Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis

A new program to characterize polyethylene glycol-modified (PEGylated) proteins is outlined using capillary zone electrophoresis (CZE). PEGylated ribonuclease A and lysozyme were selected as examples. Five separation procedures were compared to select out the mixed buffer of acetonitrile-water (1:1, v/v) at pH 2.5 as the best to characterize the PEGylated proteins without sample pretreatment. Polyethylene oxide (PEO) with a high molecular mass of 8X10(6) was applied to rinse the capillary to form a dynamic coating which would decrease the undesirable proteins adsorbed to the inner wall of the silica. The electroosmotic flow (EOF) mobility of the five procedures was determined, respectively. It is found that acetonitrile is mainly responsible for the good resolution of PEGylated proteins with the help of PEO coating in the semi-aqueous system. The low EOF mobility and current in the semi-aqueous system might also have some responsibility for the high resolution. The semi-aqueous procedure described in this paper also demonstrates higher resolution of natural proteins than aqueous ones. (C) 2001 Elsevier Science B.V. All rights reserved.

Spatial temperature gradient capillary electrophoresis for DNA mutation detection

A continuous spatial temperature gradient was established in capillary electrophoresis by using a simple temperature control device. The temperature profile along the capillary was predicted by theoretical calculations. A nearly linear spatial temperature gradient was established and applied to DNA mutation detection. By spanning a wide temperature range, it was possible to perform simultaneous heteroduplex analysis for various mutation types that have different melting temperatures.

Determination of sialic acids in the serum of cancer patients by capillary electrophoresis

A novel method for the determination of N-acetylneuraminic acid (NANA) and N-glycolylneuraminic acid (NGNA) was developed by using high-performance capillary electrophoresis (HPCE) with UV detection at 195 nm. NANA and NGNA were separated directly and analyzed without pre- or postcolumn derivation. The detection limit of NANA is 9.6 x 10(-6) mol L-1 and for mass 3.879 x 10(-14) mol (39 fmol). This method was applied for the determination of NANA in 30 normal human and 72 cancer patients. The results demonstrated that NANA in the sera of cancer patients increased significantly as compared with the normal human (P < 0.001). The new method is simple and sensitive, and is suitable for basic research and clinical application to malignant tumors.

A collision model for DNA separation by capillary electrophoresis in dilute polymer solution

A theoretical description. based on chemical kinetics and electrochemistry, is given of DNA separation in dilute polymer solution by capillary electrophoresis. A self-consistent model was developed leading to predictions of the DNA electrophoretic velocity as a function of the experimental conditions - polymer concentration, temperature, and electric field strength. The effect of selected experimental variables is discussed. The phenomena discussed are illustrated for the example of 100 bp DNA ladder separation in dilute HPMC solution by capillary electrophoresis. This model is the first single model that can fully explain the dependence of DNA electrophoretic velocity on electrophoretic conditions.

Analysis of urinary 8-hydroxydeoxyguanosine by capillary electrophoresis and solid-phase extraction

Urinary 8-hydroxydeoxyguanosine (80HdG) has been considered as an excellent marker of individuals at high risk of developing cancer. Until now, urinary 80HdG has largely been measured by high-performance liquid chromatography with electrochemical detection. A new method for the analysis of urinary 80HdG by high-performance capillary electrophoresis has been developed and optimized in our laboratory. A single step solid-phase extraction procedure was optimized and used for extracting 80HdG from human urine. Separations were performed in an uncoated silica capillary (50 cm x 50 tm i.d.) using a P/ACE MDQ system with UV detection. The separation of 80HdG from interfering urinary matrix components is optimized with regard to pH, applied voltage, pressure injection time and concentration of SDS in running buffer. The detection limit of this method is 0.4 mug/ml, the linear range is 0.8-500 mug/ml, the correlation coefficients levels is better than 0.999. The developed method is simple, fast and good reproducibility, furthermore, it requires a very small injection volumes and low costs of analysis, which makes it possible to provide a new noninvasive assay for an indirect measurement of oxidative DNA damage.

On the evaluation of diffusivities in gels using the diffusion cell technique

Modeling of the gel-immobilized cell system requires accurate measurement of diffusion coefficients. Three methods of the quasi-steady-state (QSS) method, the time-lag (TL) method and a variant quasi-steady-state (VQSS) method were critically assessed and compared for the evaluation of diffusivities using the diffusion cell technique. Experimental data from our laboratory were used for the analysis of the influence of crucial theoretical assumptions not being fulfilled in each method. The results highlighted a risk in obtaining highly variable diffusion coefficients by not validating the QSS and the accuracy of the measurements. In the TL method, the estimation of diffusivities based on the plot intercept that was mostly used in the literature, results in a many fold lower value when compared to that based on the plot slope. The comparison with the QSS and VQSS methods confirmed similar diffusivity obtained by the TL method based on the plot slope. It thus suggested that the correct estimation of diffusivities by the TL method could be based on the plot slope only. Furthermore, the errors associated with the solute mass in the gel, the sample withdrawal and the non-negligible concentration changes in the chambers were also discussed. It is concluded that diffusion cell technique has to be employed cautiously for a correct evaluation of diffusivities. (C) 2001 Elsevier Science B.V. All rights reserved.

Determination of total iron binding capacity of serum by capillary electrophoresis

A capillary electrophoresis (CE) technique for determining total iron binding capacity (TIBC) of serum has been developed. The optimum serum pretreatment involves the following major steps: at first, saturate serum transferrin with Fe+3; then, dissociate them completely after removing excess unbound Fe. Finally, complex the released iron with phenanthroline, a chromophore, to make suitable for the CE analysis. Ammonium acetate (pH = 5.0) was used as CE background electrolyte solution. In this system, a good linear correlation coefficient was maintained over the range 0.5 similar to 10 mu M (r = 0.9979, n =12). Seven adult serum samples were studied and the TIBC parameters measured. In the present system, 10 similar to 30 mu L serum is sufficient for determination. The study shows that the CE technique described is a powerful method for rapid, efficient, sensitive and reliable analysis and hence particularly suitable for clinical application.