Direct On-Bead 18O Labeling Coupled with SP3 Sample Preparation (SP-18O) and DIA-MS for Quantitative Proteomics

Quantitative proteomics requires robust, accurate labeling strategies that minimize sample handling and preserve analytical sensitivity. Enzymatic 18O labeling offers a simple, universally applicable alternative to other isotopic labeling methods, but has been limited by incomplete labeling, back-exchange, and reliance on post-digestion, in-solution 18O labeling workflows that are impractical and introduce cumulative sample losses. Here, we present a fully integrated platform combining single-pot solid-phase-enhanced sample preparation (SP3) with direct on-bead trypsin-catalyzed C-terminal 18O labeling (SP-18O) during proteolytic digestion and data-independent acquisition mass spectrometry (DIA-MS). By performing 18O incorporation directly on-bead during digestion, the SP-18O approach eliminates the need for a separate post-digestion labeling step and associated sample losses compared to conventional enzymatic labeling workflows. Integration with DIA-MS further improves identification confidence and quantitative performance through distinctive y-ions. The platform is validated on two biologically distinct sample types, extracellular vesicles (EVs) secreted from model cancer cells and immunoaffinity-enriched protein complexes from Drosophila head tissue, demonstrating robustness and generalizability. This integrated strategy offers a practical, cost-effective solution for accurate quantitative proteomics while minimizing sample loss and analysis time.