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Ultrasensitive Phosphoproteome Analysis of Microscale Samples via Rapid In-Situ One-Tip IMAC Enrichment Strategy

ABSTRACT: In this study, we developed a novel phosphopeptide enrichment tip (Ti-PAN) based on highly hydrophilic textile PAN fibers, enabling rapid in-situ One-Tip IMAC enrichment within 8 minutes. Using this approach, we identified 2,139 to 17,546 phosphosites across 645 to 3,531 phosphoproteins from samples ranging from 100 to 100,000 cells. This high sensitivity enabled comprehensive mapping of phosphosites in microsamples, including the ERK interactome and mouse brain slices. Furthermore, we successfully applied this strategy to analyze the phosphoproteome of mouse oocytes at different developmental stages using as few as 5 oocytes per sample, identifying 2,709 phosphosites on 701 phosphoproteins. To our knowledge, this represents the deepest coverage of phosphoproteomes from such minimal oocyte material reported to date, offering valuable mechanistic insights into developmental controls during oocyte maturation. This Ti-PAN-based method provides a robust, versatile, and automatable platform for large-scale, highly sensitive phosphopeptide enrichment, substantially advancing phosphoproteomic studies in complex biological systems.

ORGANISM(S): Homo Sapiens Mus Musculus

SUBMITTER: Ci Wu

PROVIDER: PXD071174 | iProX | Tue Nov 25 00:00:00 GMT 2025

REPOSITORIES: iProX

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

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