biostudies-literatureUnknown5(1)Kale VMetaproteomics is an essential approach to analyze the <i>in situ</i> metabolic activity of microbes across various environments. In such highly diverse environmental samples, the functionality of specific microorganisms of importance often remains underexplored due to the protein inference problem arising from sequence similarities between organisms. One approach to overcome this challenge is the enrichment of uncultured target organisms. However, this often results in samples with low protein content. In this study, we have developed a workflow that combines fluorescence <i>in situ</i> hybridization (FISH) and fluorescence-activated cell sorting (FACS) with mass spectrometry-based proteomics to analyze proteins from uncultured bacteria directly from environmental samples. We demonstrate ISME communicationsycaf145https://www.ebi.ac.uk/biostudies/studies/S-EPMC12452284biostudies-literatureFISH-FACS proteomics: enhanced label-free quantitative proteome analysis from low cell numbers of uncultured environmental microorganisms.PMC12452284Kale VBecher DHo GYGFuchs BMTrautwein-Schult AMaaß SBartosik DSchweder TfalseFISH-FACS proteomics: enhanced label-free quantitative proteome analysis from low cell numbers of uncultured environmental microorganisms.Metaproteomics is an essential approach to analyze the <i>in situ</i> metabolic activity of microbes across various environments. In such highly diverse environmental samples, the functionality of specific microorganisms of importance often remains underexplored due to the protein inference problem arising from sequence similarities between organisms. One approach to overcome this challenge is the enrichment of uncultured target organisms. However, this often results in samples with low protein content. In this study, we have developed a workflow that combines fluorescence <i>in situ</i> hybridization (FISH) and fluorescence-activated cell sorting (FACS) with mass spectrometry-based proteomics to analyze proteins from uncultured bacteria directly from environmental samples. We demonstrate 2025-01-01T00:00:00Z2025 Jan2026-06-03T19:19:18.104Z2026-05-30T03:06:50.796ZS-EPMC124522844098990710.1093/ismeco/ycaf145