Measurements of stable carbon isotope ratios (δ¹³C) are widely used in biology to address questions regarding food sources and metabolic pathways used by organisms. The analysis of these so-called stable isotope fingerprints (SIFs) for microbes involved in biogeochemical cycling and microbiota of plants and animals has led to major discoveries in environmental microbiology. Currently, obtaining SIFs for microbial communities is challenging as the available methods either only provide low taxonomic resolution, such as the use of lipid biomarkers, or are limited in throughput, such as nanoscale secondary ion MS imaging of single cells. Here we present “direct protein-SIF” and the Calis-p software package (https://sourceforge.net/projects/calis-p/), which enable high-throughput measurements of accurate δ¹³C values for individual species within a microbial community. We benchmark the method using 20 pure culture microorganisms and show that the method reproducibly provides SIF values consistent with gold-standard bulk measurements performed with an isotope ratio mass spectrometer. Using mock community samples, we demonstrate that SIF values can also be obtained for individual species within a microbial community. Finally, a case study of an obligate bacteria–animal symbiosis shows that direct protein-SIF confirms previous physiological hypotheses and can provide unexpected insights into the symbionts’ metabolism. This confirms the usefulness of this approach to accurately determine δ¹³C values for different species in microbial community samples.

稳定的碳同位素比率（δ测量¹³ C）关于食物来源和由生物体中使用的代谢途径广泛用于生物学到地址的问题。对涉及生物地球化学循环和动植物微生物区系的微生物的所谓稳定同位素指纹（SIF）的分析导致了环境微生物学的重大发现。当前，获得微生物群落的SIF具有挑战性，因为可用的方法要么仅提供低分类学分辨率，例如使用脂质生物标志物，要么产量有限，例如单细胞的纳米级二次离子MS成像。在这里，我们介绍了“直接蛋白质SIF”和Calis-p软件包（https://sourceforge.net/projects/calis-p/），它们可以实现高通量的准确δ测量微生物群落中单个物种的¹³ C值。我们对使用20种纯培养微生物的方法进行了基准测试，结果表明该方法可重复提供的SIF值与使用同位素比质谱仪进行的金标准批量测量一致。使用模拟社区样本，我们证明了微生物群落内单个物种也可以获得SIF值。最后，对专性细菌与动物共生的案例研究表明，直接蛋白SIF证实了以前的生理假设，并且可以为共生体的代谢提供意想不到的见解。这证实了该方法的有效性，以准确地确定δ ^13个为微生物群落样品中不同种类的C值。