Epitope mapping by mass spectrometry: determination of an epitope on HIV-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 functional epitope on HIV-1 IIIB p26 recognized by a mAb. In this procedure, the intact protein is affinity bound to an immobilized mAb under physiologic conditions. A combination of proteolytic enzymatic cleavages was then performed to remove unprotected residues. Protected residues were identified by matrix-assisted laser desorption mass spectrometry based on their m.w. With this approach, an 11-residue sequence was identified as the most tightly affinity-bound fragment. in addition, two less tightly bound segments were observed. These latter two residues may contain elements of a discontinuous epitope or may be residues involved in a wider contact area. The combination of matrix-assisted laser desorption 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 on a native protein in its natural folded conformation.
Parker
C E
CE
Laboratory of Molecular Biophysics, National Institute of Environmental Health Science, Research Triangle Park, NC 27709, USA.
Papac
D I
DI
Trojak
S K
SK
Tomer
K B
KB
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000915:Antibody Affinity; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D015488:HIV Antigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D006868:Hydrolysis; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D054301:gag Gene Products, Human Immunodeficiency Virus
D000911:Antibodies, Monoclonal; D000939:Epitopes; D015683:Gene Products, gag; D015488:HIV Antigens; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C065558:gag protein p26, Human immunodeficiency virus; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase