## 5 Materials and Methods
### Proteomic Analysis
#### 5.2.3 In-Solution Digestion and Mass Spectrometry Analysis  
Analysis #1  
One milligram of the _D. quadriceps_ venom was solubilized in 1.0 mL of 0.1% formic acid, and a-250 \(\upmu\)L aliquot was concentrated using a 10-kDa cut-off centrifugal filter (Amicon(r) Ultra-4 Centrifugal Filter Unit, Sigma-Aldrich, St. Louis-MO, USA). The retentate (peptide fraction >10 kDa) was dried and submitted to in-solution digestion according to [PERSON] and colleagues [67]. Briefly, the venom protein samples in 8 M urea were reduced using TCEP-HCl (20 mM Tris(2-carboxyethyl)phosphine hydrochloride, TCEP) at room temperature (RT) for 1 h and then alkylated (10 mM iodoacetamide, IAA) at RT for 1 h in the absence of light. Thereafter, the samples were diluted to a urea concentration of <2 M (with 100 mM Tris-HCl, pH 8.5), followed by the addition of 10 \(\upmu\)L proteomic-grade trypsin (10 ng/uL in 100 mM Tris-HCl, pH 8.5). The incubation was performed at 30 \({}^{\circ}\)C overnight, and the enzymatic reaction was stopped by adding 50% ACN/5% TFA. The sample was lyophilized, desalted, and concentrated using a ZipTip(r) C-18 pipette tips (Millipore Co., Burlington, MA, USA). We repeated this step twice, pooling the material of the same samples and drying. Finally, we resuspended the dried material in 5 \(\upmu\)L of 0.1% formic acid, and 1 \(\upmu\)L was automatically injected in the EASY Nano LCII system (Thermo Fisher Scientific) into the top 5 cm of a 10-\(\upmu\)m Jupiter C-18 trap column (100 \(\upmu\)m I.D. \(\times\) 360 \(\upmu\)m O.D.) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham,-MA, USA). The chromatographic separation was performed on a 15-cm long column (75 \(\upmu\)m I.D. \(\times\) 360 \(\upmu\)m O.D.) packed in-house with 3-\(\upmu\)m ReproSil-Pur C-18 beads (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) in a binary system: (A) water:formic acid (999:1, _v/v_) and (B) ACN:formic acid (999:1, _v/v_). The column was eluted at a constant flow rate of 300 nL min\({}^{-1}\) with a 5% to 35% linear gradient of solvent B for 75 min. The spray voltage was set at 2.2 kV, and the mass spectrometer was operated in the data-dependent mode, in which one full MS scan was acquired in the _m/z_ range of 300-1,600, followed by an MS/MS acquisition using the collision-induced dissociation of the ten most intense ions from the MS scan. The MS spectra were acquired in the Orbitrap analyzer at 30,000 resolution (at 400 _m/z_), whereas the MS/MS scans were acquired in the linear ion trap. The minimum signal threshold to trigger the fragmentation event, isolation window, activation time, and normalized collision energy were set to 1000 cps, 2 _m/z_, 10 ms, and 35%, respectively. We applied a dynamic peak exclusion list to avoid the same _m/z_ of being selected for the next 20 s.  
Analysis #2  
One hundred micrograms of the _D. quadriceps_ venom was analyzed by in-solution digestion, as described in the previous section. The solution of tryptic-digested venom polypeptides was lyophilized, desalted, and concentrated using a ZipTip(r) C-18 pipette tips (Millipore, Co., Burlington, MA, USA). This step was repeated twice, and the resulting peptide solutions were pooled and dried. Finally, the dried tryptic peptides were solubilized with 5 \(\upmu\)L of 0.1% formic acid, and 1 \(\upmu\)L was automatically injected into a 15 cm x 50 \(\upmu\)m Acclaim PepMap(tm) C-18 column (Thermo Fisher Scientific, Waltham,-MA, USA) assembled in a nano chromatographer (EASY-nLC 1200 system, Thermo Fisher Scientific, Waltham,-MA, USA) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham,-MA, USA). A binary solvent system was used: (A) water:formic acid (999:1, _v/v_) and (B) ACN:water:formic acid (800:199:1, _v/v/v_). The peptides were eluted at a constant flow rate of 300 nL/min with a 4% to 40% linear gradient of solvent B for 100 min.