Epitope mapping of the gastrin‐releasing peptide/anti‐bombesin monoclonal antibody complex by proteolysis followed by matrix‐assisted laser desorption ionization mass spectrometry

Abstract

We have developed a method to rapidly identify the antigenic determinant for an antibody using in situ proteolysis of an immobilized antigen-antibody complex followed by matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDIITOF). A mouse anti-bombesin monoclonal antibody was immobilized to agarose beads and then the antigen, gastrin-releasing peptide (GRP), was allowed to bind. Direct analysis of the immobilized antigen-antibody complex by MALDIITOF is demonstrated and allows identification of ca. 1 pmol of the bound GRP. To identify the epitope, the immobilized antigen-antibody complex was subjected to proteolysis with trypsin, chymotrypsin, thermolysin, and aminopeptidase M. Following proteolysis, the part of the antigen in contact with the antibody and protected from proteolysis was identified directly by MALDUTOF. Subsequently, the epitope was eluted from the immobilized antibody with 0.1 M glycine buffer (pH 2.3), separated by reversed-phase HPLC, and its identity confirmed by MALDIITOF. Using this approach, the epitope for the anti-bombesin monoclonal antibody was shown to comprise the last 7-8 residues (HWAVGHLM-NH2) of GRP.

Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

“…Affinity mass spectrometry approaches using proteolytic epitope excision and epitope extraction have been previously described in detail and applied in epitope identifications [14][15][16][17][18][19]. In the general analytical method [14][15][16][17]19], the immobilised ligand-binder (antigen-antibody) complex is subjected to limited proteolytic digestion, followed by mass spectrometric analysis of the eluted affinity-bound epitope peptide fragment (s). In the proteolytic step, the epitope is protected from digestion owing to the shielding of the ligand-binder interaction structure; hence, epitope excision on an immobilised immune complex is essential for isolation, specific dissociation and identification of the bound epitope.…”

Cryptic Antigenic Determinants on the Extracellular Pyruvate Dehydrogenase Complex/Mimeotope Found in Primary Biliary Cirrhosis

“…The amount of product generated in the non-PBC liver cells when probed with C150 was significantly higher than that detected for the non-PBC liver cells probed with C355. a mAb to the complement component C3a and mapping of the gastrin-releasing peptide/anti-bombesin monoclonal antibody complex (34,37). Epitope extraction and direct identification of a single immune complex by mass spectrometry has been shown to be a sensitive and rapid method of high molecular specificity in the analysis of protein antigens.…”

Comparative characteristic of the methods of protein antigens epitope mapping

“…Перші роботи, що передбачали даний методологічний підхід, проводилися із викори-станням фрагментації АГ хімічним протеолізом [17,18], пізніше почали використовувати ензи-матичне розщеплення протеїнів [19][20][21]. Загаль-на методологія такого епітопного картування передбачає (рис.…”