*5.5. Human Plasma Depleted of 20 Most Abundant Proteins*

Depletion of the 20 most abundant proteins from human plasma was performed with the ProteoPrep 20 Plasma Immunodepletion Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's instructions. Three independent experiments were carried out with plasma samples from three individuals. Briefly, samples of 8 μL of human plasma were diluted to 100 μL with phosphate-buffered saline (PBS) and loaded to a micro-spin column, previously equilibrated with PBS. After incubation for 20 min at room temperature, the non-bound protein fraction was recovered by centrifugation at 2000 rpm for 1 min and the flow-through, containing plasma depleted of the 20 most abundant proteins P(20-MAP-D), was collected in a clean tube. The remaining unbound proteins were washed twice by adding 100 μL of PBS, centrifuging, and collecting the wash in the same tube. The depletion procedure was repeated five times, and flow-throughs were pooled and then concentrated using a Centricon YM-3 filter (Millipore). After each depletion procedure, the micro-spin column was regenerated with 2 mL of 0.1 M Glycine-HCl, pH 2.5, and TWEEN 20 in order to elute bound proteins, and stored at 5 ◦C in 5 mL of PBS with the addition of 10 μL of ProteoPrep Preservative Concentrate. Human plasma depleted of 20 most abundant proteins was designated as P(20-MAP-D).

#### *5.6. Human Plasma Enriched in Low-Abundance Proteins*

The enrichment of low-abundant proteins in human plasma was performed with the ProteoMiner Protein Enrichment Kit (Bio-Rad) [37] according to the manufacturer's instructions. Three independent experiments were carried out with plasma samples from three individuals. In brief, 200 μL of human plasma was loaded onto the column previously conditioned with PBS, pH 7.4, and incubated for 2 h at room temperature. After centrifugation, the flow-through fraction was stored for further analysis. The fraction that bound to the resin, containing the low-abundant proteins, was eluted with 8 M urea containing 2% CHAPS. For desalinization, the proteins in solution were precipitated by the addition of eight volumes of cold acetone and one volume of cold methanol, and stored for 12 h at −20 ◦C. After centrifugation for 10 min at 14,000× *g* at 4 ◦C, the precipitate was washed

with cold methanol and resuspended in 60 μL of 100 mM NaOH and 340 μL of 50 mM HEPES, pH 7.5. Plasma enriched of low-abundant proteins was designated as P(LAP-E).

#### *5.7. Proteolytic Activity of HF3 on Human Plasma*

For each of the four types of human plasma preparations, three biological replicates were performed. For each experiment, P(Alb-D), P(20-MAP-D), and P(LAP-E); 50 μg were separately incubated with 0.5 μg HF3 (1:100; *w/w*) in 250 mM ammonium acetate containing CaCl2 1 mM for 2 h at 37 ◦C. P(W) (200 μg) was incubated with 2 μg HF3 under the same conditions. Samples of P(Alb-D), P(20-MAP-D), P(LAP-E), and P(W) were incubated without HF3, as a control, under identical conditions. Reactions were stopped by adding eight volumes of cold acetone and one volume of cold methanol, and incubated for 12 h at −20 ◦C. Peptide fractions (supernatant) were obtained by centrifugation at 14,000 g for 10 min at −4 ◦C, and subsequently dried using a SpeedVac concentrator. The protein fraction (precipitate) was separated and stored at −20 ◦C until use.

### *5.8. LC-MS/MS Analysis of the Plasma Peptide Fraction*

Previously to LC-MS/MS analysis, plasma peptide fractions containing hydrolysis products resulting from the proteolytic activity of HF3 were subjected to removal of traces of detergent using Macro Spin Columns (Harvard Apparatus). Samples were then desalted with Sep-Pak Light C18 (Waters) cartridges, vacuum dried, and resuspended in 20 μL of 0.1% formic acid. Aliquots of 10 μL were separated by RP-HPLC on an EASY-nLC II (Thermo Scientific, Waltham, MA, USA) using a column (75 μm i.d. × 10 cm) packed with 5 μm C18 beads (Phenomenex), and coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The gradient was 5–40% acetonitrile in 0.1 M formic acid over 90 min, at a flow rate of 300 nL/min. The mass spectrometer was operated in data dependent mode, in which one full MS scan was acquired in the m/z range of 400–2000 at 60,000 resolution, followed by MS/MS acquisition using high-energy collision dissociation of the six most intense ions from the MS scan, at 15,000 resolution. A dynamic peak exclusion was applied to avoid the same m/z of being selected for the next 25 s, using a ± 1 Da mass tolerance window around the precursor ion mass.
