• N-glycosylation exhibits both macro- and microheterogeneity, with thousands of possible glycan structures at each site, necessitating site- and structure-specific analysis.
• Advanced mass spectrometry techniques, including chemical derivatization, LC separation, ion mobility, and tandem MS, enable detailed structural characterization of N-glycopeptides.
• AI-based software and search engines have improved the interpretation of intact N-glycopeptide fragmentation patterns, facilitating clinical biomarker discovery.
• Structure-specific N-glycoproteomics has broad biomedical applications, including early diagnosis of hepatocellular carcinoma and understanding immunotherapy responses.