*5.11. Validation of HF3 Substrates in Human Plasma by Incubation with HF3*

Apolipoprotein A-IV, clusterin, α-2-antiplasmin, kininogen, and transthyretin (2 μg; Sigma-Aldrich, St. Louis, MO, USA) were incubated with HF3 (200 ng) (1:10 (*w/w*) enzymeto-substrate ratio) in 0.05 M Tris-HCl, pH 8.0, 1.0 mM CaCl2 for 2 h at 37 ◦C. Apoliprotein E (4 μg; Sigma-Aldrich, St. Louis, MO, USA) was incubated with HF3 (400 ng) (1:10 (*w/w*) enzyme-to-substrate ratio) in the same buffer. A sample of each protein was incubated without enzymes under identical conditions. Reactions were stopped by adding a Laemmli sample buffer, and subjected to SDS-PAGE.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10.3 390/toxins13110764/s1, Supplementary Tables S1–S8: Peptides identified by LC-MS/MS analysis of the peptide fraction of human plasma after incubation with HF3. Supplementary Table S9: Peptides identified by LC-MS/MS analysis of the peptide fraction of human fibrinogen after incubation with HF3.

**Author Contributions:** Conceptualization, S.M.T.S.; experimental design, A.Z., L.B. and S.M.T.S.; data collection: A.F.A.C., A.K.O., A.Z., L.B. and M.C.M.; data analysis, A.F.A., A.F.A.C., A.K.T., A.Z., L.B. and S.M.T.S.; manuscript writing, S.M.T.S.; funding acquisition, S.M.T.S.; manuscript revising, all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by São Paulo Research Foundation (FAPESP 2011/16623-1; 2011/08514-8; 2013/07467-1; 2020/12317-2) and the National Council for Scientific and Technological Development (CNPq 308133/2015-3 to SMTS).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All mass spectrometry peptidomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org), via the PRIDE partner repository [110] with the dataset identifier: PXD027997.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

