Sequences of antigenic determinants were identified by limited proteolysis of peptide antigens bound to an immobilized monoclonal antibody and direct molecular weight determination of the monoclonal antibody-bound peptide fragments by 252Cf plasma desorption mass spectrometry. The epitope peptides to the monoclonal antibody h453 [Burger, R., Zilow, G., Bader, A., Friedlein, A. & Naser, W. (1988) J. Immunol. 141,[553][554][555][556][557][558] were isolated from immobilized antigen-antibody complexes by partial trypsin digestion. A synthetic eicosapeptide comprised of the C-terminal sequence of the human complement component polypeptide des-Arg77-C3a as well as guinea pig des-Arg78-C3a was used as an antigen. Conditions were developed under which trypsin specifically degraded the antigens without inactivation of the immobilized antibody. After proteolysis, epitope peptides were dissociated from the antibody with 4 M MgCl2. The antigenic peptides were purified by HPLC and identified by 252Cf plasma desorption mass spectro! retry. The epitope recognized by h453 resides on the C-terminal tryptic peptides of human (residues 70-76) and guinea pig (residues 70-77) C3a. As an estimation of accuracy this method is able to provide, trypsin digestion of immune complexes caused cleavage of the antigen within a distance of two amino acid residues upstream from the epitope.A variety of methods have been applied to the study of monoclonal antibody (mAb)-antigen interactions and the characterization of their respective epitopes. Two major approaches that have been widely employed for epitope characterization are competitive binding analysis using synthetic peptides and fine specificity studies with panels of evolutionary variant or recombinant proteins (1). Although well established, these methods have major limitations; e.g., discontinuous or conformationally defined epitopes may not be detectable by using peptide probes (2). Site-directed mutagenesis experiments in epitope studies could be greatly facilitated if some information about the putative epitope is available in advance. A direct approach of epitope mapping, which seems promising in this respect, has been more recently introduced based on the finding that (i) mAbs exhibit remarkable resistance towards proteolytic enzymes, (ii) in immune complexes, antigenic determinants can be protected from proteolytic degradation, and (iii) proteolysis does not lead to dissociation of immune complexes (3-6). Limited proteolytic cleavage of immune complexes has been used for epitope characterization by means of PAGE (7) and by HPLC (6) of the respective peptide digests. However, HPLC separation of complex digest mixtures followed by amino acid analysis or peptide sequencing may not enable unambiguous epitope identification due to unresolved peptides.The feasibility of fast atom bombardment mass spectrometry (FABMS) and 252Cfplasma desorption mass spectrometry (PDMS) for accurate molecular weight determination of polypeptides has been established in several bioanalytical ...

In 1980 the C-terminal pentapeptide LGLAR (C3a 73-77) was described (Caporale, L. H. et al. J. Biol. Chem. 1980, 255: 10758) as the minimal sequence inducing a C3a-specific activity. We have synthesized C3a-analogue peptides connected to non-peptidic acyl residues known to potentiate biological activity. Starting from the acylated hexapeptide fluorenylmethoxycarbonyl(Fmoc)-aminohexanoyl(Ahx)-ALGLAR+ ++, a related series of shorter peptides was synthesized. C3a-specific activity was measured as ATP release from guinea pig platelets. Even the tripeptide LAR, acylated with Fmoc-Ahx, exhibited C3a-specific activity. With 0.34% C3a activity, it was even more potent than the native LGLAR sequence which has 0.01% activity. N-terminal extension of the acylated tripeptide LAR by adding one to three alanines increased activity tenfold up to 3.26% (Fmoc-Ahx-AAALAR), while N-terminal addition of three glycine residues (Fmoc-Ahx-GGGLAR) only increased activity to 0.83% of native C3a. Furthermore, a stimulus-specific desensitization could be observed. Fmoc-Ahx-R and Fmoc-Ahx-AR exhibited neither activity nor desensitizing capacity, but the addition of four alanines to the dipeptide AR led to a sequence (Fmoc-Ahx-AAAAAR) with a C3a-specific activity of 0.14%. Even arginine prolonged N-terminally with five glycines (Fmoc-Ahx-GGGGGR) exhibited some C3a-specific activity so that for biological activity only the appropriate presentation of arginine seems to be essential.

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