Comprehensive Proteomic Analysis Revealed a Large Number of Newly Identified Proteins in the Small Extracellular Vesicles of Milk from Late-Stage Lactating Cows

SIMPLE SUMMARY: Bovine milk provides excellent nutrition for infants and contains significant bioactive compounds, including small extracellular vesicles. Small extracellular vesicles are nanosized particles that have long been recognized to carry a variety of cargos, including proteins, nucleic acids, lipids, and other biomolecules. Previous research has shown that the proteomic profile in milk small extracellular vesicles differed in accordance with the lactation stages of cows, such as early- and mid-stage lactation. However, the protein profile in milk small extracellular vesicles from late-stage lactation is mostly unknown. In this study, a comprehensive proteomic analysis was performed to explore the proteins in milk small extracellular vesicles from late-stage lactating cows. The present study identified a total of 2225 proteins in milk small extracellular vesicles from late-stage lactating cows. Notably, a total of 429 proteins were newly identified after comparing with the previously published bovine milk small extracellular vesicles proteomic datasets. These findings indicate that the newly identified proteins could be helpful in better understanding the proteomic dynamics in milk small extracellular vesicles during the late-stage lactating cows. ABSTRACT: Bovine milk contains small extracellular vesicles (sEVs) that provide proteins, miRNAs, mRNAs, DNAs, and lipids to target cells and play a role in intracellular communications. Previous studies have characterized proteins in milk sEVs from early- and mid-stage lactation. However, the proteins in milk sEVs from late-stage lactation are mostly unexplored. The aim of this study was to determine the proteomic profile of milk sEVs from late-stage lactating cows. A comprehensive nanoliquid chromatography–tandem mass spectrometry (nanoLC-MS/MS) approach was carried out to reveal the proteins in milk sEVs. Additionally, bioinformatics analysis was carried out to interpret the molecular signatures of newly identified proteins in milk sEVs from three late-stage lactating cows. NanoLC-MS/MS analysis revealed a total of 2225 proteins in milk sEVs from cows. Notably, after comparing these identified proteins with previously deposited datasets of proteins in bovine milk sEVs, 429 proteins were detected as newly identified. Bioinformatic analysis indicated that these newly identified proteins in milk sEVs were engaged in a diverse range of molecular phenomena relevant to mammary gland physiology, milk production, immunity, and immune response. These findings suggest that the newly identified proteins could expand the inventory application of molecular cargos, nutritional status, and immune modulation of sEVs in milk during the late-stage lactation.