Selective enrichment of glycopeptides using ground eggshell materials

•Application of natural eggshell in enrichment of glycopeptides.•Eggshell exhibited excellent sensitivity, selectivity, and size-exclusion effect.•More than 153 glycopeptides were captured by eggshell from the serum. The current research of protein glycosylation is focused on develop various functionalized hydrophilic materials that can effectively enrich glycopeptides. However, most of these materials require complex synthesis steps, plenty of chemical reagents, and high cost. In this study, we employed the natural eggshell for glycopeptides enrichment for the first time. Using horseradish peroxidase (HRP) tryptic digest as a standard sample, eggshell exhibited excellent sensitivity (0.05 fmol μL−1), good selectivity [HRP tryptic digest:bovine serum albumin (BSA) tryptic digest = 1:1000], excellent size-exclusion effect (HRP tryptic digest:BSA protein = 1:10,000), good loading capacity (75 mg g−1), and recovery (97.6 ± 0.3%). In addition, 153 and 114 glycopeptides were captured by eggshell from the serum tryptic digests of normal humans and diabetic patients, respectively. Benefiting from the singular porous structure and abundant biomass, eggshell performed excellently in the capture and separation of glycopeptides. These results demonstrated the potential of environmentally friendly eggshell in glycosylation proteomics analysis.