PRObing The Efficacy of Commercial Stage Storage Buffers and Evaluating Gut Metaproteome Variability Between Individuals

A. Introduction:

Metaproteomics studies the entire protein complement of a microbial community and offers insight into intricate ecosystems like the gut microbiome. Metaproteomics allows researchers to delve deep into gut microbiomes, providing insights into these microbial inhabitants, abundance and functional activities. This knowledge has been instrumental in elucidating the mechanisms underlying various diseases, such as inflammatory bowel disease, and obesity. Researchers can develop targeted strategies for personalized medicine and precision healthcare based on the underlying biology by identifying key protein signatures.

In gut microbiome studies, standardized sample collection and preservation are particularly crucial to preserve the integrity of the microbial community and its proteome. Indeed, to obtain reliable results it must be considered the susceptibility to environmental influences like temperature and oxygen amount. For gut microbiome metaproteomic studies, non-invasive or minimally invasive methods are preferred to minimize disruption, or contamination, and preserve the native microbial composition and protein profiles. Also, the sample handling and storage strategies affect the degradation of proteins and the microbial population stability. For this reason, immediate freezing, or preservation at low temperatures (e.g., -80°C) is often recommended to maintain sample integrity until analysis. However, using a cold chain for storage and transportation is not compatible with an on-site sampling strategy that is necessary for large-scale and delocalized studies. Moreover, it is less ideal, more expensive, and logistically tedious than potential room temperature options.

Furthermore, present knowledge of the fecal sample collection/preservation methods is based on preserving nucleic acids or metabolites during the transportation to the laboratory for analysis. However, protein preservation requires different strategies and studies are scarce and rely on adopting the devices or reagents used in the case of nucleic acids or metabolites analysis. Thus, tailored, and standardized protocols together with metadata collection are essential for ensuring reproducibility and comparability across metaproteomic studies.

The present study aims to evaluate the fitness of several commercial and non-hazardous buffers for the storage of human gut fecal samples over time. Fecal samples will be stored in five different buffers and will be benchmarked with directly frozen samples. Using metaproteomics the investigators will compare the proteomic, taxonomic, and functional identifications to understand the potential of these buffers as a reliable storage solution for human feces. This research is unique and to the best of our knowledge, no similar study has addressed this demanding question.