Aromatic cage-functionalized porous separation material: Design, synthesis and application in selective extraction of...

Protein crotonylation is a critical post-translational modification closely associated with the pathogenesis of diseases including atherosclerosis. As it holds potential for development as a biomarker or therapeutic target, sensitive and specific detection methods are urgently required. However, direct analysis of crotonylated proteins in human plasma is hampered by their low abundance and severe matrix interference. Inspired by the YEATS domain's precise recognition of crotonylated proteins via its aromatic cage, we developed a porous aromatic cage-functionalized separation material using post-modified 4‑tert-butylcalix[4]arene as the monomer. This material was fabricated into a polymeric monolithic column via copolymerization, which leverages multi-site synergistic interactions (hydrophobic effects, hydrogen bonding, π-π stacking, and dipole-dipole interactions) to achieve the selective recognition of crotonylated proteins. It features a hierarchical pore structure, high specific surface area, and excellent biocompatibility, thus enabling efficient mass transfer and specific binding with crotonylated proteins. Coupled with a Bio-C18 column, we established an SPE-HPLC-MS method for the selective extraction and detection of crotonylated proteins in human plasma. For atherosclerotic patients' and healthy volunteers' plasma, this workflow detected 8 and 25 crotonylated proteins in 10' and 7* chromatographic fractions, respectively-more than either Bio-C18-only separation followed by MS or direct MS analysis.Vs. traditional protein ligand-based affinity methods, our approach eliminates complex ligand coupling steps and achieves enhanced stability and repeatability via covalent binding of recognition sites.