Protein Protocols Protein Protocols

Enzymatic Digestion 519
Another approach that works extremely well is to use an SDS polyacrylamide gel containing
a funnel-shaped well that allows samples to be loaded in volumes as large as 300 µL (9).
7. In general, the sample should be run in as few SDS-PAGE lanes as possible to maximize
the substrate concentration and to minimize the total gel volume present during the digest.
Whenever possible, a 0.5–0.75-mm thick gel should be used and at least 0.5–0.75 µg of
the protein of interest should be run in each gel lane so the density of the protein band is at
least 0.05 µg/mm 3 . As shown in Table 1, the in-gel procedure has an average success rate
of close to 98%. The least amount of an unknown protein that we have successfully
digested and sequenced was 5 pmol of a protein that was submitted at a comparatively
high density of 0.1 µg/mm 3. We believe the latter factor contributed to the success of
this sample.
Although several enzymes (i.e., trypsin, chymotrypsin, lysyl endopeptidase, and
endoproteinase GluC) may be used with the in gel procedure, nearly all our experience has
been with trypsin. In general, we recommend using 0.5 µg enzyme/15 mm 3 of gel with the
only caveat being that we use a corresponding lower amount of enzyme if the mole ratio
of protease/substrate protein would exceed unity.
8. Since high concentrations of Coomassie blue interfere with digestion, it is best to use the
lowest Coomassie blue concentration possible and to stain for the minimum time necessary
to visualize the bands of interest. In addition, the gel should be well destained so that
the background is close to clear.
9. As shown in Table 1, the in-gel digestion procedure outlined in this chapter appears to
have a success rate of nearly 98% with unknown proteins. Surprisingly, this success rate
does not appear to vary significantly over the range of protein extending from an average
of about 37–323 pmol. Obviously, however, the quality of the resulting sequencing data is
improved by going to the higher levels, and this is probably reflected by the increased
number of positively called residues/peptide sequenced that was observed at the >200 pmol
level. One critical fact that has so far not been noted is that 71% of the proteins on which
the data in Table 1 are based were identified via data base searching of the first peptide
sequence obtained. Hence, all internal peptide sequences obtained should be immediately
searched against all available databases to determine if the protein that has been digested
is unique.
10. Although most estimates of protein amounts are based on relative staining intensities, our
data suggest there is a 5–10-fold range in the relative staining intensity of different proteins.
Obviously, when working in the 50–100 pmol level, such a 5–10-fold range could
well mean the difference between success and failure. Hence, we routinely subject an
aliquot of the SDS-PAGE gel (usually 10–15% based on the length of the band) to
hydrolysis and ion-exchange amino acid analysis prior to proceeding with the digest. As
these analyses will often contain