Fraction collector model 2

Peptide purification was performed by semi-preparative RP-HPLC. Separation of peptides was performed on an HPLC system (Waters Corp., Mildford, MA, USA) equipped with two pumps (module Delta 600), a pump controller (module 600), an autosampler (module 717 plus), and a diode array detector (module 996). The data-processing software was Empower 2 (Waters Corp.). A 250 × 21.5 mm Hi-Pore 318 reverse phase column (BioRad, Hercules, CA, USA) was used. Peptide fractions were dissolved in solvent A (water: trifluoroacetic acid (TFA), 1000:1, v/v) at a concentration of 30 mg/mL. Fractions (400 μL) were injected and eluted at a flow rate of 10 mL/min, with a linear gradient of solvent B (acetonitrile:TFA, 1000:0.8, v/v) in A, going from 5% to 45% B in 50 min. Each chromatographic run was repeated 15–20 times and the subfractions were collected automatically with a Fraction Collector (Model II, Waters Corp.). The collection times were from min 6 to 12 for F1, min 12 to 16 for F2, min 16 to 22 min for F3, and min 22 to 35 for F4. The collected fractions were pooled, freeze-dried, and stored at −20 °C until further analysis. Quantification of peptides in each subfraction was performed by the Quantitative Colorimetric Peptide Assay, according to the manufacturer’s protocol.