Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

180 Shaw
3.3.3. Purification
1. Connect the semipreparative reversed-phase column to the instrument along with
the 5-mL injection loop.
2. Equilibrate the column with 0.05% eluent A at a flow rate of 5 mL/min until a
constant baseline is observed.
3. Inject the entire dissolved peptide sample onto the column and wait approx 5 –7 min
for the void volume to elute.
4. Initiate the elution gradient. For peptides eluting near 35% eluent B, the following
gradient is useful: time 0, 100% A; linear gradient 1% B for 28 min;
linear gradient 0.1% B for 120 min; rapid linear gradient to 100% B (7.5% B/min
for 8 min).
5. Using a programmable fraction collector, collect fractions at a rate of 1–2 mL/min
during the 0.1% gradient period.
3.3.4. Peptide Analysis
In most cases the desired peptide will correspond to the largest peak in the
chromatogram obtained in Subheading 3.3.2. Use this chromatogram as a reference
for the following analysis.
1. Choose 3–4 fractions from the purification run and analyze these individually
using the analytical column and the 50-µL injection loop.
2. For each sample mix 10–20 µL of each fraction with an equivalent amount of
HPLC grade H 2O.
3. Equilibrate the column with eluent A.
4. Inject the sample and run a 2% B/min linear gradient. Monitor the run at 210 nm.
5. Choose samples that have similar retention times corresponding to that of the
largest peak in the analytical chromatogram in Subheading 3.3.2. (see Note 4).
6. Choose two samples that appear to correspond to the same peptide. Mix appropriate
amounts of each (10–20 µL) based on their relative peak heights obtained
in the chromatograms in step 5.
7. Inject and monitor this sample. If the two peaks arose from the same peptide, a
single peak will be obtained. Two peaks indicates the peptides are not the same
species.
8. Repeat step 7 as necessary to identify fraction tubes containing the same
peptide.
9. Pool the fractions and analyze by mass spectrometry for the correct mass. A convincing
mass spectrum will have a mass within ±1 atomic mass unit (amu) from
the calculated peptide mass.
4. Notes
1. In our experience purifications done with HPLC grade H2O are more reproducible.
Deionized H2O can be used and is less expensive; however, the H2O must be
passed through a 0.22-µm filter before use.