*4.4. SDS-PAGE and LC-MS/MS Analysis of C. iheringi*

*C. iheringi* venom was analyzed by SDS-PAGE (12% acrylamide resolution and Pierce, USA) under reducing conditions [67]. After protein separation by electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 (GE Healthcare) (0.1% coomassie R-250, 40% ethanol; 10% acetic acid) and bleached with a bleach solution (20% methanol, 5% acetic acid) for which a molecular weight standard for proteins (Middle Range protein Ladder, ready-to-use—Thermo Fisher Scientific, Waltham, MA, USA) was used. Electrophoresis occurred at room temperature, using a voltage of 150 V and a current of 40 mA per gel. The regions of interest were cut out from the gel manually with the aid of a sterile scalpel on a clean surface and placed in Eppendorf tubes washed with methanol and Milli-Q water. To remove the Coomassie dye, the gel bands containing the venom proteins were incubated under agitation for 10 min in a bleaching solution (50% acetonitrile; 25 mM NH4HCO3; pH 8), followed by a 10 min rest; the procedure was repeated until complete dye removal. After removing the dye, the fragments were washed with 100% acetonitrile and dried in a vacuum centrifugation system for 25 min.

During enzymatic digestion, the gels were rehydrated with trypsin solution (10–15 μg/mL trypsin; 25 mM NH4HCO3). The enzymatic solution was incubated (4 ◦C), the samples remained in an ice bath for 30 min, then 25 mM NH4HCO3 was added and kept at 37 ◦C for 20 h. After this period, the digestion tube supernatant was transferred to a methanol treated Eppendorf tube. To extract the peptides, the gel fragments were covered with a 50% acetonitrile solution; 5% TFA, and gently homogenized for 30 min, the supernatants were reduced to a volume of 5 μL in a vacuum centrifugation system and then purified in C18 ZIP TIPs micro columns following the manufacturer's instructions.

The total venom was filtered through a 0.22-micron filter solubilized in ammonia bicarbonate (Sigma–Aldrich, St. Louis, MO, USA # A-6141) pH 8.0, containing a phosphatase inhibitor. The protein content of the samples was quantified using the BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). The protein disulfide bridges were reduced by adding 10 mM DTT (Sigma–Aldrich, St. Louis, MO, USA # D-5545), followed by incubation for 30 min at 56 ◦C. The proteins were alkylated with 55 mM IAA (Sigma–Aldrich, St. Louis, MO, USA # I-1149), at room temperature, in the dark, for 30 min. Protein digestion was performed using trypsin (Promega # V5111), in a 1:20 ratio, at 37 ◦C, for 18 h. Columns C 18 (Harvard Apparatus, Holliston, MA, USA) were used for cleaning and the desalination of the sample. Digestion products were loaded onto a tandem system containing a pre-column that separates the products and passes the effluent automatically into an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The enzymatic digestion with trypsin and desalination of the gel bands, as well as the crude venom were carried out at CEFAP: Center for research support facilities at the University of São Paulo, USP.

#### *4.5. LC-MS/MS Data Analysis*

After analyzing the samples by LC-MS/MS, the raw data were collected and passed through the Mascot platform (Matrix Science, Boston, MA, USA), using carbamidomethylation and methionine oxidation as variable modifications. The resulting files were exported in the .dat extension and processed in the Scaffold Q + software version 4.0 (Proteome Software, Portland, OR, USA), using as selection criteria of the presence of at least three peptide fragments, a probability rate of 95% for protein identification and a false discovery rate (FDR) of 5%. In addition, for the analysis carried out on the Scaffold Q + and Mascot platforms, a FASTA database was built containing the amino-acid sequences obtained through the predicted proteins from assembled transcripts of the transcriptome venom gland, together with sequences of possible sample contaminants such as trypsin and human keratin, to avoid alignment and coverage errors. Based on the data on the probability of protein identification and percentage of coverage, a contig for each band of venom was identified.
