A Clinical Metaproteomics Workflow Implemented within Galaxy Bioinformatics Platform to Analyze Host-Microbiome Interactions Underlying Human Disease.

Clinical metaproteomics reveals host-microbiome interactions underlying diseases. However, challenges to this approach exist. In particular, the characterization of microbial proteins present in low abundance relative to host proteins is difficult. Other significant challenges are attributed to using very large protein sequence databases, which impedes sensitivity and accuracy during peptide and protein identification from mass spectrometry data in addition to retrieving taxonomy and functional annotations and performing statistical analysis. To address these problems, we present an integrated bioinformatics workflow for mass spectrometry-based metaproteomics that combines custom protein sequence database generation, peptide-spectrum match generation and verification, quantification, taxonomic and functional annotations, and statistical analysis. This workflow also offers characterization of human proteins (while prioritizing microbial proteins), thus offering insights into host-microbe dynamics in disease. The tools and workflow are deployed in the Galaxy ecosystem, enabling the development, optimization, and dissemination of these computational resources. We have applied this workflow for metaproteomic analysis of numerous clinical sample types, such as nasopharyngeal swabs and bronchoalveolar lavage fluid. Here, we demonstrate its utility via the analysis of residual fluid from cervical swabs. The complete workflow and accompanying training resources are accessible on the Galaxy Training Network to equip non-experts and experienced researchers with the necessary knowledge and tools to analyze their data.