Comprehensive TCM molecular networking based on MS/MS in silico spectra with integration of virtual screening and affinity MS screening for discovering functional ligands from natural herbs.

Accessing the rich source of compounds from natural herbs for use in the pharmaceutical industry using conventional bioassay-based screening platforms has low efficiency and is cost-prohibitive. In this study, we developed a new method involving traditional Chinese medicine (TCM) molecular networking and virtual screening coupled with affinity mass spectrometry (MN/VS-AM) for the efficient discovery of herb-derived ligands. The in silico MS/MS fragmentation database (ISDB) generated by molecular networking of TCM can rapidly identify compounds in complex herb extracts and perform compound activity mapping. Additionally, the pre-virtual screening conveniently includes candidate herbs with potential bioactivity, while affinity MS screening completely eliminates the requirement for a tedious pure compound preparation at the initial screening phase. After applying this approach, two types of compounds, isoamylene flavanonols and 20(s)-protopanoxadio saponins, which were confirmed to interact with the small GTPase of Ras, were successfully identified from a dozen anti-cancer TCM herbs. The results demonstrate that the modified screening strategy dramatically improved the accuracy and throughput sensitivity of ligand screening from herbal extracts. Graphical abstract.