Rapid isolation and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography–electrospray ionization mass spectrometry

Abstract

The isolation of staphylococcal extracellular toxins and enzymes (exoproteins) usually requires time-consuming purification steps such as repeated chromatographic separations and isoelectric focusing. We performed rapid isolation, quantification and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography–electrospray ionization mass spectrometry (LC–ESI/MS) followed by the determination of N-terminal amino acid sequences of separated peaks. We identified two novel exoproteins as well as previously reported antigens ORF-1 and ORF-2, glutamyl endopeptidase in Staphylococcus aureus NCTC8325 and protein A, staphylococcal enterotoxin C3 (SEC3), toxic shock syndrome toxin-1 (TSST-1) and α-toxin in a clinical isolate methicillin-resistant S. aureus (MRSA) 3543. MRSA3543 secreted 5.33 and 1.45 μg of SEC3 and TSST-1 per 20 μg total exoproteins ml −1, respectively. The capillary LC treatment of the exoprotein fraction separated at least 12 peaks, indicating its high-resolution power. We found that when a protein was once determined by its N-terminal sequence, its mass spectrum and the obtained molecular mass was applicable for the assignment of the protein.