Data Independent Acquisition Pipeline for Microbiome Samples (Microbe-DIA)

The functional complexity inherent in microbiomes complicates analytical approaches aimed at defining phenotype. As proteins are the functional effectors of microbiome phenotypes, improving the performance of mass spectrometry-based metaproteomics is critical to achieving the functional characterization of these systems. Data-independent acquisition (DIA) improves protein coverage and reduces data missingness when compared to data-dependent acquisition (DDA) in metaproteomics. However, the application of DIA to complex microbial systems remains constrained by analytical throughput and computational scalability. Here, we optimized LC–MS/MS acquisition parameters for both DDA and DIA using a model microbiome, demonstrating how DIA enables increased sample throughput without compromising quantitative performance. In addition, we demonstrated a computationally efficient, library-free DIA workflow that overcomes reliance on empirical spectral libraries. Our analytical and computational innovations establish a scalable and cost-effective pipeline for metaproteomics of complex microbial communities.