Permethylated Sample, and Analysis of the Partially Methylated Sugars

Degradation of the Permethylated Sample, and Analysis of the Partially Methylated Sugars 

Depolymerization of the permethylated carbohydrate is achieved by hydrolysis with acid. Under these conditions, the amino sugar residues are N-deacetylated, and the aminohexosidic linkages become resistant to hydrolysis. Stellner and coworkers29 showed that, when the acid degradation is conducted in 95% acetic acid, the amino sugar residues are also liberated, and can be analyzed by the methylation technique.29 Therefore, acetolysis followed by acid hydrolysis is now commonly used, as it allows the analysis both of hexose and hexosamine residues. 

The partially methylated monosaccharides obtained on depolymerization of the permethylated sample are preferably analyzed as acetates by g.l.c.-m.s., as shown by Bjomdal and coworkers.41,42 The neutral sugars and the amino sugars obtained in acetolysis-acid hydrolysis are reduced, and acetylated for the analysis, and the amino-hexitol and the neuraminic acid residues are acetylated after methanolysis. Identification with the aid of g.l.c.-m.s. has been described for all of the common components of protein- and lipid-linked glycans and oligosaccharides from animal cells, namely, the neutral sugars,41-43 hexitols,44 hexosamines,29,43,45,46 aminohexitols,31,32 and neuraminic acids.33,34,47 

Separation of the partially methylated sugars by g.l.c. gives evidence on the methyl-substitution pattern complementary to that obtained from the mass spectrum. The electron-impact, mass spectra do not differentiate between the sugar isomers (such as the galactose, 

The methyl-substitution pattern of the neutral sugar residues can be reliably identified from their mass spectra.41-43 Identification of the substitution pattern from the electron-impact, mass spectra rarely presents any major problems. It should, however, be noted that, in some cases, the occurrence of symmetrical molecules jeopardizes the identification. An example of this is the symmetrical, 3,4-di-O-methyl derivative (6) of mannose, which is commonly found in the analysis of 

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