Fully automated two-dimensional capillary electrophoresis for high sensitivity protein analysis

We report a system for automated protein analysis. In the system, proteins are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde, which reacts with lysine residues and creates a highly fluorescent product. These labeled proteins are analyzed by submicellar capillary electrophoresis at pH 7.5 to perform a first dimension separation. Once the first components migrate from the capillary, a fraction is transferred to a second dimension capillary, where electrophoresis is performed at pH 11.1 to further separate the proteins. Laser-induced fluorescence is used as an ultrasensitive detector of the separated proteins. Successive fractions are transferred from the first dimension capillary to the second dimension capillary for further separation to generate, in serial fashion, a two-dimensional electropherogram. The transfer of fractions is computer-controlled; there is no operator intervention once the sample has been injected. Zeptomoles of labeled proteins are detected, providing exquisite sensitivity.     

Application of capillary zone electrophoresis in cephalosporin analysis

Cephalosporins have structures and antibiotic activity similar to those of penicillins which represent a class of compounds with closely related structures. Most of the cephalosporins contain aromatic groups and show distinctive UV spectra. Separating the different types of cephalosporins is a challenging task for HPLC, but the resolving power of capillary zone electrophoresis (CZE) makes this separation fast and simple. The present study reports the application of CZE for cephalosporin analysis and the separation of cephalosporins from plasma. Both field strength and temperature were shown to influence the plate number. The influence of injection time on the peak height was studied. Furthermore, the influence of pH value on the separation of cephalosporins by CZE was investigated. The low sample amount required and the relatively short analysis time are the main advantages of this method.     

Characterization of latex allergenic components by capillary zone electrophoresis and N-terminal sequence analysis

In a previous study, protein components purified from latex gloves that elicited allergenic reactions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and yielded apparent molecular weights of 14, 22, 30, 34, 46, and 58 kD. These allergenic components were isolated for further characterization by capillary zone electrophoresis and N-terminal amino acid sequence analysis. These components all migrated at approximately 25 and 35 min on capillary zone electrophoresis. Diode array spectral analysis detected indistinguishable characteristics between these two protein peaks. In addition, complex formation of these components with patients' immunoglobulin was demonstrated by capillary zone electrophoresis. Analysis of components separated by SDS-PAGE on a polyvinylidene difluoride membrane showed that the first 13 residues were identical to the sequence of hevein. Based on the criteria of charge-to-mass ratio and N-terminall amino acid sequence, our results suggest that these components of latex proteins are similar in the primary structure.     

Application of capillary electrophoresis for DNA-protein binding tests

The combination of affinity chromatography and capillary electrophoresis (CE-SDS) has been found to be a useful tool to analyse populations of proteins which specifically bind to ssor dsDNA. Proteins were extracted from tissue, cytosol or nuclei of meristems of Pisum sativum seedlings and separated on cellulose column functionalized with ss-, dsDNA (calf thymus) and ssDNA (P. sativum) at 2M concentration of sodium chloride. Electropherograms of the crude protein extracts show two fractions of proteins specific for dsDNA (calf thymus) and three fractions specific for ssDNA (calf thymus). Four and five fractions of proteins specific for ssDNA (P. sativum) were identified in the material isolated from cytosolic and nuclear extracts, respectively. Both ds- and ssDNA (calf thymus) form complexes with ca. 4.0 % of the total amount of proteins, while ssDNA (P. sativum) binds to ca. 11.0 % of cytosolic and 5.0 % of nuclear proteins.     

Three methods of capillary electrophoresis compared with high-resolution agarose gel electrophoresis for serum protein electrophoresis

We assessed the BioFocus 2000 capillary electrophoresis instrument for use in a routine clinical laboratory. We examined 210 serum samples received for serum protein electrophoresis by four methods: (1) The Bio-Rad HR015EC high-resolution serum protein kit on the BioFocus; (2) the Jenkins–Guerin (JG) method on the Applied Biosystems 270A HT Capillary Electrophoresis System (JG-ABI); (3) the Jenkins–Guerin method using the BioFocus (JG-BF); and (4) the quantitation of monoclonal bands found in 76 of the 210 samples was assayed by Helena Titan Hi-Res agarose gel electrophoresis (HRAGE). The correlation coefficient between the three sets of capillary electrophoresis monoclonal band results and the Helena quantitation was 0.92 or better. Although the quantitative comparison of monoclonal bands by HR015EC was very good, the lack of sharpness of monoclonal bands using the HR015EC kit meant our preference was to use the JG method on either the ABI or on the Biofocus.     

HiTRACE: high-throughput robust analysis for capillary electrophoresis

MOTIVATION: Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment. RESULTS: We propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. We illustrate the application of HiTRACE on 13 datasets representing 4 different RNAs, 3 chemical modification strategies and up to 480 single mutant variants; the largest datasets each include 87 360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate datasets. Furthermore, while the smallest of these datasets required 7-10 h of manual intervention using prior approaches, HiTRACE quantitation of even the largest datasets herein was achieved in 3-12 min. The HiTRACE method, therefore, resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands.     

Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis

Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.     

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.     

High-resolution glycoprotein analysis using capillary electrophoresis

The high-resolution separation achievable with capillary electrophoresis has been applied successfully to the analysis of glycoproteins. Inherent in the implementation of this technology for glycoprotein analysis is the use of specific buffer additives. Bifunctional cationic reagents, such as simple alkyl chains bearing terminal amino or quaternary ammonium groups, have been particularly useful for the analysis of ovalbumin, an excellent model glycoprotein. Although dynamic coating of the capillary wall and the subsequent decrease in protein-wall interactions is known to be key in the effectiveness of these additives, much remains to be learned regarding the mechanism through which they function.     

Oligonucleotide analysis by gel capillary electrophoresis

Several million oligonucleotides are synthesized each year for a broad variety of molecular biology applications. Steady improvements in the synthesis chemistry efficiency and the automated DNA synthesizers have made production of oligonucleotides routine and reliable. Many applications, such as PCR and sequencing, are often successful when the primers have not been rigorously purified. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary for the dozens of oligonucleotides produced each day by a DNA synthesis laboratory. Traditional methods of analysis have been HPLC and polyacrylamide slab tel electrophoresis (PAGE). Gel capillary electrophoresis is a new option, combining the advantages of the HPLC and PAGE, with unprecedented resolution and speed.     

Serum lamotrigine analysis by capillary electrophoresis

Lamotrigine, a new antiepileptic drug, is analyzed by capillary zone electrophoresis. Samples were deproteinized with acetonitrile containing an internal standard, acidified with dilute acetic acid and injected into the capillary. The drug migrated rapidly with the cationic compounds in about 3.5 min far from any interfering substances. The test was linear between 0.5–10 mg/l. The analysis time was about 5 min. The CE values correlated well with an HPLC method (r=0.97; n=35). The mean serum concentration of 121 patients on this drug was 3.7 mg/l. Incubating the serum with ß-glucuronidase for 1 h increased the peak height of lamotrigine by about 24%.     

Rapid detection of DNA sequence variants by conformation-sensitive capillary electrophoresis

The identification of novel sequence variants, which may be either disease-causing mutations or silent polymorphisms, in large numbers of samples is becoming the rate-limiting step in associating diseases with specific genes. This is particularly true in light of the imminent arrival of the complete reference sequence of the human genome. A number of techniques have been developed to analyze DNA samples for sequence variants rapidly. We describe a new technique, capillary-based conformation-sensitive gel electrophoresis (capillary CSGE) that transfers mutation detection from acrylamide gel to capillary electrophoresis. Capillary CSGE was able to detect 7/7 short insertion/deletions and 16/22 base substitutions in a series of random single-nucleotide polymorphisms and known variants in the lipoprotein lipase and BRCA2 genes. This technique has the potential to screen many megabases of DNA in a single day.     

Determination of protein charge by capillary zone electrophoresis

The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a "charge ladder" approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein.     

Application of polyacrylamide gel electrophoresis/neutron activation analysis for protein quantification

Biological Trace Element Research - A combination of two methods, polyacrylamide gel electrophoresis (PAGE) and neutron activation analysis (NAA), has been applied to solutions containing...     

High-performance capillary electrophoresis for determining HIV-1 Tat protein in neurons

The HIV-1 protein, Tat has been implicated in AIDS pathogenesis however, the amount of circulating Tat is believed to be very low and its quantification has been difficult. We performed the quantification of Tat released from infected cells and taken up by neurons using high performance capillary electrophoresis. This is the first report to successfully measure the amount of Tat in neurons and places Tat as a key player involved in HIV-associated neurocognitive disorders.     

Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures

Tau is a microtubule-associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double-stranded DNA (dsDNA) but not to bind single-stranded DNA (ssDNA) [Cell. Mol. Life Sci. 2003, 60, 413-421]. Here, we prove that tau binds not only dsDNA but also ssDNA. This finding was facilitated by using two kinetic capillary electrophoresis methods: (i) non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM); (ii) affinity-mediated NECEEM. Using the new approach, we observed, for the first time, that tau could induce dissociation of strands in dsDNA by binding one of them in a sequence-specific fashion. Moreover, we determined the equilibrium dissociation constants for all tau-DNA complexes studied.