Capillary electrophoresis protein separations

Dubin et al. used capillary electrophoresis to separate the complexes of proteins with synthetic polyanions from the free protein in the complex solution and to determine the complex composition. The binding isotherms were studied in relation to the pH value [70], the chain length of the poly-... 

Huang YF, Huang CC, Hu CC, Chang HT. Capillary electrophoresis-based separation techniques for the analysis of proteins. Electrophoresis 2006 27 3503-22. 

Capillary electrophoresis is primarily limited to small molecules that are water solul le because of their compatibility with the run buffer. Other similar techniques such as isoelectric focusing and capillary zone elec-trophoresis ° have aided in the separation of proteins by allowing for the separation of larger proteins. In addition these techniques can separate isoforms of proteins and peptides by using an extraordinarily low pH range. However, capillary electrophoresis cannot separate neutral compounds, and... 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

P. D. Grossman, J. C. Colburn, H. H. Lauer, R. G. Nielsen, R. M. Riggin, G. S. Sittampalam and E. C. Rickard, Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides . Anal. Chem. 61 1186-1194 (1989). 

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). 

Enantioresolution in capillary electrophoresis (CE) is typically achieved with the help of chiral additives dissolved in the background electrolyte. A number of low as well as high molecular weight compounds such as proteins, antibiotics, crown ethers, and cyclodextrins have already been tested and optimized. Since the mechanism of retention and resolution remains ambiguous, the selection of an additive best suited for the specific separation relies on the one-at-a-time testing of each individual compound, a tedious process at best. Obviously, the use of a mixed library of chiral additives combined with an efficient deconvolution strategy has the potential to accelerate this selection. 

Chemical surface modifications The first surface modification for the purpose of eliminating EOF and protein adsorption was recommended by Hjerten.28 The attachment of vinyl silanes allowed the polymerization of a variety of molecules to the surface. Most of the chemical modifications used for preparing capillaries for electrophoresis originated from the experience acquired over the years preparing GC and LC stationary phases. Chemical modification should conform to certain requirements, including the prevention of adsorption, the provision of stable and constant EOF over a wide pH range, chemical stability, ease of preparation, and reproduciblity of preparation. The effects of silanization of the inner surface of capillaries on electrophoretic separations have been extensively studied.26-29... 

High performance capillary electrophoresis was introduced originally as an analytical tool. Now that instruments are equipped with automated fraction collection, however, capillary electrophoresis can be used for micropreparative collection of individual peaks separated from a mixture. Using the fraction collection feature, nanomolar amounts of solute such as proteins, peptides, oligonucleotides can be collected in amounts sufficient for microsequencing. An intersample washing procedure and use of well-formed capillaries aid in the prevention of artifacts.44... 

Capillary electrophoresis employing chiral selectors has been shown to be a useful analytical method to separate enantiomers. Conventionally, instrumental chiral separations have been achieved by gas chromatography and by high performance liquid chromatography.127 In recent years, there has been considerable activity in the separation and characterization of racemic pharmaceuticals by high performance capillary electrophoresis, with particular interest paid to using this technique in modem pharmaceutical analytical laboratories.128 130 The most frequently used chiral selectors in CE are cyclodextrins, crown ethers, chiral surfactants, bile acids, and protein-filled... 

Wiktorowicz, J. E. and Colburn, J. C., Separation of cationic proteins via charge reversal in capillary electrophoresis, Electrophoresis, 11, 769, 1990. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Li, J., Kelly, J.F., Chernushevich, I., Harrison, D.J., and Thibault P., Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectro-spray mass spectrometry, Anal. Chem. 72, 599, 2000. 

Widhalm et al. (1991) reported the use of noncrosslinked polyacrylamide for protein separation in fused silica capillaries. This matrix has low viscosity and can be replaced between separations, greatly facilitating automation of the separation. A wide range of noncrosslinked polymers has been used for size-based protein separations. Noncrosslinked polymers do not form a gel, and it is inappropriate to refer to this separation as gel electrophoresis. A number of names have been used for the method. In an effort to standardize nomenclature, IUPAC has used the term capillary sieving electrophoresis. 

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

FIGURE 15.4 Computer record of a two-dimensional capillary electrophoresis analysis of a protein homogenate prepared from ahiopsy obtained from the fundus of a Barrett s esophagus patient. The data were generated hy performing 1 s transfers between capillaries and a 9 s second-dimension separation. The first-dimension separation employed the same buffer as the CSE separation in Fig. 15.1 and the second-dimension separation employed the same buffer as the MECC separation in Fig. 15.1. 

Larmann, J.P.Jr, Lemmo, A.V., Moore, A.W. Jr, Jorgenson, J.W. (1993). Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis. Electrophoresis. 14, 439-447. 

High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are two instrumental separation techniques that are applicable to the separation of proteins and peptides. The advantage of HPLC and CE techniques is that they afford the analyst the freedom to resolve a complex mixture by different routes employing different... 

Issaq, H.J., Chan, K.C., Cheng, S.L., Qingho, L. (2001). Multidimensional high performance liquid chromatography-capillary electrophoresis separation of a protein digest an update. Electrophoresis 22, 1133-1135. 

Over the past two decades, capillary electrophoresis (CE) and related techniques have rapidly developed for the separation of a wide range of analytes, ranging from large protein molecules to small inorganic ions. Gas chromatography has been considered as a powerful tool due to its sensitivity and selectivity, especially when coupled with mass spectrometry. Nevertheless, liquid chromatography is the most used method to separate and analyze phenolic compounds in plant and tissue samples. 

Capillary electrophoresis separates by differences in charge while reversed-phase HPLC separates on the basis of hydrophobicity. It turns out that these proteins have wide differences in hydrophobicity. 

Capillary electrophoresis systems are also likely to play an increasingly prominent analytical role in the QC laboratory (Figure 7.2). As with other forms of electrophoresis, separation is based upon different rates of protein migration upon application of an electric field. 

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