Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Ultra-Sensitive Protein-SIP to Quantify Activity and Substrate Uptake in Microbiomes with Stable Isotopes


Manuel Kleiner1* (, Angela Kouris2, Marlene Violette1, Grace D’Angelo1,3, Yihua Liu2,4, Abigail Korenek1, Nikola Tolić5, Timo Sachsenberg6, Janine McCalder2, Mary S. Lipton5, and Marc Strous2


1North Carolina State University; 2University of Calgary; 3Max Planck Institute for Marine Microbiology; 4Max Planck Institute for Biology Tübingen; 5Environmental Molecular Science Laboratory; and 6University of Tübingen



The project’s goal is to use stable isotope probing (SIP) to address the question of how microbes and minerals make necromass that persists.


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 nanoscale secondary ion mass spectrometry, allow researchers 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.

The team has developed an ultra-sensitive, high-throughput protein-based SIP 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 measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software. Research demonstrates sensitivity, precision, and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, the team benchmarks the approach against two existing Protein-SIP approaches and shows that in the low labeling range, the team’s approach is the most sensitive and accurate. Finally, researchers 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 persample, with nine 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.

In conclusion, research demonstrates the 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.


Kleiner, M., et al. 2023. “Ultra-Sensitive Isotope Probing to Quantify Activity and Substrate Assimilation in Microbiomes,” Microbiome 11, 24.

Funding Information

This work was supported by the U.S. Department of Energy under Grant number DE-SC0022996, by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM138362, and the U.S. National Science Foundation grant OIA #1934844. The funders had no role in the design of the study, data generation, analysis, and interpretation of study results.