Temporal N-glycoproteomic differences between COVID-19 survivors and non-survivors at hospital admission and beyond

COVID-19 has proven to have a wide range of symptoms in human patients, prompting investigation into relevant biomarkers and underlying biological mechanisms. In this study, we aimed to develop a robust quantitative N-glycoproteomics approach for complex plasma samples and to characterize the altered glycosylation profiles associated with COVID-19 severity. We analyzed plasma samples from 16 survivors and 15 non-survivors, collected at three distinct time points following hospital admission. Using data-dependent acquisition (DDA) mass spectrometry, combined with a spectral library and the "match-between-runs" strategy, we profiled 397 unique glycopeptides and 62 glycan species, derived from 50 proteins across 86 glycosylation sites. By assessing both cross-sectional differences and longitudinal changes, we identified several site-specific glycosylation alterations that may serve as potential markers of disease severity. The most notable site-specific change was in the complement C1q A-chain (C1QA) at Asn146, with non-survivors having less fucosylation at hospital admission. We also observed increased bisection of immunoglobulins in non-survivors (IgG3/4 Asn177 and IgM Asn146) and glycosylation pattern changes in acute-phase proteins. Longitudinally, the fucosylation levels of C1QA increased in the non-survivors, whereas they decreased in the survivors. These findings highlight the potential value of site-specific glycoproteomic profiling for improving the molecular understanding of COVID-19 severity.