A high-throughput <i>N</i> -glycan analysis strategy with targeted mass spectrometry (HTnGQs-target) for liver disease diagnosis

Hepatocellular carcinoma (HCC), a global leading cause of cancer-related mortality, is critically hindered by delayed diagnosis due to the lack of sensitive early biomarkers. Alterations in <i>N</i>-glycan composition and structure, which are closely associated with HCC pathogenesis, hold great promise for early detection; however, conventional mass spectrometry-based glycomic methods are limited by low sample throughput. To address this, we developed HTnGQs-target-a high-throughput targeted mass spectrometry approach integrating methylamine derivatization and 6-plex aminoxy TMT labeling. This approach enables robust quantitative analysis of up to 144 serum samples per day with high sensitivity and reproducibility. Applied to a cohort of 320 serum samples encompassing the full spectrum of liver disease-from healthy controls to chronic hepatitis B (CHB), liver cirrhosis, and HCC-our targeted MS method identified a novel panel of <i>N</i>-glycan biomarkers that effectively discriminates between benign and malignant stages. Furthermore, isomer-specific analysis revealed 12 sialylated <i>N</i>-glycan isomers with significant differential expression, further enhancing diagnostic specificity and underscoring the potential for clinical application in early HCC detection.