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.
Kientz
C E
CE
TNO Prins Maurits Laboratory, Rijswijk, The Netherlands.
Hulst
A G
AG
Wils
E R
ER
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D019075:Electrophoresis, Capillary; D004768:Enterotoxins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009862:Online Systems / Q000295:instrumentation*; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013211:Staphylococcus aureus / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D004768:Enterotoxins; D010446:Peptide Fragments; C031818:enterotoxin B, staphylococcal; D014357:Trypsin