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Isolation, characterisation and proteomics profiling of porcine milk exosomes

Milk exosomes are gaining interest in research due to their potential as drug vehicles, carriers of presumptive biomarkers, and of mediators involved in various patho-physiological functions. Exosome isolation from milk is particularly troublesome, especially for OMIC analyses, due to its complex nature, the high lipid and protein content, and the presence of milk fat globules. This study aimed at establishing an efficient method for isolation of exosomes from porcine milk, and at the characterisation of their proteome by differential ultracentrifugation coupled with size exclusion chromatography (SEC) and high-throughput LC-MS/MS. Skimmed milk was sequentially centrifuged at 12, 000×g for 30 min at 4 °C, at 100, 000×g for 1 h at 4 °C and at 150, 000×g for 2 h at 4 °C. The exosome pellet was collected, suspended in PBS, and loaded on a SEC column (Izon qEV). After the void volume, 4 fractions of 500 µl were collected. Fractions 2 and 3 contained exosomes, substantiated by verification of the presence of a marker (Western Blotting of TSG101), concentration and size distribution by Nanoparticle Tracking Analysis (NanoSight NS300) and morphology by Transmission Electron Microscopy. In-gel digestion was performed after concentrating exosome fractions (10 kDa cut-off column), resolving the proteins by 12% SDS-gel at 90 V for 15 min, fixing with 25% isopropanol/10% acetic acid and staining with PAGE Blue. Bands were excised and shrunk with acetonitrile (ACN), reduced with 10 mM DTT/50 mM TEAB at 56 °C, alkylated (55 mM iodoacetamide/50 mM TEAB), and digested (trypsin + TEAB/10% ACN ; incubated overnight at 37 °C for maximum peptide recovery). Peptides were extracted (5% formic acid/ACN), dried and stored at -80 °C until LC-MS/MS analysis. Proteomic analysis enabled the identification of 1, 306 proteins in qEV fraction 2 and 419 in qEV fraction 3 (minimum 2 unique peptides). The proteome of porcine milk exosomes characterised herein provides new information on the composition of this milk protein fraction which is important not only for basic physiology but may also reveal potential industrial applications.

exosomes, porcine milk, proteomics

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