Comprehensive Analysis of Proteins and Their Phosphorylation in Milk-derived Exosomes From Different Species

Objective Exosomes are microvesicles which could be secreted by all cell types with diameters between 30 and 150 nm. It was widely distributed in body fluids including blood, urine, and breast milk.Exosomes are considered as potential biomarkers and drug carriers by reason of containing nucleic acids, lipids,proteins and other bioactive molecules. Milk-derived exosomes have been widely used as drug delivery carriers to treat targeted diseases with a lower cost, higher biocompatibility and lower immunogenicity. Until now, there is no research about the milk-derived exosomes phosphorylation to reveal the difference of protein phosphorylation in different species of milk. To investigate the pathways and proteins with specific functions, phosphorylated proteomic analysis of milk-derived exosomes from different species is performed, and provide new ideas for exploring diversified treatments of disease.Methods Whey and exosomes derived from bovine, porcine and caprine milk were performed for proteomics and phosphoproteomics analysis. The relationship between milk exosome proteins from different species and signaling pathways were analyzed using bioinformatics tools. Results A total of 4191 global proteins,1640 phosphoproteins and 4064 phosphosites were identified from 3species of milk-derived exosomes, and the exosome proteins and phosphoproteins from different species were significantly higher than those of whey. Meanwhile, some special pathways were enriched like Fc gamma-mediated phagocytosis from bovine exosomes, pathways related with neural and immune system from caprine exosomes,positive and negative regulation of multiple activities from porcine exosomes.Conclusion In this study, the proteomic and phosphoproteomic analyses of exosomes and whey from bovine, porcine and caprine milk were carried out to reveal the difference of composition and related signaling pathways of milk exosome from different species. These results provided powerful support for the application of exosomes from different milk sources in the field of disease treatment.