*2.2. Peptidomic Profiling of Plasma Samples Incubated with HF3*

The peptide fractions of control and treated samples of P(W), P(Alb-D), P(20-MAP-D), and P(LAP-E) were subjected to LC-MS/MS, and analyses of peptide identification and spectra counting were carried out by: (i) a Mascot database search and validation of results using the PeptideProphet and ProteinProphet tools of the TPP platform (the results of this approach were designated as 'Mascot/TPP'); and (ii) de novo sequencing, a database search, and validation of results by Peaks Studio 7 program (the results of this approach were designated as 'PEAKS') (Supplementary Tables S1–S8).

Figure 3 shows Venn diagrams comparing the total numbers of plasma proteins identified as cleaved by HF3 using both identification approaches. The analysis of whole plasma [P(W)] incubated with HF3 showed 36 proteins as cleaved by HF3 using both identification approaches (Table 1), whereas five proteins were identified exclusively by PEAKS. In the plasma depleted of albumin [P(Alb-D)], we identified 21 proteins cleaved by HF3 using both identification approaches (Table 2), including three proteins exclusively identified by Mascot/TPP, and two proteins exclusively by PEAKS. In the plasma depleted of the 20 most abundant proteins [P(20-MAP-D)], 30 proteins were identified as cleaved by HF3 using both identification approaches (Table 3), with one protein identified exclusively by Mascot/TPP, and five by PEAKS. The incubation of HF3 with the plasma enriched of low-abundance proteins [P(LAP-E)] revealed 38 substrates by both approaches (Table 4), and 16 proteins identified only by PEAKS.

**Figure 3.** Summary of numbers of plasma proteins identified as degraded by HF3, by LC-MS/MS analysis of the peptide fraction, using four different plasma preparations. Comparison of results obtained using Mascot/TPP and PEAKS Studio 7 for peptide identification.

**Table 1.** HF3 substrates revealed by the analysis of P(W) peptide fraction by LC-MS/MS. Only proteins that showed peptides identified exclusively in the sample of plasma treated with HF3 were considered. The boldface indicates the ten most degraded proteins according to the number of identified spectra.



\* Only spectra identified exclusively in the HF3-treated samples were considered.

**Table 2.** HF3 substrates revealed by the analysis of P(Alb-D) peptide fraction by LC-MS/MS. Only proteins that showed peptides identified exclusively in the sample of plasma treated with HF3 were considered. The boldface indicates the ten most degraded proteins according to the number of identified spectra.


\* Only spectra identified exclusively in the HF3-treated samples were considered.

**Table 3.** HF3 substrates revealed by the analysis of P(20-MAP-D) peptide fraction by LC-MS/MS. Only proteins that showed peptides identified exclusively in the sample of plasma treated with HF3 were considered. The boldface indicates the ten most degraded proteins according to the number of identified spectra.


\* Only spectra identified exclusively in the HF3-treated samples were considered.

**Table 4.** HF3 substrates revealed by the analysis of P(LAP-E) peptide fraction by LC-MS/MS. Only proteins that showed peptides identified exclusively in the sample of plasma treated with HF3 were considered. The boldface indicates the ten most degraded proteins according to the number of identified spectra.



**Table 4.** *Cont.*

\* Only spectra identified exclusively in the HF3-treated samples were considered.

Regarding the different methods of depletion of abundant plasma proteins and enrichment of low-abundant proteins, the four-way Venn diagrams displayed in Figure 4 show that the analysis of the peptide fraction of P(LAP-E) provided a higher number of identified HF3 substrates, with 11 substrates indicated by the approach of Mascot/TPP, and 19 substrates indicated by PEAKS. Unexpectedly, the analysis of P(W) incubated with HF3 also provided results on HF3 substrates which were identified exclusively by this method (six substrates by the Mascot/TPP identification approach, and seven substrates by PEAKS), however, it is worth mentioning that the initial amount of proteins used in the incubation of P(W) with HF3 was higher (200 μg) compared to 50 μg for P(Alb-D), P(20-MAP-D), and P(LAP-E). This result can be attributed to the fact that there was less manipulation of the whole plasma sample compared to other approaches, which involved at least one more protein depletion/enrichment step, performed separately (biological replicates), before incubation with the proteinase.

In general, in each method of preparation of human plasma for incubation with HF3, most proteins considered as a target of HF3 for proteolysis were identified by both bioinformatics approaches used, reinforcing the results (Figure 4). Interestingly, both approaches resulted in a higher number of substrates identified in the P(W) and P(LAP-E) plasma samples. Moreover, although the PEAKS approach revealed a higher number of HF3 substrates (70), 53 proteins were identified by both bioinformatics approaches.

**Figure 4.** Summary of proteins identified as HF3 substrates. Upper panels: Four-way Venn diagrams of substrate identification using four samples of human plasma, and two methods of protein identification. Lower panel: Summary of proteins identified by each method of protein identification.

#### *2.3. Proteins Degraded by HF3 in the Human Plasma*

Table 1 shows the list of 41 proteins identified as substrates of HF3 in the P(W) sample. The ten most degraded substrates are proteins involved in functions in the coagulation cascade, complement system, protein transport, and proteinase inhibition.

The identification of substrates of HF3 in the plasma depleted of albumin resulted in only 26 proteins (Table 2). The ten most degraded substrates are proteins involved in functions in the coagulation cascade, complement system, lipid transport, hormone transport, and proteinase inhibition.

In the plasma depleted of the 20 most abundant proteins, 36 proteins were identified as degraded by HF3 (Table 3), whereas in the plasma submitted to enrichment of the low-abundant proteins, 54 proteins were detected as cleaved by HF3 (Table 4). Despite the higher number of substrates identified in the latter, the ten most degraded proteins in both types of plasma preparations were nearly the same. Considering the bioinformatics approaches applied for peptide identification, overall, when using PEAKS, higher numbers of spectra were identified for all degraded proteins.

Overall, a total of 61 proteins (Table 5) were detected as cleaved by HF3, including 18 that were identified in all types of plasma preparations: alpha-2-antiplasmin; alpha-2-HSglycoprotein; apolipoproteins A-I, A-II, A-IV, C-II, C-III, E, and L1; clusterin; complement C3; fibrinogen alpha and beta chains; inter-alpha-trypsin inhibitor heavy chains H2 and H4; kininogen-1; prothrombin; and transthyretin.


**Table 5.** 61 proteins identified as cleaved by HF3 in human plasma.

We further investigated the newly identified proteins as HF3 substrates in the human plasma. For this analysis, we selected some proteins that are commercially available and that were detected as cleaved by HF3 using LC-MS/MS analysis. The proteins were incubated at a 1:10 (*w/w*) enzyme-to-substrate ratio with HF3 for 2 h and subjected to SDS-PAGE (Figure 5). Apolipoprotein A-IV was completely degraded by HF3. Apolipoprotein E was almost completely degraded by HF3 to generate a fragment of ~15 kDa. After incubation with HF3, clusterin showed a slight reduction of molecular mass, indicating that HF3 may have promoted its limited proteolysis. HF3 also promoted the limited proteolysis of α-2-antiplasmin, resulting in fragments of ~68 kDa and 55 kDa. The 120 kDa band of high molecular weight kininogen was almost completely degraded by HF3, resulting in a stable fragment of ~68 kDa. In the case of transthyretin, its dimer and monomer bands remained unchanged after incubation with HF3.

**Figure 5.** Proteolytic activity of HF3 upon isolated plasma proteins. Apolipoprotein A-IV, apolipoprotein E, clusterin, α-2-antiplasmin, kininogen, and transthyretin were incubated at a 1:10 (*w/w*) enzyme-to-substrate ratio with HF3 for 2 h, as described in Materials and Methods, and subjected to SDS-PAGE. Arrows indicate the band of HF3. Numbers on the right and left indicate molecular mass marker mobility. Proteins were silver stained.

Figure 6 shows the protein-protein interaction network of 61 proteins cleaved by HF3 in the human plasma, visualized using STRING analysis [25], evidencing that most of them (53) have connected molecular functions related mainly to the activation and control of the coagulation and complement systems.

**Figure 6.** Protein-protein interactions of proteins identified as degraded in human plasma by HF3 according to the STRING database (61 proteins; 401 edges (expected 27); PPI enrichment *p*-value < 1.0−16). The connecting lines between protein nodes indicate protein-protein interactions.
