期刊
GLYCOBIOLOGY
卷 22, 期 3, 页码 389-399出版社
OXFORD UNIV PRESS INC
DOI: 10.1093/glycob/cwr138
关键词
dolichol-linked precursor; graphitic carbon; LC-MS; N-glycan; oligomannosidic
资金
- Austrian Science Fund (FWF) [P22274, P20817]
- Osterreichische Forschungsforderungsgesellschaft (FFG) within the Laura-Bassi Center
- Austrian Science Fund (FWF) [P22274, P20817] Funding Source: Austrian Science Fund (FWF)
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 alpha-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(3)Man(9)GlcNAc(2) precursor oligosaccharide to Man(5)GlcNAc(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.
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