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Treatment of antibiotic-resistant infections is dependent on the detection of specific bacterial genes or proteins in clinical assays. Identification of methicillin-resistant  Staphylococcus aureus  (MRSA) is often accomplished through the detection of penicillin-binding protein 2a (PBP2a). With greater dependence on mass spectrometry (MS)-based bacterial identification, complementary efforts to detect resistance have been hindered by the complexity of those proteins responsible. Initial characterization of PBP2a indicates the presence of glycan modifications. To simplify detection, we demonstrate a proof-of-concept tandem MS approach involving the generation of N-terminal PBP2a peptide-like fragments and detection of unique product ions during top-down proteomic sample analyses. This approach was implemented for two PBP2a variants, PBP2a mecA  and PBP2a mecC , and was accurate across a representative panel of MRSA strains with different genetic backgrounds. Additionally, PBP2a mecA  was successfully detected from clinical isolates using a five-minute liquid chromatographic separation and implementation of this MS detection strategy. Our results highlight the capability of direct MS-based resistance marker detection and potential advantages for implementing these approaches in clinical diagnostics.

Rapid MRSA detection via tandem mass spectrometry of the intact 80 kDa PBP2a resistance protein