N-Glycoproteomics sample preparation using commercially available cellulose magnetic particles

Glycopeptide preparation is a critical step in bottom-up glycoproteomics. In this study, a robust method for N-glycopeptide preparation was developed using low-cost, commercially available magnetic particles. These particles function as a solid phase for hydrophilic interaction liquid chromatography (HILIC) to enrich N-glycopeptides from mixtures of N-glycopeptides and non-glycosylated peptides. Two types of magnetic particles, carboxylated polymer beads and cellulose resin, were compared. The latter demonstrated higher efficiency in N-glycopeptide recovery and non-glycopeptide removal. Biological samples often contain impurities such as salts and detergents that reduce efficiency and reproducibility of trypsin digestion and subsequent HILIC purification. To remove these impurities, we combined an optimized HILIC extraction protocol with a single-pot solid-phase-enhanced sample preparation protocol (SP3) for N-glycopeptide enrichment. Blood-derived samples (serum/plasma) were first tested. Compared with in-solution digestion, the method improved digestion efficiency and achieved N-glycopeptide recoveries comparable to those of conventional HILIC solid-phase extraction in a single tube. This method can be used for N-glycopeptide preparation not only from albumin-immunoglobulin-depleted serum but also from tissue extracts. Furthermore, the method has been successfully applied to glycoproteomic analysis of clinical serum and tissue samples from patients with gastric cancer, enabling detection of cancer-related alterations in glycoproteins.