Comparative Proteomic Analysis of Milk-Derived Extracellular Vesicles from Dairy Cows with Clinical and Subclinical Mastitis

SIMPLE SUMMARY: Extracellular vesicles in milk may play important roles in the development of clinical and subclinical mastitis. Milk-derived extracellular vesicles of mastitis and healthy cows were extracted by a combination method of acetic acid/ultracentrifugation and density gradient ultracentrifugation and analyzed using a Shotgun proteomic by data-independent acquisition mode. Our data indicated that mammary inflammation altered milk-derived EV (Extracellular vesicle) protein cargos, especially antimicrobial peptides. These results expanded the repertoire of bovine milk-derived extracellular vesicles proteins and conduced more attention to the functions and the pathways they might impact. ABSTRACT: Extracellular vesicles (EVs) are membranous vesicles found in biological fluids with essential functions. However, milk-derived EV proteins from clinical mastitis (CM) and subclinical mastitis (SM) cows have yet to be studied in detail. In this study, milk-derived EVs of CM, SM, and Healthy cows were extracted using a combination of acetic acid/ultracentrifugation and density gradient ultracentrifugation and analyzed using a shotgun proteomic by data-independent acquisition mode. A total of 1253 milk exosome proteins were identified and quantified. Differently enriched (DE) proteins were identified as given a Benjamini–Hochberg adjusted p < 0.05 and a fold change of at least 2. There were 53 and 1 DE proteins in milk-derived EVs from CM and SM cows compared with healthy cows. Protein S100-A9, Protein S100-A8, Chitinase-3-like protein 1, Haptoglobin, Integrin beta-2, and Chloride intracellular channel protein 1 were more abundant in the CM group (adjusted p < 0.05). Still, their enrichment in the SM group was not significant as in the Healthy group. The enrichment of DE proteins between CM and Healthy group was consistent with elevated GO (Gene Ontology) processes—defense response, defense response to Gram-positive bacterium, granulocyte chemotaxis also contributed to Reactome pathways—neutrophil degranulation, innate immune system, and antimicrobial peptides in the CM group. These results provide essential information on mastitis-associated proteins in milk-derived EVs and indicate the biological functions of milk-derived EVs proteins require further elucidation.