Determination of staphylococcal enterotoxin B by on-line (micro) liquid chromatography-electrospray mass spectrometry

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.