FISH-FACS proteomics: enhanced label-free quantitative proteome analysis from low cell numbers of uncultured...

Metaproteomics is an essential approach to analyze the in situ 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 in situ 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 that 1 × 10⁵ bacterial cells are sufficient for reliable qualitative protein identifications, while 5 × 10⁵ to 1 × 10⁶ cells allow for both reproducible protein identification and quantification after FISH and FACS. In addition, the use of a clade-specific database enhances data analysis by improving peptide mapping, especially when compared to metaproteomics results.