Introduction: Milk exosomes are known to play an important role in immune function in mammary glands, act in intercellular communication and have recently been explored as drug delivery vehicles. This study aimed to assess the suitability of our bovine milk exosome isolation method on human milk and compare the proteomic content of bovine and human milk exosomes.
Methods: Exosomes were isolated from both human and bovine milk by ultracentrifugation and size exclusion chromatography (utilising a published method by our team on bovine milk). The exosomes were characterised and validated by electron microscopy, nanoparticle quantification and immunoblotting; exosomal protein was quantified by BCA Assay. Proteomic profiling of both human and bovine milk was conducted using Mass Spectrometry (LC-MS/MS). Exosomal protein was reduced and alkylated, tryptic digested and desalted. Information Dependant Acquisition (IDA, 90min) was used to identify proteins from both groups.
Results: Exosomal markers tetraspanin CD9 and tetraspanin CD81 were identified in both human and bovine milk exosomes. Mass spectrometric analyses revealed many of the abundant milk proteins e.g. lactadherin, butyrophilin, xanthine Oxidase and perillipin-2 were common to both human and milk exosomes. However, a number of unique proteins were also identified in human milk exosomes (274 unique proteins) and bovine milk exosomes (129 proteins).
Conclusion: Analyses of exosomal profiles from both human and bovine milk may be exploited for their potential uses as drug delivery vehicles and the diagnosis of inflammation such as in mastitis.