FIGURE 3.

Glycan analysis of Notch EGF1-5 by mass spectrometry. pgsA-745 and pgsI-208 cells were transiently transfected with a construct encoding EGF repeats 1-5 of mouse Notch1. Tryptic peptides of the purified secreted product were analyzed by liquid chromatography-MS/MS for the presence of peptides modified with O-glucose. A, identification of the O-glucose trisaccharide form of a peptide from the pgsA-745 sample. The top panel shows an MS spectrum of material eluting at 52.9 min. The ions labeled [M + 3H]³⁺ and [M + 4H]⁴⁺ match the predicted mass for triply and quadruply charged forms of the O-glucose trisaccharide form of ¹³⁷SCQQADPCASNPCANGGQCLPFESSYICR¹⁶⁵, a tryptic peptide from EGF 4 previously demonstrated to be modified with O-glucose trisaccharide.³ Collision-induced dissociation fragmentation of the triply charged form of this peptide resulted in the MS/MS spectrum shown in the bottom panel. The major product ions at m/z 1210.9, 1167.1, and 1112.9 correspond to sequential losses of a pentose, a second pentose, and a hexose. Numerous sequence fragment ions (y-ions are shown) are observed that confirm the identity of the peptide. Ions selected for fragmentation in the MS spectrum are identified by red diamonds. The position of the parent ion fragmented in the MS/MS spectrum is identified with a blue diamond. B, identification of the O-glucose monosaccharide form of the peptide from the pgsI-208 sample. The top panel shows an MS spectrum of material eluting at 53.7 min, slightly later than the trisaccharide form (consistent with the loss of two hydrophilic xylose residues). The ions labeled [M + 3H]³⁺ and [M + 4H]⁴+ match the predicted mass for triply and quadruply charged forms of the glycopeptide. Collision-induced dissociation fragmentation of the triply charged form resulted in the MS/MS spectrum shown in the bottom panel. The major product ion, m/z 1112.8, matches the mass for the unglycosylated peptide. C, the trisaccharide form is present in samples from pgsA-745 but not pgsI-208. The data from both samples was searched for the ion corresponding to the triply charged form of the trisaccharide form, m/z 1255.1 (see A). D, the monosaccharide form is present in samples from psgI-208 but not pgsA-745. The data from both samples was searched for the ion corresponding to the triply charged form of the monosaccharide form, m/z 1166.7 (see B).