Microbiomes play a pivotal role in human health, environmental ecosystems, and agriculture. While genomic approaches dominate microbiome research, metaproteomics offers unique insights into the microbiomes functionality. However, challenges persist, including universal sample preparation, analysis depth, and efficient database search strategies.

Here, we introduce a universal metaproteomic sample preparation method. Our approach ensures lysis of both gram-positive and gram-negative bacteria without the use of detergents, chaotropic agents, or any other mass spectrometry-incompatible reagents. The streamlined 96-well format enables rapid protein extraction, purification, and digestion. Coupled with Evosep LC and data-independent acquisition (DIA) mass spectrometry, we achieves highly efficient metaproteomic analysis of up to 60 samples per day while maintaining deep metaproteomic coverage.

As shotgun proteomics spectral matching requires some prior knowledge, proteome databases and/or spectral libraries are required. Since its inception, this simple reality has plagued metaproteomics and several strategies have been developed to address this; these are typically not applicable to DIA-based metaproteomics. Although we have previously successfully used 16s analysis coupled with a DIA-NN library free search, these analyses rely on prior genomic analysis. To achieve a truly unbiased, comprehensive means to independently acquire quantitative taxonomic and metaproteomic data directly from a single mass spectrometry run, we employ a multi-tiered search strategy which progressively increases specificity while preserving sensitivity without compromising search the FDR using all Uniprot bacterial proteins as an intital reference proteome database. To validate the sensitivity and specificity of this approach, we analysed a well-characterized commercial microbiome preparation and achieving nearly 100% identification of bacterial biomass of a gut microbiome in a single injection.

Thus, our universal, rapid, and unbiased DIA-based metaproteomics requires less than 2 hours of hands-on sample preparation and identifies nearly 100% of microbial biomass along with deep proteome coverage facilitating adaptable and unbiased metaproteomic analysis with applications in clinical, agricultural, and environmental metaproteomics.