In plants, a large diversity of polysaccharides comprise the cell wall. Each major type of plant cell wall polysaccharide, including cellulose, hemicellulose, and pectin, has distinct structures and functions that contribute to wall mechanics and influence plant morphogenesis. In recent years, pectin valorization has attracted much attention due to its expanding roles in biomass deconstruction, food and material science, and environmental remediation. However, pectin utilization has been limited by our incomplete knowledge of its structure. Herein, we present a workflow of principles relevant for the characterization of polysaccharide primary structure using nature’s most complex polysaccharide, rhamnogalacturonan-II (RG-II), as a model. We outline how to isolate RG-II from celery and duckweed cell walls and from red wine using chemical or enzymatic treatments coupled with size-exclusion chromatography. From there, we applied mass spectrometry (MS)-based techniques to determine the glycosyl residue and linkage compositions of the intact RG-II and derived oligosaccharides including special considerations for labile monosaccharides. In doing so, we demonstrated that in the duckweed Wolffiella repanda the arabinopyranosyl (Arap) residue of side chain B is substituted at O-2 with rhamnose. We used electrospray-MS techniques to identify non-glycosyl modifications including methyl-ethers, methyl-esters, and acetyl-esters on RG-II-derived oligosaccharides. We then showed the utility of proton nuclear magnetic resonance spectroscopy (1H-NMR) to investigate the structure of intact RG-II and to complement the RG-II dimerization studies performed using size-exclusion chromatography. The complexity of pectic polysaccharide structures has hampered efforts aimed at their valorization. In this work, we used RG-II as a model to demonstrate the steps necessary to isolate and characterize polysaccharides using chromatographic, MS, and NMR techniques. The principles can be applied to the characterization of other saccharide structures and will help inform researchers on how saccharide structure relates to functional properties in the future.

中文翻译：

---

从植物细胞壁中分离和表征多糖的方案：使用鼠李糖半乳糖醛酸-II 的案例研究

在植物中，大量多样的多糖构成细胞壁。每种主要类型的植物细胞壁多糖，包括纤维素、半纤维素和果胶，都具有不同的结构和功能，有助于细胞壁力学并影响植物形态发生。近年来，果胶增值因其在生物质解构、食品和材料科学以及环境修复中的作用不断扩大而备受关注。然而，果胶的利用受到我们对其结构不完全了解的限制。在此，我们介绍了使用自然界最复杂的多糖、鼠李糖半乳糖醛酸-II (RG-II) 作为模型来表征多糖一级结构的相关原理工作流程。我们概述了如何使用化学或酶处理结合尺寸排阻色谱从芹菜和浮萍细胞壁以及红酒中分离 RG-II。从那里，我们应用基于质谱 (MS) 的技术来确定完整 RG-II 和衍生寡糖的糖基残基和连接组成，包括对不稳定单糖的特殊考虑。在这样做时，我们证明了在浮萍中，侧链 B 的阿拉伯吡喃糖基 (Arap) 残基在 O-2 处被鼠李糖取代。我们使用电喷雾质谱技术来鉴定非糖基修饰，包括 RG-II 衍生寡糖上的甲基醚、甲基酯和乙酰酯。然后，我们展示了质子核磁共振波谱 (1H-NMR) 的效用，以研究完整 RG-II 的结构并补充使用尺寸排阻色谱进行的 RG-II 二聚化研究。果胶多糖结构的复杂性阻碍了旨在使其价值化的努力。在这项工作中，我们使用 RG-II 作为模型来演示使用色谱、MS 和 NMR 技术分离和表征多糖的必要步骤。这些原理可以应用于其他糖类结构的表征，并将帮助研究人员了解糖类结构如何与未来的功能特性相关。果胶多糖结构的复杂性阻碍了旨在使其价值化的努力。在这项工作中，我们使用 RG-II 作为模型来演示使用色谱、MS 和 NMR 技术分离和表征多糖的必要步骤。这些原理可以应用于其他糖类结构的表征，并将帮助研究人员了解糖类结构如何与未来的功能特性相关。果胶多糖结构的复杂性阻碍了旨在使其价值化的努力。在这项工作中，我们使用 RG-II 作为模型来演示使用色谱、MS 和 NMR 技术分离和表征多糖的必要步骤。这些原理可以应用于其他糖类结构的表征，并将帮助研究人员了解糖类结构如何与未来的功能特性相关。