Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA.
Methods Mol Biol. 2021;2271:249-271. doi: 10.1007/978-1-0716-1241-5_18.
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.
多糖和寡糖是一组具有重要生物学功能的天然聚合物，具有多样性。碳水化合物聚合物的多样性非常广泛，从定义组成的、修饰蛋白质的小寡糖，到包含植物、真菌和细菌细胞壁成分的大型复杂杂多糖。在对样品中未知碳水化合物结构进行阐明的过程中，重要的一步是评估存在的各种键合。这通过对样品进行键合分析来完成。分析过程是一系列化学步骤，其中未键合的碳水化合物羟基用甲基标记，将样品水解成单糖并还原为糖醇，最后游离羟基被乙酰化。气相色谱-质谱联用（GC-MS）分析用于分析得到的部分甲基化糖醇乙酸酯（PMAAs）。本文综述了与本文所述碳水化合物键合分析的具体方案相关的主要文献。综述详细介绍了完成糖醛酸键合分析以及分析含甲壳素聚合物所需的附加步骤。它还给出了常见错误结果的色谱示例，这些结果是第一次进行该实验的研究人员需要注意的。我们希望该方案将成为一个明确的指南，使新手研究人员能够在自己的实验室中进行碳水化合物的键合分析。
