University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Website: @unf

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Classification: Bacteria -> staphylococcus

Citations 1

"Determination Of Staphylococcal Enterotoxin B By Online (micro) Liquid Chromatography-electrospray Mass Spectrometry"
J. Chromatogr. A 1997 Volume 757, Issue 1-2 Pages 51-64
Charles E. Kientz*, Albert G. Hulst and Eric R. J. Wils

Abstract: A solution of staphylococcal enterotoxin B (I) was dialysed vs. 0.2% formic acid for 24 h. For mol. wt. determination, dialysate was injected into an aqueous 50% acetonitrile stream (10 µl/min) which passed through a fused-silica tube (30 cm x 0.1 mm i.d.) prior to electrospray MS. Alternatively, I was analyzed on a microcolumn (40 cm x 0.5 mm i.d.) packed with TSKgel Phenyl-5PW with gradient elution from aqueous 10^-80% acetonitrile containing 0.2% TFA in 25 min and electrospray MS detection (operating details given). In addition I solution (20 µM) was digested at 37°C with L-1-tosylamide-2-phenylethyl chloromethyl ketone-treated trypsin in 0.1 M ammonium bicarbonate of pH 8. Then portions (10 µL) of digest were analyzed on a microcolumn (60 cm x 0.3 mm i.d.) packed with LiChrosorb RP-18 (5 µm) as described above. Both methods allowed analysis of I at 3 nM levels. Further structural information was obtained by FIA-MS (as described above) of dialysate subjected to disulfide-bond cleavage with mercaptoethanol (details given). For amino-acid sequencing, the use of micro-LC columns for LC-tandem MS (details given) enabled 30-40-fold increases in sensitivity compared to conventional LC columns. The use of (micro) liquid chromatography electrospray mass spectrometry (LC-ES-MS) was investigated as potential technique for the determination of the high-molecular-mass protein toxin Staphylococcal Enterotoxin B (SEB). The molecular mass was determined (28,366.3±1.1) by flow injection analysis and micro-LC-MS with a TSK-gel Phenyl-5PW column packing. Both methods allowed molecular-mass determination of SEB at levels down to 3 pmol/ml. Additional evidence of identification was obtained by detecting the presence of a disulfide bridge by the addition of 2-mercaptoethanol and by tryptic digestion. Collision induced dissociation spectra were recorded from the major tryptic fragments resulting in a sufficient number of sequence ions to allow for the determination of the amino acid sequence. On the analysis of the tryptic digests with C18 reversed-phase columns the use of micro-LC-MS-MS resulted in an 30-40-fold increase in sensitivity as compared with conventional-size LC-MS-MS.
Enterotoxin B Mass spectrometry Dialysis