Analysis of Staphylococcus enterotoxin B using differential isotopic tags and liquid chromatography quadrupole ion trap mass spectrometry.

Staphylococcus aureus produces enterotoxins, which are causative agents of foodborne intoxications. Enterotoxins are single-chain polypeptides and have a molecular weight of about 26-28 kDa. The consumption of food contaminated with Staphylococcus aureus enterotoxins results in the onset of acute gastroenteritis within 2-6 h. The objective of this study was the development of a new method for the quantification of Staphylococcal enterotoxin B (SEB) in food matrices. Tryptic peptide map was generated and nine proteolytic fragments were clearly identified (sequence coverage of 35%). Among these, three specific tryptic peptides were selected to be used as surrogate peptides and internal standards for quantitative analysis using an isotopic tagging strategy along with analysis by LC-MS/MS. The linearity of the measurement by LC-MS/MS was evaluated by combining mixtures of both isotopes at 0.1, 0.2, 0.5, 1.0 and 2.0 ¹H/²H molar ratios with a slope near to 1, values of R² above 0.98 and %CV obtained from six repeated measurement was below 8%. The precision and accuracy of the method were assessed using SEB spiked in chicken meat homogenate samples. SEB was fortified at 0.2, 1 and 2 pmol/g. The accuracy results indicated that the method can provide accuracy within a 84.9-91.1% range. Overall, the results presented in this manuscript show that proteomics-based methods can be effectively used to detect, confirm and quantify SEB in food matrices.