Session: Session 4

Highly complex microbiomes offer a spectacular playground for new tandem mass spectrometry technologies

Microbial communities are challenging biological systems due to the diversity of their components, their dynamics in time and space, intricate and redundant functional capabilities, and their myriad of possible interactions and networks. By identifying proteins and monitoring their quantities, metaproteomics provides crucial information on the structural components, enzymes, and informational messengers of microorganisms, as well as on the host response, if any. The peptide diversity contained in most microbiome samples is so enormous that the analytical system can only scratch the surface. We designed a real-life metaproteomics sample consisting of peptides extracted from a human fecal sample and spiked with peptides from two known but atypical bacteria. With this sample, we explored the performances of the next-generation of high-resolution tandem mass spectrometer with MS/MS spectra acquisition combining DDA and DIA acquisition to maximize the taxonomical and functional information. The pipeline for interpreting the high-density acquired data comprises: (i) a DDA survey of the sample to proteotype organisms without any a priori at the genus or species level using a generic database derived from the NCBInr database, (ii) the construction of a dedicated database representing, as far as possible, the organisms that contribute the most to protein biomass, based on the first list of peptide sequences that can trace back to the specific organism present in the sample, and (iii) the interpretation of large DIA datasets with a sample-specific database of limited size. We achieved a coverage of more than 122,000 unique peptides and 38,000 protein groups within a 30-min DIA run. This marks a significant departure from current state-of-the-art metaproteomics methodologies, resulting in species level resolution and broader coverage of the metabolic pathways governing the biological system. References: DOI: 10.1186/s40168-024-01766-4; doi: 10.1111/1462-2920.16238.