Abstract
Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of the whole milk proteome and illustrates that milk-derived EV are macromolecular components with a unique functional proteome.
Highlights
From the ‡Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands; §Division of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, the Netherlands; ¶Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht, the Netherlands; ʈNutricia Research, Utrecht, the Netherlands
Isolation of Milk-derived extracellular vesicles (EV) and Workflow of the Proteomics Approach—Isolation of milk-derived EV from breast milk of seven donors was performed using the protocol by Zonneveld et al [10] (Fig. 1A)
To get more insight into the subcellular origin of the identified proteins, functional enrichment analysis for the cellular component was done. These analyses showed that in both milk-derived EV and high-density complexes a high percentage of proteins linked to Gene Ontology (GO) terms like “exosomes”, “lysosome,” and “cytoplasm” (Fig. 2B)
Summary
Breast Milk Collection—Breast milk was collected as previously described [10]. Briefly, fresh, mature milk samples were collected by seven healthy mothers (between 3 and 9 months after delivery) who were not actively terminating breast feeding. In gel digestion fractions were dried, reconstituted in 10% FA and delivered to a trap column (ReproSil C18, (Dr Maisch GmbH, Ammerbuch, Germany); 20 mm ϫ 100 m inner diameter, packed in-house (at the Netherlands Proteomics Centre) at 5 l/min in 100% solvent A (0.1 M acetic acid in water). Functional enrichment analysis for Gene Ontology (GO) terms and comparison of data sets (Fig. 2, Fig. 3, Fig. 5, and Fig. 6) was done using FunRich (with the human FunRich database as background; as recent as November 2015) [21]. Proteins without a common gene name (“N/A”), or entries no longer existing in the UniProt database, or listed under cDNA entries were excluded from the milk proteome data set. The common gene names from all studies were imported into FunRich as a data set and FunRich was used to compare this data set in a Venn diagram
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