction experiments sugge st that the epitope is discontinuous. For the further characterization of t he epitope, amino groups in the epitope-containing fragment were acetylated in both the affinity bound and free states followed by mass spectrometric analysis. Two successive acetylation reactions were performed: (1) the firs t used a low molar excess of acetic anhydride, and (2) the second, after se paration from the antibody, a high molar excess of its hexadeuteroderivativ e. This isotopic labeling procedure, in combination with high resolution ma ss spectrometry, allowed the precise determination of relative reactivities of amino groups. In this study, no differences were observed in the rankin g of the relative reactivities of five lysine residues. However, the N-term inal amino group was found to be part of the discontinuous epitope.