John E. Wiktorowicz

Isoelectric focusing by free solutioncapillary electrophoresis

A reproducible, quantitative isoelectric focusing method using capillary electrophoresis that exhibits high resolution and linearity over a wide pH gradient was developed. RNase T1 and RNase ba are two proteins that have isoelectric points (pIs) at the two extremes of a pH 3-10 gradient. Site-directed mutants of the former were separated from the wild-type form and pIs determined in the same experiment. The pIs of RNase T1 wild-type, its three mutants, and RNase ba were determined for the first time as 2.9, 3.1, 3.1, 3.3, and 9.0, respectively. The paper describes the protocol for isoelectric focusing by capillary electrophoresis, as well as presenting data describing the linearity, resolution, limits of mass loading, and reproducibility of the method.

Characterization of polyethylene glycol modified proteins using charge-reversed capillary electrophoresis

Abstract A capillary electrophoretic method employing a charge reversal technique [J. E. Wiktorowicz and J. C. Colburn, Electrophoresis, 11 (1990) 769] has been developed to characterize polyethylene glycol (PEG)—proteins. A removable coating is applied to a standard fused-silica capillary in order to change the negative charge of the capillary surface to a positive charge. This prevents the adsorption of basic and PEG—proteins. The positive electrode is positioned at the detector end of the capillary, orienting electroosmotic flow towards the detector. Automated reconditioning procedures prior to each analysis give relative standard deviations in migration time and area of less than 2%, with most analysis times under 20 min. As the number of PEGs conjugated to a protein increases, the net positive charge and migration time of the copolymer decrease. Resulting peak widths are broad, reflecting the broad molecular mass distribution of PEGs and the heterogeneous nature of the PEG conjugates. This method can be used to monitor process steps, optimize reaction conditions, determine extent of modification, or assess product quality and consistency. Examples of PEG derivatized molecules characterized in our laboratory by charge-reversed capillary electrophoresis are tryptophan, Lys-Trp-Lys, lysozyme, myoglobin, RNase and immunoglobulin G; the size of the attached monomethoxy-PEG molecules varied from 0.15 kDa to 10 kDa (103 dalton).

The use of capillary electrophoresis in a micropreparative mode: methods and applications.

The ability to collect sufficient quantities of analytes from capillary electrophoresis for subsequent analyses is demonstrated. Fractions collected have been analyzed using the following techniques: capillary electrophoresis, mass spectrometry, and protein sequencing. Fractions can be collected directly into small volumes of buffer or directly onto membrane surfaces. Relevant parameters such as capillary diameter, mass loading, and separation parameters are addressed.

Methods Optimization for the Analysis of Peptides Using Capillary Electrophoresis

Publisher Summary This chapter discusses the optimization methods for the analysis of peptides using capillary electrophoresis (CE). CE is a relatively new analytical technique in which separations are performed in narrow inside diameter columns at high field strengths. Separations in narrow, silica capillaries at high field strengths are defining features of the technique. The narrow diameters permit the use of high fields because of the efficient heat dissipation in the narrow columns. The nature of the separation media and the structure of the analytes influence the observed mobility of the analytes under a given set of conditions. The observed mobility is a sum of the electroosmotic mobility (EOF) and the analyte mobility. The EOF is the bulk flow of liquid because of the surface charge on the capillary. The effect of a number of separation parameters on Joule heating, EOF, and analyte mobility are described in the chapter with respect to the optimization of separation conditions. A decrease in capillary diameter will result in a decrease in the current flow as well as an increase in the thermal efficiency of the system.

High Resolution Full-Range (pI = 2.5 to 10.0) Isoelectric Focusing of Proteins and Peptides in Capillary Electrophoresis

Publisher Summary This chapter discusses high-resolution full-range isoelectric focusing (IEF) of proteins and peptides in capillary electrophoresis (CE). IEF is a method that separates proteins on the basis of their isoelectric points (PI). It is accomplished by the electrophoresis of proteins or peptides through a stable pH gradient until their net charge is zero. At this pH, mobility is zero, and the isoelectric point is achieved. This method has been widely used in research, pharmaceutical, and clinical areas to separate, isolate, and analyze proteins from various biological products. The chapter discusses the effects of sample buffer matrix, surfactants, denaturant, and narrow pH range ampholyte using the same CE-IEF method. The study indicated that the CE-IEF method developed by Chen and Wiktorowicz has proven to be very flexible. This CE-IEF method used on a free solution separation can be used to perform rapid high-resolution analysis of proteins and peptides with various sample buffer matrices and additives.

Estimation of Protein Isoelectric Points by Capillary Electrophoresis Using MICRO-COAT™

Publisher Summary Information on the isoelectric points of proteins is important for a number of reasons. This chapter discusses the estimation of protein isoelectric points by capillary electrophoresis using MICRO-COAT™. The ability of capillary electrophoresis to provide separations of charged molecules rapidly, with a minimum sample requirement, and in the absence of denaturant would make it a likely candidate for a simpler and quicker method. This potential is complicated by the tendency of the capillaries above pH 4 to bind positively charged proteins via the unprotonated silanol groups of the fused silica wall. A solution to this complication has been offered via the method of reversing the charge on the capillary wall by applying a coating of a polycationic reagent MICRO-COAT™. When a capillary is treated in this manner, the walls become positively charged at neutral and low pH, allowing proteins below their pI to move through the capillary without being attracted to the wall via electrostatic interaction.