Recent developments in electrophoretic methods

13. Given the recent extended review by Vesterberg [J. Chromatogr., 480 (1989) 3-19] of electrokinetic methods, this survey has been restricted to the last decade, which has seen tremendous progress in several fields. DNA electrophoresis has experienced strong developments, both in the sequencing strategies (which have been largely automated with the use of fluorescent probes) and in pulsed field analysis of mega-DNA fragments, which has seen such developments as inverse-field, contour-clamped and rotating gel platforms, all allowing for straight band migration in each lane. Chromosome size mapping has now become a reality. Two-dimensional (2D) maps have also shown a dramatic improvement in performance, largely through the development of immobilized pH gradients, giving highly reproducible protein spots in the 2D plane and allowing the exploration of very narrow pH regions. Blotting techniques, combined with 2D mapping, allow sequence analysis and fingerprinting of a single polypeptide spot in a complex sample without resorting to lengthy chromatographic purification steps. Chromatophoresis generates a novel type of 2D mapping, based on hydrophobicity vs. size, rather than on charge vs. size, by direct coupling of a reversed-phase high-performance liquid chromatographic (HPLC) eluate to sodium dodecyl sulphate electrophoresis. The new rising star, capillary zone electrophoresis, offers speed, a large number of theoretical plates, selectivity and small sample requirements in a highly automated equipment.     

Cyclic electrophoretic and chromatographic separation methods

A review is given of the application of cyclic analytical methods in capillary electroseparation (CE) and liquid chromatography (LC) systems. Cyclic methods have been used since the early sixties in chromatographic systems to overcome pressure limitations to resolution. From the early nineties on they have also been applied in capillary electroseparation systems to overcome voltage limitations. Some basic theory is given, outlining the temporal development of resolution in cyclic CE and LC systems and calculating the maximal resolution that can be obtained as a function of the operational parameters of pressure and electrical field. Simple equations are given for the temporal change in the peak capacity and the loss of peaks from the systems as it occurs in some cyclic systems. Finally, a circular open tubular chromatographic system is proposed using integrated pumping and continuous detection. The performance of such a system is discussed using magnetohydrodynamic and alternating current electroosmotic pumping as examples of integrated pumps and Shah Convolution Fourier transform detection as an example of a continuous detection method.     

Novel quantitative electrophoretic analysis of endotoxins on microchips

A novel microchip electrophoresis method was developed and applied for sensitive detection and quantitative analysis of endotoxins extracted from Gram-negative bacteria. The method provides a fast and quantitative differentiation of smooth and rough endotoxins based on the solubilization and complexation of the lipopolysaccharides with dodecylsulfate, and then with a fluorescent dye. The migration of the complexes was followed by LIF detection. The novel method is able to replace the SDS-PAGE with the advantage of high speed and better sensitivity, and by avoiding the laborious gel-preparation and silver staining.     

Recent innovations in enantiomer separation by electrochromatography utilizing modified cyclodextrins as stationary phases.

Enantiomer separation by electrochromatography employing modified cyclodextrins as stationary phases is performed in two ways. (i) Polysiloxane-linked permethylated beta-cyclodextrin (Chirasil-Dex 1) or related selectors are coated and immobilized onto the inner surface of a capillary column. Enantiomer separation is performed in the open tube and the method is referred to as open-tubular capillary electrochromatography (o-CEC). (ii) Silica-linked native beta-cyclodextrin, permethylated beta-cyclodextrin (Chira-Dex 2) or hydroxypropyl-beta-cyclodextrin are filled into a capillary column and the bed is secured by two frits. Enantiomer separation is performed in a packed column and the method is referred to as packed capillary electrochromatography (p-CEC). In a unified instrumental approach, method (i) as well as method (ii) can be operated both in the electro- and pressure-driven modes (o-CEC vs. open-tubular liquid chromatography (o-LC) and p-CEC vs. p-LC). It is demonstrated that the electro-driven variant affords higher efficiencies at comparable elution times. Employing a single open-tubular column coated with Chirasil-Dex 1, a unified enantioselective approach can be realized in which the same selectand is separated using all existing chromatographic modes for enantiomers, i.e., gas chromatography (GC), super-critical fluid chromatography (SFC), o-LC and o-CEC. As the chiral selector is utilized as a stationary phase, an additional chiral selector may be added to the mobile phase. In the resulting dual chiral recognition systems, enhancement of enantioselectivity (matched case) or compensation of enantioselectivity (mismatched case) may be observed. The overall enantioselectivity is dependent on the sense of enantioselectivity of the selectors chosen and their influence on the electrophoretic and electroosmotic migration of the enantiomers of a selectand.     

Nanotube-grafted polyacrylamide hydrogels for electrophoretic protein separation

Multiwalled carbon nanotube-modified polyacrylamide gels have been employed for the electrophoretic separation of proteins. Two approaches are compared in this investigation, one using nanotubes only as fillers inside the gel matrix and the other using nanotubes as catalyst for polymerization of acrylamide. In both the cases, polymerization of acryl-amide/bisacrylamide has been carried out in situ in the presence of nanotubes dispersed in the gel buffer containing monomer and cross-linker. In the former case, initiator and catalyst have been added after ultrasonication of nanotubes in the gel buffer mixture where the nanotubes play the role of filler. On the other hand, the second approach precludes use of catalyst and involves addition of initiator alone during ultrasonication of nanotubes in the gel buffer containing monomer and cross-linker, which leads to the formation of nanotube-grafted gel after 25 min. When nanotubes are used as a catalyst instead of N,N,N',N'-tetramethylethylenediamine, pore size distribution of the gel matrix and linearity of molecular weight calibration plots are found to be improved. In addition, other issues associated with the use of an external catalyst like handling the moisture-sensitive and corrosive reagent and associated irreproducibility are addressed in this approach.     

DNA analysis on electrophoretic microchips: effect of operational variables

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.     

翻译后修饰蛋白质组学分离方法的研究进展

蛋白质翻译后修饰（PTMs）是调节细胞内生理活动的重要途径。该文总结了近年来PTMs蛋白质组学相关的分离方法,包括反相（RP）色谱法、离子交换（IEX）色谱法、亲水相互作用色谱（HILIC）法、多孔石墨化碳（PGC）色谱法、毛细管电泳（CE）法及分子筛色谱（SEC）法等。这些新方法为磷酸化、乙酰化、糖基化等PTM肽段或蛋白质的鉴定提供了更高的分离度和灵敏度。此外,该文也介绍了蛋白质领域其他重要分离方法的研究进展,这些方法可能被进一步应用于PTMs蛋白质组学的研究中。     

A method for UV-bonding in the fabrication of glass electrophoretic microchips.

This paper presents an approach for the development of methodologies amenable to simple and inexpensive microchip fabrication, potentially applicable to dissimilar materials bonding and chip integration. The method involves a UV-curable glue that can be used for glass microchip fabrication bonding at room temperature. This involves nothing more than fabrication of glue "guide channels" into the microchip architecture that upon exposure to the appropriate UV light source, bonds the etched plate and cover plate together. The microchip performance was verified by capillary zone electrophoresis (CZE) of small fluorescent molecules with no microchannel surface modification carried out, as well as with a DNA fragment separation following surface modification. The performance of these UV-bonded electrophoretic microchips indicates that this method may provide an alternative to high temperature bonding.     

Recent advances in proteolysis and peptide/protein separation by chromatographic strategies

This review gives a broad glance on the progress of recent advances on proteolysis and peptide/protein separation by chroma-tographic strategies in the past ten years, covering the main research in these areas especially in China. The reviewed research focused on enzymatic micro-reactors and peptide separation in bottom-up approaches, and protein and peptide separation in top-down approaches. The new enzymatic micro-reactor is able to accelerate proteolytic reaction rate from conventionally a couple of hour...     

Advances of modern chromatographic and electrophoretic methods in separation and analysis of flavonoids

Flavonoids, one of the largest groups of secondary metabolites, are widespread in vegetable crops such as herbs, fruits, vegetables, grains, seeds and derived foods such as juices, wines, oils, etc. They receive considerable attention due to their biological and physiological importance. Hundreds of publications on the analysis of flavonoids have appeared over the past decade. Traditional and more advanced techniques have come to prominence for sample preparation, separation, detection, and identification. This review intends to provide an updated, concise overview on the recent development and trends of separation, identification and quantification for flavonoids by modern chromatographic and spectrophotometric analytical techniques, including gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE). The sample preparation before analysis is also briefly summarized.     

Increase of separation resolution through field enhancement in microchips

An enhanced ability to separate charged species from neutral compounds in a microfluidic chip is demonstrated using a chip design with low-resistance electrode channels operating with a multiport pressure/voltage controller. A factor of 2.7 improvement in resolution was obtained from chips made using identical mask designs but different etch depth protocols. Greater separation power allows one to cover a wider dynamic range for compounds with different electrophoretic mobilities.     

Achiral and chiral analysis of duloxetine by chromatographic and electrophoretic methods,a review on the separation methodologies

Duloxetine (DLX) is a widely used antidepressant drug belonging to the class of selective serotonin and norepinephrine reuptake inhibitors (SNRIs); its efficacy has been demonstrated in the treatment of not only major depressive disorders but also diabetic neuropathic pain, generalized anxiety disorder, fibromyalgia or stress urinary incontinence. It is a chiral substance and is used in therapy in the form of the enantiopure S‐DLX, which is twice as active as R‐DLX. Several methods have been published for the achiral and chiral determination of DLX in pharmaceuticals, biological materials and environmental samples, the majority using liquid chromatography and capillary electrophoresis coupled with different detection techniques (UV detection, fluorescence, mass spectrometry). The aim of the current review is to provide a systematic survey of the analytical techniques used for the determination of DLX from different matrices.     

Major histocompatibility complex (MHC) protein analysis by optimised two-dimensional electrophoretic methods

Human histocompatibility molecules HLA-Class I and Class II (DR, DQ, DP) were analysed using three two-dimensional protocols: nonequilibrium pH gradient electrophoresis (NEPHGE), isoelectric focusing-acidic gradient (IEF-AG) and isoelectric focusing-basic gradient (IEF-BG). The three methods differ in their carrier ampholyte combinations and electrophoretic conditions. They provide different pH gradients and therefore different electrofocusing profiles. The NEPHGE protocol was adequate for separating proteins across a broad range of pI mobilities, i.e. 4.4 pH units between the acidic and the basic end. In contrast, the IEF-AG and the IEF-BG protocols gave a separation power across a narrow pH range, 1.9 and 1.7 pH units respectively. Thus, whereas the NEPHGE protocol provides a tool for a global major histocompatibility complex (MHC) antigen profile analysis, the IEF-AG and -BG allows one to investigate subcomponents of the individual MHC chains. For example, NEPHGE analysis of the HLA Class I heavy chain revealed a single spot. However, IEF-BG revealed the presence of six equidistantly spaced spots spanning a short pH gradient with identical molecular weight. Similar improved resolution was seen for the HLA-DR, DQ, and DP molecules. The IEF acidic gradient was adequate for separating the alpha chain; the IEF basic gradient gave better resolution of the beta chains. This data provides a baseline set of conditions for both analytical and preparative MHC protein studies prior to amino acid sequencing.     

Glucose microfluidic biosensors based on immobilizing glucose oxidase in poly(dimethylsiloxane) electrophoretic microchips

Here we reported a novel microfluidic biosensor with an on-column immobilized enzyme microreactor. The fabrication approach of this biosensor is simple and the enzyme microreactors with controlled sizes can be placed at any desired position on the microchip. Taking glucose oxidase (GOx) as an example, electroosmotic flow (EOF) as a driving force and amperometry as a detection method, the performance of biosensors were modulated by changing the length of enzyme reactor from 0.5 cm to 3 cm, and the linear ranges were changed from 0-8.0 mM to 0-30.0 mM with the detection limits from 42 microM to 6.5 microM. The enzyme reactor remained its 65% activity after 23 days storage. It also showed good anti-interference ability and was used to quantify glucose in human serum samples.     

Recent developments in electrophoretic separations on microfluidic devices

Research combining the areas of separation science and microfluidics has gained popularity, driven by the increasing need to create portable, fast, and low analyte-consumption devices. Much of this research has focused on the developments in electrophoretic separations, which use the electrokinetic properties of analytes to overcome many of the problems encountered during system scale-down. In addition, new physical phenomenon can be exploited on the microscale not available in standard techniques. In this study, the innovative developments, including electrophoretic concentration, sample preparation/conditioning, and separation on-chip are reviewed, along with some introductory discussions, from January 2008 to July 2010.     

Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.     

Multistep purification of an antifreeze protein from Ammopiptanthus mongolicus by chromatographic and electrophoretic methods

Antifreeze proteins (AFPs) are known as thermal hysteresis proteins, which can depress the freezing points of the solution by noncolligative effects, but do not affect the melting points. Although some AFPs have been found in some plants, the identity of most proteins remains unclear, owing to insufficient quantity and quality to characterize them. In this report, we describe the purification of an AFP from the winter leaves of Ammopiptanthus mongolicus using a combination of column chromatography and gel electrophoresis. After homogenization in ascorbate-acid-containing Tris buffers (pH 7.4) the soluble proteins are captured by (diethylamino)ethyl-cellulose 52 material. An elution with 0.1-0.3M KCl leads to a crude active fraction. The crude fraction is further purified on a Superdex 75 prep-grade column and finally a Poros 20HP2 column. A complex, consisting of two proteins with relative molecular masses of 34,700 and 37,100, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, is obtained by this protein purification protocol. The recovery of two proteins from the gel is carried out by electrophoresis. The purified protein, with a molecular mass of 37,100, shows thermal hysteresis activity (THA) and can modify the normal growth of ice crystals. The THA of this purified antifreeze protein is 0.24 degrees C at the concentration of 5 mg/mL.     

The protein composition of ferret parotid saliva as revealed by high-resolution electrophoretic methods.

Ferret parotid saliva has been analysed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) to determine, for the first time, its protein composition. SDS-PAGE, in combination with Coomassie Brilliant Blue (CBB) staining, revealed up to 20 bands and the patterns were characterised by major protein constituents of Mr 105000, 51000, 47000, 33000, 22000 and 16 400 common to all samples from all animals. Sequential samples collected from the same animal during prolonged stimulation of the parasympathetic nerve (40 min at 40 Hz) showed subtle but reproducible protein changes. Saliva collected from different animals varied widely in the amount of a protein Mr 66000. 2-DE, in combination with silver staining, revealed up to 300 spots and the patterns were characterised by major protein constituents of Mr 105000 (pI 6.3-7.2), Mr 66000 (pI 4.7-5.3), Mr 51000 (pI 5.0-5.7), Mr 47000 (pI 6.0-7.5), and Mr 33000 (pI 4.7-6.0). Many of the polypeptide spot clusters consisted of one or more horizontal strings of spots suggesting extensive microheterogeneity. Both SDS-PAGE and 2-DE indicated that the protein patterns of ferret parotid saliva evoked by electrical stimulation of the parasympathetic nerve in the absence or presence of atropine are similar, i.e., the protein composition of the atropine-resistant nonadrenergic, noncholinergic (NANC) secretion is similar to that of saliva evoked in the absence of muscarinic receptor blockade.     

Recent advances in monolithic columns for protein and peptide separation by capillary liquid chromatography

Capillary liquid chromatography (cLC) has great potential for protein and peptide separation, with advantages of high efficiency, high resolution, low sample consumption, and high sensitivity when coupled with mass spectrometry. In recent years, monoliths have been widely used as the stationary phases for capillary columns, owing to easy preparation, high permeability, fast mass transfer, and low backpressure. This review summarizes recent advances (2007–2012) in monolithic columns for protein and peptide separation by cLC. After a brief introduction on the preparation of monolithic capillary columns, the emphasis of this review is focused on the recent application of such columns for protein and peptide separation by cLC. Furthermore, the challenges and potential hot points of monolithic capillary columns in the future are discussed.