Fine-Scale Characterization of Plant Diterpene Glycosides Using Energy-Resolved Untargeted LC-MS/MS Metabolomics Analysis

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.