Two-dimensional analysis of proteins in unprocessed human urine using double stain.

In the present study, a revised two-dimensional (2-D) map of proteins in unprocessed human urine was established. Spot urines were obtained from ten healthy men and ten healthy women each in Dewar flasks over a mixture of protease inhibitors. The specimens were sieved, desalted and freeze-dried, and identical amounts were pooled according to sex. The resulting samples were dissolved in urea mix and subjected to 2-D gel analysis employing the ISO-DALT system: the electrophoretograms were developed by double stain. We visualized more protein spots than reported previously, though most of them as yet remain unidentified. With the exception of similar relative positions of major protein spots, the samples demonstrated different patterns: urine of women, in general, had more spots and a greater proportion of low molecular weight proteins. Besides alpha 1-acid glycoprotein and the most acidic urinary proteins (MAUP), which were discerned earlier only in the ACIDO-DALT system, the ones known to precipitate while processing the samples were also detected. The 2-D profiles we established could be used as a diagnostic aid to study the excretion of proteins under various disease conditions.