Computational Mass Spectrometry-Based Reassessment of European Amanita sect. Phalloideae Cyclopeptidome

An advanced metabolomic workflow featuring expert knowledge-tailored mass spectral queries and multi-informative molecular networking was applied to French DNA-barcoded collections of Amanita species belonging to the section Phalloideae. The study aimed to clarify the toxin content of these mushrooms, focusing on chemical differences between A. virosa and A. amerivirosa which lacked available state-of-the-art analytical data. We validated the inability of A. amerivirosa to produce α-amanitin but revealed that it synthesizes amaninamide, quantified herein for the first time. Careful evaluation of the MS/MS fragmentation patterns associated with ama-, phallo-, and virotoxins led to interoperable MassQL queries (viz., compatible with MS/MS spectra from both QTOF and Orbitrap) for identifying these toxins in MS/MS datasets, with varying degrees of structural accuracy. The ability of these queries to convey structural information was highlighted by the MS/MS prediction of six new toxins, with structural proposals validated after isolation and structure elucidation by NMR. Among the reported phallotoxins, ameriphalloidin (1) displayed an unprecedented viroidin-like amino acid sequence, supporting the often-inferred biosynthetic relationship between these toxin classes. The discovery of new phallo- and virotoxins is significant, as these are the first Amanita toxins to be reported since the pioneering work of Wieland and Faulstich between 1930 and 1980.