Microorganism characterization for the clinics using SRM
Résumé
Background: Recently, proteomics delivered its' first clinical application in routine with identification of microorganisms using MALDI-TOF. However this technology, based on protein fingerprints, provides only a probability of identification and is not well suited for antibiotic susceptibility testing (AST) or virulence detection. To overcome MALDI-TOF limitations, we propose to use a triple-quadrupole mass spectrometer working in Specific Reaction Monitoring (SRM) mode, coupled with a conventional bore chromatography. Methods : Multiplexed SRM methods enabling an absolute identification of the microorganism as well as the determination of mechanisms of resistance, virulence and strain typing have been developed in our lab. After tryptic digestion either a colony or crude sample, is injected through a short chromatographic gradient (24 min) into a triple quadrupole mass spectrometer working in SRM mode. Results: As an example, methicillin-resistant Staphylococcus aureus (MRSA) strains could be fully characterized in 24 minutes using the multiplexed detection of 36 peptides from 14 proteins, including Penicillin-Binding Protein 2a (PBP-2a) and Panton-Valentine Leucocidin (PVL) toxin. Candida albicans yeast pathogenicity could be characterized, as well, by quantification of Ergosterol metabolite for resistance prediction and Lipase-8 protein for virulence estimation. Conclusions: Multiplexed and quantitative capabilities of SRM offer a unique possibility to develop targeted methods for characterization of microorganisms either after culture or directly on crude sample. The power of this state-of-the-art approach is the development speed, which one can adapt methods to capture the rapid microorganism evolution in biological mechanisms. This technology is expected to bring new proteomics applications into the clinic in the near future.