Peptidomics: LC-MS operational parameters do matter.

The sensitive and specific detection of peptides at low levels in biofluids is critical to increase the lab-to-human translation of peptidomic research. An interesting group of peptides with increasing evidence for involvement in human diseases are quorum sensing peptides. To obtain more reliable conclusions on peptide measurands in biofluids, a selection of often neglected parts of the analytical process using LC-MS were investigated, with novel approaches recommended for each part. Quorum sensing peptides were used as the main model-peptides. The peptidomic parts investigated and discussed here are: Our work addresses aQbD-approached solutions to these challenges, encompassing sample stabilization measures, a suitable peptide anti-adsorption tool, judicious choice of injection solvent versus gradient system and optimal duty cycle parameters. Our recommendations will improve the peptidomics bio-analytics of not only quorum sensing peptides, but can also be of value for other measurands at low concentrations.

Bacterial Quorum-Sensing Peptides as Immune Modulators Present in Systemic Circulation.

Quorum-sensing peptides (QSPs) are bacterial peptides traditionally considered only as inter-bacterial communication molecules. Recently, their involvement in microbiome-host interactions influencing host diseases such as cancer and sarcopenia were explored. However, it is still unknown to what extent these peptides have the potential to modulate the immune system. In this proof-of-concept study, we screened 89 QSPs for their potential to induce IL-6 and TNFα in murine splenocytes and J774 macrophages. Confirmatory experiments on the positive screening-hits were conducted using murine splenocytes and human PBMCs of different ages. Finally, to investigate the biological relevance of immunomodulatory QSPs, we analysed plasma in a human cohort for the presence of the immunomodulatory QSP Q010. To do this, we used a newly developed UHPLC-MS/MS method. Our findings indicated that specific QSPs activate immune cells in vitro, with Q007, Q010, Q017 and Q212 being the top four screening hits. Q007 and Q010 were affirmed in subsequent confirmatory experiments using murine splenocytes and human PBMCs. Finally, Q010 was detected in human plasma, demonstrating for the first time the presence of an immunomodulatory QSP in human circulation. In conclusion, our data are the first evidence indicating the potential of biologically relevant quorum-sensing peptides to modulate the immune system.

A fit-for-purpose LC-MS/MS method for the analysis of selected Streptococcal quorum sensing peptides in human saliva.

The development of analytical methods for the detection of peptides at the nanomolar level can be challenging. Peptides can suffer from adsorption, rendering the detection of peptides at these low levels difficult. A subset of peptides are the quorum sensing peptides, which are bacterial communication molecules demonstrating possible host effects as well. However, their direct presence in human biofluids has only rarely been reported. Therefore, a UHPLC-MS/MS method capable of detecting 15 selected Streptococcal competence stimulating quorum sensing peptides at the nanomolar level in human saliva was developed. This method, using an anti-adsorption diluent, was applied on saliva samples obtained from 38 healthy donors. Six donors did have a positive hit for at least one of three competence stimulating quorum sensing peptides using a triple quadrupole assay. These observations indicate that Streptococcus species produce quorum sensing peptides in the human oral cavity.