High-throughput site-specific N -glycoproteomics reveals glyco-signatures for liver disease diagnosis.

The glycoproteome has emerged as a prominent target for screening biomarkers, as altered glycosylation is a hallmark of cancer cells. In this work, we incorporated tandem mass tag labeling into quantitative glycoproteomics by developing a chemical labeling-assisted complementary dissociation method for the multiplexed analysis of intact N -glycopeptides. Benefiting from the complementary nature of two different mass spectrometry dissociation methods for identification and multiplex labeling for quantification of intact N -glycopeptides, we conducted the most comprehensive site-specific and subclass-specific N -glycosylation profiling of human serum immunoglobulin G (IgG) to date. By analysing the serum of 90 human patients with varying severities of liver diseases, as well as healthy controls, we identified that the combination of IgG1-H3N5F1 and IgG4-H4N3 can be used for distinguishing between different stages of liver diseases. Finally, we used targeted parallel reaction monitoring to successfully validate the expression changes of glycosylation in liver diseases in a different sample cohort that included 45 serum samples.