Epitope mapping factor VIII A2 domain by affinity-directed mass spectrometry: residues 497-510 and 584-593 comprise a...

The murine monoclonal antibody R8B12 recognizes the C-terminal region (residues 563-740) of the A2 subunit of factor VIIIa [J Biol Chem 266: 1991; p. 20139], as judged by Western blotting. However, the location of the epitope within this region is not known. In the present study, we used affinity-directed mass spectrometry to map the epitope. A2 subunit was digested with trypsin or chymotrypsin and then subjected to immunoprecipitation (IP) using R8B12 IgG. Masses of the affinity-selected peptides were determined directly from the immune complexes by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Proteolysis of A2 with the two proteases generated a pre-IP peptide fingerprint that covered approximately 70% of the A2 domain sequence. Analysis of the post-IP tryptic peptide fingerprint showed two masses, 1309 and 1653 Da representing residues 584-593 and 497-510, respectively, determined from a theoretical database search and confirmed by direct sequencing. Results using a chymotryptic digest yielded a single, weakly reactive fragment consistent with residues 577-586, suggesting the importance of residues Ser584-Tyr586 in forming the epitope. A synthetic peptide to residues 584-593 was immunoprecipitated by the IgG and blocked R8B12-directed blotting to A2 subunit. The 497-510 and 584-593 segments were observed to be adjacent and surface exposed in the A2 domain model, and together with the above results suggest that A2 domain residues 497-510 and 584-593 represent a discontinuous epitope for R8B12. Furthermore, based upon blotting specificity, we speculate that residues 584-593 make a substantially greater contribution to the binding energy for this interaction.