Preparation of a cucurbit[6]uril-containing monolithic material for specific recognition and enrichment of multi-phosphopeptides

Supramolecular affinity chromatography based on host-guest interactions represents a promising approach for achieving highly specific separation and purification of biomacromolecules. Among the various macrocyclic molecules, cucurbit[n]urils (CB[n]) have attracted significant attention in recent years due to their unique molecular recognition capabilities. Notably, recent studies have revealed the specific binding of CB[n] to aromatic amino acids and short peptides with aromatic residues or N-terminal motif. In this work, we report the specific recognition of protein phosphorylation sites by cucurbit[6]uril (CB[6]), and propose a CB[6]-based supramolecular affinity chromatography (CSAC) strategy for the enrichment of multi-phosphopeptides. A polymeric monolith was prepared by polymerization of perallyloxyl CB[6] and ethylene dimethacrylate. In order to study the recognition mechanism of CB[6] on phosphate groups, the chromatographic conditions of CSAC, including loading, washing and elution, were separately evaluated utilizing the CB[6] monolithic material as a platform. Molecular recognition of phosphorylation sites was achieved in the solution of high organic solvent content (90% ACN), which was rarely reported. The established method exhibited specific recognition on multi-phosphopeptides and was employed for enrichment of multi-phosphopeptides, which showed good specificity (1:1000 of β-casein: BSA). Furthermore, CSAC can effectively isolate multi-phosphopeptides from mouse liver and improve the coverage of phosphopeptides. The recognition of phosphate groups by CB[6] is observed in a high-organic solvent environment, which is distinctly different from the recognition effect previously reported in aqueous phases. The established CSAC strategy holds promise as an effective technique for biological separations.