OSaMPle workflow for salivary metaproteomics analysis reveals dysbiosis in inflammatory bowel disease patients

The human oral microbiome has been associated with multiple inflammatory conditions including inflammatory bowel disease (IBD). Identifying functional changes in oral microbiome by metaproteomics helps understanding the factors driving dysbiosis related to intestinal diseases. However, enriching bacterial cells from oral samples (such as saliva and mouth rinse) rich in host proteins is challenging. Here, we present an Optimized Salivary MetaProteomic sample analysis workflow (OSaMPle) to enrich salivary bacteria and reduce host-derived interferences for in-depth analysis of the oral metaproteome. Compared to a conventional approach, OSaMPle improved the identification of bacterial peptides and proteins by 3.2 folds and 1.7 folds, respectively. Furthermore, applying OSaMPle to analyze mouth rinse samples from IBD patients revealed significant alterations in bacterial protein expressions under disease conditions. Specifically, proteins involved in the fatty acid elongation pathway in Peptostreptococcus were significantly less abundant in IBD patients, whereas proteins associated with the TCA cycle in Neisseria were significantly more abundant. The OSaMPle workflow is capable of processing small-volume oral samples and adaptable to high-throughput automation. It holds promise as a strategy for investigating the functional responses of oral microbiomes under disease conditions and identifying disease-associated microbes with their proteins, providing critical insights for detecting disease-related biomarkers within the oral microbiome.