Protein Protocols Protein Protocols

810 Hounsell, Davies, and Smith
9. Repeat step 8 and evaporate the combined supernatants under a stream of nitrogen.
10. Dry over phosphorus pentoxide before adding 20 µL TMS reagent.
11. Heat at 60°C for 5 min, evaporate remaining solvent under a stream of nitrogen, and add
20 µL dry toluene.
12. Inject onto a capillary GC column with 14 psi He head pressure and a temperature program
from 130 to 230°C over 20 min and held at 230°C for 20 min.
13. Calculate the total peak area of each monosaccharide by adding individual peaks and
dividing by the peak area ratio of the internal standard. Compare to standard curves for
molar calculation determination.
4. Notes
1. The use of methanolic HCl for cleavage of glycosidic bonds and oligosaccharide-peptide
cleavage yields methyl glycosides and carboxyl group methyl esters, which gives acid
stability to the released monosaccharides, and thus, monosaccharides of different chemical
lability can be measured in one run. If required as free reducing monosaccharides (e.g.,
for HPLC), the methyl glycoside can be removed by hydrolysis. The reagent can be
obtained from commercial sources or made in laboratory by bubbling HCl gas through
methanol until the desired pH is reached or by adding a molar equivalent of acetyl chloride
to methanol.
2. An equilibrium of the α and β methyl glycosides of monosaccharide furanose (f) and
pyranose (p) rings is achieved after 18 h so that a characteristic ratio of the four possible
(fα, fβ, pα, pβ) molecules is formed to aid in unambiguous monosaccharide assignment.
3. Solid-silver carbonate has a pink hue in an acidic environment, and, therefore, neutrality
can be assumed when green coloration is achieved.
4. The acidic conditions remove N-acetyl groups, which are replaced by acetic anhydride.
This means that the original status of N-acetylation of hexosamines and sialic acids is not
determined in the analysis procedure. If overacetylation occurs, the time can be reduced.
5. Direct re-N-acetylation by the addition of pyridine-acetic anhydride 1:1 in the absence of
silver carbonate can be achieved, but this gives more variable results.
References
1. Hounsell, E. F. (1993) A general strategy for glycoprotein oligosaccharide analysis, in
Methods in Molecular Biology, vol. 14: Glycoprotein Analysis in Biomedicine (Hounsell,
E. F., ed.), Humana, Totowa, NJ, pp. 1–15.
2. Hounsell, E. F. (1994) Physicochemical analyses of oligosaccharide determinants of glycoproteins.
Adv. Carbohyd. Chem. Biochem. 50, 311–350.