Compared to advancements in microscale sample proteomics, phosphoproteomics sensitivity has lagged behind due to low abundance, complex sample preparation, and substantial sample input requirements. We developed a simple and rapid in-situ one-tip immobilized metal affinity chromatography (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 [bovine serum albumin (BSA)/standard phosphopeptide=2000:1)], and enabled 10332 phosphosites assigned to 1992 phosphoproteins from 20000 HeLa cells. Furthermore, a large scale of 36521 phosphosites assigned to 4452 phosphoproteins with 8952 new sites could be identified by using 30 µg of HeLa cell lysates as starting material via combination with high-pH reversed-phase (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.
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