High-throughput intact Glycopeptide quantification strategy with targeted-MS (HTiGQs-target) reveals site-specific IgG N-glycopeptides as biomarkers for hepatic disorder diagnosis and staging.

Liver disease is one of the leading causes of global mortality, and identifying biomarkers for diagnosing the progression of liver diseases is crucial for improving its outcomes. Targeted mass spectrometry technology is a powerful tool with unique advantages for verifying biomarker candidates and clinical applications. It is particularly useful in validating protein biomarkers with post-translational modifications, eliminating the need for site-specific antibodies. Especially, targeted mass spectrometry technique is particularly critical for translation of glycoproteins into clinical applications as there are no site-specific antibodies for N-glycosylation. Nevertheless, its limitation in analyzing only one sample per run has become apparent when dealing with a large number of clinical samples. Herein, we developed a high-throughput intact N-glycopeptides quantification strategy with targeted-MS (HTiGQs-Target), which allows the validation of 20 samples per run with an average analysis time of only 3 min per sample. We applied HTiGQs-Target in a cohort of 461 serum samples (including 120 healthy controls (HC), 127 chronic hepatitis B (CHB) cases, 106 liver cirrhosis (LC) cases, and 108 hepatocellular carcinomas (HCC) cases) and found that a panel of 10 IgG N-glycopeptides have strong clinical utility in evaluating the severity of the liver disease.