Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Synthetic Calcium-Binding Peptides 179
and distill in the desired volume of HF keeping the reaction vessel at –4°C for 1 h.
Remove the HF under vacuum using the apparatus.
6. Extract the peptide resin three times with 25 mL diethyl ether to remove organic
impurties generated from side-chain deprotection.
7. Extract the resin three times with 25 mL glacial acetic acid to solubilize the peptide.
8. Dilute the acetic acid extract with at least 150 mL deionized water, transfer the
solution to a lyophilization flask, and lyophilize until dry.
3.2. Multiple Sequences from One Synthesis
This procedure is useful for synthesis of calcium-binding peptides that have
identical N- and C-termini (i.e., the helices), but different intermediate regions
(“loops”) as shown in Fig. 2 (3).
1. Stop the synthesis after the identical C-terminal residues have been added.
2. Divide the resin into four equal portions corresponding to 0.5 mmol scale each.
3. For each portion of resin, carry out the next series of amino acid couplings
according to the peptide sequence desired (10 residues in Fig. 2).
4. When complete, place a 0.16-mmol resin aliquot for each peptide in separate
polypropylene bags (2.5 cm × 3.5 cm) and thermally seal the bag. Mark each bag
with a distinguishing tag according to the sequence it contains (see Note 3).
5. Place the bags back in the synthesizer and carry out the remainder of the sequence.
6. Continue with step 4 (see Subheading 3.1.).
3.3. Peptide Purification
3.3.1. Sample Preparation
1. Dissolve 40–60 mg of lyophilized crude peptide in 3–4 mL of 50% aqueous
trifloroacetic acid (TFA).
2. After a maximum of the peptide has dissolved, spin the sample in a benchtop
centrifuge for 5 min. Transfer the clear supernatant to a clean tube.
3.3.2. HPLC Preparation
1. Connect the analytical reversed-phase column to the instrument along with the
50 µL injection loop.
2. Equilibrate the HPLC system with the desired solvents. Popular solvents for peptide
purification include 0.05% TFA/H2O (eluent A) and 0.05% TFA/CH3CN (eluent B).
3. Run one or two “blank” runs to ensure the column and system are clean. Use a
flow rate of 1.0 mL/min and a linear gradient of 5% eluent B/min. Monitor the
system at 210 nm.
4. Re-equilibrate the system with eluent A. Load 20 µL of the dissolved peptide
solution and inject onto column. Run a linear gradient of 2% eluent B/min.
5. Record the spectrum to determine the retention time (i.e., % eluent B) the peptide
mixture elutes. For the 34-residue calcium-binding peptides shown in Fig. 2 this
is typically near 35% B.