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 alpha-toxin in a clinical isolate methicillin-resistant S. aureus (MRSA) 3543. MRSA3543 secreted 5.33 and 1.45 microg of SEC3 and TSST-1 per 20 microg 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.