COMPARISON OF PROTEIN SEPARATIONS IN CAPILLARY ZONE ELECTROPHORESIS AND CAPILLARY ISOELECTRIC-FOCUSING INTERFACING WITH ELECTROSPRAY MASS-SPECTROMETRY

On-line capillary zone electrophoresis/electrospray ionization mass sp ectrometry (CZE-ESMS) and capillary isoelectric focusing/electrospray ionization mass spectrometry (CIEF/ESMS) were employed for protein ana lysis. The separation mechanisms and tile detection limits of CZE/ESMS and CIEF/ESMS were compared by using model proteins including cytochr ome c (horse heart), ribonuclease A (bovine pancreas), myoglobin (hors e heart), carbonic anhydrase I (human erythrocytes) and beta-lactoglob ulin A (bovine milk). The effect of a moving ionic boundary inside the electrophoresis capillary on the separation resolution of model prote ins by CZE/ESMS and CIEF/ESMS is described. In CZE/ESMS, the formation of a moving ionic boundary due to the replacement of background elect rolyte counterions with sheath liquid counterions can be eliminated by using a common counterion in both the background electrolyte and the sheath liquid. Additionally, the moving ionic boundary in CIEF/ESMS is minimized by combining cathodic mobilization with a gravity-induced h ydrodynamic Baiu. The concentration detection limits of model proteins in a full-scan CIEF/ESMS analysis are in the region of 10(-7) M, simi lar to 20-50 times lower than that achievable using CZE/ESMS. Sample p reconcentration taking place during the focusing step in CIEF is respo nsible for the improved detection limits.