Figure 2.

TMT-based quantitation of glycopeptides of transferrin spiked into MCF10A cells in defined ratios. (A) The number of all identified glycopeptides of transferrin at four glycosylation sites are indicated in the bar chart. Four glycosylation sites (Asn⁴³², Asn⁵²³, Asn⁶³⁰, and Asn⁶³⁷) are plotted on the x-axis, with the corresponding consensus motif indicated in parentheses. Asn⁴³² and Asn⁶³⁰ had consensus motifs of NXS and NXT, respectively, where X is any amino acid but proline. Asn⁵²³ and Asn⁶³⁷ contained the NXC motif, which is relatively rare in mammalian N-glycopeptides. The y-axis shows the number of identified glycopeptides. (B) A scatter plot with the average reporter ion intensity of all glycopeptides from three replicates of spiked-in transferrin from the 1× group (x-axis) and the 2× group (y-axis). A trendline was drawn, and R² was shown on the plot (0.99). One glycopeptide with discordant values is represented by a red circle. (C) Filtered list of identified glycopeptides of transferrin at the four sites that have unique glycan compositions, where H = hexose, N = N-acetyl hexosamine, F = fucose, and S = N-acetylneuraminic acid.