With the ongoing rise of antibiotic-resistant pathogens such as Methicillin-resistant Staphylococcus aureus, and the loss of their effective treatment, novel approaches to tackle this superbug crisis are urgently needed. The exploitation of bacterial quorum sensing (QS), i.e. the regulation of gene expression in response to fluctuations in cell-population density, has previously been discussed as a potential leverage point to combat pathogens. By interfering with the bacterial communication process, also known as quorum quenching, the goal is to shut down virulence production without developing resistance mechanisms. S. aureus employs oligopeptides, so-called autoinducer peptides (AIPs), as QS signaling molecules. AIPs consist of about 8 amino acids (AA) with a thiolester linkage between the central cysteine and the C-terminal carboxyl group. AIPs released by different species have divergent AA sequences but conserved thiolactone ring structure. After the detection of a minimal threshold concentration of AIPs, S. aureus expresses phenol-soluble modulins (PSMs), a toxin family usually composed of 21-26 AA arranged in an alpha-helix.

To facilitate monitoring of AIPs and PSMs in bacterial communities, the purpose of this project was to establish a high-throughput mass spec method for screening of staphylococcal isolates. To establish multiple reaction monitoring (MRM) methods, selected AIP and PSM peptides were custom-synthesized and used to experimentally compile a Q1/Q3 transition list and optimize collision energies. Finally, we have been able to squeeze the separation of the physico-chemically very different peptides in a 4.5 min gradient on an Agilent Poroshell column. Screening efforts are currently underway to sensitively detect and quantify AIP and PSM peptides in culture media filtrates from bacterial isolates. Furthermore, gathered knowledge about the fragmentation behavior of cyclic peptides will aid to generate transition lists in silico in the future.