Epitope mapping factor VIII A2 domain by affinity-directed mass spectrometry: Residues 497-510 and 584-593 comprise a discontinuous epitope for the monoclonal antibody R8B12

Abstract

Themurinemonoclonal antibodyR8B12 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 affinitydirectedmass spectrometry tomap 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 ∼70% of the A2 domain sequence. Analysis of the post-IP tryptic peptide fingerprint showed twomasses, 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. © 2006 International Society on Thrombois and Haemostais.