Post-Synthetic Modification of Phenylboronic Acid-Functionalized Magnetic Covalent Organic Frameworks for Specific Enrichment of N-Linked Glycopeptides

Mass spectrometry (MS)-based in-depth glycoproteomics demands additional sample pretreatment to remove interference from nonglycoproteins and to enhance the precise identification of low-abundance glycopeptides. Herein, an attractive strategy was proposed for preparing core–shell-structured phenylboronic acid-immobilized magnetic covalent organic frameworks (MCNCs@COF@PBA), which were comprised of magnetite colloid nanocrystal clusters (MCNCs) adopted as a core and phenylboronic acid-modified covalent organic frameworks (COFs) served as the shell via a facile azide–alkyne "click" reaction and the first application in the specific enrichment of N-linked glycopeptides. The resultant MCNCs@COF@PBA composites exhibited high magnetic responsiveness, uniform mesoporous structure, and high surface area, as well as abundant binding sites, which could act as an efficient adsorbent for specific capture of the N-linked glycopeptides from the digested glycoprotein and protein mixture along with simultaneous protein repelling. These results confirmed that the MCNCs@COF@PBA composites had some distinct advantages in the enrichment of the N-linked glycopeptides, which included outstanding selectivity (HRP:BSA = 1:600), good sensitivity (100 amol), high enrichment recovery (∼93% ± 3%) and rapid magnetic separation (∼1 min). Furthermore, the developed MCNCs@COF@PBA-based MS method could be successfully applied to analyze glycopeptides in exosomes secreted from HeLa cell. This study not only provides a potential selective enrichment platform for comprehensive N-glycoproteome profiling, but also opens up a new avenue for universal functionalization of COFs-based materials.