Self-assembly of DNA tetrahedral multifunctionalized nanomaterial and its application in N-glycoproteins ultra-fast analysis in human serum

Abnormal proteins glycosylation is associated with kinds of diseases. A thorough investigation of protein glycosylation can provide valuable insights into the mechanisms underlying disease development. In this work, DNA tetrahedral (DNA TET) functionalized hydrophilic magnetic nanosphere (Fe₃O₄@PDA@AuNPs@DNA TET-Trypsin) was proposed for in-deep glycoprotein analysis. The good biocompatibility, good hydrophilicity, and abundant functional groups endowed the nanosphere with excellent performance on N-glycoprotein analysis. Surprisingly, the material showed excellent selectivity (HRP: BSA = 1/10,000) and low detection limit (0.1 amol/μL) in complex samples. Besides, the nanosphere was applied in human serum, after enrichment of the sample with Fe₃O₄@PDA@AuNPs@DNA TET-Trypsin, 302 glycopeptides corresponding to 94 glycoproteins from the serum of healthy individuals and 103 glycoproteins corresponding to 364 glycopeptides from the serum of gastric cancer (GC) patients were detected by nano-LC-MS/MS, respectively. Finally, a comprehensive Gene Ontology (GO) analysis was carried out on the glycopeptides that were enriched in the serum samples. In contrast to the normal control group, the findings revealed aberrant expression of innate lymphocytes and extracellular vesicles, as well as specific molecules or compounds associated with endopeptidases in GC patients. More importantly, this strategy reduces the digestion time of proteins from 18 h to 5 min, greatly improving analysis efficiency.