Figure 4.

Intact glycopeptide analysis allows comparison of glycan composition caused by treatment with the sugar analog 1,3,4-O-Bu₃ManNAc. Overall, this analog caused a shift toward increased sialylation within identified glycopeptides, including secreted recombinant human erythropoietin (rhEPO). (A) Combination violin and dot plot depicting the distribution of Log2 treated/control ratios (y-axis) for all detected intact glycopeptides, grouped by the number of sialic acid residues in their glycan moieties (0–4, x-axis). The log-transformed intensity ratio for each intact glycopeptide is depicted by a gray dot scaled to the total intensity for that glycopeptide and the mean intensity ratio for each number of sialic acid residues is depicted by a white diamond. The distribution and mean intensity of glycopeptides containing zero or one sialic acid residue decreased in samples treated with 1,3,4-O-Bu₃ManNAc and those containing two to four residues increased. The decrease for glycopeptides containing zero sialic acid residues and the increase those containing two sialic acid residues was statistically significant as measured by an un-paired t-test (statistical significance * p < 0.05, un-paired t-test). (B) Lollipop chart depicting all detected glycopeptides, grouped by glycan moiety (y-axis). For each shared glycan, glycopeptide intensities were summed, and the Log2 treated/control ratios calculated (x-axis). The color of each dot depicts the number of sialic acid residues within the glycan and the size of the dot depicts their overall intensity. (C) A table showing glycosites on two representative proteins bearing out the overall trend. The NDTQHI glycosite on endogenous endoplasmin and the NITTGC (Asn24) and NSSQPW (Asn83) glycosites on rhEPO (Ectopic Human Erythropoietin) exhibiting intensity ratio decreases in glycans with 0 or 1 sialic residue and increases in those with 2–4 sialic residues.