High-performance capillary electrophoresis of hydrophobic membrane proteins

Hydrophobic membrane proteins, extrinsic and intrinsic ones, were separated by high-performance capillary zone electrophoresis (HPCZE) and high-performance capillary isotachophoresis (HPCITP). In the case of HPCZE with both coated and uncoated quartz capillaries the addition of 7 M urea to the separation buffers was necessary to achieve reproducible results. In the HPCITP experiments PTFE capillaries were used. When spacers were used, e.g., ampholytes, additional splitting of peaks was observed. The splitting was caused by the microheterogeneity of the investigated proteins, which are differently glycosylated and/or phosphorylated.     

High-performance capillary electrophoresis of histones.

A high-performance capillary electrophoresis (HPCE) system was developed for the fractionation of histones. This system involves electroinjection of the sample and electrophoresis in 0.1 M phosphate buffer (pH 2.5) in a 35 cm x 50 micron I.D. coated capillary. Electrophoresis was accomplished in 9 min, separating a whole histone preparation into its components in the following order of decreasing mobility: (MHP) H3, H1 (major variant), H1 (minor variant), (LHP) H3, (MHP) H2A (major variant), (LHP) H2A, H4, H2B and (MHP) H2A (minor variant), where MHP is the more hydrophobic component and LHP is the less hydrophobic component. This order of separation is very different from that found in acid-urea polyacrylamide gel electrophoresis and in reversed-phase high-performance liquid chromatography and, thus, brings the histone biochemist a new dimension for the qualitative analysis of histone samples.     

High-performance capillary electrophoresis of proteins. Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins.

Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins. An excellent linear relationship (r greater than 0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10,000-100,000 and 40,000-200,000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively. The relative standard deviation (R.S.D.) of the peak migration time is ca. 1%. Theoretical plates of 5 X 10(5)-1 X 10(6) per metre are routinely being obtained. Calibration graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear (r greater than 0.999) and the proteins may be quantified with an R.S.D. of ca. 3-7%. As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca. 1500 less than that of the parent compound (ca. 60,000 dalton) may be differentiated by HPCE with a gel-filled capillary column.     

High-performance capillary electrophoresis of gangliosides

Gangliosides are sialic acid-containing glycosphingolipids. In aqueous media, these glycolipids have been shown to exist as stable micelles. Ganglioside micelles could be analyzed by high-performance zonal capillary electrophoresis in uncoated fused-silica capillaries within 10 min. The mass sensitivity determined by monitoring the absorption of ultraviolet light at 195 nm was in the order of 10(-11) mol. Increasing the pH of the running buffer from 3.0 to 7.4 or the voltage from 10 to 30 kV increased the relative mobilities of gangliosides. By contrast, increasing the ionic strength of the buffer decreased the migration and broadened the elution peak widths of gangliosides. Ganglioside* micelles including GM1, GD1b, and GT1b were resolved into separate peaks by capillary electrophoresis at physiological pH shortly after mixing. Upon prolonged incubation, the ganglioside peaks merged to form a single species. The fusion process was temperature-dependent. At 50 degrees C, formation of mixed micelles between polysialogangliosides GD1b and GT1b was complete within 30 min. In contrast, no fusion of the ganglioside peaks was observed at 0 degrees C even after 75 h. Formation of mixed micelles between GD1b and other polysialogangliosides including GD1a, GT1b, and GQ1b at 37 degrees C required 1.5, 3.0, and 2.0 h, respectively. Formation of mixed micelles between monosialoganglioside GM1 and polysialogangliosides were 6- to 36-fold slower. No fusion was observed between monosialogangliosides GM1 and GM2 after 2 days of incubation. These findings indicate that polysialogangliosides may have higher propensities than monosialoganglioside to form mixed micelles.     

High-performance liquid chromatography and capillary electrophoresis for the analysis of maize proteins

Methods for the analysis of maize proteins using HPLC and CE are reviewed. Most of the references cited in this review concern HPLC methods. Size-exclusion HPLC and especially RP-HPLC methods have been developed for characterization of normal and genetically modified maize, cultivar differentiation, and prediction of quality. Few CE methods for the analysis of maize proteins were found in the existing literature. Most of these methods focus on optimization of the separation of maize proteins using CZE and SDS-capillary gel electrophoresis.     

High-performance capillary electrophoresis of meat, dairy, and cereal proteins

Food proteins play important roles in food functionality, nutrition, and human health. For these reasons, new analytical methods are continually being developed to separate and characterize these important proteins. High-performance capillary electrophoresis (HPCE) is one of the latest analytical methods to be applied to the separation of food proteins. This review covers methods and applications for the separation of three major groups of food proteins, meat, dairy, and cereal proteins.     

High-performance genetic analysis using microfabricated capillary array electrophoresis microplates.

This review focuses on some recent advances in realizing microfabricated capillary array electrophoresis (microCAE). In particular, the development of a novel rotary scanning confocal fluorescence detector has facilitated the high-speed collection of sequencing and genotyping data from radially formatted microCAE devices. The concomitant development of a convenient energy-transfer cassette labeling chemistry allows sensitive multicolor labeling of any DNA genotyping or sequencing analyte. High-performance hereditary haemochromatosis and short tandem repeat genotyping assays are demonstrated on these devices along with rapid mitochondrial DNA sequence polymorphism analysis. Progress in supporting technology such as robotic fluid dispensing and batched data analysis is also presented. The ultimate goal is to develop a parallel analysis platform capable of integrated sample preparation and automated electrophoretic analysis with a throughput 10-100 times that of current technology.     

Recent developments in capillary zone electrophoresis of proteins.

This review article with 125 references describes recent developments in capillary zone electrophoresis of proteins. It encompasses approximately the last two years, from the previous review (V. Dolník, Electrophoresis 1997, 18, 2353-2361) through Spring 1999. Topics covered include modeling of the electrophoretic properties of proteins, sample preconcentration and derivatization, wall coatings, improving selectivity, special detection techniques, and applications.     

Electroosmotic pump-assisted capillary electrophoresis of proteins

A new method for protein analysis, that is, electroosmotic pump-assisted capillary electrophoresis (EOPACE), is developed and demonstrated to possess several advantages over other CE-based techniques. The column employed in EOPACE consists of two linked sections, poly(vinyl alcohol) (PVA)-coated and uncoated capillaries. The PVA-coated capillary column is the section for protein electrophoresis in EOPACE. Electroosmotic flow (EOF) is almost completely suppressed in this hydrophilic polymer coated section, so protein electrophoresis in the PVA-modified capillary is free of irreversible protein adsorption to the capillary inner wall. The uncoated capillary section serves as an electroosmotic pump, since EOF towards cathode occurs at neutral pH in the naked silica capillary. By the separation of a protein mixture containing cytochrome c (Cyt-c), myoglobin and trypsin inhibitor, we have demonstrated the advantages of EOPACE method over other relevant ones such as pressure assisted CE, capillary zone electrophoresis (CZE) with naked capillary and CZE with PVA-coated capillary. A significant feature of EOPACE is that simultaneous separation of cationic, anionic and uncharged proteins at neutral pH can be readily accomplished by a single run, which is impossible or difficult to realize by the other CE-based methods. The high column efficiency and good reproducibility in protein analysis by EOPACE are verified and discussed. In addition, separation of tryptic digests of Cyt-c with the EOPACE system is demonstrated.     

Whole-column imaging capillary electrophoresis of proteins with a short capillary

Whole-column imaging capillary electrophoresis with a short capillary is discussed. A short capillary (3-6 cm) coated with either fluorocarbon or polyacrylamide was used as a separation capillary. The whole capillary was illuminated with 280 nm light, and the transmitted light was monitored by a linear charge-coupled device (CCD). For the short capillary, hydrodynamic flow caused by a subtle height difference between the anodic and cathodic reservoirs affected the sample migration in the capillary greatly. Several sample injection methods, including use of a cross connection, sealing of the capillary ends with a gel, and use of a gel-filled capillary, have been discussed. The experimental results showed that the peak height decreased and peak width increased with the electromigration distance. Therefore, higher sensitivity was obtained in a short capillary rather than a long capillary. The whole-column imaging CE with the short capillary has been applied for the study of conjugation reactions of protein cytochrome c with sodium dodecyl sulfate (SDS) and the dye Congo Red. The method has also been used for in situ monitoring of the electrophoretic protein desorption process. Our technique is a unique tool for the study of protein binding reactions and the interaction between analyte and inner wall of the capillary.     

Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins

The number of proteins produced by recombinant DNA technology continues to grow at a rapid pace. In this review, the emphasis is on proteins that are of therapeutic interest. Aspects of protein analysis, such as glycoform separation of proteins produced in mammalian cells and the separation of oligosaccharides for structure elucidation, are covered. The use of antibodies as therapeutic proteins is growing and currently antibodies are the largest class of proteins produced by biotechnology. This has merited a separate section on analysis of antibodies by capillary electrophoresis (CE). Applications of mass spectrometry as an ancillary technique, used in conjunction with CE, are also covered briefly. This review covers the literature since 1999.     

High-performance liquid chromatography of proteins on short capillary columns

A microchromatographic procedure for highly sensitive analysis of proteins has been developed by using short capillary columns with reverse-phase, ion-exchange, and hydrophobic interaction sorbents. The high analytical mass sensitivity on the level of several nanograms was attained by the miniaturization of the column and the optimization of gradient steepness.     

High-sensitivity laser-induced fluorescence detection of native proteins in capillary electrophoresis.

A highly sensitive laser-induced (LIF) detection scheme for native, tryptophan- or tyrosine-containing proteins in capillary electrophoresis (CE) has been demonstrated. The 275.4 nm line from an argon-ion laser is used to excite native protein fluorescence. A limit of detection (LOD) (S/N = 2) of 1 x 10(-10) M for conalbumin represents a 140-fold improvement over earlier reports. With stacking at injection, the LOD is 3 x 10(-12) M. Linear dynamic ranges of at least 5 and 4 orders of magnitude for, respectively, tryptophan and bovine serum albumin are found. The practical performance and blueprint of an easily constructed, rugged, compact and user-friendly LIF detector for CE are shown.     

Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries.

Numerous efforts have been made to separate proteins by capillary zone electrophoresis (CZE). The most common optimization techniques are changing the pH of the running buffer, coating the capillary surface with a hydrophilic polymer, or using additives in the sample solution. Surface coatings and solution additives can reduce the adsorption of the protein onto the capillary surface, but they diminish the separation efficiency and the resolution of CZE. This paper reports the successful separation of proteins in a untreated fused-silica capillary by raising the pH of the running buffer and washing between runs with 1.0 M sodium hydroxide. Under these conditions, model proteins and proteins in human serum have been determined by CZE. It is shown that the results from CZE are compatible with those of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.     

High-performance chiral separation of fourteen triazole fungicides by sulfated beta-cyclodextrin-mediated capillary electrophoresis

In this paper, sulfated beta-cyclodextrin-mediated capillary electrophoresis (CE) is evaluated as a new approach for the chiral separation of triazole-type fungicides. The 14 fungicides investigated were bitertanol, cyproconazole, difenoconazole, diniconazole, flutriafol, hexaconazole, myclobutanil, paclobutrazol, penconazole, propiconazole, tebuconazole, tetraconazole, triadimefon and triadimenol. Under the optimal conditions, excellent enantioseparation was achieved for all the 14 fungicides, including those fungicides containing two chiral centers. To our knowledge, this is the only system to date that offers outstanding enantiodiscrimination towards all triazole-type fungicides. The impact of the molecular structures of the triazole compounds on their migration behavior was studied. Similar to other chemical systems involving host-guest complexation, the interaction between sulfated beta-cyclodextrin and the triazole compounds was found to be affected by a variety of factors, including electrostatic force, hydrogen bonding, steric effect and hydrophobicity. These factors, coupled with the countercurrent electroosmotic flow (EOF), were believed to be the major forces behind the exceptional chiral selectivity.     

Polyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis

A hydrophilic, positively charged, durable coating has been developed for capillary electrophoresis of macromolecules. Polyethyleneimine is adsorbed to the inner wall of fused silica capillaries and the adsorbed coating cross-linked into a stable layer. Capillaries of polyethyleneimine-coated silica gave unique separations owing to the reversal of electro-osmotic flow caused by the positively charged coating. The resulting coating was stable from pH 2-12 and could be used over a wide pH range without substantial change in electro-osmotic flow. High-molecular-weight polymers were needed to give thick coatings which mask silanol groups on the wall. Proteins were resolved quickly and efficiently with good recovery using capillaries of 50 cm in length.     

The analysis of human amniotic fluid using capillary electrophoresis

This study has investigated the composition of amniotic fluid (AF) using capillary electrophoresis (CE). A detailed optimisation investigation was undertaken to obtain the best resolution of the major peaks in amniotic fluid. In the final method, capillary zone electrophoresis (CZE) of AF was performed on a Hewlett Packard3D CE instrument using a fused-silica capillary of 44 cm total length (36 cm to the detector) with in internal diameter of 50 microm. The background electrolyte was 20 mM sodium tetraborate containing 0.8 mM EDTA adjusted to pH 9.0. AF was diluted 1 plus 1 with deionised water prior to hydrodynamic injection for 3 s at 50 mbar. The separation was performed at +22.5 kV and resulted in a current of 65 microA. The capillary temperature was 28 degrees C. Using this CZE method, some eight peaks were consistently resolved in AF samples and several other more transient peaks have been separated from AF in less than 10 min. A scheme for the identification of peaks once they had been separated was also developed. Four peaks have been identified as proteins, i.e., gamma-globulin, alpha1-antitrypsin, transferrin and albumin. Surprisingly, one major peak was shown to be the purine catabolite, xanthine.     

Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins

The separation of the four major whey proteins by sodium dodecyl sulphate (SDS)-capillary gel electrophoresis (CGE) is described. Whilst commercially purified whey proteins could be analysed using the recommended protocol, the more complex nature of an acid whey and a reconstituted whey protein concentrate (WPC) powder necessitated considerable refinement of the CGE sample buffer. Individual whey proteins in the acid whey and WPC samples were then also separated and quantitated using capillary zone electrophoresis, polyacrylamide gel electrophoresis (PAGE) and HPLC methods and the results were compared. The values obtained for -lactalbumin (-Lac) and β-lactoglobulin (β-Lg) were consistent throughout the various methods, although size-exclusion HPLC, SDS-PAGE and SDS-CGE could not separate the two β-Lg variants or the glycosylated form of -Lac from the β-Lg. There was considerable variation in the values for the bovine serum albumin and immunoglobulin determined by the different methods and it was concluded that none of the methods could satisfactorily quantitate all four whey proteins.