Human Milk Site-specific N-Glycoproteome Changes Across Lactation

N-glycosylation is an essential post-translational modification (PTM) of human milk proteins for the promotion of infant health. N-glycosylation is relevant regarding proteolytic susceptibility and functions as a competitive inhibitor of pathogen binding and immunomodulators. Due to the individual uniqueness of human milk, the overall complexity and temporal changes of N-glycosylation, the analysis for site-specific information of this PTM must take these factors into consideration. Here we demonstrate, by adapting the automated platform and hydrophilic-interaction chromatography (HILIC)-based cartridge with 150 μg of milk protein digestion, that proteome-wide N-glycopeptides can be reproducibly enriched in technical triplicates and biological samples across lactation. With higher energy c-trap dissociation (HCD) triggered electron-transfer/higher-energy collision dissociation (EThcD), we were able to map, to our knowledge, the most comprehensive human milk N-glycoproteome to date. We found 1697 glycopeptides from 110 glycoproteins and 191 glycosites of which 43 novel sites were not yet reported in experimental evidence. We next quantified the glycopeptides in targeted monitoring mode with scheduled selective ion monitoring (SIM) and parallel reaction monitoring (PRM). Co-trending HCD fragment ions enabled us to pinpoint the MS1 extracted ion chromatogram (XIC) of intact glycopeptide. In a 90 min gradient, we could measure 318 glycopeptides from 51 glycoproteins, catching at least 10 data points in their chromatographic peaks. We observed distinctive quantitative site-specific glycosylation trends, indicating the potential of specific temporal changes associated with functions that support the developing infant’s health.