Mesoporous covalent organic framework microspheres with dual-phase separation strategy for high-purity glycopeptide...

High-performance porous materials and rational enrichment strategies are crucial during sample pretreatment process in glycoproteomics analysis. Herein, we report a dual-phase separation strategy based on hydrophobic and hydrophilic mesoporous covalent-organic framework (COF) microspheres for high-purity glycopeptide enrichment for the first time. The COF microspheres (about 1.8±0.5 μm) with hydrophobic mesopores (2.5 nm) were prepared by a facile method at room temperature. Through the post-synthesis modification strategy, hydrophilic mesopores were obtained by modifying the vinyl ligands with glutathione (GSH), and the hydrophilic properties of COF microspheres were further enhanced by the introduction of Au nanoparticles and GSH to obtain the hydrophilic COF microspheres (denoted as COF@Au-GSH). The low-abundance hydrophilic glycopeptides could be efficiently enriched by the hydrophilic COF@Au-GSH microspheres in low polar solutions after the high-abundance hydrophobic non-glycopeptides were removed with the hydrophobic COF microspheres in high polar solutions. With the help of dual-phase separation strategy and inherent properties of the COF structure, the as-prepared COF microspheres showed splendid enrichment performance for glycopeptides, including ultrahigh sensitivity (2 fmol, IgG digests), extremely high specificity (1:10000, IgG digests/BSA digests), excellent size selectivity (1:500:500, IgG digests/BSA/IgG), and large binding capacity (200 mg g^-1, IgG digests). In addition, a total of 1993 glycopeptides could be enriched and identified from the rat liver digests after enrichment by the COF microspheres. As a proof of concept application, the proposed strategy was successfully used in sample pretreatment process for plasma glycoproteomic analysis.