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Abstract BackgroundStable 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. ResultsHere, 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 using18O 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 severalBacteroidesspecies known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. ConclusionsWe 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.
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This content will become publicly available on July 1, 2026
Comparing Raman and NanoSIMS for heavy water labeling of single cells
ABSTRACT Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543Escherichia colicells grown in M9 minimal medium in the absence or presence of heavy water (2H2O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of2H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The12C2H/12C1H and12C22H/12C21H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the2H/1H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive2H quantification. Importantly, despite its higher mass resolution requirements, the use of C22H/C21H may be a viable alternative to the use of C2H/C1H due to lower background and higher overall count rates. Furthermore, using an empirical approach in determining Raman wavenumber ranges via the second derivative improved the data equivalency of2H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems.IMPORTANCEAccurate and reliable measurements of cellular properties are fundamental to understand the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope (2H through incorporation of2H2O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with2H2O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research.
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- Award ID(s):
- 2125748
- PAR ID:
- 10629414
- Editor(s):
- Rotaru, Amelia-Elena
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- Microbiology Spectrum
- Volume:
- 13
- Issue:
- 7
- ISSN:
- 2165-0497
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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