Purification of peptides
Peptides were purified by solid-phase extraction (SPE)
using LC-18 Silica-Based Packing (Sigma-Aldrich Co.) [15].
Columns were prepared by transferring at 0.1% TFA in acetonitrile
(ACN) – 5 times for 10 ml, then 0.1% TFA in ACN
and 0.1% TFA in water (1 : 1v/v) – 5 times for 10ml and 0.1%
TFA in water also 5 times for 10ml. Each time 100 μl of the
peptide was added to the mixture. As a liquid phase we
applied a linear gradient from 0-100% of phase B (phase A:
0.1% TFA in water, phase B: 0.1% TFA in ACN). After se pa -
ration the columns were regenerated by washing 5 timesfor 10 ml of 0.1% TFA in ACN, 5 times for 10 ml of MeOH
and 5 times for 10 ml for 0.1% TFA in water