Biology subjects

Awan, A.

Publications and source records attributed to Awan, A..

8 recordsLinked to original sources

bioRxiv2026

Diet-derived peptides mediate the effects of dietary protein source on gastrointestinal health

Plant-based diets support gastrointestinal (GI) health while animal-based diets can disrupt gut homeostasis. Although multiple aspects of these diet types are believed to confer their respective effects, the role of their protein component is less well understood. Here, we conducted a randomized crossover-controlled feeding trial wherein healthy subjects consumed 70% of their daily protein intake in the form of pea protein (PP) or egg white protein (EWP) isolate (NCT05619939). Individuals who consumed EWP reported increased GI symptoms and exhibited elevated intestinal permeability. In contrast, these endpoints did not change following PP consumption. Fecal analysis showed increased diet-derived peptides only following EWP consumption, which was associated with resistance of EWP isolate to degradation by digestive enzymes in vitro. Metagenomic, metaproteomic and metabolomic analyses of stool after the EWP-based diet showed reduced abundance of multiple gut-protective bacterial species and increased bacterial amino acid utilization compared to samples following the PP-based diet. Dietary peptides in the gut luminal content of EWP-fed subjects reduced metabolic function of intestinal epithelial cell in culture. Providing an amino acid-based diet mimicking EWP composition to mice prevented colonic accumulation of diet-derived proteins and GI dysfunction associated with EWP diet consumption. Collectively, these findings demonstrate that dietary protein source is a key mediator of GI function, revealing a modifiable lifestyle factor that impacts human health.

physiology↗

bioRxiv2026

Metagenomics-enabled proteomics reveals how AMF and PSB co-inoculation reshapes tomato rhizosphere dynamics across growth stages

Urban agriculture increasingly relies on compost-based substrates for sustainable production, yet we lack a clear characterization of how these systems respond to biological amendments aimed at introducing beneficial microbiota. Here we investigated how developmental stage and co-inoculation with arbuscular mycorrhizal fungi (AMF) and phosphate-solubilizing bacteria (PSB) reshape rhizosphere microbial function in Solanum lycopersicum grown in compost-based urban farm substrate. Using plant physiology assays, 16S rRNA amplicon sequencing, and metagenome-informed metaproteomics, we characterized tomato physiological responses and rhizosphere microbial activity during flowering and fruiting across control, single AMF, single PSB, and AMF and PSB co-inoculation treatments. Co-inoculation synergistically enriched beneficial taxa, improved fruit nutrient accumulation, elevated nutrient transporter and quorum sensing protein production, and drove stress-driven dormancy in competitively excluded taxa, with responses varying between developmental stages. Our findings establish metagenome-informed metaproteomics as essential for resolving stage-specific rhizosphere microbiome functional responses to tomato development and AMF and PSB co-inoculation.

bioinformatics↗

bioRxiv2026

Area under the curve quantification outperforms spectral counting in metaproteomics, but matching between runs is detrimental

Metaproteomics enables the functional characterization of complex microbial communities by detecting and quantifying thousands of proteins. Accurate quantification is essential for deriving meaningful biological inferences from metaproteomes. In mass spectrometry-driven metaproteomics, protein quantification is performed using either MS1-based area under the curve (AUC) or MS2-based peptide spectral counts (SpC). Systematic benchmarking of AUC vs. SpC quantification for metaproteomics, however, is lacking. Additionally, the impact of matching identifications between runs (MBR) on AUC quantification accuracy has not been evaluated. Here, we tested whether AUC or SpC is better suited for quantification in metaproteomics using data-dependent acquisition mass spectrometry data and investigated the impact of MBR on AUC using defined metaproteomics samples with known composition as ground truth. We found that MBR incorrectly transferred peptide and protein identifications and led to a substantial number of falsely identified proteins in samples of known composition. Additionally, when comparing MBR-free AUC data to SpC, AUC data had a wider dynamic range, higher quantitative accuracy, and higher sensitivity for detecting abundance differences. Based on these findings, we recommend MBR-free AUC quantification for metaproteomics analysis, while SpC-based identifications can be used to increase the number of identified proteins to obtain a more comprehensive view of expressed functions. Significance of the studyAlthough accurate quantification of proteins is crucial for characterizing microbial functions in microbiomes, quantification strategies in metaproteomics are mostly selected based on convention rather than evidence. With this study, we provide an evidence-based framework for the informed selection of quantification strategies in metaproteomics. We highlight the strengths and limitations of the tested approaches with respect to dynamic range, quantitative accuracy, and proteome coverage. Based on our results, we recommend using AUC data without MBR for quantitative metaproteomics.

molecular biology↗

bioRxiv2025

Assessing the diversity and functional profile of the microbial proteome in fermented foods

Fermented foods are staples in diets worldwide and are known for their health benefits. Microorganisms are the key to fermented food production as they convert raw substrates into digestible, nutritious, and health-promoting products. While microbes are essential for fermented food production, their contribution to the dietary protein profile of the final food product in terms of microbial biomass is largely unknown. We analyzed proteins from 17 fermented foods using metaproteomics to identify and quantify microbial and food-derived proteins. We found that microbial proteins contribute up to 11% of the total protein content in fermented foods and comprise as much as 60% of the total number of identified proteins. These microbial proteins included many for central functions in microbial cells, such as glycolysis enzymes, translation machinery, and chaperones, as well as proteins for specialized functions that are important for the ecological niches in food fermentation, such as carbohydrate degrading enzymes and proteases. Some of these microbial proteins, such as proteases, could impact gut physiology. These findings highlight the substantial contribution of microbial proteins to the nutritional and functional profile of fermented foods, which may have important implications for interactions with the gut microbiota and health outcomes.

microbiology↗

bioRxiv2025

Source of dietary protein alters the abundance of proteases, intestinal epithelial and immune proteins both directly and via interactions with the gut microbiota

Dietary protein source differentially impacts long-term health outcomes, yet the specific host responses underlying these health effects remain unclear, including which are direct effects of protein source and which are microbiota-mediated. Using metagenome-informed metaproteomics, we determined that protein source altered the abundance of host proteins related to proteolysis, immunity, and the gut barrier as well as microbial function in mice. These stool host responses further reflected site-specific responses throughout the intestinal tract. Additionally, while some host responses were microbiota-dependent, others were microbiota-independent. These effects of dietary protein source on the host and microbiota translated to humans. Notably, microbial enzymes related to mucin degradation increased consistently in response to egg white protein in both mice and humans. Our results show that protein source impacts multiple aspects of host physiology through microbiota-dependent and independent routes, providing essential insights into mechanisms underpinning protein source-driven differences in health outcomes.

microbiology↗

bioRxiv2024

Dietary protein from different sources escapes host digestion and is differentially modified by the microbiota

Protein is an essential macronutrient and variations in its source and quantity have been shown to impact long-term health outcomes. Differential health impacts of dietary proteins from various sources are likely driven by differences in their digestibility by the host and subsequent availability to the intestinal microbiota. However, our current understanding regarding the fate of dietary proteins from different sources in the gut, specifically how component proteins within these sources interact with the host and the gut microbiota, is limited. To determine which dietary proteins are efficiently digested by the host, and which proteins escape host digestion and are used by the gut microbiota, we used high-resolution mass spectrometry to quantify proteins that constitute different dietary protein sources before and after digestion in germ-free and conventionally raised mice. We detected proteins from all sources in fecal samples of both germ-free and conventional mice suggesting that even protein sources with high digestive efficiency make it to the colon where they can serve as metabolic substrate for gut microbiota. Additionally, we found that specific component proteins of dietary protein sources were degraded to a greater extent in the presence of the microbiota. We found that specific proteins with functions that could potentially impact host health and physiology were differentially enriched in germ-free or conventionally raised mice. These findings reveal large differences in the fate of dietary protein from various sources in the gut which could explain some of their differential health impacts.

microbiology↗

bioRxiv2024

Dietary protein source strongly alters gut microbiota composition and function

The source of protein in a persons diet affects their total life expectancy. However, the mechanisms by which dietary protein sources differentially impact human health and life expectancy are poorly understood. Dietary choices have major impacts on the composition and function of the intestinal microbiota that ultimately modulate host health. This raises the possibility that health outcomes based on dietary protein sources might be driven by interactions between dietary protein and the gut microbiota. In this study, we determined the effects of seven different sources of dietary protein on the gut microbiota of mice using an integrated metagenomics-metaproteomics approach. The protein abundances measured by metaproteomics can provide microbial species abundances, and evidence for the molecular phenotype of microbiota members because measured proteins indicate the metabolic and physiological processes used by a microbial community. We showed that dietary protein source significantly altered the species composition and overall function of the gut microbiota. Different dietary protein sources led to changes in the abundance of microbial proteins involved in the degradation of amino acids and the degradation of glycosylations conjugated to dietary protein. In particular, brown rice and egg white protein increased the abundance of amino acid degrading enzymes. Egg white protein increased the abundance of bacteria and proteins usually associated with the degradation of the intestinal mucus barrier. These results show that dietary protein sources can change the gut microbiotas metabolism, which could have major implications in the context of gut microbiota mediated diseases.

microbiology↗

bioRxiv2023

Effective seed sterilization methods require optimization across maize genotypes

Studies of plant-microbe interactions using synthetic microbial communities (SynComs) often require the removal of seed-associated microbes by seed sterilization prior to inoculation to provide gnotobiotic growth conditions. A diversity of seed sterilization protocols have been developed in the past and have been used on different plant species with various amounts of validation. From these studies it has become clear that each plant species requires its own optimized sterilization protocol. It has, however, so far not been tested if the same protocol works equally well for different varieties and seed sources of one plant species. We evaluated six seed sterilization protocols on two different varieties (Sugar Bun & B73) of maize. All unsterilized maize seeds showed fungal growth upon germination on filter paper, highlighting the need for a sterilization protocol. A short sterilization protocol with hypochlorite and ethanol was sufficient to prevent fungal growth on Sugar Bun germinants, however a longer protocol with heat treatment and germination in fungicide was needed to obtain clean B73 germinants. This difference may have arisen from the effect of either genotype or seed source. We then tested the protocol that performed best for B73 on three additional maize genotypes from four sources. Seed germination rates and fungal contamination levels varied widely by genotype and geographic source of seeds. Our study shows that consideration of both variety and seed source is important when optimizing sterilization protocols and highlights the importance of including seed source information in plant-microbe interaction studies that use sterilized seeds.

microbiology↗