Comparing and optimizing protein extraction methods with different lysis buffers for the analyses of human fecal microbiome via metaproteomics approach

Metaproteomic analysis offers critical insights into gut microbiome function; however, efficient microbial protein extraction from fecal samples remains challenging due to the complexity of different types of bacterial cell walls in the microbiome. In this study, we systematically compared three representative detergent-based lysis buffers (sodium dodecyl sulfate_urea, dodecyl β-D-maltoside_urea, sodium dodecyl sulfate_ dodecyl β-D-maltoside_urea) for metaproteomics sample preparation. After multiple levels of analyses, we identified SDS_DDM_urea as the most efficient option for extracting diverse microbial proteins, peptides, and identifying microbial species. Applying this optimized method to samples from a community based dietary intervention study (Summer Harvest Adventure), we found minimal group-level microbial diversity shifts during this type of intervention, but substantial individual-specific variations reflected by metaproteomics results. Functional analyses also revealed microbial protein changes, especially proteins related to metabolic adaptations, including enhanced carbohydrate metabolism, amino acid biosynthesis, vitamin transport, and increased expression of membrane-associated proteins. Our results highlighted the personalized microbiome response to dietary interventions and underscored the importance of selecting appropriate protein extraction methods to accurately capture microbiome functional dynamics in microbiome analyses via metaproteomics.