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 host-microbiome 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.

中文翻译：

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简化的人类肠道微生物组中小蛋白的富集和鉴定。

大小小于100个氨基酸的小蛋白质（sProteins）参与主要的生物过程，并在不同细菌中起重要作用。尽管人们对它们的兴趣日益浓厚，但细菌群落（如人类肠道微生物组）中sProteins的数据却很少。在这项研究中，我们使用扩展的简化的人类肠道菌群（SIHUMIx）作为人类肠道微生物组的模型系统，以检测sProteins并比较不同的sProtein富集方法。我们观察到，通过我们的测试方法，C8碳粉盒富集导致检测到的sProteins数量最多（n = 295），并且在复制分析中具有很高的重现性。但是，为了进一步增加sProteins的总数，C8柱富集与GelFree富集相结合是受青睐的，因为与C8柱方法相比，后者对n = 343个sProteins的补充比后者多了n = 48个sProteins。在所有检测到的sProteins中，我们能够鉴定出79种迄今未表征的sProteins，在当前发布的数据库中没有描述的蛋白质证据。总共，这些未表征的sProtein中的34个位于不同细菌物种之间保守的基因簇中，从而可以进行功能预测。这项研究改进了sProtein检测的评估，并使其在未来的实验中得以表征。意义：小蛋白具有巨大的潜力，因此引起了人们的极大兴趣。特别是在人类微生物群中 许多未发现和未表征的小蛋白促进了对复杂的宿主-微生物组和细菌-细菌相互作用的理解。但是，主要挑战在于使用自下而上的蛋白质组学技术检测小蛋白质。我们的研究导致细菌群落中小蛋白的鉴定得到改善。它进一步描述了已鉴定的，先前未表征的小蛋白的性质和潜在功能。总而言之，它改善了对sProtein检测的评估，并使其能够在未来的实验中进行功能表征。它进一步描述了已鉴定的，先前未表征的小蛋白的性质和潜在功能。总而言之，它改善了对sProtein检测的评估，并使其能够在未来的实验中进行功能表征。它进一步描述了已鉴定的，先前未表征的小蛋白的性质和潜在功能。总而言之，它改善了对sProtein检测的评估，并使其能够在未来的实验中进行功能表征。