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Muscle wasting diseases, such as cancer cachexia and age-associated sarcopenia, have a profound and detrimental impact on functional independence, quality of life, and survival. Our understanding of the underlying mechanisms is currently limited, which has significantly hindered the development of targeted therapies. In this study, we explored the possibility that the streptococcal quorum sensing peptide Competence Stimulating Peptide 7 (CSP-7) might be a previously unidentified contributor to clinical muscle wasting. We found that CSP-7 selectively triggers muscle cell inflammation in vitro , specifically the release of IL-6. Furthermore, we demonstrated that CSP-7 can traverse the gastrointestinal barrier in vitro and is present in the systemic circulation in humans in vivo. Importantly, CSP-7 was associated with a muscle wasting phenotype in mice in vivo. Overall, our findings provide new mechanistic insights into the pathophysiology of muscle inflammation and wasting. • Streptococcal quorum sensing peptide CSP-7 emerges as a previously unrecognized factor in skeletal muscle homeostasis. • CSP-7 triggers the production of IL-6 in muscle cells in vitro. • CSP-7 induces muscle wasting in mice in vivo. • CSP-7 is present in human systemic circulation. [ABSTRACT FROM AUTHOR]

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: 1. Sample collection: obtaining and storing biofluids such as plasma/serum, feces or saliva from mice and humans up to the laboratory handling (sample preparation) should ensure no degradation/metabolization of the peptide measurand (yielding false negatives) or of proteins (yielding false positives). 2. Sample preparation: to remove interfering compounds as well as to release peptides from the adsorbing matrix-components and to preconcentrate them; this is a crucial step which should assure analytical stability and adsorption-minimization. 3. Chromatography: not only the separation power and orthogonality of the complementary systems is critical, but a careful characterisation of the gradient-system, such as the starting conditions, need to be addressed as well because of worsened detection limits (yielding false negatives) or carry-over (false positives). 4. MS detection: operational parameters such as duty cycle characteristics applied are critical in obtaining low-level, reliable results. 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. [Display omitted] • LC-MS operational parameters are crucial to minimize false negative/positive results of peptides in low levels in biofluid. • Increasing the eluent strength of the start mobile phase in the gradient will significantly decrease carry-over. • Duty cycle must be optimized in function of the number of m/z transitions in multiple reaction monitoring (MRM) mode. [ABSTRACT FROM AUTHOR]

• Selected Streptococcal Competence Stimulating peptides were detected in saliva by UHPLC-MS/MS. • A fit-for-purpose UHPLC-MS/MS method wasdeveloped and verified for the analysis of selected Streptococcal CompetenceStimulating peptides in human saliva. • The sample preparation incorporates ananti-adsorption diluent to prevent loss due to adsorption of the quorum sensingpeptides. • Presence of Streptococcal Competence StimulatingPeptides in saliva was demonstrated by UHPLC-MS/MS. [Display omitted] 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. [ABSTRACT FROM AUTHOR]