No abstract

Staphylococcus aureus is an important human pathogen frequently resistant to a wide range of antibiotics. Methicillin-resistant S. aureus (MRSA) strains are common nosocomial pathogens that pose a world-wide problem. Rapid and accurate discrimination between methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus is essential for appropriate therapeutic management and timely intervention for infection control. We report here the application of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for monitoring the bacterial fingerprints expressed by two well characterized S. aureus strains ATCC 29213 (MSSA) and ATCC 43330 (MRSA). Consistent strain-specific data were obtained from subcultures analyzed over a period of three months as well as after changing the growth media from Mueller-Hinton to blood agar indicating the reliability of the method. The bacterial fingerprints of these two strains were compared to independent clinical isolates of S. aureus. A uniform signature profile for MRSA could not be identified. However, the bacterial fingerprints obtained proved to be specific for any given strain. This study demonstrates that MALDI-TOF MS is a powerful method for rapid identification of clonal strains of S. aureus, which might be useful for tracking nosocomial outbreaks of MRSA and for epidemiologic studies of infections diseases in general.

Matrilin-4 is the most recently identified member of the matrilin family of von Willebrand factor A-like domain containing extracellular matrix adapter proteins. Full-length matrilin-4 was expressed in 293-EBNA cells, purified using affinity tags, and subjected to biochemical characterization. The largest oligomeric form of recombinantly expressed full-length matrilin-4 is a trimer as shown by electron microscopy, SDS-polyacrylamide gel electrophoresis, and mass spectrometry. Proteolytically processed matrilin-4 species were also detected. The cleavage occurs in the short linker region between the second von Willebrand factor A-like domain and the coiled-coil domain leading to the release of large fragments and the formation of dimers and monomers of intact subunits still containing a trimeric coiled-coil. In immunoblots of calvaria extracts similar degradation products could be detected, indicating that a related proteolytic processing occurs in vivo. Matrilin-4 was first observed at day 7.5 post-coitum in mouse embryos. Affinity-purified antibodies detect a broad expression in dense and loose connective tissue, bone, cartilage, central and peripheral nervous systems and in association with basement membranes. In the matrix formed by cultured primary embryonic fibroblasts, matrilin-4 is found in a filamentous network connecting individual cells.

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.