Completely monodisperse, highly repetitive proteins for bioconjugate capillary electrophoresis: development and characterization

Protein-based polymers are increasingly being used in biomaterial applications because of their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for free-solution conjugate electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis.     

Protein glycosylation analysis with capillary-based electromigrative separation techniques

An impressive complexity is associated with glycoproteins due to the microheterogeneity of glycosylation as posttranslational modification giving rise to a vast number of isoforms. The full characterization of glycoproteins is difficult to achieve, and a number of analytical methods have to be combined for a detailed understanding of glycosylation. In this review, we focus on capillary electromigrative separation techniques in the formats capillary electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. These separation techniques can be applied to all levels of glycosylation analysis including intact glycoproteins, glycopeptides, and released glycans. We here discuss the separation characteristics for each method and the information that they can provide for each level. Detection issues, especially laser-induced fluorescence detection and mass spectrometry are taken into account. In addition, tables provide an overview on the achievements made from the very beginning of glycosylation research by electromigrative separation techniques. From the literature presented here it is clear, that glycosylation analysis by electromigrative separation techniques is on the edge of transition of basic research and method development towards applications. First proof-of-principle studies for in-depth glycoprotein characterization and clinical diagnosis are described. However, this overview also shows that many basic aspects of separation have not yet been fully understood and more research is necessary to be able to fully use the capabilities of electromigrative separation techniques.     

Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays

Enzyme-amplified lanthanide luminescence (EALL) is a new method which has been developed for enzymatically amplified signal detection in ultrasensitive bioanalytical assays where an enzyme is used as label or is itself the analyte of interest. Signal generation is performed by enzymatically transforming a substrate into a product which forms a luminescent lanthanide chelate; the product chelate can then be detected using time-resolved or normal fluorescence methods. Alkaline phosphatase substrates have been developed and demonstrated in a model immunoassay in microwell format. The method has also been demonstrated for detection of a variety of other hydrolytic and oxidative enzymes. Thus the EALL method shows promise for use in a wide variety of bioanalytical applications.     

Chemiluminescence determination of pharmacologically active compounds by capillary electrophoresis.

A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory.     

Separation and detection methods for covalent drug-protein adducts

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.     

Synchrotron radiation for direct analysis of metalloproteins on electrophoresis gels

Metalloproteomics requires analytical techniques able to assess and quantify the inorganic species in metalloproteins. The most widely used methods are hyphenated techniques, based on the coupling of a high resolution chromatographic method with a high sensitivity method for metal analysis in solution. An alternative approach is the use of methods for solid sample analysis, combining metalloprotein separation by gel electrophoresis and direct analysis of the gels. Direct methods are based on beam analysis, such as lasers, ion beams or synchrotron radiation beams. The aim of this review article is to present the main features of synchrotron radiation based methods and their applications for metalloprotein analysis directly on electrophoresis gels. Synchrotron radiation X-ray fluorescence has been successfully employed for sensitive metal identification, and X-ray absorption spectroscopy for metal local structure speciation in proteins. Synchrotron based methods will be compared to ion beam and mass spectrometry for direct analysis of metalloproteins in electrophoresis gels.     

Bioseparation and bioanalytical techniques in environmental monitoring

The growing use of antibody-based separation methods has paralleled the expansion of immunochemical detection methods in moving beyond the clinical diagnostic field to applications in environmental monitoring. In recent years high-performance immunoaffinity chromatography, which began as a separation technique in biochemical and clinical research, has been adapted for separating and quantifying environmental pollutants. Bioaffinity offers a selective biological basis for separation that can be incorporated into a modular analytical process for more efficient environmental analysis. The use of immunoaffinity chromatography for separation complements the use of immunoassay for detection. A widely used immunochemical detection method for environmental analyses is enzyme immunoassay. The objective of this paper is to review the status of bioaffinity-based analytical procedures for environmental applications and human exposure assessment studies. Environmental methods based on bioaffinity range from mature immunoassays to emerging techniques such as immunosensors and immunoaffinity chromatography procedures for small molecules.     

Application of microfluidic chip with integrated optics for electrophoretic separations of proteins

This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.     

A Comparative Structural and Bioanalytical Study of IVIG Clinical Lots

Intravenous immunoglobulin are important bio-therapeutics used in the replacement therapy for primary and secondary immunodeficiencies, chronic inflammatory disorders and several autoimmune haematologic disorders. Currently, a number of immunoglobulin intravenous (IVIG) products have been approved by the Food and Drug Administration (FDA) and are available commercially. It is known that small differences in the manufacturing processes as well as in the formulations may affect their clinical efficacy and tolerability. Therefore, given the complexity of the multi-step process required for the isolation of IVIG from human plasma, it is necessary to ensure a rigorous quality control of final products. We show here that a set of different bioanalytical techniques can be conveniently used to comparatively characterize, at a quantitative and qualitative level, different lots of IVIG preparations and to unveil randomly occurring impurities which can also affect the overall product stability. We have used circular dichroism, surface plasmon resonance and two-dimensional electrophoresis (2DE), and have demonstrated that this combination of bioanalytical approaches is very useful to improve the quality control of antibodies and to monitor the reliability of the IVIG manufacturing process.     

Capillary electrophoresis of peptides and proteins with plug of Pluronic gel

Electromigration capillary methods are promising techniques in proteomics and they are still under research. We used a partial filling approach, i.e. a combination of gel and non-gel separation mechanisms in a single dimension. We tried using an interesting gel, Pluronic F 127, which can be considered as a surfactant capable of self-association both with isotropic and anisotropic gels. The Pluronic was inserted inside the capillary as a plug at the start of the capillary, and it provided separation at the first time. Separation by this gel was achieved according to molecular weight and/or hydrophobicity. The applicability of this method was demonstrated in the separation of real samples-peptides arising from collagen after CNBr or collagenase cleavage and albumin after trypsin cleavage (peptide mapping). Some peptides and proteins were selectively retained by the Pluronic gel. These interactions with the gel did not depended on their molecular weight alone, but they probably depend on a combination of both principles. It was confirmed that capillary electrophoresis with Pluronic plug can give us another new separation option, complementary to free solution capillary electrophoresis. The CE method presented here, consisting of a partial filling approach with combine gel and non-gel separation mechanisms seemed to be a promising method for the separation of complex mixtures of peptides.     

Stopped-flow analysis of Ru(bpy)33+chemiluminescent reactions

The stopped-flow technique was employed to measure chemiluminescent emission from the reaction of a mixture of oxalate and proline with a chemiluminescence reagent, tris(2,2′-bipyridine)ruthenium(III), or Ru(bpy)₃³⁺. Ru(bpy)₃³⁺ is a versatile reagent and is often used in bioanalytical applications, including the detection of certain drugs and their metabolites, for example. Unfortunately, Ru(bpy)₃³⁺ has not yet been fully examined as a possible chemiluminescence reagent for simultaneous kinetic determinations. In this work, a differential reaction rate method, based on simple least squares regressions of the pseudo-first order decay data, was used to resolve two compounds, oxalate and proline, reacting simultaneously with Ru(bpy)₃³⁺. Our results indicate that stopped-flow analyses with Ru(bpy)₃³⁺ could provide a viable method for simultaneous determinations of unresolvable analytes of environmental and pharmaceutical importance. © 1998 John Wiley & Sons, Ltd.     

LC/CE-MS tools for the analysis of complex arabino-oligosaccharides

Recently, various branched arabino-oligosaccharides as present in a sugar beet arabinan digest were characterized using NMR. Although HPAEC often has been the method of choice to monitor the enzymatic degradation reactions of polysaccharides, it was shown that HPAEC was incapable to separate all known linear and branched arabino-oligosaccharides present. As this lack of resolution might result in an incorrect interpretation of the results, other separation techniques were explored for the separation of linear and branched arabino-oligosaccharides. The use of porous-graphitized carbon liquid chromatography with evaporative light scattering and mass detection as well as capillary electrophoresis with laser-induced fluorescence and mass detection demonstrated the superiority of both the techniques toward HPAEC by enabling the separation and unambiguous identification of almost all the linear and branched arabino-oligosaccharides available. The elution behavior of all arabino-oligosaccharides for the three tested separation techniques was correlated with their chemical structures and conclusions were drawn for the retention mechanisms of the arabino-oligosaccharides on the different chromatographic and electrophoretic systems. The combination of the elution/migration behavior on LC/CE and the MS fragmentation patterns of the arabino-oligosaccharides led to the prediction of structures for new DP6 arabino-oligosaccharides in complex enzyme digests.     

Features and applications of blue-native and clear-native electrophoresis

1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.     

High-pressure gel loader for capillary array electrophoresis microchannel plates.

Microfabricated capillary array electrophoresis (microCAE) microchannel plates are the next generation of bioanalytical separation devices. To fully exploit the capabilities of microCAE devices, supporting technology such as robotic sample loading, gel loading, microplate washing, and data analysis must be developed. Here, we describe a device for loading gel into radial capillary array electrophoresis microplates and for plate washing and drying. The microplates are locked into a loading module, and high-pressure helium is used to drive aqueous separation media or wash solutions into the microchannels through fixtures connected to the central anode reservoir. Microplates are rapidly (30 s to 5 min) loaded with separation media, such as 3%-4.8% linear polyacrylamide or 0.7%-3.0% hydroxyethyl cellulose, for electrophoresis. The effective and rapid gel-filling and plate-cleaning methods together with short electrophoretic analysis times (2-30 min) make microCAE systems versatile and powerful nucleic acid analysis platforms.     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.     

Improved mutation detection in GC-rich DNA fragments by combined DGGE and CDGE.

Denaturing gradient gel electrophoresis (DGGE) has proven to be a powerful pre-screening method for the detection of DNA variants. If such variants occur, however, in DNA fragments that are very rich in G and C, they may escape detection. To overcome this limitation, we tested a novel gel system which combines DGGE and constant denaturant gel electrophoresis (CDGE), as it might have the advantages of both methods. Indeed, this combination had the advantages of both methods, good separation of hetero-duplex molecules and prevention of total strand dissociation, and it proved successful in the detection of DNA variants in several GC-rich fragments.     

The quantitative characterization of free radical sources and traps by electromigration applications

Nowadays, very diverse human activities generate urgent demands for fast, sensitive reliable innovative tools capable of detecting major industrial, military, and other dangerous products. An important part of these compounds are free radicals. Capillary electrophoresis (CE), especially in its miniaturized format (lab-on-a-chip), and other electromigration methods offer special possibilities to resolve this problem. These measurements have a great opportuness because of very wide chemical and biological role of free radicals. Several compounds, e.g. monomers and some biologically important groups (as are nitrones) oppose oxidative challenges by virtue of their trap very rapidly oxygen- or carbon-centered radicals and generating other radical species which are stable and biochemically less harmful than the original ones. In many cases, conventionally, the relative trap capacity is measured against tert.-butylhydroperoxide (TBH). In this lecture are presented numerous important free radical species (active oxygen–, nitrogen- and carbon-centered ones, as HO, NO etc) and their adequate in vitro and in vivo applied bioanalytical methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence analysis. A simple and highly sensitive method is the capillary zone electrophoresis with amperometric detection (CZE-AD); It was introduced to determine indirectly OH by analysing its reaction products with salicylic and dihydroxybenzoic acids. Hydroxylated radical products of these acids are often used as a relative measurement in free radical research. Accurate determination of pK(a) values is important for proper characterization of newly synthesized molecules. CZE method was used for determination of their values. Are initiated new research fields as Fenton-, electro-Fenton and photoelectro-Fenton chemistry and foreseen their perspectives.

Nitric oxide is an important cell signaling molecule in physiology and pathophysiology. An indirect method for monitoring nitric oxide (NO) by determining nitrate and nitrite by microchip capillary electrophoresis (CE) with electrochemical (EC) detection has been developed. The amount of nitrite formed in this reaction (analyzed by capillary electrophoresis) was compared with the amount of oxygen consumed (measured by polarography). Were observed a linear relationship between the amount of consumed oxygen and the amount of nitrite formed in the measured range. These results demonstrate that polarographic measurements of the amount of oxygen consumed in the reaction with NO could be used to estimate the concentration of dissolved NO in authentic media. Polarography is an adequate method also to quantitative kinetic study of the free radical activity and of the trapping capacity of different compounds. This method is based on measure of the catalytic polarografic current of Fe(III) in the presence of free radical sources (TBH, hydrogen-peroxydes), and their traps. Personal contribution of the authors in this field is discussed.     

Strategies for revealing lower abundance proteins in two-dimensional protein maps

One of the most challenging contemporary research endeavors is the mapping of proteins and establishing their linkages to normal and pathological conditions. The availability of current proteomics technologies has greatly facilitated the separation and identification of proteins in a complex protein mixture by standard two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy number is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. It is the current limits of detection of the methods that are used that prevents the detection of these proteins not the proteins themselves. More importantly, considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understand biological changes, and two-dimensional gel electrophoresis remains the only technique that can offer sufficient resolution to address this issue at a functional level. Cellular fractionation techniques followed by specific affinity probes for tracking target proteins have been developed to deplete the proteome of high abundance proteins in order to increase the sample loading for achieving greater sensitivity for proteins present in low abundance. Those applications can entail the removal of one protein or a class of proteins that interferes with the resolution of proteins in a 2-DE map. Moreover, the use of better solubilizing detergents in combination with an overlapping narrow immobilized pH gradients, results in higher resolution by stretching the protein pattern in the first dimension. In this review we will discuss strategies to remove high abundance proteins that can result in the visualization and detection of low abundance proteins in biological samples. The potential use of these strategies, as a means of developing diagnostic tools for early screening of diseases and identification of drug targets for therapeutic intervention, will also be discussed.     

Presence of polyphosphate of low molecular weight in zygomycetes.

Polyphosphate of average chain length corresponding to 10 phosphate units was detected in the mycelial extract of zygomycetes. Gel electrophoresis techniques commonly used for the separation and characterization of acidic mucopolysaccharides were successfully used for the detection, purification, and characterization of the polyphosphate.     

Quantitative analysis of chemiluminescence signals using a cooled charge-coupled device camera

Chemiluminescence has been considered an alternative to radioisotopic detection of materials in the life sciences. One application of this nonradioisotopic method is the electrophoretic mobility shift assay. The essential requirement for quantitative applications is that the chemiluminescence signal is linearly proportional to the concentration. However, the generation of chemiluminescence is a multi-step process, therefore linearity cannot be assumed. Therefore, it is important to verify linearity before applying the method as a quantitative tool. We used a commercial chemiluminescence generating system to evaluate the validity of quantitative measurements of biotin-labeled tRNA and single-stranded DNA. The results indicate that the relationship between the chemiluminescence signals and the quantity of biotin-labeled nucleic acids is hyperbolic rather than linear. However, it was found that with less than 50 fmol of biotin-labeled nucleic acid, which corresponds to 2.5 nM in 20 microl, linearity can be demonstrated within 5% error. Therefore, chemiluminescence-based quantitative measurements are a reliable method within these limitations.