It was crucial to choose the appropriate dissociation method and collision energy for large-scale glycoproteomics studies. HCD has been successfully applied to large-scale glycoproteomics experiments31. We know that TMT-labelled glycopeptides require higher collision energies for identification and quantitative accuracy because glycosidic bonds can absorb part of the energy32. Therefore, we systematically evaluated the performance of different HCD collision energies in characterizing TMT-labelled intact N-glycopeptides based on the StrucGP high HCD energy identification of peptides, and low HCD energy identification of glycans by fragmentation strategy.