DOI: 10.5061/dryad.47d7wm398
title: Data from: Exhaustive extraction of cyclopeptides from Amanita phalloides: guidelines for working with complex mixtures of secondary metabolites

description: Understanding plant-insect interactions is an active area of research in
 both ecology and evolution. Much attention has been focused the impact of
 secondary metabolites in the host plant or fungi on these interactions.
 Plants and fungi contain a variety of biologically active compounds, and
 the secondary metabolite profile can vary significantly between individual
 samples. However, many experiments characterize the biological effects of
 only a single secondary metabolite or a subset of these compounds. Here,
 we develop an exhaustive extraction protocol using an accelerated solvent
 extraction protocol to recover the complete suite of secondary metabolites
 found in Amanita phalloides (death cap mushrooms) and compare its efficacy
 to the ‘Classic’ extraction method used in earlier works. We demonstrate
 that our extraction protocol recovers the full suite of secondary
 metabolites in A. phalloides unlike the ‘Classic’ method that favors polar
 secondary metabolites. Based on these findings, we provide recommendations
 for how to optimize protocols to ensure exhaustive extracts and also the
 best practices when using natural extracts in ecological experiments.
description: <i>Amanita phalloides</i> extracts were analyzed
 using Agilent 1260 Infinity II system, comprised of an Infinity II Binary
 Pump, Infinity Multisampler, Multicolumn Thermostat, Diode Array Detector
 (DAD, UV spectrometer), Agilent’s Instrument Control Framework, and Mass
 Selective Detector (MSD, mass spectrometer). The analysis was performed
 using Pursuit 5C18 150 x 4.6 mm column at 23 °C. The mobile phase
 comprised (A) water containing 0.2% formic acid and (B) acetonitrile
 containing 0.2 % formic acid. The following elution gradient was applied:
 0-4 min 10% B; 4-20 min 10-55% B; 20-25 min 55% B; 25-30 min 55-100% B;
 30-33 min 100% B; 33-35 min 100-10% B; 35-40 min 10% B. Five µL of the
 extract was injected onto the column and run at a 1.0 ml/min mobile phase
 flowrate. The eluate was monitored by DAD at 214 and 295 nm, and UV-VIS
 spectra within the range of 190-400 nm were stored for all the peaks. All
 the samples were analyzed on the MSD in electrospray positive ionization
 mode with 10 l/min drying gas flow rate, 50 psig nebulizer pressure, 350
 °C drying gas temperature, and 4000 V capillary voltage. For
 fingerprinting, full scan acquisition mode was used with scan range:
 100-1200. Standard samples of α-amanitin, β-amanitin,
 phalloidin, and phallacidin obtained from SantaCruz Biotechnology
 (α-amanitin) and Cayman Chemical (all others). These four cyclopeptides
 are the only ones with commercially available standards to the best of the
 author’s knowledge. They were dissolved in double distilled water
 (α-amanitin, β-amanitin) or methanol (phalloidin, phallacidin) at a
 concentration of 1 µg/µL. These stock solutions were subsequently diluted
 ten-fold and used to confirm identification of selected cyclopeptides in
 the analyzed extracts. Other potential cyclopeptides were identified by
 their ionization pattern.