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

Abstract

The binding epitope structure of a protein specifically recognized by an antibody provides key infonnation 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 polycldnal 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.

A Novel Mass Spectrometric Epitope Mapping Approach without Immobilization of the Antibody

“…Our experimental procedure shows that incubation at a 3:1 ratio of antibody to peptides is well suitable for epitope mapping. This rather fixed antibody/peptide ratio differs from those reported in the literature where excess of antibody varies in broader ranges (Pimenova et al, 2009;Dhungana et al, 2009;Zhao and Chait, 1994) while in other studies peptides were used in excess (Bílková et al, 2005;Parker and Tomer, 2002;Peter and Tomer, 2001; Kiselar and Downard, 1999;Papac et al, 1994).…”

“…In epitope excision studies, mass spectrometry was used to identify epitope peptides released from an immobilized antibody-antigen complex after limited proteolysis (Pimenova et al, 2009;Parker and Tomer, 2002;Suckau et al, 1990). Here, the antibody prevents either proteolysis (Jemmerson, 1996) or chemical modification (Burnens et al, 1987) of sites of the antigen that are situated in the antibody binding pocket.…”

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.…”

Mass spectrometric epitope mapping

“…Immobilization was mostly carried out such that the antibody was covalently attached to a carrier material or a solid support. Carrier materials were tresylactivated sepharose (Suckau et al, 1990), agarose gels (Papac, Hoyes, & Tomer, 1994;Griffiths et al, 2011), polystyrene beads (Pimenova et al, 2009), magnetic microcellulose beads (Yu, Gaskell, & Brookman, 1998), Perloza MT 500 carriers (Jankovicova et al, 2008), sepharose beads (20 papers; therefrom CNBr-activated: 12 papers). The use of so many different carrier materials represents the versatility of epitope mapping procedures and the associated adaptability of the method to particular in-solution handling conditions, if necessary.…”