Novel boronate material affords efficient enrichment of glycopeptides by synergized hydrophilic and affinity...

Development of novel materials for enrichment of glycopeptides is the key to a comprehensive analysis of the glycoproteome, which is closely related to several major diseases and biomarker findings. We synthesized phenylboronic acid (PBA) bound to SiO₂ microspheres by a thiol-ene click chemistry method (this material was denoted as click PBA) and used it to separate cis-diol-containing molecules and enrich glycopeptides in hydrophilic interaction chromatography mode. Successful preparation of click PBA was confirmed by elemental analysis, X-ray photoelectron spectroscopy, N₂ adsorption-desorption isotherms, and high-resolution scanning electron microscopy. Click PBA showed stronger retention towards glycopeptides under alkaline, higher content of organic solution conditions than under acidic, higher content of organic solution or alkaline aqueous solution conditions. Click PBA exhibited high selectivity for both neutral and acidic glycopeptides, which could resist interference from 100 molar fold of bovine serum albumin digests. The high enrichment efficiency can be ascribed to the synergetic effects of affinity interaction and hydrophilic interaction. The application of click PBA to 1 μL human serum resulted in the identification of 101 unique glycosylation sites from 71 glycoproteins. Such material will facilitate comprehensive glycoproteome analysis.