BackgroundMicroRNAs (miRNAs) play essential roles in gene regulation. A substantial fraction of miRNAs in tissues and body fluids is encapsulated in exosomes (exos), thereby conferring protection against degradation. Traditionally, miRNAs have been considered endogenous regulators of gene expression. Recently, we have made the paradigm‐shifting discovery that humans absorb dietary miRNAs from cow's milk (encapsulated in exos) by endocytosis, and dietary miRNAs have biological activity in humans. Inhibitor and competitor studies implicated glycoproteins on the surface of exos and cells in intestinal transport.ObjectiveIdentify glycoproteins on cow's milk exos as putative substrate recognition feature in intestinal transport.MethodsExos were isolated from cow's milk using ultra centrifugation; identity, purity, and integrity were confirmed using nanoparticle tracker, western blot, and transmission electron microscopy. Where applicable, exos were treated with the proteases Glu‐C (targeting glu), trypsin (targeting arg and lys), or proteinase K (non‐specific); controls were not treated with proteases. For transport studies, exos were labeled using fluorophore (FM‐4), and uptake rates were analyzed in human intestinal Caco‐2 cells. Surface peptides released by treatment with trypsin were identified using LC/MS‐MS and Mascot, and glycoproteins were identified using SwissProt. One way Anova and Bonferroni's Multiple comparison was used to test for statistical significance.ResultsWe identified 417 exosomal surface proteins, including N‐glycans (4), O‐glycans (2), and C‐glycans (2). When exos were treated with Glu‐C or trypsin, rates (Vmax) decreased compared with controls (arbitrary units): 88±1 for control, 40±0.6 for trypsin, 19±0.3 for Glu‐C (P<0.05 vs. control). For comparison, we identified 504 proteins in breast milk exos (6‐N‐glycans, 4 O‐glycans, and 3 C‐glycans). The identities of surface glycoproteins were distinct in bovine and human milk exos.ConclusionEight glycoproteins were identified on surface of cow's milk exos, which might be essential for intestinal transport. We propose that glycan features, as opposed to protein features, are important for exo recognition by intestinal cells.Future directionsWe will use site‐directed mutagenesis and gene knockout protocols in cell cultures and mice to provide unambiguous evidence for the importance of distinct glycoprotein features on the mucosal surface in exo recognition.Support or Funding InformationSupported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award number 2015‐67017‐23181, NIH 1P20GM104320 and P30GM103335, the Gerber Foundation, the Egg Nutrition Center, the University of Nebraska Agricultural Research Division (Hatch Act), and USDA multistate group W3002.