Polysaccharides and oligosaccharides are a diverse group of natural polymers with important biological functions. The diversity of carbohydrate polymers is vast, ranging from small oligosaccharides of defined composition decorating proteins, to large, complex heteropolymers comprising integral cell wall components of plants, fungi and bacteria. An important step in the elucidation of unknown carbohydrate structures in a sample is the assessment of the various linkages present. This is accomplished by performing linkage analysis of the sample. The analysis proceeds as a successive series of chemical steps in which unlinked carbohydrate hydroxyls are marked with methyl groups, the sample is hydrolyzed into monosaccharides and reduced to alditols, and finally free hydroxyls are acetylated. Gas chromatography-mass spectrometry (GC-MS) analysis is employed to analyze the resultant partially methylated alditol acetates (PMAAs). The following paper reviews the major literature pertaining to the specific protocol for linkage analysis of carbohydrates outlined herein. The review details additional steps necessary for the completion of uronic acid linkage analysis, as well as analysis of chitin containing polymers. It also gives chromatographic examples of common erroneous results which the first time practitioner will want to be aware of. Our hope is that this protocol will serve as a definitive guide, allowing novice researchers to perform linkage analysis of carbohydrates in their own lab.
Keywords: Carbohydrate; GC-MS; Gas chromatography; Linkage analysis; PMAAs; Partially methylated alditol acetates; Polysaccharide.