Quantification of Staphylococcal Enterotoxins by Coupling Laser-Induced Dissociation and Mass Spectrometry

Staphylococcal enterotoxins (SEs) make up a superfamily of virulence factors that make <i>Staphylococcus aureus</i> a major cause of food poisoning. The amount of SEs produced by a strain may correlate with its virulence; however, their accurate quantification remains a major challenge. This difficulty arises from two main factors: SEs exhibit emetic activity at nanogram levels, and they are secreted into complex biological matrices during bacterial growth, which typically requires immunoaffinity enrichment before multiplex mass spectrometry (MS) analysis. This study presents an innovative method combining laser-induced dissociation (LID) with mass spectrometry to detect and quantify low-abundance SEs without prior immunoenrichment. To enhance detection specificity based on optical properties, a 473 nm laser was used to selectively fragment chromophore-derivatized cysteine peptides from SEs via LID-MS/MS. The derivatization strategy was first validated on synthetic peptides from five major SEs. Sample preparation was then optimized using purified toxins spiked into biological matrices. The method linearity was assessed by spiking SE synthetic peptides into the matrix across a wide concentration range. Finally, the full analytical protocol was validated by the detection and quantification of endogenous SEs produced by <i>S</i>. <i>aureus</i> strains. This LID-MS/MS approach offers a promising alternative to antibody-based methods for the precise quantification of staphylococcal enterotoxins in complex samples.