Facile synthesis of Ti⁴⁺-immobilized magnetic covalent organic frameworks for enhanced phosphopeptide enrichment

In this work, core-shell structured Ti⁴⁺-immobilized magnetic covalent organic frameworks (denoted as Fe₃O₄@TAPTDHTA-Ti⁴⁺ composites) were prepared for enhanced phosphopeptide enrichment by one-pot synthesis of COFs shell with inherent bifunctional groups on Fe₃O₄ NPs and further Ti⁴⁺ immobilization. The widely distributed bifunctional groups could provide abundant chelating sites for Ti⁴⁺ immobilizing. Combining with the high specific surface area and mesoporous structure, the Fe₃O₄@TAPTDHTA-Ti⁴⁺ composites exhibited excellent enrichment efficiency for phosphopeptides, such as low detection limit (0.05 fmol μL^-1), high selectivity (1:5000 of molar ratio of β-casein/bovine serum albumin (BSA) tryptic digests), high adsorption capacity (62.9 μg mg^-1) and strong size-exclusive effect (1:250:250 of molar ratio of β-casein tryptic digest/β-casein/BSA). In addition, this method was general for immobilizing other metal ions (Zr⁴⁺ and Fe³⁺). Notably, the Fe₃O₄@TAPTDHTA-Fe³⁺ composites exhibited controllable affinity towards mono-phosphopeptides and multi-phosphopeptides. Furthermore, the Fe₃O₄@TAPTDHTA-Ti⁴⁺ composites were successfully applied to selectively capture phosphopeptides from complex biological samples including the tryptic digest of nonfat milk, human serum and human saliva. More significantly, 3333 phosphopeptides derived from 1409 phosphoproteins with 3492 phosphorylation sites were clearly identified from the tryptic digest of HeLa cell lysate. In addition to providing a potential excellent enrichment probe for comprehensive phosphoproteomic analysis, this study also offers a new perspective for the functionalization of COFs.