FIGURE 2.

O-Fucosylation, O-glucosylation, and O-GlcNAcylation of EGF repeats 1–36 from Notch ECD. Examples of mass spectra for the three major types of O-glycosylation are shown (A–D). Top panels, MS spectra; red diamonds, ions chosen for fragmentation. Parent ions corresponding to specified glycosylated peptides (A, EGF1 m/z = 885.5; B, EGF1 m/z = 952.9; C, EGF16 m/z = 828.4; D, EGF11 m/z = 743.7) are fragmented to produce MS2 spectra, shown in the bottom panels. Other ions in the MS spectra are from co-eluting material. Blue diamonds in MS2 spectra indicate the position of the parent ion. A, mass spectra showing the peptide ⁶¹NSCTSVGCQNGGTCVTQLNGKTY⁸² from EGF1 in the +3 charge state. The peptide generated in the absence of Fringe shows a neutral loss of a fucose. B, mass spectra showing the same peptide from EGF1 in the +3 charge state, now generated in the presence of Fringe, shows sequential neutral losses of GlcNAc and fucose. Loss of fucose from the same peptide in the +2 charge state is seen to the right of the parent ion in the MS2 spectrum. A and B, asparagine residue before amino acid 61 comes from the vector and is not part of the Notch sequence. C, mass spectra showing the peptide ⁶³⁵ICQKQINECESNPCQF⁶⁵⁰ from EGF16 in the +3 charge state. Sequential neutral loss of two xyloses and a glucose is seen in the MS2 spectra. D, mass spectra showing the peptide ⁴⁷⁴CNCSQGFTGPR⁴⁸⁴ from EGF11 in the +2 charge state. A neutral loss consistent with that of a GlcNAc is seen in the MS2 spectra. Spectra analyzing O-glucosylation and O-GlcNAcylation were taken from Notch generated in the absence of Fringe. Red triangle, fucose (deoxyhexose); blue circle, glucose (hexose). Orange star, xylose (pentose). Blue square, GlcNAc (HexNAc).