Proteome characterization of extracellular vesicles from human milk: Uncovering the surfaceome by a lipid‐based protein immobilization technology

Breast milk is an essential source of nutrition and hydration for the infant. In addition, this highly complex fluid is rich in extracellular vesicles (EVs). Here, we have applied a microfluidic technology, lipid-based protein immobilization (LPI) and liquid chromatography with tandem mass spectrometry (LC-MS/MS) to characterize the proteome of human milk EVs. Mature milk from six mothers was subjected to EV isolation by ultracentrifugation followed by size exclusion chromatography. Three of the samples were carefully characterized; suggesting a subset enriched by small EVs. The EVs were digested by trypsin in an LPI flow cell and in-solution digestion, giving rise to two fractions of peptides originating from the surface proteome (LPI fraction) or the complete proteome (in-solution digestion). LC-MS/MS recovered peptides corresponding to 582 proteins in the LPI fraction and 938 proteins in the in-solution digested samples; 400 of these proteins were uniquely found in the in-solution digested samples and were hence denoted "cargo proteome". GeneOntology overrepresentation analysis gave rise to distinctly different functional predictions of the EV surfaceome and the cargo proteome. The surfaceome tends to be overrepresented in functions and components of relevance for the immune system, while the cargo proteome primarily seems to be associated with EV biogenesis.