Fig. 7.

Evidence that the glycan-associated phosphate is linked to the distal HexNAC residue. (A) MS² (HCD) spectrum of the peptide ⁹⁷LAPTVSSGAITW¹⁰⁸ carrying the pentasaccharide with the extended version of 224.067 Da at m/z 799.6713+ (triply charged potassium adduct) using the Orbitrap XL mass spectrometer. The successive loss of the glycan moiety subunits detected by the decreasing fragment ion signals at m/z 1,078.465, 985.930, 884.390, 722.337, and 620.797 unambiguously determine that the pentasaccharide is attached in the orientation HexNAc-Hex-Hex-HexNAc-HexNAc to the peptide backbone. Furthermore, it suggests that the distal HexNAc is phosphorylated, indicated by its detected β-elimination (dehydro-HexNAc; mass difference, 185.069 instead of 203.086 Da).