*4.3. Reduction, Alkylation, and Digestion of TmC4-47.2 Toxin*

The purified toxin was used for the identification of disulfide bridges, according to Yang, Liu, and Liu [18]. The toxin was reduced by 10 μL of 5 mM DTT in 25 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) and heated at 60 ◦C for 30 min. For alkylation, 10 μL of 100 mM iodoacetamide in 25 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) was added and kept for 30 min in the dark. For enzymatic digestion, 2 μL of ultra-grade trypsin (Proteomics grade; Sigma, St. Louis, MO, USA) solution at 40 ng·μL−<sup>1</sup> was added to 8 <sup>μ</sup>L of the toxin solution and incubated 18 h at 37 ◦C. The samples were analyzed using a nanoflow LC/MS/MS system customized with a PepFinder Kit. Aliquots of 10 μL were initially charged onto a reversed-phase peptide trap column in a of 10 μL/min flow rate for 3 min. Then peptides were eluted and partitioned on a reversed-phase capillary column (PicoFritTM; 5 μm BioBasic® C18, 300 Å pore size; 75 μm × 10 cm; tip 15 μm, New Objective, Woburn, MA, USA). Solution A comprised 0.1% formic acid, and Solution B acetonitrile and 0.1% formic acid. The flow rate began at 100% A at 10 μL/min for 3 min. Next, it was raised to 70 μL/min for 6.9 min at 100% A and the gradient commenced at 100% A and 0% B. The gradient was increased to 50% B in 60 min, then to 90% B in 5 min and later diminished to 0% B in 5 min and sustained at 100% A for 10 min. The complete program duration was 110 min. By the PepFinder Kit, the flow was separated in a 1:100 ratio. Hence, the factual flow rate injected into the mass spectrometer was 0.5 μL/min. The HPLC was coupled to a Finnigan LCQ Deca XP Plus ion trap mass spectrometer supplied with a nanospray ionization font. Spray voltage was established at 2.5 kV, and the equipment was managed in a data-dependent method, in which one MS scan was captured in the 300−1600 m/z range followed by MS/MS acquisition using collision-induced segregation of the 10 most intense ions from the MS scan. Dynamic peak exclusion was used to avoid the same m/z being chosen for the following 120 s. Tandem mass spectra were extracted by Xcalibur software (version 2.0; Thermo scientific, Waltham, MA). The subsequent MS/MS spectra were searched through a MASCOT search engine (Matrix Science, London, UK) against the NCBI NR database in the taxa Chordata with a 1.20 Da parent tolerance and 0.60 Da fragment tolerance. Iodoacetamide derivatives of cysteine and oxidation of methionine were detailed in MASCOT as fixed and variable modifications, respectively. Scaffold (version Scaffold\_2\_04\_00, Proteome Software Inc., Portland, OR) was used to confirm MS/MS-based peptide and protein description. Peptide distinguishing were credited if they exceeded certain database search engine thresholds. MASCOT descriptions required ion scores higher than the associated identity scores and 20, 30, 40, and 40 for singly, doubly, triply, and quadruply charged peptides. X! Tandem identifications needed at least –Log (Expect Scores) scores of greater than 2.0. Protein identifications were considered if they had at least 2 identified peptides.
