Evaluation of a novel hydrophilic derivatized capillary for protein analysis by capillary electrophoresis —electrospray mass spectrometry

Abstract

A new type of hydrophilic derivatized capillary has been used to enable the on-line capillary electrophoresis separation and electrospray mass spectrometric detection of a mixture of proteins containing bovine cytochrome c, tuna cytochrome c and horse heart myoglobin. Less than 40 fmol of each compound were loaded into the capillary. Baseline resolution of components was achieved, as were accurate assignments of molecular masses. The hydrophilic derivatized capillaries were taken through extensive testing procedures to characterize their performance and capabilities for protein analysis. A mixture of six proteins (cytochrome c, ribonuclease A, α-chymotrypsinogen, myoglobin, carbonic anhydrase II and α-lactalbumin) in acetic acid—sodium acetate buffer was used to delineate the relationships between migration time and pH, along with migration time and buffer concentration for each protein. The variations in capillary efficiency as a function of pH and as a function of buffer concentration were also characterized for the same six proteins in the acetic acid–sodium acetate system. A pH of 4.8 was found to offer an excellent compromise between separation efficiency (up to 500 000 theoretical plates) and analysis time. Capillary efficiencies were also found to be very good when employing a Tris · HCl electrolyte adjusted to pH 4.8. Lastly, electropherogram reproducibility and capillary durability were examined with the finding that little deterioration of the capillary occurred over the course of 400 injections (200 h run time). This represents a notable improvement over previously documented derivatization procedures designed to reduce protein adsorption to fused-silica capillary walls