Protein Protocols Protein Protocols

Enzymatic Digestion of Membrane-Bound Protein 529
3.3. Extraction of the Peptides
1. After digestion, vortex the sample for 5–10 s, sonicate for 5 min by holding in a sonicating
water bath, spin in a centrifuge (~1800g) for 2 min, and transfer the supernatant to a
separate vial that will be used directly for HPLC analysis.
2. Add a fresh 50 µL of digestion buffer to the sample, repeat step 11, and pool the supernatant
with the original buffer supernatant.
3. Add 100 µL 0.1 % TFA to the sample, and repeat step 1. The total volume for injection
onto the HPLC is 200 µL. Most of the peptides (~80%) are recovered in the original
digestion buffer; however, these additional washes will ensure maximum recovery of peptides
from the membrane.
4. Terminate the enzymatic reaction by either analyzing immediately by HPLC or adding
2 µL of the DFP solution.
CAUTION: DFP is a dangerous neurotoxin and must be handled with double
gloves under a chemical fume hood. Please follow all precautions listed for this chemical.
3.4. Analysis of Samples by Reverse-Phase HPLC
and Storage of Peptide Fractions
1. Prior to reverse-phase HPLC, inspect the pooled supernatants for small pieces of membrane
or particles that could clog the HPLC tubing. If membrane or particles are observed,
either remove the membrane with a clean probe (such as thin tweezers, a thin wire, thin
pipet tip, and so forth), or spin in a centrifuge for 2 min and transfer the sample to a clean
vial. A precolumn filter will help increase the life of HPLC columns, which frequently
have problems with clogged frits.
2. Sample is ready to be fractionated by HPLC (see Chapter 78). Fractions can be collected
in capless 1.5-mL plastic tubes, capped, and stored at –20°C until sequenced (see Note 9).
A typical fractionation is shown in Fig. 1.
4. Notes
1. This procedure is generally applicable to proteins that need to have their primary structure
determined and offers a simple method for obtaining internal sequence data in addition to
amino terminal sequence analysis data. The procedure is highly reproducible and is suitable
to peptide mapping by reverse-phase HPLC. Proteins 12–300 kDa have been successfully
digested with this procedure with the average size around 100 kDa. Types of
proteins analyzed by this technique include DNA binding, cystolic, peripheral, and integral-membrane
proteins, including glycosylated and phosphorylated species. The limits
of the procedure appear to be dependent on the sensitivity of both the HPLC used for
peptide isolation and the protein sequencer.
2. There are several clear advantages of this procedure over existing methods. First, it is
applicable to PVDF (especially high-retention PVDF membranes), which is the preferred
membrane owing to higher recovery of peptides after digestion, as well as being applicable
to other structural analysis. The earlier procedures (1,2) have not been successful
with PVDF. Second, because of the RTX-100 buffer, recovery of peptides from nitrocellulose
is higher than earlier nitrocellulose procedures (5). Third, the procedure is a onestep
procedure and does not require pretreatment of the protein band with PVP-40. Fourth,
since the procedure does not require all the washes that the PVP-40 procedures do, there is
less chance of protein washout. Fifth, the time required is considerably less than with the
other procedures. Overall, the protocol described here is the simplest and quickest method
to obtain quantitative recovery of peptides.