**4. Materials and Methods**

#### *4.1. Thalassophryne maculosa Venom and Isolation of TmC4-47.2 Toxin*

All necessary permits for the capture of *T. maculosa* and to collect the venom were obtained from the Escuela de Ciencias Aplicadas del Mar, Núcleo Nueva Esparta, Universidad de Oriente, Isla Margarita; Venezuela (Permit Number: 30/07). *T. maculosa* individuals were transported to the laboratory and were anesthetized with 2-phenoxyethanol before sacrifice. Venom was shortly obtained from the tip of the spines by applying pressure at their bases. After centrifugation, venom was pooled and stored at −80 ◦C before use. Protein content was examined using bovine serum albumin (Sigma Chemical Co., St Louis, MO, USA) as standard. Endotoxin content was evaluated (resulting in a total dose < 0.8 pg LPS) with QCL-1000 chromogenic Limulus amoebocyte lysate assay (Bio-Whittaker) according to the manufacturer's instructions. Aliquots of crude venom from *T. maculosa* (5 mg mL−1) was submitted to a reversed-phase (RP) LC10A high-performance liquid chromatography system (HPLC) Shimadzu (Äkta, Amersham Biosciences, Sweden) starting in a C18 semi-preparative column (Shimadzu-Shim Pack-CLC-ODS: 4.6 mm ID × 25 cm, 5 μm, and 100 Å). For elution, a binary gradient of 0.1% TFA aqueous solution (A) and 0.1% TFA 90% acetonitrile solution (B) was used, with a flow rate of 5.0 mL/min for 60 min, and detection at 214 nm absorbance. The chromatographic fractions were manually collected, vacuum dried, and stored at −20 ◦C. All fractions containing the toxin were identified by 12% SDS-PAGE [53]. The chromatographic fraction containing the toxin was submitted to 3 chromatographic steps using a Jupiter C8 4.60 mm × 250 mm, 10 μm, 300 Å, column (Phenomenex). The elution method was 20% to 80% B solution from 0 to 35 min at 1 mL/min, followed by a gradient of 10% B solution per minute until reaching a maximum concentration of 50% acetonitrile. The presence of the toxin in the eluates was confirmed by absorbance and SDS-PAGE.

#### *4.2. TmC4-47.2 Toxin Mass Determination*

To determine the toxin mass, we used 10 μL of the sample injected into an LC-MS Surveyor MSQ Plus system (Thermo Finnigan) by direct infusion in a flow of 50 μL/min of aqueous acetonitrile solution (1:1) containing 0.1% formic acid under capillary voltage conditions 3.1 kV, 75 V cone, scan lasting 1 s in the *m*/*z* range 200–2000. The equipment was previously calibrated with sodium iodide (*m*/*z* range 22.98 to 1971.61). To obtain the molecular weight of the sample, the deconvolution of the mass spectrum obtained was performed using the Mag Tran program version 1.0 beta 8 [54]. For MALDI-ToF mass spectrometry, analyzes were performed in a MALDI-ToF/PRO instrument (Amersham). The samples in solution were mixed 1:1 (*v*/*v*) with a supersaturated matrix solution for

proteins (sinapic acid), deposited on the sampling plate (0.4–0.8 μL), and allowed to dry at room temperature. The spectrometer was operated in reflection mode, and P14R ([M+H+]<sup>+</sup> 1533.85) and angiotensin II ([M+H+]<sup>+</sup> 1046.54) (Sigma, St. Louis, MO, USA) were used as external calibrants.
