Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants

• DOI: 10.1021/acs.jproteome.1c00886
• Publication Date: 2021-12-30T19:41:46Z
• Authors: Donatien Lefebvre; François Fenaille; Déborah Merda; Kevin Blanco-Valle; Cécile Feraudet-T...; Stéphanie Simon; Jacques-Antoine H...; Yacine Nia; François Becher
• Keywords: 0301 basic medicine; variants; Staphylococcus aureus; enterotoxins; [SDV]Life Sciences [q-bio]; absolute quantification; 610; immunoaffinity enrichment; Mass Spectrometry; 576; intact protein; 3. Good health; Enterotoxins; 03 medical and health sciences; top-down; Food Microbiology; mass spectrometry; bacterial toxins

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by Staphylococcus aureus and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.