Permethylated, structure analysis

N. Viseux, E. de Hoffmann, B. Domon, Structural analysis of permethylated oligosaccharides byESI-MS-MS, Anal. Chem., 69 (1997) 3193. 

A most useful application of methylation is for the structural analysis of polysaccharides. Permethylation, followed by hydrolysis of the product and identification of the individual methylated monosaccharides, indicates the original positions of the linkage points, as these are denoted by hydroxyl groups in the methylated monosaccharides. Considerable work has been done on the separation and identification of methylated monosaccharides by gas-liquid chromatography and mass spectrometry this topic is treated in detail in this Volume by Lbnngren and Svensson (see p. 41), Clearly, for such structural analyses to be valid, complete methyl-... 

The use of mass spectrometry in the structural analysis of carbohydrates, first reported in 1958 (114), was developed in detail by Kochetkov and Chizhov (115). They showed that, under electron impact, the acetylated and methyl ether derivatives of monosaccharides provided a wealth of structural information through analysis of typical fragmentation pathways of the initial molecular ion. This has proved of enormous utility in the structural elucidation of polysaccharides and complex oligosaccharides sequential permethylation, hydrolysis, reduction to the alditol, and acetylation, affords mixtures of peracetylated, partially methylated alditol acetates that can be separated and analyzed by use of a gas chromatograph coupled directly to a mass spectrometer (25). The mass spectra of stereoisomers are normally identical, while the gas chromatographic retention times readily permit differentiation of stereoisomers. 

ViSEUX, N. deHofemann, E. Domon, B. Structural analysis of permethylated oligosaccharides by electiospray tandem mass spectrometry. Anal. Chem. IW1,69, 3193-3198. 

A detailed study of the 0-linked oligosaccharides present on the surface of normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells has been completed. Structures were elucidated by f.a.b.-m.s. after permethylation, and methylation analysis before and after specific exo-glycosidase treatments. Some of the components were shown by f.a.b.-m.s. to be poly(N-acetyllactosaminyl) oligosaccharides, for example, 29. 

A second method uses permethylation of the dephosphated (48% aqueous HF, 48 h, 4°C) and 2H-reduced fipid A. This approach allowed the assignment of amide-bound fatty acids linked to GlcN(I) and GlcN(II), as well as the identification of the backbone structure as a HexpN disaccharide (85). Mass-spectrometric analysis of the products was performed by using either a short g.l.c. column (0.3 X 5 cm) or by direct insertion-probe analysis (87). In the case of C. violaceum (85), the mass spectra obtained from the permethyl-ated HexpN disaccharide bearing attached TV-methylacyl residues revealed unequivocally that both amino groups carried 12 0(3-OH). 

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