Jiaqi Wu

Optimizing separation conditions for proteins and peptides using imaged capillary isoelectric focusing

Separation conditions for antibodies, glycoproteins and peptides were optimized to fully realize the potential of automated imaged capillary isoelectric focusing (imaged cIEF) for protein analysis. Two commercially available capillary coatings, polyacrylamide and fluorocarbon, were found to provide reproducible results for cIEF separations. Both coatings could last more than 100 runs under normal cIEF conditions. Up to 30 mM salts (Na+) could be added to samples to prevent protein precipitation before and during isoelectric focusing performed under imaged cIEF. Short analysis time of the imaged cIEF also aided in the prevention of protein precipitation. High current at the beginning of the focusing for samples in salt could be avoided by applying a voltage gradient. Additions of up to 6 M urea and 20% glycerol could enhance solubility of proteins and peptide. Imaged cIEF was applied to the quantitation of monoclonal antibodies.

Capillary isoelectric focusing with whole column detection and a membrane sample preparation system

Abstract Progress of the research on capillary isoelectric focusing (cIEF)–whole column detection is reviewed. Three kinds of whole column detectors have been developed for cIEF: universal refractive index gradient imaging detector, laser-induced fluorescence (LIF) imaging detector, and optical absorption detector. Principle, applicability and detection limit of these detectors are discussed. A capillary column cartridge specially designed for cIEF with whole column detection is introduced. The column cartridge uses microdialysis hollow fiber membranes to isolate the sample solution from the electrolytes. Sample can be directly injected into the column without disturbing the electrolytes using a syringe pump or a liquid chromatography autosampler. The cartridge incorporates an on-line desalting, carrier ampholytes mixing device which also uses a microdialysis hollow fiber membrane. The on-line device adapts cIEF to a variety of analytical matrices. Examples of application of different whole column detectors to cIEF are shown in the review.

Absorption spectra and multicapillary imaging detection for capillary isoelectric focusing using a charge coupled device camera

Two absorption imaging detectors using charge coupled device (CCD) cameras are designed for capillary isoelectric focusing (CIEF). In the first detector, a light beam passes through a 4 cm capillary and is dispersed by a grating onto a CCD camera. The two-dimensional CCD in the camera records the light absorption at different positions along the capillary in one dimension, and at different wavelengths in the second dimension, simultaneously. The resolution in wavelength is about 1 nm. Since the separation time in the 4 cm long capillary column is only 4 min, the complete analysis takes 4 min, which is much faster than conventional CIEF methods. In the second detector, a light beam passes through a capillary array and then onto a CCD camera. Isoelectric focusing separation and detection of several samples can be completed in about 4 min, and the focusing processes in all capillaries can be observed simultaneously by the real-time, on-line imaging detector. In both detectors, images are normalized by light intensity, recorded simultaneously with the images, to compensate for intensity fluctuation of the light source. The detection limit of the detector is 1.5 × 10–3 absorbance units. The pH resolution of the instrument with the 4 cm long capillaries is 0.01 which is the same or better than that of conventional CIEF instruments with much longer capillaries. The deviation in pH of replicate zone positions in different capillaries of the capillary array is less than 0.01 which is much better than capillary array IEF methods using the mobilization process.

Application of capillary isoelectric focusing with absorption imaging detection to the analysis of proteins

A capillary isoelectric focusing instrument with an on-line optical absorption imaging detector was used to analyse protein samples. The separation column was a 4 cm x 100 microns I.D. capillary. The light source of the imaging detector was a argon ion laser. The light beam from the laser was focused into the capillary by a cylindrical lens. An 1024-pixel charge-coupled device (CCD) measured intensity of light beam passing through the capillary. The optical alignment of the detector was optimized to eliminate interference produced by the refractive index gradient created by sample zones inside the capillary. The signal-to-noise ratio of the detector was enhanced by averaging 30 scans of the CCD every 3 s. The on-line imaging detector allows simultaneous separation and detection so that the analysis time for a sample is only 2-4 min. Several protein samples were analyzed by the instrument, including human hemoglobin variants, cytochrome c, myoglobin and transferrin.

Capillary isoelectric focusing with imaging detection

Abstract A simple on-line absorbance imaging detection system was constructed for detection of capillary isoelectric focusing (CIEF). Because of the use of the CIEF-imaging detection system, separation and detection of protein sample could be completed in 2 minutes. The use of the imaging detection system also allowed for studying the focusing process. When a 4 cm long, 200-pm i. d. square capillary was used as the separation column, the concentration detection limit of the detectoir reached 5 pg/mL. This is the first report of applying absorbance imaging1 detection system to capillary electrophoretic techniques. The absorbance imaging detection system was compared with the refractive index glradient imaging detection system. For the capillary with large i. d., the absorbance imaging system showed higher sensitivity than the refractive index gradient imaging detector. However, the refractive index gradient imaging system was more suitable for narrow capillaries because it applied a laser as the light sour...

In vitro observation of interactions of iron and transferrin by capillary isoelectric focusing with a concentration gradient imaging detection system

Abstract A capillary isoelectric focusing—concentration gradient imaging detector system was used for in vitro observation of dynamics of interaction between iron and bovine transferrin. Transferrin was first focused inside the capillary by isoelectric focusing. A plug of ferric ion was then introduced into the capillary. The iron-free and the iron-complexed transferrins have different isoelectric points, hence they were focused at different positions inside the capillary. Concentration changes of different isoforms of iron-free and iron-complexed transferrins were monitored in an on-line fashion during the interaction by the imaging detector. One advantage of using this detector for studying the protein interaction is that the 633 nm wavelength probe beam used in the detector does not pump energy into the reaction system, which will interfere with the reaction. The results show that iron- binding and dissociation rates of different isoforms are different. This is the first report that the reaction speed of different isoforms of a protein can be observed in an on-line fashion.

Moving boundary capillary electrophoresis with concentration gradient detection

Abstract A rugged and inexpensive Schlieren optics detection system consisting of a laser, focusing lens and light beam position sensor was combined with moving boundary electrophoresis performed in a 10 cm × 20 μm I.D. capillary. This system facilitated rapid analysis and universal detection of sample components. The laser beam, which is focused directly into the separation capillary, probes the refractive index gradients produced by the arrival of new boundaries. The frontal injection method used in this approach introduces samples into the capillaries without discrimination and provides higher sensitivities because of the lack of dilution during the separation process compared with the zone technique. A specially designed cartridge accommodates a short piece of the separation capillary and reservoirs for buffer and sample. The separation in this method is completed usually within 30 s. During this short separation time only a small dispersion associated with diffusion of analytes along the flow direction occurs, which ensures that high gradients are generated in the detection volume. The detection limits in this approach are sub-micromolar concentrations of analyte injected for low-molecular-weight compounds such as sucrose. This system has been applied to the separation of amino acids and carbodydrates.

Diode laser-based concentration gradient imaging detector for capillary isoelectric focusing

Abstract A diode laser-based, real-time, universal concentration gradient imaging detector was constructed and tested for capillary isoelectric focusing (CIEF). The detector indicates sample zones by measuring refractive index gradients created by the concentration gradients of samples. An imaging detection system is the best detection approach for CIEF because all samples are focused inside the capillary column after separation. A diode laser was found to be an ideal light source for the imaging detector because of its small size, low cost, and light beam characteristics. The detector can be operated at any wavelength and does not need a high-intensity light beam. In order to focus the non-circular, non-Gaussian diode laser beam through the capillary, the optical geometry of the imaging detector was optimized. Because of the short analysis time (2–4 min), good reproducibility for determining isoelectric points, and the universality of the detector, the CIEF-imaging detector system has many possible applications, such as analysis of human hemoglobin variants and peptide mapping. This is the first report of a diode laser-based imaging detection system for analytical chemistry.

Application of capillary isoelectric focusing with universal concentration gradient detector to the analysis of protein samples

The design of a new capillary isoelectric focusing (cIEF) instrument, composed of a rugged cartridge holding a short piece of capillary and a universal, inexpensive concentration gradient detector, was optimized and applied to the analysis of various protein samples. High-efficiency cIEF separations with sub-femtomole detection limits for absolute amounts were obtained using 10 microns I.D. capillaries with large O.D.-to-I.D. ratios. An electric field strength of 1 kV/cm applied in the focusing step resulted in a 10(-8) M on-column concentration detection limit, which corresponded to 10(2) amol absolute amount of proteins. The detection volume was estimated to be 2 pl, which is among the smallest values reported to date for any optical or spectroscopic detector. When a 6-cm long capillary was used, proteins with isoelectric points ranging from 4.7 to 8.8 could be analyzed in about 5 min, the shortest analysis time ever reported for cIEF. Compared with commercial cIEF instruments with UV-visible absorbance detectors, the instrument is easier to use and has lower detection limits and better resolution. Several protein mixtures and real samples were separated with this instrument.

Protein analysis by isoelectric focusing in a capillary array with an absorption imaging detector

Isoelectric focusing (IEF) was successfully performed in capillary arrays with up to four capillaries. Separated proteins in the capillary array were detected by an UV absorption imaging detector. The whole analysis time for all samples in the capillary array was only 3 min due to the real-time imaging detector. The instrument was applied to analyse several protein samples including different human hemoglobin variants, myoglobin, transferrin, carbonic anhydrase and a monoclonal antibody to fluorescein. Because of good reproducibility of the focused pattern, unknown samples can be run simultaneously with a standard in the multichannel instrument and the components of unknown samples can be identified by comparing their zone positions to those of the standard. Minor components can be determined by the instrument in the presence of major components with 100 times higher concentrations in human hemoglobin samples. This instrument could be a powerful analytical tool for clinical analysis and for quality control in pharmaceutical companies.