Abstract：
Plant diterpene glycosides are essential for diverse physiological processes. Comprehensive structural characterization proved to be a challenge due to variations in glycosylation patterns, diverse aglycone structures, and the absence of comprehensive reference databases. In this study, a method for fine-scale characterization was proposed based on energy-resolved (ER) untargeted LC-MS/MS metabolomics analysis using steviol glycosides as a demonstration. Energy-dependent fragmentation patterns were unveiled by a series of model compounds. Distinct glycosylation sites were discerned by leveraging varying fragmentation energies for the precursor ions. The sugar moiety linkage at C19OOH (R1) exhibited facile and intact cleavage at low collision energies, while the sugar moiety at C13-OH (R2) demonstrated consecutive cleavage with increasing energy. Aglycone ions exhibited a higher relative intensity at NCE 50, with relative intensities ranging from 95% to 100%. Subsequently, aglycone candidates, R1 sugar composition, and R2 sugar sequence were deduced through ER-MS/MS analysis. The developed method was applied to Stevia rebaudiana leaves. A total of 91 diterpene glycosides were unambiguously identified, including 16 steviol glycosides with novel acetylglycosylation patterns. This method offers a rapid alternative for glycan analysis and the structural differentiation of isomers. The developed method enhances the understanding of diterpene glycosides in plants, providing a reliable tool for the in-depth characterization of complex metabolite profiles.
摘要：
植物二萜苷是多种生理过程所必需的。由于糖基化模式的变化，全面的结构表征被证明是一个挑战，不同的糖苷配基结构，以及缺乏全面的参考数据库。在这项研究中,基于能量分辨(ER)非靶向LC-MS/MS代谢组学分析,以甜菊醇糖苷为示范,提出了一种精细表征方法.一系列模型化合物揭示了能量依赖的碎片模式。通过利用前体离子的不同碎裂能量来辨别不同的糖基化位点。C19OOH（R1）上的糖部分键在低碰撞能量下表现出容易和完整的裂解，而C13-OH（R2）处的糖部分显示出随着能量增加的连续裂解。Aglycone离子在NCE50处表现出更高的相对强度，相对强度范围为95％至100％。随后，aglycone候选人，R1糖成分，通过ER-MS/MS分析推导R2糖序列。将开发的方法应用于甜叶菊叶。共鉴定出91个二萜苷，包括16种具有新型乙酰糖基化模式的甜菊醇糖苷。该方法为聚糖分析和异构体的结构区分提供了快速的替代方法。所开发的方法增强了对植物中二萜苷的理解，为深入表征复杂代谢物谱提供了可靠的工具。
