Phosphorylated polymer-grafted magnetic COFs for efficient enrichment of endogenous and tryptic phosphopeptides

A phosphorylated polyethyleneimine-functionalized magnetic covalent organic framework (mCOF-PEI-PO 3 -Ti 4+ ) was engineered for highly selective and efficient enrichment of phosphopeptides from complex biological samples. The nanocomposite was constructed by growing a vinyl-functionalized covalent organic framework on Fe 3 O 4 microspheres, followed by grafting of hyperbranched polyethyleneimine and subsequent phosphorylation to introduce a high-density Ti 4+ -chelating interface. By integrating immobilized metal ion affinity chromatography (IMAC) with the enhanced hydrophilicity and multi-site hydrogen-bonding capability of the phosphorylated PEI layer, the composite demonstrated outstanding enrichment performance. This included an ultralow detection limit (0.5 fmol), outstanding selectivity (1:2000), high loading capacity (50 mg·g −1 ), and excellent reusability (10 cycles). The material successfully enriched endogenous phosphopeptides from human saliva and serum. Furthermore, it enabled the comparative phosphoproteomic profiling of serum samples from colorectal cancer (CRC) patients, revealing a distinct CRC-associated phosphopeptide signature. Gene Ontology analysis indicated that the identified phosphoproteins were significantly enriched in lipid metabolism and receptor signaling pathways, whereas serine protease inhibitor activity was markedly downregulated in CRC samples. These findings suggest that dysregulated lipid metabolism may contribute to CRC progression through inflammatory modulation. This study establishes the mCOF-PEI-PO 3 -Ti 4+ composite as a robust and reliable platform for clinical phosphoproteomic analysis.