Review of Methodological Approaches to Human Milk Small Extracellular Vesicle Proteomics

Abstract

Proteomics can map extracellular vesicles (EVs), including exosomes, across disease states between organisms and cell types. Due to the diverse origin and cargo of EVs, tailoring methodological and analytical techniques can support the reproducibility of results. Proteomics scans are sensitive to in-sample contaminants, which can be retained during EV isolation procedures. Contaminants can also arise from the biological origin of exosomes, such as the lipid-rich environment in human milk. Human milk (HM) EVs and exosomes are emerging as a research interest in health and disease, though the experimental characterization and functional assays remain varied. Past studies of HM EV proteomes have used data-dependent acquisition methods for protein detection, however, improvements in data independent acquisition could allow for previously undetected EV proteins to be identified by mass spectrometry. Depending on the research question, only a specific population of proteins can be compared and measured using isotope and other labelling techniques. In this review, we summarize published HM EV proteomics protocols and suggest a methodological workflow with the end-goal of effective and reproducible analysis of human milk EV proteomes.

Beneficial Effects of Bovine Milk Exosomes in Metabolic Interorgan Cross-Talk

“…Among these, exosome precipitation solutions (ExoQuick TM , System Biosciences, Palo Alto, CA, USA), and membrane affinity spin columns (ExoEasy TM , QIAGEN, Hilden, Germany) have been applied to milk extracellular vesicles [ 43 , 44 , 45 , 46 , 47 ]. These kits provide rapid EVs isolation but are susceptible to high protein contamination and can promote exosome aggregation [ 31 , 48 ]. Particularly, the incapacity to differentiate different EVs subsets is a common disadvantage of most available methods.…”

“…Particularly, the incapacity to differentiate different EVs subsets is a common disadvantage of most available methods. Newer techniques, such as asymmetrical flow field-flow fractionation (AF4), have been shown to enable the differentiation between different EVs populations up to 1 nm increments [ 48 , 49 ], albeit low yield and high costs may hinder their implementation [ 50 ].…”

Extracellular vesicles in human milk

“…The protein composition of MDEVs was determined by various proteomics analyses [39,40,41]. The exosome markers CD9 and CD81 tetraspanin proteins were consistently detected by proteomic analysis in human MDEVs [39].…”

Protein and Lipid Content of Milk Extracellular Vesicles: A Comparative Overview

“…Acid precipitation accelerates casein micelles aggregation, but there is also evidence that EVs can be damaged by the treatment [ 30 ]. Chelating agents such as EDTA have also been used to remove calcium and destabilize casein micelle structure, but EVs may be altered [ 31 ]. Ultracentrifugation at intermediate speed (20,000 up to 70,000× g ) can be used to remove casein micelles, but the removal is partial [ 24 ].…”