Comprehensive peptidomic analysis of cell culture models and exosomes

Cellular endogenous peptides may harbour important signalling functions regulating health and disease. The identification of functional peptides by mass spectrometry is very challenging but it allows identification and quantification of peptides in a large-scale and can provide experimental evidence of bioactive peptides. Quantitative approaches can provide insights in the regulation of endogenous peptides, however, they need a robust peptide extraction methodology. We demonstrated that different approaches for peptidomic sample preparation introduce variation and strongly influence the identification of different sets of peptides, showing the need of a critical evaluation of peptide extraction protocols. We identified a global peptidome, stable throughout different cell lines and within different organisms. And high numbers of identification allows us to target cellular fractions.
An interesting source of bioactive peptides are extracellular vesicles (EVs). EVs transfer signalling molecules for cell-to-cell communication with surrounding but also distant cells. In cancer, EVs are involved in tumour progression, metastasis and treatment resistance. Their purity for proteomic and peptidomic analysis is very crucial and we consequently described strategies to assess the vesicle purity by mass spectrometry and selected a robust sample preparation method for further application. We identified many nuclear proteins enriched in EVs upon hypoxic treatment, even though general cellular markers were overall highly reduced in EV samples compared to cell samples. We thus conclude that the enriched cargo of the EVs is the result of targeted exocytosis of important proteins for tumour progression, rather than random co-exportation. An increased export of nuclear proteins involved in DNA replication, repair or chromatin remodelling suggests a proliferation-related signature of hypoxic EVs.
So far, most EV studies are limited to the EVs’ nucleic acid, lipid or proteomic cargo, but little is known about the peptidomic cargo. Challenges of EV peptidomics lie in the low starting material EVs provide, compared to the high starting material which is usually needed for peptidomic studies. However, the exploitation of EVs regarding their peptidomic cargo might pave the way for better understanding of signalling pathways. Thus we aimed at comprehensively studying the cellular and EV peptidome. By combining different peptide extraction methodologies, we detected a vesicle- and a cell-origin related signature in the peptidome and we showed the feasibility of a parallel peptide and protein extraction and it’s advantage for precious EV-enriched samples.