Single-Tube Mass Spectrometry-Based Workflow for Multi-Omics Profiling of Diverse Biomolecules.

Advances in mass spectrometry-based omics technologies enable detailed exploration of biological processes. However, robust detection of multiple biomolecule types from the same biological sample remains challenging. We developed a multi-omics platform for the simultaneous detection of metabolites (polar and semi-polar), lipids, proteins, and native peptides, all detected in a high-throughput manner from a single tube. As a proof-of-concept for the platform, we profiled the profound and dynamic molecular changes occurring in tomato during fruit development. This included optimizing sample collection, standardizing data acquisition from the chromatographic systems, and developing a bioinformatic pipeline to integrate data from the diverse omics technologies. Across omics layers, we detected thousands of biomolecules whose abundances shifted significantly during fruit development in peel and flesh tissues, providing temporal signatures that differentiate fruit developmental stages. This includes the characterization of numerous naturally produced endogenous peptides that exhibit dynamic changes in composition and cleavage preferences. Finally, we demonstrate the use of a machine learning framework for unsupervised integration of this type of high-throughput data. Collectively, our multi-omics platform enables a comprehensive exploration of diverse biological tissues and biofluids with broad applicability across research fields.