Amino-acetylation and -succinylation reactions in combination with mass spectrometric peptide mapping of tryptic peptide mixtures have been employed for surface topology-probing of lysine residues in bovine ribonuclease A, lysozyme, and horse heart myoglobin as model proteins of different surface structures. Direct molecular weight determinations identifying the precise number of acyl groups in partially modified proteins were obtained by electrospray and 252Cf-plasma desorption mass spectrometry. Electrospray mass spectra of multiply protonated molecular ions and deuterium exchange experiments provided a relative conformational characterization of protein derivatives and enabled the direct determinations of intact, partially acylated heme-myoglobin derivatives. Tryptic peptide mapping analysis, using plasma desorption and fast atom bombardment mass spectrometry, ascertained by mass spectrometric characterization of HPLC-separated modified peptides, yielded the exact identification of acylation sites. Relative reactivities of the amino acylation were derived from the peptide mapping data and from quantitative estimations of modified peptides upon acetylation/trideuteroacetylation and provided direct correlations with the relative surface accessibilities of lysine-epsilon-amino groups taken from X-ray crystallographic structure data of the proteins. The reactive lysine-41 residue in ribonuclease A which is part of the substrate binding site was directly identified from the mass spectrometric data. These results indicate tertiary structure-selective acylation combined with mass spectrometric peptide mapping as an efficient approach for the molecular characterization of surface topology and reactive fundamental lysine residues in proteins.

Monoclonal antibodies are widely used analytical tools in biochemical research. The knowledge of their corresponding epitopes is of major interest. One possible approach for epitope characterization is the application of protein antigen proteolysis in combination with mass spectrometric peptide mapping analysis. Two complementary analytical strategies were applied: (a) limited proteolysis of antibody-bound antigen followed by removal of nonbound peptides and detachment of the antigenic peptides (epitope excision) and (b) enzymatic digest of the antigen followed by extraction of the antigenic peptides with the antibody and detachment of antigenic peptides after removal of nonbinding fragments (epitope extraction). In the few examples published so far, immobilized antibodies were used for these studies. In this study we present a method for characterization of the epitope sequences without prior immobilization of the monoclonal antibody. The separation of nonepitope peptides from antibody-bound peptides was carried out by ultrafiltration. The epitope and nonepitope fractions were analyzed by MALDI-MS without further purification, and the epitope sequences were identified. The method was developed using a model system consisting of the synthetic C-terminal cyanogen bromide fragment CB3 of myoglobin and the commercial monoclonal anti-myoglobin MG1. In further investigations the epitope sequence of a synthetic 32 amino acid peptide derived from heart muscle protein troponin T toward a monoclonal antibody MAb-M7, which was raised against the intact protein, was characterized. With this approach the epitope binding site of this antibody was determined, and selective shielding of potential cleavage sites in the immune complex could be observed. Furthermore, statements about the three-dimensional structure of the bound antigen were made.

A new approach for the characterization of conformationally dependent epitope structures in protein antigens is described using differential chemical modification of immune complexes in combination with mass spectrometric peptide mapping analysis. Well-established methods for epitope characterization are frequently not applicable to conformationally dependent epitopes, and direct methods of structure analysis such as X-ray crystallography of immune complexes have been successful only in a few cases. Our approach combines tertiary structure-selective chemical modification of immune complexes with the molecular characterization of reaction products by mass spectrometric peptide mapping. The comparison of the modification pattern of free and antibody-bound antigen provides the identification of residues protected from modification by the antibody. These residues hence are characterized as part of the epitope structure. The well-characterized hen egg white lysozyme and a corresponding monoclonal IgM-type antibody were investigated as a model system. Specific modification reactions for arginine, lysine, and tyrosine residues were performed, and the modification sites in free and antibody-bound antigen were determined by mass spectrometric peptide mapping. The R14 residue and residues K13 and K96 in the antibody-bound lysozyme were found to be protected from modification, comprising a surface of spatially adjacent residues by folding of the native protein. In contrast, other K and R residues as well as Y20 and Y23 showed no significant shielding from modification in the immune complex. These results provided an estimation of the molecular epitope surface area of native lysozyme.

The hexane extract from the leaves of Dasymaschalon sootepense Craib (Annonaceae) showed strong cytotoxic activity against the L1210 tumor cell line. Activity‐directed fractionation of the extract by column chromatography on silica gel and high‐pressure liquid chromatography led to the isolation of the acetogenins 1 – 4 as the main active principles. The structures of the two novel structures named sootepensin A (1) and sootepensin B (2) were elucidated by spectroscopic analysis (UV, EI‐ and ESI‐MS, 1D‐ and 2D‐1H‐ and 13C‐NMR). The absolute configurations were established by 2D‐NMR experiments utilizing Mosher esters. Two recently described compounds, tonkinin C (3) and tonkinesin C (4), were also isolated and are new to the genus Dasymaschalon. All four acetogenins were found to be highly cytotoxic against the L1210 tumor cell line.

The hexane extract from the leaves of Dasymaschalon sootepense Craib ( Annonaceae) showed strong cytotoxic activity against the L1210 tumor cell line. Activity-directed fractionation of the extract by column chromatography on silica gel and high-pressure liquid chromatography led to the isolation of the acetogenins 1 ± 4 as the main active principles. The structures of the two novel structures named sootepensin A (1) and sootepensin B (2) were elucidated by spectroscopic analysis ( UV, EI-and ESI-MS, 1D-and 2D-1 H-and 13 C-NMR ). The absolute configurations were established by 2D-NMR experiments utilizing Mosher esters. Two recently described compounds, tonkinin C (3) and tonkinesin C (4), were also isolated and are new to the genus Dasymaschalon. All four acetogenins were found to be highly cytotoxic against the L1210 tumor cell line.