Electrophoretic method for separating small peptides in serum without extraction of macromolecules: application to...

We optimized the electrophoretic separation using sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and silver staining to detect small peptides in serum, without extraction of major proteins and applied this method to the search for specific peptides that may be used as early markers of preeclampsia during pregnancy. Appropriate separation of peptides was possible by modifying several parameters: increasing total proportion of monomers (%T) from 3 to 5% in the stacking gel; decreasing total proportion of crosslinker (%C) from 2.7 to 1.5% in the separating gel; selecting alkaline stacking gel buffer (pH 8.8) and alkaline sample buffer (pH 8.0); adding glycerol to both gels; and processing with weak current (15 mA). Silver nitrate staining was improved by using glutaraldehyde as the fixing agent, by reducing cross-linker concentration and by eliminating oxidizing and reducing agents. Sensitivity of the improved silver nitrate staining was 50 ng. We compared the electrophoretic pattern of serum peptides in the range of 2-14 kDa of 20 preeclamptic women with and without proteinuria and of 25 women in their third trimester who later developed preeclampsia to that of 24 normal pregnant women at term. No differences were found in the electrophoretic patterns among the groups and there was no correlation between the intensity of the bands and the severity of preeclampsia. We conclude that there is no characteristic electrophoretic pattern associated with preeclampsia.