Isomeric analysis of oligomannosidic N-glycans and their dolichol-linked precursors.
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 α-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.
Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
Martin
M
Pabst
Josephine
J
Grass
Stefan
S
Toegel
Eva
E
Liebminger
Richard
R
Strasser
Friedrich
F
Altmann
D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D004286:Dolichols / Q000031:analogs & derivatives* / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry; D006108:Graphite / Q000737:chemistry; D006801:Humans; D008361:Mannosidases / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D027805:Phaseolus; D010843:Pichia; D018515:Plant Leaves / Q000737:chemistry; D016062:Porosity; D061251:Primary Cell Culture; D021241:Spectrometry, Mass, Electrospray Ionization; D009626:Terminology as Topic
D029681:Arabidopsis Proteins; D004286:Dolichols; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; C028569:oligomannoside; D006108:Graphite; D008361:Mannosidases