REFERENCE POINTS FOR COMPARISONS OF 2-DIMENSIONAL MAPS OF PROTEINS FROM DIFFERENT HUMAN CELL-TYPES DEFINED IN A PH SCALE WHERE ISOELECTRIC POINTS CORRELATE WITH POLYPEPTIDE COMPOSITIONS

A highly reproducible, commercial and nonlinear, wide-range immobilize d pH gradient (IPG) was used to generate two-dimensional (2-D) gel map s of [S-35]methionine-labeled proteins from noncultured, unfractionate d normal human epidermal keratinocytes. Forty one proteins, common to most human cell types and recorded in the human keratinocyte 2-D gel p rotein database were identified in the 2-D gel maps and their isoelect ric points (pI) were determined using narrow-range IPGs. The latter es tablished a pH scale that allowed comparisons between 2-D gel maps gen erated 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. T he variance calculated for the discrepancies between calculated and ex perimental 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 th e calculated values depended on the buffer capacity of the proteins, a nd on average, it improved with increased buffer capacity. As shown he re, the widely available information on protein sequences cannot, a pr iori, be assumed to be sufficient for calculating pI values because po st-translational modifications, in particular N-terminal blockage, pos e a major problem. Of the 36 proteins analyzed in this study, 18-20 we re 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 bl ocked. Only 1 in 10 proteins containing other N-terminal groups were b locked.