Separation of mixtures of acidic and basic peptides at neutral pH

Mixtures of acidic and basic peptides composed of the phosphorylated and nonphosphorylated forms of peptide substrates for kinases and a phosphatase were separated by capillary electrophoresis (CE) in buffer conditions compatible with live mammalian cells. The separation of such mixtures was especially challenging given the high salt and neutral pH of the requisite physiologic buffers. Due to poor peak reproducibility in bare capillaries, several strategies were implemented to improve the electrophoretic separation of the peptide mixtures. Covalent coating of the capillary with the neutral polymer poly(dimethylacrylamide) (PDMA) resulted in a 2-fold improvement in the migration time RSD, but required the use of hydrodynamic flow to overcome the differing electrophoretic mobilities (μeo) of the peptides at neutral pH. This parabolic fluid flow diminished separation efficiency almost 5-fold. Polarity switching during the CE run was used to overcome the opposed μeo, but required the retention of hydrodynamic flow and consequent reduction in separation efficiency. The most efficient separations were seen with the use of covalently-linked, charged polymer coatings to maintain electroosmotic flow and to reduce wall interactions. Two such coatings were tested in the current study. Relative to the PDMA coating, an anionic poly(acrylate) improved the average migration time RSD of six peptides from 1.3 to 0.85% and average separation efficiency from 4.8 to 18.0 (·104 plates/m). Likewise, cationic poly([3-(methacryloylamino)propyl]-trimethylammonium) improved the average migration time RSD of eight peptides from 1.2 to 1.1% and average separation efficiency from 4.8 to 33.9 (·104 plates/m). These findings will be of value to the growing number of applications for analytical techniques utilizing CE for cellular analysis and biochemical studies.