*4.4. Proteomic Analyses*

The proteins (~10 ug) contained in the patient blisters and the serum of normal volunteers were submitted to reduction with 10 mM dithiothreitol (DTT) in 0.1 M ammonium bicarbonate and alkylation with 50 mM iodoacetamide in 0.1 M ammonium bicarbonate (both room temperature for 0.5 h). The samples were then digested overnight at 37 ◦C with 1 μg trypsin in 50 mM ammonium bicarbonate. The samples were acidified with acetic acid to stop digestion and were then spun down. The supernatant was evaporated to 20 μL for liquid chromatography–mass spectrometry (LC–MS) analysis.

The LC–MS system utilized for proteomic analyses was a Thermo Electron Q Exactive HF mass spectrometer system with an Easy Spray ion source connected to a Thermo 75 μm × 15 cm C18 Easy Spray column (through pre-column). Samples of 1 μg were injected and the peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.3 μL/min over 2.0 h. The nanospray ion source was operated at 1.9 kV. The analysis produces approximately 25,000 MS/MS spectra of ions ranging in abundance over several orders of magnitude. The data were then analyzed by database searching using the Sequest search algorithm against Uniprot Human. An analysis of the spectra generated was performed using carbamidomethylation on cysteine as a fixed modification, with oxidation of methionine as a variable modification. For the analysis of the results and validation of peptide and protein identifications, data obtained were exported to Scaffold (version 4.3.2, Proteome Software Inc., Portland, OR, USA).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/toxins13110800/s1, Supplementary Table S1: Protein identification in blister fluids of *B. atrox* snakebite patients and serum of control volunteers by LC-MS.

**Author Contributions:** Conceptualization, S.N.C.G., W.M.M., A.M.M.-d.-S. and J.W.F.; methodology, S.N.C.G., M.C., L.A.F.-d.-S., J.-J.P. and N.E.S.; formal analysis, J.A.G.S., A.G.C., L.C.L.F. and F.H.W.; resources, J.A.G.S., H.N.S.I. and M.F.S.; writing—original draft preparation, S.N.C.G.; writing review and editing, A.M.M.-d.-S., J.W.F. and W.M.M.; supervision, A.M.M.-d.-S. and J.W.F.; funding acquisition, A.M.M.-d.-S. and J.W.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Fundação de Amparo à Pesquisa de São Paulo (FAPESP 2016/50127-5; 2017/24546-3; 2018/13108-8; 2019/08208-6); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant number 303958/2018-9), and Fundação de Amparo à Pesquisa do Estado do Amazonas-FAPEAM (PRO-ESTADO; FAPEAM PPSUS 287/2013); Office of Research Core Administration, University of Virginia. A.M.M.-d.-S. and W.M.M. are CNPq productivity fellows.

**Institutional Review Board Statement:** Ethical approval for human information collection was obtained from the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (approval number CAAE 19380913.6.0000.5016/2013 approval date 13 December 2013).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

### **References**

