Milk glycopeptide across lactation

ABSTRACT: Protein N-glycosylation is an essential post-translational modification (PTM) frequently occurring on human milk proteins. These PTMs assist in protecting the infant's health and function amongst others as competitive inhibitors of pathogen binding and immunomodulators. Due to the individual uniqueness of each mother’s milk and the overall complexity and temporal changes of protein N-glycosylation, analysis of the human milk N-glycoproteome requires longitudinal personalized approaches, providing protein- and N-site-specific quantitative information. Here we describe an automated platform using HILIC-based cartridges enabling the proteome-wide monitoring of N-glycopeptides using just 150 μg of breast milk protein digestion. Reproducibility in enrichment and quantitation is demonstrated in triplicate measurements of milk samples taken along an extended lactation period. Using higher energy c-trap dissociation (HCD) triggered electron-transfer higher-energy collision dissociation (EThcD), we were able to map around 1700 glycopeptides from 110 glycoproteins occurring on 191 glycosites, of which 43 sites were not before reported with experimental evidence. We next quantified many of these glycopeptides using a targeted proteomics approach with scheduled selective ion monitoring (SIM) and parallel reaction monitoring (PRM). In a 90 min gradient, we could quantitatively monitor 318 glycopeptides originating from 51 glycoproteins. Although each glycoprotein, each N-glycosylation site and each glycan attached revealed its own dynamic changes, we did observe a few general trends. Overall fucosylation especially terminal fucosylation increased across the lactation period.

ORGANISM(S): Homo Sapiens

SUBMITTER: Jing Zhu  

PROVIDER: PXD022415 | panorama | Sat Nov 07 00:00:00 GMT 2020

REPOSITORIES: PanoramaPublic