Proteins zone electrophoresis

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). 

D. Eigeys, Y. Zhang and R. Aebersold, Optimization of solid phase microexti action-capillaiy zone electi ophoresis-mass specti ometi y for liigh sensitivity protein identification , Electrophoresis 19 2338-2347 (1998). 

E. J. Cole and R. T. Kennedy, Seleaive preconcenti ation for capillary zone electrophoresis using protein G immunoaffinity capillary chi omatography . Electrophoresis 16 549-556(1995). 

Lauer, H. H. and McManigill, D., Capillary zone electrophoresis of proteins in untreated fused silica tubing, Anal.Chem., 58, 166, 1986. 

Honda, S., Taga, A., Suzuki, K., Suzuki, S., and Kakehi, K., Determination of the association constant of monovalent mode protein-sugar interaction by capillary zone electrophoresis, /. Chromatogr., 597, 377, 1992. 

Green, J. S. and Jorgenson, J. W., Minimizing adsorption of proteins on fused silica in capillary zone electrophoresis by the addition of alkali metal salts to the buffers, /. Chromatogr., 478, 63, 1989. 

Bateman, K.R, White, R.L., Yaguchi, M., and Thibault, R, Characterization of protein glycoforms by capillary-zone electrophoresis-nanoelectrospray mass spectrometry, /. Chromatogr. A, 794, 327, 1998. 

Liu H., Cho B.-Y., Strong R., Krull I.S., Cohen S., Chan K.C., and Issaq H.J., Derivatization of peptides and small proteins for improved identification and detection in capillary zone electrophoresis (CZE), Anal. Chim. Acta, 400, 181, 1999. 

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. 

These systems rely on various combinations of size-exclusion chromatography, reversed-phase chromatography, and zone electrophoresis to characterize amines, peptides, and proteins (Yamamoto etal., 1989 Bushey and Jorgenson 1990 Larmann et al., 1993, Moore and Jorgenson, 1995 Optick and Jorgenson, 1997). Haleem Issaq reviews these separations in Chapter 16 of this book. 

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. 

Janini, G.M., Chan, K.C., Conrads, T.P., Issaq, H.J., Veenstra, T.D. (2004). Two-dimensional liquid chromatography-capillary zone electrophoresis—sheathless electrospray ionization-mass spectrometry evaluation for peptide analysis and protein identification. Electrophoresis 25, 1973-1980. 

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). 

Finally, work that may facilitate understanding the role of oq-acid glycoprotein variants in inter-individual variations in plasma protein binding, pharmacokinetic behavior, and drug action has been described. A capillary zone electrophoresis method that allows for the determination of 11 intact forms (i.e., isoforms, glycoforms) of oq-acid glycoprotein has been described [84],... 

Smithies, O., Zone electrophoresis in starch gels group variations in the serum proteins of normal human adults. Biochem. J. 61, 629 (1955). 

Gel electrophoresis provides a simple method for separating complex protein mixtures. Because proteins are visualized using stains that may not be linearly incorporated in the gel, the intensity of the stained bands may be poorly correlated with the amount of protein. For this reason, gel electrophoresis is at best a semiquantitative technique capable of generating relative purity results. In CE, separations are commonly performed in free solution, i.e., in the absence of any support such as gel matrices. This allows the replacement of the capillary s content in between analyses and therefore the automation of the process. The use of UV-transparent fused-silica capillaries enables direct on-line optical detection of focused protein zones, eliminating the requirement for sample staining. The detection systems available to CE provide true quantitative capabilities. 

One of the major advantages of CE as a separation technique is the wide variety of separation modes available. Analytes can be separated on the basis of charge, molecular size or shape, pi, or hydrophobicity. The same CE instrument can be used for zone electrophoresis, IEF, sieving separations, isotachophoresis, and chromatographic techniques such as MEKC and capillary electrokinetic chromatography. This section provides a brief description of each separation mode. Zone electrophoresis, IEF, and sieving are the primary modes used for protein separations, and these will be discussed in detail in the following sections. 

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. ... 

Zone Electrophoresis in Starch Gels and Its Application to Studies of Serum Proteins O. Smithies... 

D. Capillary Zone Electrophoresis for Protein Analysis METHOD TRANSFER... 

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