Polystyrene beads as an alternative support material for epitope identification of a prion-antibody interaction using proteolytic excision–mass spectrometry

• Published in: Analytical and bioanalytical chemistry Vol. 395; no. 5; pp. 1395 - 1401
• Main Authors: Pimenova, Tatiana, Meier, Lukas, Roschitzki, Bernd, Paraschiv, Gabriela, Przybylski, Michael, Zenobi, Renato
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.11.2009; Springer
• Subjects: Amino Acid Sequence; Analytical Chemistry; Animals; Antibodies; Antibodies - immunology; Antigens; Beads; Binding; Biochemistry; Cattle; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chymotrypsin - metabolism; Epitopes - analysis; Epitopes - chemistry; Exact sciences and technology; Food Science; Immobilization; Laboratory Medicine; Mass Spectrometry - methods; Molecular Sequence Data; Molecular Structure; Monitoring/Environmental Analysis; Original Paper; Polystyrene resins; Polystyrenes - chemistry; Prions - analysis; Prions - chemistry; Prions - immunology; Spectrometric and optical methods; Spectrometry; Spectroscopy; Epitope excision; Antibody–antigen interaction; Polystyrene beads; Prion protein; Mass spectrometry; Elution; Peptides; Immobilization; Monoclonal antibody; Digestion; Aldehyde; Protein; Antibody-antigen interaction; Proteolysis
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-009-3119-8

The binding epitope structure of a protein specifically recognized by an antibody provides key information to prevent and treat diseases with therapeutic antibodies and to develop antibody-based diagnostics. Epitope structures of antigens can be effectively identified by the proteolytic epitope excision–mass spectrometry (MS) method, which involves (1) immobilization of monoclonal or polyclonal antibodies, e.g., on N -hydroxysuccinimide-activated sepharose, (2) affinity binding of the antigen followed by limited proteolytic digestion of the immobilized immune complex, and (3) elution and mass spectrometric analysis of the remaining affinity-bound peptide(s). In the epitope analysis of recombinant cellular bovine prion protein (bPrP C ) to a monoclonal antibody (mAb3E7), we found that epitope excision experiments resulted in extensive nonspecific binding of bPrP to a standard sepharose matrix employed. Here, we show that the use of amino-modified polystyrene beads with aldehyde functionality is an efficient alternative support for antibody immobilization, suitable for epitope excision–MS, with complete suppression of nonspecific bPrP binding.