We describe an immunoaffinity-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) protocol for the direct (i.e., without prior culture), sensitive and specific detection of Yersinia pestis in complex matrices. Immunoaffinity enables isolation and concentration of intact bacterial cells from food and environmental samples. After protein extraction and digestion, suitable proteotypic peptides corresponding to three Y. pestis-specific protein markers (murine toxine, plasminogen activator and pesticin) are monitored by targeted LC-MS/MS using the selected reaction monitoring (SRM) mode. This immuno-LC-MS/MS assay has a limit of detection of 2 × 104 CFU/mL in milk or tap water, and 4.5 × 105 CFU in 10 mg of soil.
Keywords: Bacteria; Detection; Immunoaffinity; Mass spectrometry; Matrices; Targeted proteomic.