Computational Mass Spectrometry-Based Reassessment of European <i>Amanita</i> sect. <i>Phalloideae</i> 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 <i>Amanita</i> species belonging to the section <i>Phalloideae</i>. The study aimed to clarify the toxin content of these mushrooms, focusing on chemical differences between <i>A. virosa</i> and <i>A. amerivirosa</i> which lacked available state-of-the-art analytical data. We validated the inability of <i>A. amerivirosa</i> 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 data sets, 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 (<b>1</b>) 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 <i>Amanita</i> toxins to be reported since the pioneering work of Wieland and Faulstich between 1930 and 1980.