Analytical method

Spider venoms

The lyophilized spider venoms (gained by spider milking) were obtained from the following three providers: Agelena orientalis (Agelenidae), Agelenopsis potteri (Agelenidae), Eratigena agrestis (Agelenidae), Hololena curta (Agelenidae), Pireneitega luctuosa (Agelenidae), Eresus sp. 2 (Eresidae), Stegodyphus sp. 4 (Eresidae), Drassodes sp. 1 (Gnaphosidae), Alopecosa sp. 1 (Lycosidae), Alopecosa sp. 4 (Lycosidae), Alopecosa sp. 5 (Lycosidae), Desertosa sp. 3 (Lycosidae), Hippocosa sp. 1 (Lycosidae), Lycosa praegrandis (Lycosidae), Lycosa sp. 1 (Lycosidae), Lycosa sp. 3 (Lycosidae), Lycosa sp. 4 (Lycosidae), and Lycosa sp. 5 (Lycosidae) from Fauna Laboratories, Ltd. (Almaty, KAZ). Hololena sp. (Agelenidae), Larinioides cornutus (Araneidae), Nephila clavipes (Araneidae), Diguetidae canities (Diguetia), Geolycosa sp. (Lycosidae), Hogna caroliensis (Lycosidae), Peucetia vir-idans (Oxyopidae), Physocyclus mexicanus (Pholcidae), Plectreurys tristis (Plectreuridae), Ariadna sp. (Segestriidae), Latrodectus mactans (Theridiidae), and Steatoda grossa (Theridiidae) from Spider pharm (Yarnell AZ, USA). Atrax robustus (Atracidae) and Heteropoda davidbowie (Sparassidae) from Alpha Biotoxine (Montroeul-au-bois, BEL).

The venom of Parawixia bistriata (Araneidae) was extracted from the venom glands in H2O/MeCN 9:1 (gland/solvent 1:5 [m/v]), centrifuged (14,000 rpm, 10 min, 4 °C) and the supernatant was lyophilized. The work was done in the laboratory of Prof. Dr. Wagner Ferreira dos Santros, Dept. of Biology, University of São Paulo, Ribeirão Preto (BRA).

Sample preparation

The lyophilized spider venom was dissolved in H₂O/MeCN 3:1 + 0.05% TFA to a concentration of 1 μg/μl. Reference compounds were dissolved in H₂O/MeCN 3:1 + 0.05% TFA to a concentration of 10 μg/ml. All samples were stored at –20 °C prior to their use.


The UHPLC-ESI-MS and MS/MS experiments were performed on a Dionex UltiMate 3000 HPLC instrument (Thermo Scientific, Germering, Germany) equipped with an autosampler, a pump and a diode-array detector (DAD) of the same producer series. The UHPLC system was connected to a QExactive Orbitrap FT mass spectrometer (Thermo Scientific, Waltham, MA, USA) equipped with an HESI source.

UHPLC method

The samples were chromatographed at a flow rate of 0.30 ml/min on a RP-C18+ column (CortecsTM UPLC® C18+ 1.6 μm, 2.1x150 mm, Waters, Milford, MA, USA) with solvents A and B consisting of H₂O + 0.05% TFA and MeCN/H₂O 8:2 [v/v] + 0.05% TFA, respectively. The column chamber temperature was set to 25 °C. The samples were injected at a volume of 1 μl. The strong wash was MeOH and weak wash H₂O/MeCN 95:5 [v/v]. The gradient went from 3.8 to 25% of eluent B over 22 min. Finally, the column was regenerated at 100% B for 4 min and equilibrated at 3.8% B over 6 min.


The MS conditions were: sheath gas flow rate (N₂, 48), aux gas flow rate (N₂, 11), aux gas heater temperature (320 °C) and sweep gas flow rate (N₂, 2), spray voltage (3.5 kV), S-lens RF level (55), capillary temperature (256 °C). Full scan MS were performed in positive ion mode at 70’000 resolutions and was detected between m/z 100 to 1500. The AGC target setting for full scan MS experiment was set to 10⁶ with a maximum of 30 injection times. The mass spectrometer was calibrated for mass accuracy once a day according to the manufacturer’s instructions. The relative mass error being typically lower than 3 ppm (externally).

The MS/MS experiments were acquired in Full MS/dd-MS2 (Top N) mode with a resolving power of 17’500. The acquisition was performed with an isolation window set to 0.8 m/z. The AGC target setting was set to 10⁵. The normalized collision energy (NCE) was set to a gradient (30, 35, 40. The MS/MS spectra were recorded of the top five ions.

Hydrogen/Deuterium exchange

For chromatography, the solvents A and B consisted of D₂O + 0.05% d₁-TFA and MeCN/D₂O 8:2 [v/v] + 0.05% d₁-TFA, respectively. The column chamber temperature was set to 40 °C. All other parameters remained the same.


HPLC grade H₂O (< 5ppb) was obtained by purification of deionized H₂O with a MilliQ gradient apparatus (Millipore, Milford, MA, USA). Acetonitrile (MeCN, ULC/MS grade) and trifluoroacidic acid (TFA) were obtained from Biosolve (Valkenswaard, Netherland), D₂O (99.9 atom % D) from Sigma-Aldrich (Steinheim, Germany) and d₁-TFA (D 99.5%) from Cambridge Isotop Laboratories, Inc. (Andover, MA, USA).