Proteins capillary electrophoresis

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). 

H. Yamamoto, T. Manabe and T. Okuyama, Gel permeation chromatography combined with capillary electrophoresis for microanalysis of proteins , 7. Chromatogr. 480 277-283 (1989). 

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

D. Eigeys, A. Dua et and R. Aebersold, Identification of proteins by capillary electrophoresis-tandem mass specti ometiy . Evaluation of an on-line solid-phase extraction device , J. Chromatogr. A 763 295-306 (1997). 

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. 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

Oda RP, Clark R, Katzman JA, Landers JP (1997) Capillary electrophoresis as a clinical tool for the analysis of protein in serum and other body fluids. Electrophoresis 18 1715-1723. 

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... 

In the previously described electrophoretic methods, the capillary was filled with electrolytes only. Another mode of operation in capillary electrophoresis involves filling the capillary with gel or viscous polymer solutions. If desired, a column can be packed with particles and equipped with a frit.68 This mode of analysis has been favorably used for the size determination of biologically important polymers, such as DNA, proteins, and polysaccharides. The most frequently used polymers in capillary gel electrophoresis are cross-linked or linear polyacrylamide,69 cellulose derivatives,70-75 agarose,76 78 and polyethylene glycols. 

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... 

Bushey, M. M. and Jorgenson, J. W., Capillary electrophoresis of proteins in buffers containing high concentrations of zwitterionic salts, /. Chromatogr., 480, 301, 1989. 

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

Bullock, J.A. and Yuan, L.-C., Free solution capillary electrophoresis of basic proteins in uncoated fused silica capillary tubing, ]. Microcol. Sep., 3,241,1991. 

Lee, K.-J. and Heo, G. S., Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries, ]. Chromatogr., 559, 317, 1991. 

Swedberg, S. A., Characterization of protein behavior in high-performance capillary electrophoresis using a novel capillary system, Anal. Biochem., 185, 51, 1990. 

Rickard, E. C., Strohl, M. M., and Nielsen, R. G., Correlation of electrophoretic mobilities from capillary electrophoresis with physicochemical properties of proteins and peptides, Anal. Biochem., 197, 197, 1991. 

Josic, D., Zeilinger, K., Reutter, W., Bottcher, A., and Schmitz, G., High-performance capillary electrophoresis of hydrophobic membrane proteins, /. Chromatogr., 516, 89, 1990. 

Benedek, K. and Thiede, S., High performance capillary electrophoresis of proteins using sodium dodecyl sulfate-poly(ethylene oxide), J. Chromatogr., 676, 209, 1994. 

Ganzler, K., Greve, K. S., Cohen, A. S., Karger, B. L., Guttman, A., and Cooke, N. C., High-performance capillary electrophoresis of SDS-protein complexes using UV-transparent polymer networks, Anal. Chem., 64, 2665, 1992. 

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. 

Tong W., Link A., Eng J.K., and Yates, J.R. Ill, Identification of proteins in complexes by solid-phase microextraction/multitep elution/capillary electrophoresis/ tandem mass spectrometry, Anal. Chem. 71, 2270, 1999. 

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