Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides

Abstract

The application of free solution capillary electrophoresis (FSCE) to the separation of protein and peptide mixtures is presented. Both qualitative and quantitative aspects of FSCE separations are considered. In addition, a brief introduction describing the separation principle behind FSCE separations and a discussion of electrophoretic mobility are included. The applications were chosen in order to highlight the selectivity of FSCE separations and to demonstrate applications of potential practical interest to the bioanalytical chemist. Comparison of FSCE relative to traditional analytical separation alternatives is stressed throughout. The examples are presented in three broad categories: protein separations, peptide separations, and the application of both to the analysis of recombinant protein products. In the first section, FSCE separations of peptide mixtures are presented which demonstrate the suitability of FSCE for the analysis of the purity of peptide samples, the homogeneity of peptide samples prior to sequencing, the identity of peptides by using electrophoretic mobility values, and the reduction of an intrachain disulfide bridge. In the second section, protein separations are presented that show the resolution of glycoproteins having the same primary structure and the separation of immune complexes from free unreacted antibody and antigen. In the final section, highly purified and well-characterized samples of biosynthetic human insulin (BHI), biosynthetic human growth hormone (hGH), and their derivatives were used to evaluate FSCE as a complement and/or alternative to conventional analytical separation techniques for the determination of purity and identity of biosynthetic human proteins. In addition, the quantitative aspects of FSCE analysis such as linearity of response, precision, and limit of detection were examined.

Advances in capillary electrophoresis

“…This approach obviously requires that the migration velocity of the complexed and uncomplexed analyte are different. On the other hand with the high dependence of analyte mobility on the charge / mass ratio (in the simplest case) [96,98,99] this can be expected to occur at least in situations where the capillary separation is predominantly following the Offord's rule. In practice this approach is frequently used for revealing proteinprotein interactions (for a recent report see ref.…”

CE‐based simultaneous liquid‐phase noncompetitive enzyme immunoassay for three tumor markers in human serum using electrochemical detection

“…CE is a powerful technique for the separation of macromolecules such as proteins and immunocomplexes [14][15][16][17][18][19]. With both superior separation power and high detection sensitivity, CE can separate free antibody or antigen from bound antibody or antigen rapidly, and is especially suitable for immunoassay [20][21][22][23][24][25].…”

The separation of herbicides by micellar electrokinetic capillary chromatography

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