Development of Citric-Acid-Modified Cellulose Monolith for Enriching Glycopeptides

Prior to mass spectrometry (MS) analysis, pretreatment of low-abundance glycopeptides is vital for identifying protein glycosylation. In this study, we fabricated an environmentally friendly citric-acid-modified cellulose monolith (CCM) characterized by a coral-like porous structure and high-density hydrophilic groups using a thermally induced phase separation (TIPS) method. The CCM production leverages biomass resources, specifically cellulose and citric acid, utilizing TIPS to synthesize continuous porous materials through a straightforward heating and cooling process of polymer solutions. We demonstrated the efficacy of CCM as a hydrophilic interaction liquid chromatography (HILIC) medium for the efficient enrichment of glycopeptides. It exhibited remarkable selectivity in enriching glycopeptides from trypsin-digested immunoglobulin G (IgG), serving as a model protein, even in the presence of a significant amount of non-glycopeptide contaminants from bovine serum albumin (BSA) at a ratio of BSA/IgG of 1000/1. Additionally, CCM showed a low detection limit (0.25 fmol μL^-1) and commendable reusability in glycopeptide enrichment, successfully enriching 35 glycopeptides from IgG. Additionally, 641 unique N-glycosylation sites of 698 unique glycopeptides from 393 glycosylated proteins were identified from the triplicate analysis of 900 μg of human hepatocellular carcinoma tissue. Therefore, CCM holds significant promise as an eco-friendly stationary phase for hydrophilic interaction liquid chromatography aimed at glycopeptide enrichment.