Protein Protocols Protein Protocols

Enzymatic Digestion 517
In the case of ampholines, our experience is that even prolonged dialysis extending over
several days with a 15,000-Dalton cutoff membrane is not sufficient to decrease the
ampholine concentration to a level that permits efficient trypsin digestion. Rather, the
only effective methods we have found for complete removal of ampholines are TCA precipitation,
hydrophobic or reverse-phase chromatography, or SDS-PAGE followed by
staining and destaining.
2. One of the most common causes of “failed” digests is that the amount of protein being
subjected to digestion has been overestimated. Often this is because of the inaccuracy of
dye binding and colorimetric assays. For this reason, we recommend that an aliquot of the
sample be taken for hydrolysis and amino acid analysis prior to digestion. The aliquot for
amino acid analysis should be taken either immediately prior to drying the sample in the
tube in which it will be digested or after redissolving the sample in 8 M urea, 0.4 M
NH 4HCO 3. Although up to 10 µL of 8 M urea is compatible with acid hydrolysis/ionexchange
amino acid analysis, this amount of urea may not be well tolerated by PTC
amino acid analysis. Hence, in the latter case, the amino acid analysis could be carried out
prior to drying and redissolving the sample in urea. Although it is possible to succeed with
less material, to ensure a high probability of success, we recommend that a minimum of
50–100 pmol protein be digested. Typically, 10–15% of this sample would be taken for
amino acid analysis. In the case of a 25-kDa protein, the latter would correspond to only
0.125–0.188 µg protein being analyzed. When such small amounts of protein are being
analyzed, it is important to control for the ever-present background of free amino acids
that are in buffers, dialysis tubing, plastic tubes and tips, and so forth. If sufficient protein
is available, aliquots should be analyzed both before (to determine the free amino acid
concentration) and after hydrolysis. Alternatively, an equal volume of sample buffer
should be hydrolyzed and analyzed and this concentration of amino acids should then be
subtracted from the sample analysis.
3. Since many native proteins are resistant to enzymatic cleavage, it is usually best to denature
the protein prior to digestion. Although some proteins may be irreversibly denatured
by heating in 8 M urea (as described in the above protocol), this treatment is not sufficient
to denature transferrin. In this instance, prior carboxymethylation, which irreversibly
modifies cysteine residues, brings about a marked improvement in the resulting tryptic
peptide map (7). Another advantage of carboxymethylating the protein is that this procedure
enables cysteine residues to be identified during amino acid sequencing. Cysteine
residues have to be modified in some manner prior to sequencing to enable their unambiguous
identification. Under the conditions that are described in Subheading 3., the
excess dithiothreitol and iodoacetic acid do not interfere with subsequent digestion.
Although carboxymethylated proteins are usually relatively insoluble, the 2 M urea that is
present throughout the digest is frequently sufficient to maintain their solubility. However,
even in those instances where the carboxymethylated protein precipitates following
dilution of the 8 M urea to 2 M, trypsin and chymotrypsin will usually still provide complete
digestion. Often, the latter is evidenced by clearing of the solution within a few
minutes of adding the enzyme.
If carboxymethylation is insufficient to bring about complete denaturation of the substrate,
an alternative approach is to cleave the substrate with cyanogen bromide (1000-fold
molar excess over methionine, 24 h at room temperature in 70% formic acid). The resulting
peptides can then either be separated by SDS-PAGE (since they usually do not separate
well by reverse-phase HPLC) or, preferably, they can be enzymatically digested with
trypsin or lysyl endopeptidase and then separated by reverse phase HPLC. If this approach
fails, the protein may be digested with pepsin, which, as described above, is carried out
under very acidic conditions or can be subjected to partial acid cleavage (8). However, the