Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes

Area of Science:

• Microbiome Research
• Metaproteomics
• Stable Isotope Probing

Background:

• Stable isotope probing (SIP) is crucial for understanding microbial community function and interactions.
• Existing SIP methods like DNA-SIP and nanoSIMS have limitations in sensitivity, resolution, or throughput.
• Applications span global biogeochemical cycles to host-microbiota interactions.

Purpose of the Study:

• To introduce an ultra-sensitive, high-throughput protein-based stable isotope probing (Protein-SIP) approach.
• To reduce the cost of labeled substrates significantly, enabling larger-scale experiments.
• To provide a sensitive and accurate method for analyzing microbial activity and substrate assimilation.

Main Methods:

• Developed a novel Protein-SIP approach utilizing standard metaproteomics (LC-MS/MS).
• Created new algorithms implemented in open-source software for data analysis.
• Validated the method using bacterial cultures, mock communities, and human fecal samples.

Main Results:

• The Protein-SIP approach achieves ultra-sensitive detection (0.01-10% label) of stable isotopes in proteins.
• Demonstrated high sensitivity, precision, and accuracy, outperforming existing Protein-SIP methods.
• Quantified translational activity in a 63-species human gut community, revealing diet-dependent microbial responses.

Conclusions:

• The developed Protein-SIP method offers ultra-sensitive detection of stable isotopes in proteins using standard metaproteomics.
• This approach significantly lowers costs, enabling broader application of SIP in microbiome research.
• Findings highlight the importance of protein availability for intestinal microbes, even those primarily consuming fiber.