A Signature Peptide Strategy for Concurrent Detection of Multiple Pathogens and Toxins in Infant Formula

Zero-tolerance regulations for Salmonella and Cronobacter sakazakii in infant formula require rapid and reliable multitarget detection methods. Here, we developed a multiplex targeted-proteomics strategy based on species-specific signature peptides combined with an LC–MS/MS multiple reaction monitoring (MRM) workflow suitable for complex dairy matrices. Candidate peptides were identified through an integrated pipeline involving in silico peptide mining, shotgun proteomics, and literature evidence, and two three-peptide panels were selected for the respective pathogens. A dual identification strategy, incorporating retention-time matching and consistent transition-ion ratios, was implemented to enhance identification reliability and reduce false positives. After optimization, simultaneous detection of Salmonella and C. sakazakii in infant formula was achieved following a 10 h enrichment with a sensitivity of 10 CFU/mL. The method maintained stable performance in mixed cultures, including those containing Staphylococcus aureus . This peptide-centric MRM approach provides a rapid and reliable complement to conventional culture-based and nucleic-acid-based detection methods.