UniPhos Tip: A Streamlined StageTip for Deep Phosphoproteomic Profiling of Low Input Sample

Phosphoproteomics has emerged as a powerful strategy for dissecting oncogenic signaling networks and therapeutic response mechanisms in clinical research. However, its clinical application for low input samples often remains constrained by extensive multi-step sample handling, cumulative sample loss, and the large protein input requirements of conventional workflows. To address these limitations, we presented UniPhos Tip, an integrated 3-layer packing StageTip composed of Ni-NTA, C18, and SDB-XC that unifies phosphopeptide enrichment, desalting, and optional fractionation within a single device, completing the entire workflow within about 210 minutes. The UniPhos Tip employs a tandemly assembled resin architecture that eliminates inter-tube sample transfers, thereby reducing handling-induced loss and improving analytical robustness. Systematic optimization of washing sequence and fractionation strategy demonstrated that formic acid–based washing increased enrichment specificity from 88% to 96%. The strategy demonstrates robust phosphoproteomic depth under ultra-low input conditions, identifying 3,125 and 4,295 phosphopeptides from 5 µg and 25 µg of peptide inputs, respectively, with high quantitative reproducibility (Spearman ρ > 0.98). Application of the optimized workflow enabled comprehensive detection of phosphorylation sites across major oncogenic signaling cascades, including RTK-RAS-MAPK, PI3K-Akt-mTOR, and JAK-STAT pathways, demonstrating preservation of biologically and pharmacologically relevant network information from limited sample input. Together, UniPhos Tip provides a scalable and low-input–compatible solution for phosphoproteomic sample preparation, bridging workflow simplification with deep and reproducible phosphosite coverage for biologically and clinically relevant applications.