Metabolomics with ¹⁵N Labeling for Characterizing Missing Monoterpene Indole Alkaloids in Plants

Monoterpene indole alkaloids (MIAs) in medicinal plants remain uncharacterized owing to their complicated structure by metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) despite their pharmaceutical importance. We demonstrate an untargeted metabolome analysis with ¹⁵nitrogen (N) labeling to characterize MIAs having an indolic skeleton in the flowers, leaves, petioles, stems, and roots of Catharanthus roseus. Principal component analysis using ¹⁵N- and nonlabeled metabolome data showed that N-containing metabolites (N-metabolites) are labeled with ¹⁵N. Paring of the ¹⁵N- and nonlabeled precursor ions were performed using the criteria of retention time, difference of m/z value, and a nonlabeled product ion at m/z 144.08 that indicates an indolic skeleton. The mass shift of the m/z value of the product and precursor ions to their ¹⁵N-labeled ions identified the number of N of their ions. Finally, molecular formula of 45 MIAs was unambiguously identified using the identified N number. The alkaloid network analysis using the MS/MS similarity showed the structural commonness and uniqueness among the MIAs. Of them, antirhine was identified using an authentic standard compound. Multimetabolomics using LC-MS/MS and imaging mass spectrometry showed that antirhine accumulates considerably in the epidermis and vascular cylinder of the roots. The developed approach showed the existence of the missing MIAs. The modification of this approach will identify other MIAs that contain a hydroxylated or methoxylated indolic skeleton.