Comparative Analysis of Whey N-Glycoproteins in Human Colostrum and Mature Milk Using Quantitative Glycoproteomics.

Glycosylation is a ubiquitous post-translational protein modification that plays a substantial role in various processes. However, whey glycoproteins in human milk have not been completely profiled. Herein, we used quantitative glycoproteomics to quantify whey N-glycosylation sites and their alteration in human milk during lactation; 110 N-glycosylation sites on 63 proteins and 91 N-glycosylation sites on 53 proteins were quantified in colostrum and mature milk whey, respectively. Among these, 68 glycosylation sites on 38 proteins were differentially expressed in human colostrum and mature milk whey. These differentially expressed N-glycoproteins were highly enriched in "localization", "extracellular region part", and "modified amino acid binding" according to gene ontology annotation and mainly involved in complement and coagulation cascades pathway. These results shed light on the glycosylation sites, composition and biological functions of whey N-glycoproteins in human colostrum and mature milk, and provide substantial insight into the role of protein glycosylation during infant development.

Comparative proteomic analysis of milk-derived exosomes in human and bovine colostrum and mature milk samples by iTRAQ-coupled LC-MS/MS.

Exosomes are membranous vesicles found in biological fluids with important functions. However, milk-derived exosome proteins from humans and bovines have not been studied in detail. The advanced iTRAQ proteomic approach was used to analyze milk-derived exosomes in human and bovine colostrum and mature milk samples. A total of 920 milk exosome proteins were identified and quantified. Among these, 575 differentially expressed exosome proteins (P<0.05) were found. Multivariate analysis, gene ontology (GO) annotation and the KEGG pathway were used to interpret the identified proteins. The major biological processes involved were: response to stimulus (22%), localization (16%), establishment of localization (14%), and cellular component organization (14%). Cellular components engaged in intracellular (31%) and intracellular part (31%). The most prevalent molecular function mainly touched upon binding (52%). Milk exosome proteins participated in several KEGG pathways containing ribosome, regulation of actin cytoskeleton, glycolysis/gluconeogenesis, leukocyte transendothelial migration, aminoacyl-tRNA biosynthesis, pentose phosphate pathway, galactose metabolism and fatty acid biosynthesis. These results provide important information on human and bovine milk exosomes, and increase knowledge on the proteomes of these exosomes across different lactation stages, which could provide potential directions for newborn milk powder, biological markers and functional foods.