Peptide-Based Drug Design

Bioactive Peptides in Invertebrate Immune Systems 19
3. Elute in a stepwise fashion with increasing percentages of acetonitrile prepared in
acidified water: 40 and 80% (see Note 8) with 5–10 times the hold-up volume of
the column (see manufacturer instructions).
4. Remove the organic solvent by lyophilization/freeze-drying or centrifugation
under vacuum.
5. Reconstitute the fractions in MilliQ water (Millipore) and keep them at 4 ◦ C
for immediate biological activity screening (see Note 9) or in the freezer
until use.
3.3.1.2. ULTRAFILTRATION (UF PRE-SPE)
1. Dilute the extract in a large volume of 0.1% TFA.
2. Filter the diluted extract according to the manufacturer’s recommendations.
3. Load the filtrate onto the equilibrated SPE cartridge and pursue the SPE procedure.
3.3.1.3. ULTRAFILTRATION (UF POST-SPE)
1. Filter the reconstitute fractions eluted during SPE (see Subheading 3.3.1.1.).
2. Dry under vacuum and store at -20◦C until use.
3.3.2. Purification by HPLC
In general, the first step of purification of the fractions eluted from SPE,
SPE-UF, or UF-SPE is subjected to RP-HPLC followed either by a second RP-
HPLC or by size exclusion chromatography (SEC-HPLC). The last step is often
a highly sensitive RP-HPLC using highly selected elution conditions (gradient
with a soft slope, low flow rate, and high sensitivity detection). However,
before finalizing purification, several steps may be requested (at least three
steps are needed to get a sufficient amount [1 nmol or 100 �g] of highly pure
[>98%] material for structural characterization either by Edman chemistry or
MS strategies (enzymatic digestion, on-line or off-line micro-RP-HPLC mass
fingerprints, MSn sequencing).
3.3.2.1. STEP 1: REVERSED-PHASE HPLC (SEE NOTE 10)
1. Load the sample on an appropriate RP column (internal diameter of the column
chosen according to the starting biological material available, 300 ˚A for granulometry,
internal diameter from 3.8 to 7 mm, a C18 or C8 column is recommended
for step 1). It is recommended to equilibrate the column with 2% acetonitrile in
acidified water (0.05 % TFA) to remove the hydrophilic molecules and to properly
stack the interesting material.
2. Elute the peptides with a linear gradient of acetonitrile in acidified water at a
flow rate between 0.8 and 1.3 mL/min depending of the internal diameter of the
column. Flow rate can be 0.8–1 mL/min if the column has 4.6 mm of internal
diameter and increased to 1.3 mL/min for a semi-preparative column (7 mm of