DOI: 10.5061/dryad.hmgqnk9qz
title: Proteomic and N-glycomic comparison of synthetic and bovine whey proteins and their effect on human gut microbiomes in vitro

description: 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 β-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 distinct whey protein sources generated significant differences
 in 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.
description: 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
 <em>N</em>-glycans from each sample were labeled with a 2-AA
 tag. 50 μL of 2-AA tag (48 mg/mL<sup>−1</sup> 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 <em>N</em>-glycans was achieved by
 solid-phase extraction cartridges containing cellulose and porous
 graphitized carbon materials, as previously described by Kayili et al
 (2021). MALDI-TOF(/TOF)-MS analysis of 2-AA labeled
 <em>N</em>-glycans from bovine and synthetic whey protein
 samples was carried out on a Bruker rapifleX™ MALDI Tissuetyper™ (Bruker
 Daltonik GmbH, Bremen, Germany) equipped with a SmartBeam 3D laser system.
 On the AnchorChip MALDI-target plate, the purified
 <em>N</em>-glycans (1 μL) were spotted and allowed to dry.
 Then, 1 μL of DHB matrix (5 mg/mL<sup>−1</sup> in
 ACN/H<sub>2</sub>O, 1/1, v/v comprising 0.1% ortho-phosphoric
 acid) was added. The analysis included a 20 kV acceleration voltage, a 160
 ns extraction delay, and the summation of 8000 shots at 2000 Hz for each
 spectrum. The mass range of 1000-5000 Da was used to produce all spectra
 using a random walk pattern in negative ion and reflectron mode. Data
 obtained by MALDI-TOF(/TOF)-MS analysis were processed using Flex Analysis
 v.4.0 software (Bruker Daltonik Gmbh). Peaks of 2-AA labeled
 <em>N</em>-glycans were inserted into ProteinScape software
 including the GlycoQuest algorithm (Bruker Daltonik GmbH, Bremen, Germany)
 for glycan identification. Total area normalization was used to determine
 the relative abundance of individual <em>N</em>-glycans
 (mass-intensity based). All experiments were performed with three
 technical replicates.
description: # 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 for this work. The original file is available at [http://www.dallaslab.org/resources.](http://www.dallaslab.org/resources.) *.RAW Files - DIA analysis files. Five replicates were prepared from the same samples as the MALDI-TOF data. ## Sharing/Access information Data was derived from the following sources: Whey protein samples as described in the manuscript (Bolino et al, 2024). ## Code/Software Data was generated using Flex Analysis v.4.0 and ProteinScape software.