High-energy collision-induced dissociation mass spectrometry of synthetic mannose-6-phosphate oligosaccharides

The high-energy collision-induced dissociation spectra of a series of linear and branched synthetic mannosyl oligosaccharides that contain 6-phosphate substituents on either or both non-reducing terminal or penultimate residues have been studied. These phosphorylated structures were designed to mimic those of naturally derived N-glycans (Man-6-PO₄) on lysosomal enzymes and to probe the minimally required binding motif for the Man-6-PO₄ receptors. When a phosphate group was present, the spectra were dominated by ions that arise from cleavages at the glycosidic bonds (single and double) with charge retention on the phosphate-containing fragments. The spectra of linear structures that bear the nonreducing terminal Man-6-phosphate residues were devoid of Y-type ions, unlike those with similar phosphorylation at the penultimate residue. The location of the phosphorylated residue was deduced from the presence or absence of unique B and Y ions. In neutral branched structures, the ions were formed by cleavage at the glycosidic bond at either one or both of the branch points and the aglycon, which was attached to the disubstituted mannosyl residue. Branched oligosaccharides that contained one or two terminal Man-6-PO₄ residues also showed double cleavages with charge retention on the phosphate-containing fragment. Our investigation shows that positive mode high energy collision-induced dissociation mass spectrometry can determine the location - terminal or penultimate - of Man-6-PO₄ residues in N-linked type oligosaccharides.