Use of affinity‐directed liquid chromatography‐mass spectrometry to map the epitopes of a factor VIII inhibitor antibody fraction

Abstract

Summary Background Neutralizing factor (F) VIII antibodies develop in ~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 following 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 less than 15 residues. A number of the peptides identified mapped to known sequences involved in functionally important protein-protein and protein-membrane interactions.

Mass spectrometric and peptide chip characterization of an assembled epitope: analysis of a polyclonal antibody model serum directed against the Sjøgren/systemic lupus erythematosus autoantigen TRIM21

“…In terms of mass spectrometric epitope‐mapping procedures, epitope excision and/or extraction have been the methods of choice for studying such noncovalent molecular antigen antibody interactions . The antibody/peptide ratio differed from report to report including examples where excess of antibody over the antigen varied over broad ranges, whereas in other studies, antigens were used in excess to antibody . In most of these published methods, fairly large amounts of antibodies (about 300–6000 pmol) were consumed.…”

Hydrogen/deuterium exchange mass spectrometry applied to IL-23 interaction characteristics: potential impact for therapeutics

“…Currently, there are several software tools available that can be used to apply different calculation methods to proteins allowing the prediction of a protein structure and study of the molecular interactions between proteins. Empirically, the interactions between proteins can be studied from several experimental techniques, such as alanine scanning, peptide panning, yeast two-hybrid systems [58], affinity purification/mass spectrometry [59], and protein microarrays [60]. A number of computational methods for prediction of protein/protein interactions have been developed in the recent years [61, 62].…”

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures

“…Supporting materials, such as Sepharose beads (Suckau et al, 1990;Papac et al, 1994;Hochleitner et al, 2000;Griffiths et al, 2011) and magnetic beads (Lu et al, 1998;Jankovicova et al, 2008), are available for covalent attachment of antibodies, through which the specificity of an affinity chromatography system is provided. Alternatively, noncovalent immobilization of antibodies on protein A and/or protein G´in columns/pipette tips (Zhao and Chait, 1994;AlMajdoub et al, 2013b) or by immunoprecipitation (Ansong et al, 2006) has been applied successfully for antibody-antigen or epitope mapping studies.…”