<p class="first" id="d2110500e71">A highly reproducible, commercial and nonlinear,
wide-range immobilized pH gradient
(IPG) was used to generate two-dimensional (2-D) gel maps of [35S]methionine-labeled
proteins from noncultured, unfractionated normal human epidermal keratinocytes. Forty
one proteins, common to most human cell types and recorded in the human keratinocyte
2-D gel protein database were identified in the 2-D gel maps and their isoelectric
points (pI) were determined using narrow-range IPGs. The latter established a pH scale
that allowed comparisons between 2-D gel maps generated either with other IPGs in
the first dimension or with different human protein samples. Of the 41 proteins identified,
a subset of 18 was defined as suitable to evaluate the correlation between calculated
and experimental pI values for polypeptides with known composition. The variance calculated
for the discrepancies between calculated and experimental pI values for these proteins
was 0.001 pH units. Comparison of the values by the t-test for dependent samples (paired
test) gave a p-level of 0.49, indicating that there is no significant difference between
the calculated and experimental pI values. The precision of the calculated values
depended on the buffer capacity of the proteins, and on average, it improved with
increased buffer capacity. As shown here, the widely available information on protein
sequences cannot, a priori, be assumed to be sufficient for calculating pI values
because post-translational modifications, in particular N-terminal blockage, pose
a major problem. Of the 36 proteins analyzed in this study, 18-20 were found to be
N-terminally blocked and of these only 6 were indicated as such in databases. The
probability of N-terminal blockage depended on the nature of the N-terminal group.
Twenty six of the proteins had either M, S or A as N-terminal amino acids and of these
17-19 were blocked. Only 1 in 10 proteins containing other N-terminal groups were
blocked.
</p>