N‐glycosylation proteomic characterization and cross‐species comparison of milk whey proteins from dairy animals

Glycosylated proteins in milk have been implicated in multiple biological roles. However, the N‐glycoprotein components and their complexity in milk whey from dairy animals are not well characterized. Here, a modified proteomics approach consisting of N‐glycopeptide enrichment and identification was used to map the N‐glycoproteome profile of milk whey from Holstein and Jersey cows, buffaloes, yaks, goats, camels, and horses. A total of 233 N‐glycosylation sites, corresponding to 147 N‐glycoproteins, were detected in the studied animals. Most of the identified N‐glycosylation sites were not characterized in the database and were considered as unknown. Functional analysis of the identified glycoproteins demonstrated that response to stimulus was the most abundant GO category shared in the studied animals according to their annotation. Lysosome, glycosaminoglycan degradation, and extracellular matrix‐receptor interaction pathways were shared between Holstein and Jersey cows, and yaks. N‐glycoprotein components of milk whey from Holstein and Jersey cows, buffaloes, yaks, and goats were more similar to each other than to those of camels and horses. These results significantly extend the number of known N‐glycosylation sites and reveal in‐depth composition and potential functions of N‐glycoproteins in milk whey, which in turn provides insights to further explore N‐glycoprotein biosynthesis in the studied animals.