EPITOPE MAPPING BY MASS-SPECTROMETRY - DETERMINATION OF AN EPITOPE ONHIV-1(IIIB) P26 RECOGNIZED BY A MONOCLONAL-ANTIBODY

Matrix-assisted laser desorption mass spectrometry in combination with proteolytic protection assays has been used to identify the functiona l epitope on HIV-1(IIIB) p26 recognized by a mAb. In this procedure, t he intact protein is affinity bound to an immobilized mAb under physio logic conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Protected residues wer e identified by matrix-assisted laser desorption mass spectrometry bas ed on their m.w. With this approach, an 11-residue sequence was identi fied as the most tightly affinity-bound fragment. In addition, two les s tightly bound segments were observed. These latter two residues may contain elements of a discontinuous epitope or may be residues involve d in a wider contact area. The combination of matrix-assisted laser de sorption and proteolytic epitope footprinting has been applied to the determination of the epitope on a recombinant protein recognized by a mAb but should be equally applicable to the definition of an epitope o n a native protein in its natural folded conformation.