*2.1. Purification of TmC4-47.2 Toxin from Thalassophryne maculosa Venom*

Initially, the solution of *T. maculosa* venom chromatographed on a C18 column coupled to a high-pressure liquid chromatography system (Figure S1) generated 3 fractions, with the presence of the toxin mainly in fraction 2 that was subsequently re chromatographed 3 times on a reverse-phase C8 column. The first passage of fraction 2 through the C8 column generated 5 new fractions, with the toxin present in fractions 3, 4, and 5 (Figure S2). The second passage through the C8 column of the 3 previous pooled fractions generated 6 more fractions with the protein present only in fractions 3 to 5 (Figure S3).

These 3 pooled fractions were finally rechromatographed on C8 and generated 3 fractions with the toxin with a molecular mass of 15 kDa as shown on the 12% SDS-PAGE gel (Figure 1A). To determine the exact mass and purity of the toxin obtained, we collected the 3 fractions and applied 10 μL of the toxin to an LC/MS system by direct infusion and the deconvolution of the mass spectrum revealed two distinct proteins with very close molecular masses, one with 15,135.9 and the other with 15,634.3 Da, as observed in Figure 1B. Next, the number of cysteines in each protein was determined in the reduced and alkylated samples, a process that adds 57 Da to each cysteine residue present in the native proteins. We can observe in Figure 1C that the treatment generated proteins with masses of 15,484.8 and 15,982.8. By the difference of the masses of the pure samples and the reduced/alkylated samples, we prove the presence of 6 cysteine residues in each of the proteins according to Yang, Liu, and Liu [18].

**Figure 1.** The raw venom of *Thalassophryne maculosa* was submitted to a sequence of fractionation in a high-pressure liquid chromatography (HPLC) system using a C18 followed by C8 reversed phase HPLC columns, according to the scheme in (**A**) (top left). The gradient applied was 20–80% of buffer B in 35 min, in a 1 mL min−<sup>1</sup> flux. The absorbance was measured at 214 nm and the last pooled fractions containing the isolated toxin are presented in the chromatogram and highlighted in the 12% SDS-PAGE gel with a molecular mass of 15 kDa. 10 μL of the toxin collected from the last 3 fractions were applied to an Liquid Chromatography Mass Spectrometry (LC/MS) system by direct infusion to determine the mass and purity (**B**). The number of cysteines in each protein was determined by LC/MS in the reduced and alkylated samples (**C**).
