New strategy revolutionizes glycoproteomics research

Protein glycosylation is a fundamental biological process that regulates various cellular functions, yet its study has been hampered by the complexity and low abundance of glycoproteins. In a groundbreaking study published in National Science Review, scientists from Fudan University developed a chemical ligation-based glycopeptide enrichment strategy called HG-TCs. This method uses advanced solid-phase materials and bioorthogonal chemistry to simultaneously identify multiple glycosylation types, including N-glycosites, O-GlcNAc sites, and O-GalNAc sites, as well as N-glycans (see image below). Thiss HG-TCs strategy has the ability to enrich glycopeptides using an azide-alkyne cycloaddition reaction and release them through trypsin cleavage. This one-tube workflow minimizes sample loss while maintaining high reproducibility.

The approach offers a time-efficient workflow with exceptional scalability, identifying over 900 O-GlcNAc sites and 800 N-glycosites in HeLa cells in a single experiment, using minimal sample amounts. The team noted that “Even between the technical replicates, guaranteeing identical results remains challenging. So, in comprehensive analysis, mapping multiple glycosylations individually may introduce operational and technical variations that lead to inconsistent data, especially for certain dynamic and complex biological models or systems.” So, “HG-TCs are very conducive to the study of a highly dynamic and complex carbohydrate system, allowing not only the mapping of multiple glycosylations but also the simultaneous monitoring of multiple glycosylation alterations.“

The research team also applied this strategy to analyze HeLa cell samples under oxidative stress. They uncovered distinct spatial glycosylation patterns between the nucleus and cytoplasm, offering valuable insights into glycosylation’s dynamic roles in cellular responses. This study provides a robust tool for glycoproteomics research, aiding in the understanding of glycosylation’s dynamic roles in cellular signaling and disease mechanisms. For researchers in glycoproteomics, this method represents a significant leap forward, simplifying complex workflows while maintaining high data quality. The findings are particularly relevant for studying glycosylation alterations in cancer and other diseases.