Monosaccharide Analysis by HPAEC
- Neutral and amino sugars (fucose, xylose, Arabinose, N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose and mannose) and sialic acids (N-acetylneuraminic acid and N-glycolylneuraminic acid) from mammalian and bacterial glycoproteins are analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD).
- As low as 15 picomoles neutral and amino sugars or 120 picomoles sialic acids could be detected by HPAEC-PAD.
- About 100-150 micrograms of a glycoprotein subjected to acid hydrolysis is adequate for monosaccharide/sialic acid analysis.
Monosaccharide Analysis by GC-MS
- The method of choice, if the glycoprotein is of plant or bacterial origin because of its MS capability for determining more and unique/uncommon monosaccharide residues.
- Methyl glycosides produced by acid hydrolysis are derivatized with TMS reagent and analyzed by CCRC’s Agilent 7890 GC-MS instruments.
- Data can be obtained from 100 μg of pure carbohydrate material (~1 mg or more as glycoprotein material).
Glycosyl Linkage Analysis by GC-MS
To determine the positions of linkage of glycosyl residues in an oligosaccharide, polysaccharide, or glycoprotein. The linkage of the residues, including percentage of each, is provided with this analysis.
Linkage analysis of neutral, uronic acids or amino-residues (methylation analysis) – The polysaccharides or oligosaccharides are derivatized prior to GC-MS analysis. This procedure uses either the Hakomori, or NaOH base, for the permethylation procedure.
Amount required for sample analysis: A minimum of 100-200 micrograms of carbohydrate is required, more if available. Your sample must be pure usually free of salt and detergent.
- Exoglycosidase digestion is a strategy used for in-depth structural characterization of oligosaccharides.
- Glycosyl sequence, linkage and anomeric configuration in glycans can be determined or confirmed by digestion with specific exoglycosidases.
- First, glycans are released from a glycoprotein. Then the released glycans are treated with specific exoglycosidase enzymes such as neuraminidase, N-acetylhexosaminidase or alpha 1,6 mannosidase. Sequential removal of exterior residues is monitored by mass spectrometry.