Methylation, linkage analysis

Methylation (linkage) analysis To determine linkages between sugars. 

Figure 1. Methylation linkage analysis of PSL-I by GC/MS total ion chromatogram of partially methylated alditol and myoinositol acetates (PMAA) from PSL-I carboxyl-reduced trisaccharide by gas chromatography /mass spectrometry in...

Methylation linkage analysis of the partially methylated alditol acetates gave the following derivatives ... 

Figure 5. Methylation linkage analysis of the major tetrasaccharide from tobacco glycophosphoceramide concentrate...

Electron impact MS of open-chain monomeric derivatives was useful for both determination of the position of acylamino groups and linkage analysis. The mass spectrum of the carbonyl-reduced and methylated derivative of the monosaccharide 48 from P. aeruginosa 07a,7d (Fig. 12) showed the expected fragmentation pattern, which demonstrated the... 

GC-MS linkage analysis of partially methylated alditol acetates derived from the 50% (v v) aqueous acetonitrile fractions of the permethylated fV-glycans derived from the wildtype and Mgat4a knockout mouse spleen... 

In a useful variant of the classical methylation procedure for linkage analysis in the structural determination of oligo- and poly-sacccharides, the fully methylated oligomer is treated with triethylsilane and boron trifluoride etherate or triethylsilane and trimethylsilyl trifluoromethanesulfonate. This procedure produces partially methylated anhydroalditols. These are acetylated and analyzed by GLC-MS.222 223 This reductive-cleavage method makes possible simultaneous determination of identity, ratio, linkage position, and especially the ring size for each monosaccharide component. 

Methylation analysis is the most important procedure for linkage analysis in carbohydrate-containing polymers (Lindberg, 1972). The procedure requires that all free hydroxyl in the oligo- or polysaccharide are etheri-fied. Following depolymerization of the methylated material, the monomers are separated, identified, and quantified. The free hydroxyl groups in the partially methylated monomers mark the position(s) at which the sugar residues were substituted. 

Fig. 1 Schematic for the permethylation linkage analysis, exemplified by a hypothetical trisaccharidc, Gal-(a-l,4)-Glc-(a-l,6)-Man. The acid-hydrolyzed partially methylated monosaccharides generated are analyzed by GC-MS, typically as alditol or aldononitrile derivatives...

In this paper, we have compared two commonly used permethylation methods [4, 5] and have applied them to a model carbohydrate compound. The completeness of the methylation steps was established by matrix-assisted laser desorption/ionization tlme-of-flight mass spectrometry (MALDI-TOF MS). The methylated carbohydrate products were acid-hydrolyzed and converted to methylated partially acelylated aldononitrile derivatives (Me PAANs) for linkage analysis by gas chromatography-mass spectrometry (GC-MS) [11]. 

Practical applications are again stressed in the Chapter by Lonngren and Svensson (Stockholm) on Mass Spectrometry in Stmctural Analysis of Natural Carbohydrates. They build on the fundamentals of carbohydrate mass spectrometry, as laid down by Kochetkov and Chizhov in Volume 21, and demonstrate the profound analytical value of mass spectrometry for structural analysis of complex polysaccharides. In particular, this tool has dramatically increased the scope of the traditional methylation linkage-analysis procedure, especially when used in conjunction with gas-liquid chromatographic methods of separation. The latter topic is the subject of complementary Chapters by Dutton, one already published in Volume 28 and the other scheduled for publication in Volume 30. 

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