RP-HPLC analysis
The peptides were purified with an HP1100 HPLC system (Agilent
Technologies, Inc) on a column (ZORBAX Eclipse Plus C18 RRHD 2.1x50 mm,
1.8 μm, Agilent Technologies, Inc) that had been equilibrated in 98 %
of solution A (0.1 % v/v TFA in ultrapure water). The concentration of
solution B (TFA, 0.1 % v/v in pure ACN) in the mobile phase was
increased to 55 % in 15 minutes and reached 80 % of solution B in 0.1
minutes. The column was washed for an additional 0.5 min in 80 % of
solution B and was then equilibrated under the initial conditions for a
new separation. The flow rate was 0.35 mL/min and the absorbance was
monitored at 210 nm during the chromatographic separation. The
temperature of the column was maintained at during all chromatographic
separations.