Automated Platform for Mass-Spectrometry-Based Multiomics Analysis from Small Volumes of Biofluids

Proteins and metabolites directly reflect biological states, yet traditional mass-spectrometry-based sample preparation remains labor-intensive, manual, and necessitates separate workflows for different molecules, hindering large-scale studies and risking microsample depletion. We developed an integrated multiomics workflowdesigned for the sequential enrichment of extracellular vesicle proteins (EVPs), secreted proteins (SPs, enriched via protein corona formation), and metabolites from a single biofluid sampleby leveraging an automated liquid-handling and sample-preparation system. Applied to 1 mL of urine, it identified 2,287 EVPs, 1,759 SPs, and 1,122 metabolites, with >82% protein overlap in at least two replicates, a coefficient of variation <6%, and Pearson correlations >0.86. In 100 μL of respiratory syncytial virus-stimulated immune organoid culture media, we observed multiomics patterns that were consistent with three typical immune response stages. The automated system-based multiomics workflow provides a scalable and reliable solution for high-throughput sample preparation, offering broad utility in clinical research, biomarker discovery, and translational studies.