Serum Stability Assay

The serum stability of the peptides was tested using human male serum (Sigma, USA), as previously described (Chan et al., 2011). All peptides were tested at a final concentration of 6.6 μM after dilution from a stock solution at 200 μM. The serum was centrifuged at 17,000g for 10 min to remove the lipid component, and the supernatant incubated at 37°C for 15 min, prior to conducting the assay. The peptides were incubated in serum or phosphate-buffered saline (PBS) at 37°C, and aliquots were taken at 0, 3, 8, and 24 h (40-μl aliquots were taken in triplicate). The aliquots of serum were quenched with 40 μl of 6 M urea and incubated for 10 min at 4°C. The aliquots were then quenched with 40 μl of 20% trichloroacetic acid (TCA) and incubated for another 10 min at 4°C to precipitate serum proteins. Finally, samples were centrifuged at 17,000g for 10 min, and 90 μl of the supernatant was analyzed on reversed-phase HPLC using a Phenomenex Jupiter Proteo C₁₂ analytical column (150 mm × 2.00 mm, 4 μm, 90 Å, Phenomenex, Torrance, CA, USA) and a linear gradient of solvent B [90% acetonitrile/10% H₂O, 0.045% trifluoroacetic acid (TFA)] at a flow rate of 0.4 ml/min using an Agilent 1260 Infinity system (Agilent Technologies, Hanover, Germany). The control samples contained equivalent amounts of the peptides in PBS and were subjected to the same treatment procedure. The stability at each time point was calculated based on the area of the RP-HPLC peaks following incubation with serum at 214 nm as a percentage of the area of 0-h serum-treated peptides (Chan et al., 2013).