Gold nanoparticles immobilized hydrophilic monoliths with variable functional modification for highly selective enrichment and on-line deglycosylation of glycopeptides

The poly (glycidyl methacrylate-co-poly (ethylene glycol) diacrylate) monoliths modified with gold nanoparticles, with advantages of enhanced reactive sites, good hydrophilicity and facile modification, were prepared as the matrix, followed by variable functionalization with cysteine and PNGase F for glycopeptide enrichment and on-line deglycosylation respectively. By the cysteine functionalized monolithic column, glycopeptides could be efficiently and selectively enriched with good reproducibility based on hydrophilic interaction chromatography (HILIC). Furthermore, the enrichment was specially achieved in weak alkaline environment, with 10 mM NH₄HCO₃ as the elution buffer, compatible with deglycosylation conditions. Therefore, the glycopeptides could be on-line deglycosylated with high efficiency and throughput by directly coupling the PNGase F functionalized monolithic column with the enrichment column during elution without the requirement of buffer exchange and pH adjustment. By such a method, within only 70-min pretreatment, 196 N-linked glycopeptides, corresponding to 122 glycoproteins, could be identified from 5 μg of human plasma with 14 high-abundant proteins removed, and the N-linked glycopeptides occupied 81% of all identified peptides, achieving to the best of our knowledge, the highest selectivity of HILIC-based methods. All the results demonstrated the high efficiency, selectivity and throughput of our proposed strategy for the large scale glycoproteome analysis.