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In-depth Phosphoproteome Analysis from Microscale Samples with Rapid In-Situ One-Tip IMAC Strategy

ABSTRACT: In this study, we developed a simple and rapid in-situ one-tip IMAC phosphoproteomics workflow integrated with data dependent acquisition without exclusion mass spectrometry for microscale phosphoproteomic analysis. The phosphopeptide enrichment material, made from hydrophilic cotton fibers with immobilized titanium ions, is easy to pack into a pipette tip. It enables fast, efficient enrichment through aspiration and dispensing, minimizing sample loss and enhancing sensitivity. Ti-Cotton tip successfully enriched phosphopeptides from protein standard digests and exhibited a high selectivity (BSA/standard phosphopeptide = 2000:1), and enabled 10,332 phosphosites assigned to 1,992 phosphoproteins from 20,000 HeLa cells. Furthermore, large scale of 36,521 phosphosites assigned to 4,452 phosphoproteins with 8,952 new sites could be identified by using 30 μg HeLa cell lysates as starting material via combination with high-pH RP fractionation. Taken together, our strategy is promising to serve as a widely applicable and robust platform for achieving large-scale and highly sensitive phosphopeptide enrichment and expanding our knowledge of phosphoproteomics in complex microscale biological systems.

ORGANISM(S): Homo Sapiens

SUBMITTER: Ci Wu

PROVIDER: PXD073439 | iProX | Fri Jan 23 00:00:00 GMT 2026

REPOSITORIES: iProX

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