Figure 4. Identification of new core sequences of GAG-like O-glycans from O. dentatum by enzymatic and chemical digestions (A-G).

The HexNAc₁GlcA₁Hex₃Xyl₁-PA (m/z 1094, see Figure 3D) was analysed by positive MS and MS/MS before (A) and after β3-galactosidase (B) or β3/4-galactosidase (C) digestions; only the β3/4-galactosidase was able to remove a single hexose, as also seen by the alteration in the MS/MS spectra (see insets), thus indicating that the third hexose is a branching β4-linked galactose. The PC_1-2HexNAc₁GlcA₁Hex₂Xyl₁-PA glycans (m/z 1097 and 1262; see Figures 2 E/G and 3 M/N) were analysed by positive MS before (D, F) and after (E, G) hydrofluoric acid (HF) treatment which removed one or two phosphorylcholine residues to yield HexNAc₁GlcA₁Hex₂Xyl₁-PA at m/z 932; shifts in the MS/MS spectra (see insets) show the absence of the PC-containing B-ions (e.g., PC-HexNAc at m/z 369) and the dominance of the PA-containing Y-ions (e.g., Xyl-PA at m/z 229) after HF treatment. Some partial loss of methyl groups from the PC units during the release and work-up procedure resulted in the observation of ions at m/z 1248 in addition to the bona fide structure at m/z 1262 (F).