T1426

Icariin and compounds 1–9 were assayed against bovine trypsin (Sigma-Aldrich, T1426) at 1x and 10x the IC₅₀ concentration. Compounds 3 and 7 were also assayed at 50x and 250x the IC₅₀ concentration. In assay buffer (50 mM Tris-HCl, 20 mM CaCl₂, pH 8.19 with or without 0.5% igepal), 0.002 mg freshly made trypsin is incubated with drugs for 5 minutes at 25 °C. 1 mM of substrate (Nα-benzoyl-L-arginine 4-nitroanilide hydrochloride, Sigma-Aldrich, B3133) is added to start the reaction. Final reaction volume is 100 μl. Product formation is monitored by increasing absorbance at 410 nm at 25 °C for 30 minutes in kinetic mode using a SpectraMax M5 plate reader (Molecular Devices). All values were measured in duplicate.
To assay the effect of increasing PDE5 concentration on icariin and icariin analogs, the PDE5 inhibition assay described in the methods section was done with modifications. Compounds at approximately 1x the PDE5 IC₅₀ concentration were incubated with 1 or 2 nM PDE5, and GMP production was measured as described. All values were measured in triplicate.

Casein and BW-WM protein were added Trizma buffer (50 mM, pH 8.0) and hydrolysed using trypsin from bovine pancreas (T1426 from Sigma) at 45 °C for 4 h as recommended by Shalaby et al. [37 (link)]. The two blue whiting protein hydrolysates (BW-HA, BW-HP) were not hydrolysed with trypsin prior to analyses. Protein in hydrolysates were quantified on the Cobas c111 system (Roche Diagnostics GmbH, Mannheim, Germany) using the TP2 kit from Roche. Renin inhibition was measured using the Renin Assay Kit (MAK157, from Sigma-Aldrich) as described in the user manual. ACE-inhibition was measured using the method by Shalaby et al. [37 (link)], as previously described [20 (link)].

Renin activity assay was performed as previously described.^{9 (link)} Briefly, renin activity in urine and renal inner medulla was determined by the delta value of the Ang I generation using an ELISA kit from the sample incubating at 4 °C and 37 °C for 1 hour, respectively. Total renin content was measured with excessive angiotensinogen plus trypsinization and active renin content with excessive angiotensinogen. Urine and tissue samples were spiked with 1 µmol/L synthetic renin substrate tetradecapeptide (R8129; Sigma-Aldrich, St Louis, MO). After incubation at 37 °C for 18 hours, Ang I generation was assayed by using an Ang I enzyme immunoassay kit (S-1188; Peninsula Laboratories International, SanCarlos, CA), according to the manufacturer’s instructions. The values were expressed as nanograms per milliliter per hour of generated Ang I. For measurement of total renin content, trypsinization was performed to activate prorenin to renin.^{17 (link)} The samples were incubated with trypsin derived from bovine pancreas (100 g/L, T1426, Sigma-Aldrich) in 37 °C for 18 hours. The reaction was then terminated with soybean trypsin inhibitor (100 g/L, T6522; Sigma-Aldrich) at 37 °C for 1 hour. Renin activity was determined in the native condition, active renin content with excessive angiotensinogen, and total renin content with excessive angiotensinogen plus trypsinization.

Platelet proteomics analyses were performed, based on procedures reported earlier (13 (link), 15 (link), 23 (link)), with modifications. Well purified platelet samples (5 × 10⁸ platelets) were prepared in SDS lysis buffer, and protein concentrations were determined using a bicinchoninic acid assay (Pierce, Thermo-Fisher Scientific, Bremen, Germany). Cysteines were reduced by 30 min incubation at 56 °C with 10 mm dithiothreitol; free sulfhydryl groups were alkylated with 30 mm iodoacetamide for 30 min at room temperature. Samples of 150 μg protein were then processed using filter-aided sample preparation (FASP) with a 30 kDa molecular weight cut-off spin filter (24 (link), 25 (link)).
Proteins were digested in 50 mm triethylammonium bicarbonate (TEAB), 200 mm guanidinium hydrochloride, and 2 mm CaCl₂, pH 8.0 in the presence of trypsin (1:20 w/w, T-1426, Sigma, St. Louis, MO) at 37 °C. After incubation for 7 h, digested peptides were collected by centrifugation at 13,800 × g for 25 min. Filters were washed with 50 μl of 50 mm TEAB and 50 μl of LC-MS grade water to increase peptide yield. Trypsin digestion was monitored by monolithic RP HPLC as described (23 (link)). After lysis and digestion with trypsin, samples were used for either label-free proteome analysis, or iTRAQ-based (phospho)proteome analysis.

Casein and cod protein meals were added Trizma buffer (50 mM, pH 8.0) and hydrolyzed using trypsin from bovine pancreas (T1426, from Sigma-Aldrich) at 45 °C for 4 h as recommended by Shalaby et al. [42 (link)]. ACE-inhibition was measured using the method by Shalaby et al. [42 (link)], as previously described [43 (link)]. Renin inhibition was measured using the Renin Assay Kit (MAK157, from Sigma-Aldrich) as described in the user manual. Protein in hydrolysates were quantified on the Cobas c111 system (Roche Diagnostics GmbH, Mannheim, Germany) using the TP2 kit from Roche.