Fig. 5: Systematic characterization of surface-exposed glycoforms.

a, Intact living HEK293 cells are treated with either PNGase or proteinase K. Surface-exposed glycoforms are affected while intracellular glycoforms remain protected. b, Number of glycoforms on affected sites (at least two glycoforms changing) significantly changing in abundance upon enzymatic treatments, per subcellular compartment annotation of proteins. c,d, Characterization of surface-exposed glycoforms belonging to the zinc ion channel ZIP6 and to the cell adhesion protein DSC2. Each data point represents a unique glycoform, with the color representing the glycan class. The shape of the data point reflects significance, while the dotted line represents the effect size cutoff (log₂ 0.6 = 1.5 fold change). e, Fold change of glycoforms, separated per glycan class, on sites for which at least two glycoforms were affected by the enzymatic treatments. High-mannose glycans were significantly less exposed than the other glycan classes (two-sided t-test). The horizontal lines represent the median. f, Comparison of fold changes of glycopeptide abundance upon proteinase K (y axis) and PNGase treatments (x axis), for sites affected by both enzymes (sites with at least two glycopeptides significantly changing in abundance upon treatment) and glycopeptides identified in both experiments. g, Number of times a given glycan composition was identified on a glycopeptide significantly changing in abundance upon proteinase K (y axis) and PNGase (x axis) treatments. The size of the dot represents the frequency with which this composition was identified as surface exposed.