Kayili, H. M. | bioRxiv Science

bioRxiv2024

Identification and comparison of N-glycome profiles from common dietary protein ingredients

The N-glycome profiles purified from dietary bovine whey, egg white, pea, soy protein isolates and a recently commercialized animal-free whey is described. Purified glycoproteins resulting from centrifugation and ethanol precipitation of protein powder supplements were treated with peptide-N- glycosidase F (PNGase F) to release protein-bound N-glycans. Once released from the protein, N-glycans were labeled by procainamide labeling, purified via cotton-hydrophilic interaction liquid chromatography (HILIC), and analyzed using HILIC high performance liquid chromatography equipped with a fluorescence detector and a quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). A total of 33, 33, 10, and 10 N-glycan structures were identified from bovine whey, egg, soy, and pea glycoprotein isolates, respectively. The type of N-glycans per glycoprotein source were highly predictable, likely attributed to differences in biosynthetic glycosylation pathways. Mammalian glycoprotein sources favored a combination of complex and hybrid glycan configurations while the plant proteins were dominated by oligomannosidic N-glycans. Bovine whey glycoprotein isolate contained the most diverse N-glycans by monosaccharide composition as well as structure, while plant sources such as pea and soy glycoprotein isolates contained an overlap of oligomannosidic N-glycans. The results suggest N-glycan structure and composition is dependent on the host organism rather than protein sequence homology, likely driven by the differences in N-glycan biosynthetic pathways.

biochemistry↗

bioRxiv2024

Proteomic and N-glycomic comparison of synthetic and bovine whey proteins and their effect on human gut microbiomes

Advances in food production systems and customer acceptance have led to the commercial launch of dietary proteins produced via modern biotechnological approaches as alternatives to traditional agricultural sources. At the same time, a deeper understanding of how dietary components interact with the gut microbiome has highlighted the importance of understanding the nuances underpinning diet-microbiome interactions. Novel food proteins with distinct post-translational modifications resulting from their respective production systems have not been characterized, nor how they may differ from their traditionally produced counterparts. To address this, we have characterized the protein composition and N-glycome of a yeast-synthesized whey protein ingredient isolated from commercially available ice cream and compared this novel ingredient to whey protein powder isolate derived from bovine milk. We found that despite strong similarities in protein composition, the N-glycome significantly differs between these protein sources, reflecting the biosynthetic machinery of the production systems. Further, the composition profile and diversity of proteins found in the synthetic whey protein were lower relative to bovine whey protein, despite both being predominantly composed of {beta}-lactoglobulin. Finally, to understand whether these differences in N-glycome profiles affected the human gut microbiome, we tested these proteins in an in vitro fecal fermentation model. We found that the two whey protein sources generated significant differences among three distinct microbial compositions, which we hypothesize is a product of differences in N-glycan composition and degradation by these representative microbial communities. This work highlights the need to understand how differences in novel biotechnological systems affect the bioactivity of these proteins, and how these differences impact the human gut microbiome.

microbiology↗