Extended Data Fig. 4: The site-specific N-glycosylation of proteins in the investigated serum sample was found to quantitatively match previously reported N-glycoform distributions of the same proteins from normal human serum.

Four high abundance glycoproteins each harboring multiple N-glycosylation sites were selected for this comparison including a. alpha-1-antitrypsin (A1AT, P01009), b. ceruloplasmin (CP, P00450), c. haptoglobin (HP, P00738) and d. immunoglobulin G1 (IgG1, P01857). The glycoproteins selected for this analysis are positive acute phase proteins and hence their serum levels and glycosylation features may be altered as a result of physiological changes. The quantitative glycoprofiling (indicated as “Rel abundance (HGI)”) was manually performed using AUC-based quantitation and compared to robust literature reporting on the relative abundance of site-specific glycoforms from the same proteins. The glycoforms have been labelled according to their generic monosaccharide composition (N, HexNAc; H, Hex; F, dHex; S, NeuAc). Cartoons illustrating likely N-glycan structures have been provided for the high abundance glycoforms. Low abundance glycoforms were listed according to their relative expression level (high->low, see zoom indicated with broken boxes). Black compositions indicate the glycopeptides reported in literature and found in HGI study; Blue compositions indicate glycopeptides reported only in literature; Green compositions indicate glycopeptides found only in HGI study. The relative abundance (in %) of the individual glycoforms were plotted and correlation coefficients (R²) generated for each N-glycosylation site. The consistently high correlation between the site-specific glycoprofiles generated from the HGI sample and from the literature (R² = 0.85 – 1.00) validates the use of literature to score and rank the team performance in this study as used for the performance tests N2-N3 and O1-O2 (see Table 2 for details of performance tests).