Enrichment and identification of small proteins in a simplified human gut microbiome

Small proteins (sProteins) with a size of 100 amino acids and less are involved in major biological processes and play an important role in different bacteria. Despite the increasing interest in them, the data on sProteins in bacterial communities, like the human gut microbiome is sparse. In this study, we are using the extended simplified human intestinal microbiota (SIHUMIx) as model system of the human gut microbiome to detect sProteins and to compare different sProtein enrichment methods. We observed that with our tested methods, the C8-cartridge enrichment resulted in the highest number of detected sProteins (n = 295) with high reproducibility among replication analysis. However, in order to further increase the total number of sProteins, the combination of C8 cartridge enrichment with GelFree enrichment is favored because the latter complemented n = 48 more sProteins compared to the C8 cartridge approach resulting in n = 343 sProteins. Among all detected sProteins we were able to identify 79 so far uncharacterized sProteins, with no described protein evidence in the current released database. In total, 34 of those uncharacterized sProteins are localized in gene clusters conserved between different bacteria species allowing functional predictions. This study improves the assessment of sProtein detection and enables their functional characterization in future experiments. Significance: Small proteins have great potential and are therefore obtaining high interest. Especially in human microbiota, many undiscovered and uncharacterized small proteins promote the understanding of complex hostmicrobiome and bacteria-bacteria interactions. However, a major challenge lies in the detection of small proteins using bottom-up proteomics. Our study leads to an improved identification of small proteins in bacterial communities. It further describes properties and potential functions of identified, previously uncharacterized small proteins. Altogether it improves the assessment of sProtein detection and enables their functional characterization in future experiments.