Kruse Lars H, Bennett Alexandra A, Baruah Vishwa J, Irfan Mohammad, Moghe Gaurav D
Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.
Department of Chemistry, Institute of Analytical Chemistry, University of Natural Resources and Life Sciences, Vienna, Vienna, Austria.
Methods Mol Biol. 2025;2895:177-200. doi: 10.1007/978-1-0716-4350-1_13.
The metabolic diversity of plants, comprising over a million different metabolites across the plant kingdom, harbors enormous potential for pharmaceutical and biotechnological applications. Resin glycoside (RG) acylsugars from the Convolvulaceae are of interest due to their medicinal and agricultural potential. However, understanding the biological relevance of RGs is challenging as they exhibit a high lineage-specific structural diversity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with computational peak annotation can provide insights into this diversity. Here, we present a comprehensive protocol for the characterization of RG diversity using a sensitive LC-MS/MS instrument, a knowledge-based computational pipeline, and a web tool for peak annotation. The described experimental approach provides a step-by-step guide for RG sampling, extraction, purification for downstream analyses such as bioassays, and structural annotation using LC-MS/MS and computational metabolomics. The protocol focuses on qualitative analysis for putative annotation (Annotation Level 2 as defined by the Metabolomics Standards Initiative) of RGs and can serve as a valuable template for researchers exploring plant metabolic diversity beyond RGs and acylsugars.
植物的代谢多样性涵盖了植物界超过一百万种不同的代谢物，在制药和生物技术应用方面具有巨大潜力。旋花科的树脂糖苷（RG）酰基糖因其药用和农业潜力而备受关注。然而，由于RG表现出高度的谱系特异性结构多样性，了解其生物学相关性具有挑战性。液相色谱 - 串联质谱（LC-MS/MS）结合计算峰注释可以深入了解这种多样性。在这里，我们提出了一个全面的方案，用于使用灵敏的LC-MS/MS仪器、基于知识的计算流程和用于峰注释的网络工具来表征RG多样性。所描述的实验方法为RG采样、提取、纯化以进行下游分析（如生物测定）以及使用LC-MS/MS和计算代谢组学进行结构注释提供了逐步指导。该方案侧重于对RG进行推定注释（代谢组学标准倡议定义的注释级别2）的定性分析，并且可以作为研究人员探索RG和酰基糖以外的植物代谢多样性的有价值模板。
