Giving it a fair shake: A simple, fast, & efficient method to extract amatoxins from the death cap mushroom, Amanita phalloides

1.1.The death cap mushroom, Amanita phalloides, is well known for containing amatoxins such as alpha- and beta-amanitin, which inhibit eukaryotic RNA polymerase II. While these toxins have been used in research for almost a century, they have recently garnered attention for their role in drug-antibody conjugates. The amatoxins are still largely extracted from wild mushrooms, which cannot be made to fruit in the lab. We propose simplified extraction methods that could reduce hazardous exposures to dust and expedite sample analysis without sacrificing accuracy. We recently developed a Lateral Flow Immunoassay (LFIA), for which we identified that sample maceration was not needed to extract the amatoxins and that the incubation time for extraction could be accomplished in 1 minute. In this current work, we hypothesized that these same extraction adjustments-minimal tissue maceration and reduced incubation time-could be transferable to instrumental detection methods. To test the need for sample maceration, we utilized three different techniques: 1) traditional mortar and pestle, 2) a similarly disruptive method of bead beating, and 3) no grinding, but rather hand shaking dried mushroom tissue in extraction buffer. In addition, we performed the solvent extraction step at varying times to observe if more time allows for more toxin to be removed from the tissue. Lastly, we utilized two comparable solvent evaporation methods (rotovap or speedvac) to establish if multiple samples could be processed simultaneously, thus improving sample throughput. We adjusted aspects of the typical extraction protocol, which resulted in a rapid (1 min) incubation step, along with minimal sample handling (no grinding) of the dried mushroom tissue. We present an extraction protocol that saves time, reduces equipment contamination, and minimizes risk to the researcher. The impact of this faster, safer method may help produce these important toxins faster, for both research and medical use.