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

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 d..., Enzymatic deglycosylation of denatured protein samples was performed with PNGase F (1 Unit/μL) obtained from Promega (Madison, WI, USA). First, dried purified protein samples (1 mg) were dissolved in 50 μL of 2% SDS and denatured by incubations at 60°C. Denatured protein samples were then mixed with 2% NP-40 solution and 5X PBS and 1 U PNGase F was added and incubated at 37°C overnight. Finally, the samples were centrifuged, and supernatants were collected for further analysis. After enzymatic deglycosylation of protein samples, released N-glycans from each sample were labeled with a 2-AA tag. 50 μL of 2-AA tag (48 mg/mL−1 in DMSO/glacial acetic acid, 7/3, v/v) and 50 μL of 2-sodium cyanoborohydride (60 mg/mL in DMSO/glacial acetic acid, 7:3 w/v) were added to the released glycan samples (50 μL). Subsequently, the mixtures were incubated at 65°C for two hours. Purification of N-glycans was achieved by solid-phase extraction cartridges containing cellulose and porous graphitized carbon m..., , # Proteomic and N-glycomic comparison of synthetic and bovine whey proteins and their effect on human gut microbiomes in vitro

[https://doi.org/10.5061/dryad.hmgqnk9qz](https://doi.org/10.5061/dryad.hmgqnk9qz)

This dataset contains proteomics and paired MALDI-TOF profiling data for three replicates each of bovine-derived whey protein and yeast-manufactured whey protein.

## Description of the data and file structure

MALDI-MS_data.zip - This dataset contains a .zip file that can be decompressed to yield six folders, each corresponding to the six samples (N=3 technical replicates of each sample type) analyzed by MALDI-TOF. The folders are named correspondingly. The XML files can be imported into relevant peak calling software (see below) to recreate the analysis presented in the manuscript.

CowMilkProteinDatabase.fasta - the milk-protein specific FASTA file referenced in the manuscript. This database was prepared by Dr. David C Dallas, Oregon State University. All credit to Dr. Dallas...,