title: Use of affinity-directed liquid chromatography-mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction.
abstract: BACKGROUND
  Neutralizing factor (F) VIII antibodies develop in approximately 30% of individuals with hemophilia A and show specificity to multiple sites in the FVIII protein.
  
  
  METHODS
  Reactive epitopes to an immobilized IgG fraction prepared from a high-titer, FVIII inhibitor plasma were determined after immuno-precipitation (IP) of tryptic and chymotryptic peptides derived from digests of the A1 and A2 subunits of FVIIIa and FVIII light chain. Peptides were detected and identified using highly sensitive liquid chromatography-mass spectrometry (LC-MS).
  
  
  RESULTS
  Coverage maps of the A1 subunit, A2 subunit and light chain represented 79%, 69% and 90%, respectively, of the protein sequences. Dot blots indicated that the inhibitor IgG reacted with epitopes contained within each subunit of FVIIIa. IP coupled with LC-MS identified 19 peptides representing epitopes from all FVIII A and C domains. The majority of peptides (10) were derived from the A2 domain. Three peptides mapped to the C2 domain, while two mapped to the A1 and A3 domains, and single peptides mapped to the a1 segment and C1 domain. Epitopes were typically defined by peptide sequences of < 12 residues.
  
  
  CONCLUSIONS
  IP coupled with LC-MS identified extensive antibody reactivity at high resolution over the entire functional FVIII molecule and yielded sequence lengths of < 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Griffiths
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.forename: F K
authors.initials: FK
authors.lastname: Hagen
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D001665:Binding Sites; D002846:Chromatography, Affinity; D056148:Chromatography, Reverse-Phase; D018604:Epitope Mapping / Q000379:methods*; D005169:Factor VIII / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D016056:Immunodominant Epitopes; D047468:Immunoprecipitation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D053719:Tandem Mass Spectrometry
chemical_list: D057134:Antibodies, Neutralizing; D016056:Immunodominant Epitopes; D005169:Factor VIII