Capillary zone electrophoresis protein separation

Capillary zone electrophoresis provides effective separations of any charged species, including inorganic anions and cations, organic acids and amines, and large biomolecules such as proteins. For example, CZE has been used to separate a mixture of 36 inorganic and organic ions in less than 3 minutes.Neutral species, of course, cannot be separated. 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

A. V. Lemmo and J. W. Jorgenson, Two-dimensional protein separation by mictocolumn size-exclusion chromatography-capillary zone electrophoresis , 7. Chromatogr. 633 213-220(1993). 

Capillary zone electrophoresis, an up-to-date high resolution separation method useful for proteins and peptides, has been shown to be a useful method for determining electrophoretic mobilities and diffusion coefficients of proteins [3], Diffusion coefficients can be measured from peak widths of analyte bands. The validity of the method was demonstrated by measuring the diffusion coefficients for dansylated amino acids and myoglobin. 

Table 1 summarizes several of the experimental methods discussed in this chapter. A need exists for new or revised methods for transport experimentation, particularly for therapeutic proteins or peptides in polymeric systems. An important criterion for the new or revised methods includes in situ sampling using micro techniques which simultaneously sample, separate, and analyze the sample. For example, capillary zone electrophoresis provides a micro technique with high separation resolution and the potential to measure the mobilities and diffusion coefficients of the diffusant in the presence of a polymer. Combining the separation and analytical components adds considerable power and versatility to the method. In addition, up-to-date separation instrumentation is computer-driven, so that methods development is optimized, data are acquired according to a predetermined program, and data analysis is facilitated. 

Widhalm, A., Schwer, C., Blaas, D., Kenndler, E. (1991). Capillary zone electrophoresis with a linear, non-cross-linked polyacrylamide gel separation of proteins according to molecular mass. J. Chromatogr. 549, 446 451. 

We therefore sought to evaluate reproducibility of shotgun proteomics in studies of archival FFPE tissue. Because FFPE samples are more complex than non-cross-linked samples, we evaluated FFPE human liver for analytical reproducibility and confidence in protein assignments.20 This complexity strengthens the argument for using high-resolution separations to maximize analyte concentration and minimize matrix effects. In this case, we used transient capillary isotachophoresis/capillary zone electrophoresis (cITP/cZE) in place of IEF to help address this effect. cITP/cZE has a resolution superior even to cIEF (90% of identified peptides in 1 fraction, 95% in 2 fractions or less for cITP/cZE, vs. 75% and 80%, respectively, for cIEF). 

In CZE, the capillary, inlet reservoir, and outlet reservoir are filled with the same electrolyte solution. This solution is variously termed background electrolyte, analysis buffer, or run buffer. In CZE, the sample is injected at the inlet end of the capillary, and components migrate toward the detection point according to their mass-to-charge ratio by the electrophoretic mobility and separations principles outlined in the preceding text. It is the simplest form of CE and the most widely used, particularly for protein separations. CZE is described in Capillary Zone Electrophoresis. ... 

Cloud point extraction has been applied to the separation and preconcentration of analytes including metal ions, pesticides, fungicides, and proteins from different matrices prior to the determination of the analyte by techniques such as atomic absorption, gas chromatography, high performance liquid chromatography, capillary zone electrophoresis, etc. 

S Busch, JC Kraak, H Poppe. Chiral separations by complexation with proteins in capillary zone electrophoresis. J Chromatogr 635 119-126, 1993. 

Xu, R. J., Vidal-Madjar, C., Sebille, B., and Diez-Masa, J. C., Separation of basic proteins by capillary zone electrophoresis with coatings of a copolymer of vinylpyrrolidone and vinylimidazole, /. Chromatogr. A, 730, 289-295,1996. 

N. E. Baryla, J. E. Melanson, M. T. McDermott, and C. A. Lucy, Characterization of Surfactant Coatings in Capillary Electrophoresis by Atomic Force Microscopy, Anal. Chem. 2001, 73, 4558 M. M. Yassine and C. A. Lucy, Enhanced Stability Self-Assembled Coatings for Protein Separations by Capillary Zone Electrophoresis Through the Use of Long-Chained Surfactants, Anal. Chem. 2005, 77, 62. 

Capillary zone electrophoresis has been used for the separation of a wide variety of analytes, ranging from inorganic ions8-10 to proteins,11 peptides,1213 and nucleotides.14 Protein and peptide separations are by far the most common application areas, and CE is able to provide information complementary to that obtained by HPLC. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

Capillary zone electrophoresis (CZE), also known as free-solution CE, is the most widely used mode of CE essentially because of its versatility. Protein separation in CZE is based on the differential electrophoretic mobility of the analytes. This mobility is primarily dependent on a protein s size and net charge, the charge-to-mass ratio. Solvent properties that influence the size and charge of a protein include pH, ionic strength, viscosity, and dielectric constant.67 Manipulation of these properties, most notably pH, dictates the selectivity in CZE. Maximizing the charge difference between two proteins via pH modification optimizes their separation. 

Mazzeo, J. R., and Krull, I. S. (1991). Coated capillaries and additives for the separation of proteins by capillary zone electrophoresis and capillary isoelectric focusing. BioTechniques 10, 638-645. 

The separation medium used in the capillary dictates the type of separation that will be observed. In capillary zone electrophoresis (CZE), an aqueous buffered solution at a specific pH is used. Protein variants... 

Capillary zone electrophoresis (CZE), micellar capillary electrokinetic chromatography (MECC), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE) are CE modes using continuous electrolyte solution systems. In CZE, the velocity of migration is proportional to the electrophoretic mobilities of the analytes, which depends on their effective charge-to-hydrodynamic radius ratios. CZE appears to be the simplest and, probably, the most commonly employed mode of CE for the separation of amino acids, peptides, and proteins. Nevertheless, the molecular complexity of peptides and proteins and the multifunctional character of amino acids require particular attention in selecting the capillary tube and the composition of the electrolyte solution employed for the separations of these analytes by CZE. 

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