Construction of a magnetic covalent organic framework with synergistic affinity strategy for enhanced glycopeptide enrichment

Considering the inherent properties of glycopeptides, such as glycan structure, size, and hydrophilicity, affinity materials possessing suitable functional molecule-glycan interactions, matched channels with size exclusion, and surfaces with hydrophilic interactions are preferred for glycopeptide separation in biological samples. Here, a novel boronic-acid-functionalized magnetic covalent organic framework was prepared through epitaxial growth and multi-ligand strategies. The multi-ligand strategy was firstly employed to prepare functionalized magnetic covalent organic framework without any post-functionalization protocol. Notably, the proposed strategy was found to be time saving, robust, and reproducible. The versatile magnetic covalent organic framework nanocomposite was endowed with phenylboronic acid functional molecules, strong hydrophilic features, mesoporous channels, fast magnetic responsiveness, and a large surface area. Benefitting from multiple affinity interactions, namely, synergistic reversible covalent interactions and hydrophilic affinity interactions, the nanocomposite presented extremely high performance in the recognition of intact N-glycopeptides. The inherent properties endowed the nanocomposite with excellent enrichment performance for N-glycopeptides: excellent selectivity (1 : 2000, IgG/BSA, m/m), an ultralow detection limit (0.05 fmol μL-1), and a good size-exclusion effect (1 : 500, IgG digests/BSA, m/m). More excitingly, a total of 1921 unique intact glycopeptides assigned to 1154 glycoproteins were identified from rat liver tissue; this performance is superior to that of commercial products. Additionally, the nanocomposite was successfully applied to enrich intact glycopeptides of exosomes extracted from healthy individuals and renal failure patients, providing a novel concept for the design of materials using a synergistic affinity strategy for sample preparation in glycoproteomics.