Isomeric analysis of oligomannosidic N-glycans and their dolichol-linked precursors

Abstract

Oligomannosidic (OM) N-glycans occur as a mixture of isomers, which at early stages of glycosidase trimming also comprise structures with one to three glucose residues. A complementary set of isomers is generated during the biosynthesis of the lipid-linked precursor. Here, we demonstrate the remarkable capacity of liquid chromatography (LC) with porous graphitic carbon and mass spectrometric detection for the determination of OM isomers. Protein-linked N-glycans were released enzymatically from samples with known isomer composition such as kidney bean proteins and ribonuclease B. Lipid-linked oligosaccharides were obtained by a direct mild acid hydrolysis of microsomes thus avoiding biphasic partitioning. A parallel analysis of pyridylaminated glycans by amide-silica and reversed-phase high-performance LC, the application of branch-specific-mannosidases and work with ALG mutant plants led to the assignment of the relative retention times of the isomers occurring during the degradation of the Glc 3Man 9GlcNAc 2 precursor oligosaccharide to Man 5GlcNAc 2 and beyond. A tightly woven net of evidence supports these assignments. Noteworthy, this isomer assignment happens in the course of a comprehensive analysis of all types of a sample's N-glycans.