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

The isolation of staphylococcal extracellular toxins and enzymes (exoprotei ns) usually requires time-consuming purification steps such as repeated chr omatographic separations and isoelectric focusing. We performed rapid isola tion, quantification and identification of staphylococcal exoproteins by re verse phase capillary high performance liquid chromatography-electrospray i onization mass spectrometry (LC-ESI/MS) followed by the determination of N- terminal amino acid sequences of separated peaks. We identified two novel e xoproteins as well as previously reported antigens ORF-1 and ORF-2, glutamy l endopeptidase in Staphylococcus aureus NCTC8325 and protein A, staphyloco ccal enterotoxin C3 (SEC3), toxic shock syndrome toxin-1 (TSST-1) and alpha -toxin in a clinical isolate methicillin-resistant S. aureus (MRSA) 3543. M RSA3543 secreted 5.33 and 1.45 mu g of SEC3 and TSST-1 per 20 mu g total ex oproteins ml(-1), respectively. The capillary LC treatment of the exoprotei n fraction separated at least 12 peaks, indicating its high-resolution powe r. We found that when a protein was once determined by its N-terminal seque nce, its mass spectrum and the obtained molecular mass was applicable for t he assignment of the protein. (C) 2000 Federation of European Microbiologic al Societies. Published by Elsevier Science B.V. All rights reserved.