Search

Protein N-glycosylation on human milk proteins assists in protecting an infant’s health and functions among 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 hydrophilic-interaction chromatography (HILIC)-based cartridges enabling the proteome-wide monitoring of intact N-glycopeptides using just a digest of 150 μg of breast milk protein. We were able to map around 1700 glycopeptides from 110 glycoproteins covering 191 glycosites, of which 43 sites have not been previously reported with experimental evidence. We next quantified 287 of these glycopeptides originating from 50 glycoproteins using a targeted proteomics approach. Although each glycoprotein, N-glycosylation site, and attached glycan revealed distinct dynamic changes, we did observe a few general trends. For instance, fucosylation, especially terminal fucosylation, increased across the lactation period. Building on the improved glycoproteomics approach outlined above, future studies are warranted to reveal the potential impact of the observed glycosylation microheterogeneity on the healthy development of infants.

Quickly after birth, the gut microbiota is shaped via species acquisition and resource pressure. Breastmilk, and more specifically, human milk oligosaccharides are a determining factor in the formation of microbial communities and the interactions between bacteria. Prominent human milk oligosaccharide degraders have been rigorously characterized, but it is not known how the gut microbiota is shaped as a complex community. Here, we designed BIG-Syc, a synthetic community of 13 strains from the gut of vaginally born, breastfed infants. BIG-Syc replicated key compositional, metabolic, and proteomic characteristics of the gut microbiota of infants. Upon fermentation of a four and five human milk oligosaccharide mix, BIG-Syc demonstrated different compositional and proteomic profiles, with Bifidobacterium infantisand Bifidobacterium bifidumsuppressing one another. The mix of five human milk oligosaccharides resulted in a more diverse composition with dominance of B. bifidum, whereas that with four human milk oligosaccharides supported the dominance of B. infantis, in four of six replicates. Reintroduction of bifidobacteria to BIG-Syc led to their engraftment and establishment of their niche. Based on proteomics and genome-scale metabolic models, we reconstructed the carbon source utilization and metabolite and gas production per strain. BIG-Syc demonstrated teamwork as cross-feeders utilized simpler carbohydrates, organic acids, and gases released from human milk oligosaccharide degraders. Collectively, our results showed that human milk oligosaccharides prompt resource-sharing for their complete degradation while leading to a different compositional and functional profile in the community. At the same time, BIG-Syc proved to be an accurate model for the representation of intra-microbe interactions.

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution “fingerprint” of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before. Copyright 2000 Academic Press.