Open AccessRapid isolation and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography–electrospray ionization mass spectrometry
Author(s) -
Kawano Yasushi,
Ito Yasutomo,
Yamakawa Yoshinori,
Yamashino Takafumi,
Horii Toshinobu,
Hasegawa Tadao,
Ohta Michio
Publication year - 2000
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2000.tb09214.x
Subject(s) - chromatography , electrospray ionization , chemistry , mass spectrometry , staphylococcus aureus , microbiology and biotechnology , bacteria , biology , genetics
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.