A novel clinical metaproteomics workflow enables bioinformatic analysis of host-microbe dynamics in disease

Clinical metaproteomics has the potential to offer insights into the host-microbiome interactions underlying diseases. However, the field faces challenges in characterizing microbial proteins found in clinical samples, usually present at low abundance relative to the host proteins. As a solution, we have developed an integrated workflow coupling mass spectrometry-based analysis with customized bioinformatic identification, quantification, and prioritization of microbial proteins, enabling targeted assay development to investigate host-microbe dynamics in disease. The bioinformatics tools are implemented in the Galaxy ecosystem, offering the development and dissemination of complex bioinformatic workflows. The modular workflow integrates MetaNovo (to generate a reduced protein database), SearchGUI/PeptideShaker and MaxQuant [to generate peptide-spectral matches (PSMs) and quantification], PepQuery2 (to verify the quality of PSMs), Unipept (for taxonomic and functional annotation), and MSstatsTMT (for statistical analysis). We have utilized this workflow in diverse clinical samples, from the characterization of nasopharyngeal swab samples to bronchoalveolar lavage fluid. Here, we demonstrate its effectiveness via analysis of residual fluid from cervical swabs. The complete workflow, including training data and documentation, is available via the Galaxy Training Network, empowering non-expert researchers to utilize these powerful tools in their clinical studies.