*5.5. Sample Preparation for Proteomic Analysis*

To the cell lysates, four microliters of 10 mM dithiothreitol (DTT) were added to reduce the disulfide bonds and incubated at 56 ◦C for 1 h. After incubation with DTT, 40 μL of iodoacetamide (IAA) was added for the alkylation of the cysteines for 1 h in the dark at room temperature. After incubation, 2.5 mL of ammonium acetate and 20 μL of trypsin (Sigma Aldrich, St. Louis, MO, USA) were added enough to have an enzyme:protein ratio of 1:50 and incubated at 37 ◦C overnight. The reaction was stopped by adding 30 μL of 100% acetic acid. Samples volume were reduced in a SpeedVac (Hetovac VR-1, Heto Lab Equipment, Allerød, Denmark) until the volume has lowered to 50 μL and were further desalted on in-house manufactured stop-and-go extraction tips (Stage-Tip) with three SDB-XC (styrene-divinylbenzene, Empore, 3M, Royersford, PA, USA) membranes as previously described [147]. The stage-tips were initially conditioned with 100% methanol followed by 0.1% formic acid. Samples were applied to the stage-tips and the peptides bound to the membranes were eluted with 50% acetonitrile, 0.1% formic acid. The eluate was lyophilized for further analysis in the mass spectrometer.

#### *5.6. Mass Spectrometry Analysis*

Dried samples were resuspended in 0.1% formic acid and analyzed on LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, Bremen, Germany) coupled to an EASY II nano liquid chromatographer (Thermo Scientific) using the shotgun approach. The spectrometer was equipped with a nanospray source connected to an in-house prepared analytical column (10 cm × ID 75 μm × OD 360 μm) packaged with 7 cm of 5 μm C18 resin (Jupiter, Phenomenex, Torrance, CA, USA). Precolumn (7 cm × ID 75 μm × OD 360 μm) was also prepared in-house packed with 5 cm of 10 μm C18 resin (Acqua, Phenomenex). The LC-MS/MS analyses were carried out by injecting 5 μg of the peptide extract and the peptides eluted from the column with a gradient of 5–40% in 100 min of solvent B (acetonitrile 90%, formic acid 0.1%) at a flow rate of 200 nL/min. The nanospray source was operated at 1.8 kV. The peptide mixture was analyzed by the acquisition of spectra in the full scan mode at a resolution of 30,000 for the determination of molecular masses (MS) of up to 10,000 Da. The 10 most intense peaks were automatically selected via data dependent acquisition (DDA) for the subsequent acquisition of spectra of the ions product to MS/MS for amino acid sequence determination at a resolution of 7500, a maximum injection time of 30 ms, a range of 200 to 2000 *m*/*z*, and a dynamic exclusion of 70 s. Protein identification was performed using Mascot (Matrix Science, version 2.4.0, Boston, MA, USA), MaxQuant (version 1.4.1.2, www.maxquant.net accessed on 1 October 2019), and Peaks Studio (version 10, Bioinformatic Solutions Inc, Toronto, Canada) against the *Homo sapiens* database downloaded from the Uniprot in March 2019 (www.uniprot.org

accessed on 1 October 2019). As search parameters, oxidation of methionine was set as a variable modification and carbamidomethylation of cysteine was set as a fixed modification. Searches were performed with mass error tolerance of 10 ppm for MS and 0.3 Da for MS/MS, and trypsin was selected as the proteolytic enzyme used in the digestion of proteins, and up to two miscleavages were allowed. Mass spectrometry-based proteomic analysis of MCF7 and MDA-MB-231 cell lysates, treated or not treated with low or high doses of *B. jararaca* venom were performed in triplicate. All raw data files for these analyses were uploaded and are available at: http://massive.ucsd.edu/MSV000084138/ accessed on 5 July 2021.
