Introduction
In recent years, the importance of commensal microbes for human health has been more and more recognised. Many leading causes of morbidity and mortality, including asthma and allergies1, metabolic diseases2, autoimmune diseases3, and cardiovascular diseases4,5, have been connected to changes in the composition of the microbiome. While it is well established that diet profoundly influences gut microbial composition, far less is understood about how dietary change shapes the functional output of the microbiome6. One example of microbial metabolites that deeply affect their host are short-chain fatty acids (SCFAs), particularly butyrate, propionate, and acetate. These metabolites, which are produced through bacterial fermentation of dietary fibre, have emerged as key effectors of human health. They exert anti-inflammatory, gut-barrier-protective, and potentially cardioprotective effects7–9. However, the mechanistic link between diet, specific bacterial proteins, their enzymatic activity, and SCFA production in a clinical setting remains poorly defined.
To address this, we are establishing an integrated metaproteomics and metabolomics pipeline, with the aim of connecting microbial protein expression and metabolite production. Combined, these approaches offer a powerful window into diet–microbiome–host interactions at a functional, mechanistic level.
Materials and Methods
20-40 µg of stool samples were weighed in and extracted with methanol. Samples were centrifuged and supernatants were subjected to derivatisation with 3-NPH, enabling the chromatographic separation of SCFAs using a reversed phase column. Mass spectrometric analysis was performed in targeted mode with negative ionisation on a ZenoTOF 7600+ mass spectrometer. For proteomics analyses, 20-40 µg of stool were weighed in and lysed in SP3 buffer. Proteins were reduced and alkylated with CAA and TCEP, followed by bead-based cleanup and tryptic digestion. Peptides were separated on a Evosep Eno and analysed in data-independent mode on a ZenoTOF 7600+ mass spectrometer.
For analysis of serum samples, 30 µL of serum were treated with methanol and centrifuged to precipitate proteins. Supernatants were subjected to derivatisation with 3-NPH and derivatised metabolites were measured as described above.
Metabolomics data were analysed with Sciex Analyst. Proteomics data were analysed in Spectronaut, and further data processing was done in R using the unipept package.
We applied this pipeline to a clinical cohort of patients with cardiovascular diseases undergoing inpatient rehabilitation while adhering to a Mediterranean diet. Paired stool and serum samples of 17 patients were collected at the Hochgebirgsklinik Davos. Together with data on dietary habits before cardiac rehabilitation, this allows us to assess both local gut and systemic metabolic changes over the course of the intervention.
Results
Our metaproteomics analysis shows substantial inter-individual differences in microbiome composition in the stool of different patients, including changes in bacterial species known as butyrate producers.
Preliminary metabolomics results reveal a correlation between nutritional fibre uptake assessed by the food frequency questionnaire and butyrate levels in stool. Additionally, we observed a statistically significant increase in serum butyrate levels over the course of the rehabilitation stay.
Together, these results confirm the well-known correlation between nutrition and the microbiome and suggest that even a short-term Mediterranean diet intervention may meaningfully shift the microbiome's metabolic output towards cardioprotective metabolites.
Discussion
This work represents a step towards a mechanistic understanding of how diet shapes microbiome function. By directly linking bacterial protein expression with the production of clinically relevant metabolites, we move beyond compositional towards functional microbiome analysis. With this, we hope to lay the groundwork for evidence-based microbiome-targeted interventions for human health, including specific dietary interventions or direct metabolite supplementation.
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