Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes.
Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes.
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DOI：
10.1186/s40168-022-01454-1
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发表时间：
2023-02-09
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影响因子：
15.5
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中科院分区：
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稳定同位素探测（SIP）方法是微生物组研究中确定物种与底物之间的关联以及物种活性的关键工具。这些方法的应用范围从研究微生物群落对全球微生物地球化学循环的重要性到肠道中宿主-微生物群的相互作用。目前的SIP方法，如DNA-SIP或nanoSIMS允许分别在群落和单细胞水平上分析具有高覆盖度分类群的稳定同位素的掺入，然而它们在灵敏度、分辨率或通量方面受到限制。在这里，我们提出了一种超灵敏，高通量的基于蛋白质的稳定同位素探测方法（Protein-SIP），与其他SIP和Protein-SIP方法相比，该方法将标记底物的成本降低了50-99%，从而使同位素标记实验能够在更大的规模和更高的重复性上进行。该方法允许使用标准元蛋白质组学液相色谱-串联质谱（LC-MS/MS）测量以物种水平分辨率确定同位素掺入微生物组成员。该方法的核心是分析数据的新算法，该算法已在开源软件（https：//sourceforge.net/projects/calis-p/）中实现。我们证明了灵敏度，精密度和准确性，使用不同的标记方案的细菌培养物和模拟社区。此外，我们将我们的方法与两种现有的蛋白质-SIP方法进行基准测试，并表明在低标记范围内，我们的方法是最敏感和准确的。最后，我们测量翻译活动使用18 O重水标记的63种社区来自人类粪便样本生长在模拟两种不同的饮食介质。每个样品平均可对27个物种的活性进行量化，与高纤维饮食相比，9个物种在高蛋白饮食中显示出显著更高的活性。令人惊讶的是，在高蛋白活性增加的物种中，有几种被称为纤维消费者的拟杆菌属物种。显然，在评估肠道微生物在纤维（包括基于纤维的益生元）上的生长时，蛋白质供应是一个关键的考虑因素。我们证明，我们的蛋白质SIP方法允许使用标准元蛋白质组学数据对蛋白质中发现的元素的稳定同位素进行超灵敏（0.01至10%标记）检测。在线版本包含补充材料，可通过10.1186/s40168-022-01454-1获得。
Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput. Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software (https://sourceforge.net/projects/calis-p/). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data. The online version contains supplementary material available at 10.1186/s40168-022-01454-1.
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