biostudies-literatureKlaes SGerman Research FoundationHelmholtz-GemeinschaftEuropean Regional Development FundHelmholtz-Zentrum für Umweltforschung111249https://www.ebi.ac.uk/biostudies/studies/S-EPMC11700628biostudies-literatureUnknown27(12)Protein-based stable isotope probing (protein-SIP) can link microbial taxa to substrate assimilation. Traditionally, protein-SIP requires a sample-specific metagenome-derived database for samples with unknown composition. Here, we describe GroEL-prototyping-based stable isotope probing (GroEL-SIP), that uses GroEL as a taxonomic marker protein to identify bacterial taxa (GroEL-proteotyping) coupled to SIP directly linking identified taxa to substrate consumption. GroEL-SIP's main advantages are that (1) it can be performed with a sample-independent database and (2) sample complexity can be reduced by enriching GroEL proteins, increasing sensitivity and reducing instrument time. We applied GroEL-SIP to pure cultures, synthetic bicultures, and a human gut model using ²H-, <sup>18<iScienceRevealing taxonomy, activity, and substrate assimilation in mixed bacterial communities by GroEL-proteotyping-based stable isotope probing.PMC11700628GRK 2032/2Cooper MKlaes SMadan SDeobald DAdrian LfalseRevealing taxonomy, activity, and substrate assimilation in mixed bacterial communities by GroEL-proteotyping-based stable isotope probing.Protein-based stable isotope probing (protein-SIP) can link microbial taxa to substrate assimilation. Traditionally, protein-SIP requires a sample-specific metagenome-derived database for samples with unknown composition. Here, we describe GroEL-prototyping-based stable isotope probing (GroEL-SIP), that uses GroEL as a taxonomic marker protein to identify bacterial taxa (GroEL-proteotyping) coupled to SIP directly linking identified taxa to substrate consumption. GroEL-SIP's main advantages are that (1) it can be performed with a sample-independent database and (2) sample complexity can be reduced by enriching GroEL proteins, increasing sensitivity and reducing instrument time. We applied GroEL-SIP to pure cultures, synthetic bicultures, and a human gut model using ²H-, <sup>18<2024-01-01T00:00:00Z2024 Dec2026-04-08T19:53:24.383Z2026-04-08T14:31:27.485ZS-EPMC117006283975901010.1016/j.isci.2024.111249