Session: Parallel session 1 - environmental analysis

Coupling laser-induced dissociation in the visible range with mass spectrometry to improve the detection and quantification of Staphylococcus Aureus enterotoxins

Annabelle MEDALIN¹, Benjamin YOUENOU², Chloé DESBIOLLES², Roxane PRAT², Cedric BADIOU³, Francois VANDENESH^2,3, Jérome LEMOINE¹, Marion GIROD¹

¹Institut of Analytical Sciences ; UMR 5280 ; Université Lyon 1 et CNRS, Villeurbanne, France
²Centre National de Référence des Staphylocoques ; Institut des agents infectieux ; Hospices Civils de Lyon, LYON, France
³Centre International de Recherche en Infectiologie (CIRI) ; UMR 5308 ; ENS Lyon, INSERM, CNRS et Université Lyon 1, LYON, France

Introduction

Recent studies show that sepsis kills 11 million people each year, 50% of which are linked to infections by 5 main bacteria, among which Staphylococcus aureus.

The identification and quantification of toxin proteins secreted by infectious germs could be correlated to its virulence. However, toxins are produced in low quantities and released in complex matrix such as blood or culture media.

Methods

In order to improve the sensibility of these minor proteins in such complex matrices, an experimental setup coupling MS and laser-induced dissociation (LID) in the visible range is used, adding an optical specificity to the mass selectivity. Since proteins do not naturally absorb in the visible range, this approach relies on a specific chemical derivatization of the targeted peptides with a Dabcyl chromophore. For a better specificity, peptides containing a cysteine (Cys), rare amino acid but representative of a large set of bacterial proteins, are derivatized with the chromophore and targeted.

The PRM-LID method was developed to target derivatized Cys peptides of Staphylococcus Aureus enterotoxins (SEs). In a first time, protein extraction, digestion and derivatization protocol was optimized with Brain Heart Infusion culture media spiked with five main enterotoxins. Then, the linearity of response of these spiked SEs was assessed to establish the PRM-LID performance. After all, endogenous SEs were analyzed in 13 strains selected from genomic data: depending on the toxin, 2 to 6 strains were supposed to produce the targeted SE.

Results & Conclusion

Derivatized Cys-peptides of four endogenous toxins were systematically detected in the expected samples with coefficients of variation lower than 15% over 3 replicates. Only one targeted peptide had not been observed in 1 of the 3 expected samples. Targeted LID-MS is a promising method for the sensitive detection of low concentrated staphylococcal enterotoxins.