An N , N -Dimethylethylenediamine-Derivatized In Silico Mass Spectral Library for Comprehensive Profiling of Isothiocyanates in Brassicaceae Plants

Abstract Isothiocyanates (ITCs) are bioactive hydrolysis products of glucosinolates in Brassicaceae plants with diverse health-promoting properties. However, comprehensive profiling of ITCs remains challenging due to the limited availability of authentic standards, the underexplored structural diversity of naturally occurring ITCs, and poor mass spectrometry (MS) detection sensitivity. Here, we established a N,N-dimethylethylenediamine (DMEDA) derivatization strategy and constructed an in silico MS/MS library for systematic ITC annotation. DMEDA derivatization markedly improved LC–MS detection sensitivity, enabling the reliable detection of low-abundance ITCs with limits of detection at the nanomolar level. Detailed characterization of the MS/MS fragmentation behaviors of derivatized ITC standards revealed subclass-dependent fragmentation patterns, which were subsequently translated into spectral prediction rules for the generation of an in silico mass spectral library with 2209 simulated ITC structures. Application of the developed workflow to Brassicaceae plant samples enabled the annotation of 50 endogenous ITCs, including 4 low-abundance compounds that were previously not reported. Our work provides a valuable resource for the systematic exploration of ITC structural diversity and facilitates functional studies of glucosinolate-derived bioactive metabolites.