A major application of the permethylation reaction is linkage or “methylation analysis” for determination of carbohydrate linkages by GC/MS. It was developed in the late 1960s [153, 154] and is still in use today. The method has been the subject of many articles and reviews [141, 155-161], and several modifications have been developed. Basically, the procedure is one in which all hydroxyl groups in a polysaccharide are first permethylated after which the molecule is hydrolyzed to generate monosaccharides that contain free hydroxyl groups at the sites of linkage. These monosaccharides are then reduced in order to avoid the production of two peaks on chromatographic analysis due to a- and P-anomers, and, finally, the resulting alditols are derivatized with a different reagent such as acetic anhydride to produce partially methylated alditol acetates, known as PMAAs. The positions of the various substituents are subsequently located by GC/MS in order to determine which of the hydroxyl groups (those derivatized as OAc in this example) were originally involved in bonding. Ring size is also reflected in the results because for hexoses, additional free OH groups will be generated at C4 and C5 for furanoses and pyranoses, respectively. However, the additional OH groups can cause problems with certain sugars such as 4-linked hexopyranoses and 5-linked hexofuranoses, both of which will produce 1,4,5-triacetoxy products. However, carbohydrates derived from mammalian glycoproteins appear to contain only pyranose structures, and, consequently this difficulty should not present problems in this area. Although these procedures will identify which hydroxyl groups on a given sugar ring are involved in linkage, information as to which sugar is attached at that site is not available and must be obtained by other means.
Data on the retention times and mass spectra of these derivatives have been published in several reviews [141, 159, 160]. Fragmentation occurs primarily along the carbon chain following charge localization on a nitrogen (amino sugar) or oxygen atom. Secondary fragments are the result of the further elimination of neutral fragments such as acetic acid (60 u) ketene (42 u), formaldehyde (30 u), or methanol (32 u). The base peak is usually at m/z 43 (CH3CO+).
Derivatization of Sialic Acids
Glycans containing sialic acids are relatively unstable, particularly under MALDI conditions, and readily eliminate sialic acid. Methods for overcoming this problem are discussed in Section 18.104.22.168. One of these methods involves methyl ester or amide formation to remove the labile acidic protons that are responsible for the sialic acid losses. Permethylation not only achieves the same purpose but also blocks the hydroxyl groups preventing fragmentation in negative ion mode. More details can be found later in this chapter.