Comprehensive Characterization of Atypical Serum Albumin N-Glycosylation Reveals Hepatocellular Carcinoma Diagnostic Biomarkers

Human serum albumin (HSA), the most abundant plasma protein predominantly synthesized and secreted by the liver, harbors atypical N-glycosylation modifications at Asn68 and Asn123. Despite their potential biological relevance, the comprehensive characterization of these modifications remains limited. Here, we systematically characterized atypical N-glycosylation on HSA using a sialic acid linkage-specific derivatization strategy coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), identifying 35 N-glycans including 23 sialylated N-glycans distinguished sialic acid linkage isomers. LC-ESI-MS analysis further resolved 25 site-specific N-glycopeptides, establishing the most detailed HSA N-glycosylation profile to date and elucidating its structural diversity. Given the liver-specific origin of HSA, we further explored the clinical relevance of HSA glycosylation in serum samples from healthy controls (HC), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) patients and found that HSA glycopeptides exhibited exceptional diagnostic accuracy in distinguishing LC from HCC patients (AUC = 0.906). These findings not only decode the complexity of HSA glycosylation but also validate its clinical utility as a liver-specific biomarker for HCC detection.