DNA sequencing using 96-capillary array electrophoresis

Practical DNA sequencing in a rugged capillary array electrophoresis system coupled directly to 96-well microtiter plates is demonstrated. A CCD detector was used to monitor all capillaries simultaneously with laser-induced fluorescence at 1.75 frames per second. The reconstructed electropherograms show good signal-to-noise ratios and resolution for the entire capillary array. The system used standard dye labeling and image splitting to obtain fluorescence intensities in two wavelength regions to allow calling up to 410 bases for the DNA sequence. The use of a replaceable poly(ethylene oxide) matrix and a protective poly(vinylpyrrolidone) coating allows high separation speed and short turnaround time for high throughput DNA sequencing. Critical evaluation of the system performance over repeated runs with base calling is presented.     

On-line combination of monolithic immobilized pH gradient-based capillary isoelectric focusing and capillary zone electrophoresis via a partially etched porous interface for protein analysis

An integrated platform consisting of monolithic immobilized pH gradient-based capillary isoelectric focusing (M-IPG CIEF) and capillary zone electrophoresis (CZE) coupled by a partially etched porous interface was established. Since carrier ampholytes (CAs) were immobilized on monolith in M-IPG CIEF to form a stable pH gradient, subsequent depletion of CAs at the interface to prevent the interference on CZE separation and detection were avoided. Moreover, a partially etched porous capillary column, which was facile for fabrication and durable for operation, was exploited as the interface to combine M-IPG CIEF and CZE. The RSD values in terms of the migration time for M-IPG CIEF separation, transfer protein from the first dimension to the second dimension, and CZE separation, were 2.4%, 3.9% and 2.3%, respectively. With a 6-protein mixture as the sample, two-dimensional capillary electrophoresis (2D-CE) separation was successfully completed within 116 min, yielding a peak capacity of ～200 even with minute sample amount down to 5.0 μg/mL. The limit of detection was 0.2 μg/mL. In addition, proteins extracted from milk were used to test the performance of such a 2D-CE separation platform. We expect that such a novel 2D-CE system would provide a promising tool for protein separation with high throughput and high peak capacity.     

Multi-dimensional liquid phase based separations in proteomics

This review covers recent developments towards the implementation of multi-dimensional (MuD) liquid phase based systems for proteome investigations. Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as a standard approach in proteomics, its drawbacks including the limited dynamic range and molecular mass range, together with lack of on-line integration with biological mass spectrometery (Bio-MS) have limited its widespread use and applications in proteomics. In the meantime, various liquid-phase based multi-dimensional separation techniques have been explored. Especially, with the emergence of the combination of nanoflow capillary high-performance liquid chromatography (cHPLC) and Bio-MS, attention is again refocused on utilizing multi-dimensional liquid-phase based separation of proteins. Some remarkable applications of on-line analysis of intact proteins and on-column digested proteins, and the emergence of approaches such as multiple HPLC-electrospray ionization tandem MS and capillary array electrophoresis-matrix assisted laser desorption ionization MS, have stimulated thinking towards developing a automated multi-dimensional system (MuDSy) that integrates liquid phase based separation, digestion and identification of proteins in complex biological mixtures.     

Capillary array reversed-phase liquid chromatography-based multidimensional separation system coupled with MALDI-TOF-TOF-MS detection for high-throughput proteome analysis

A high-throughput on-line capillary array-based two-dimensional liquid chromatography (2D-LC) system coupled with MALDI-TOF-TOF-MS proteomics analyzer for comprehensive proteomic analyses has been developed, in which one capillary strong-cation exchange (SCX) chromatographic column was used as the first separation dimension and 18 parallel capillary reversed-phase liquid chromatographic (RPLC) columns were integrated as the second separation dimension. Peptides bound to the SCX phase were "stepped" off using multiple salt pulses followed by sequentially loading of each subset of peptides onto the corresponding precolumns. After salt fractionation, by directing identically split solvent-gradient flows into 18 channels, peptide fractions were concurrently back-flushed from the precolumns and separated simultaneously with 18 capillary RP columns. LC effluents were directly deposited onto the MALDI target plates through an array of capillary tips at a 15-s interval, and then alpha-cyano-4-hydroxycinnamic acid (CHCA) matrix solution was added to each sample spot for subsequent MALDI experiments. This new system allows an 18-fold increase in throughput compared with serial-based 2D-LC system. The high efficiency of the overall system was demonstrated by the analysis of a tryptic digest of proteins extracted from normal human liver tissue. A total of 462 proteins was identified, which proved the system's promising potential for high-throughput analysis and application in proteomics.     

Fast analysis of antibacterial isothiazolones by capillary electrophoresis

Some technical aspects influencing the total time of CE analysis are discussed. A high throughput electrophoretic system based on micellar electrokinetic chromatography (MEKC) is demonstrated as an example. A short capillary, strong electric field, alkaline buffer (pH 9.5) generating strong electroosmotic flow, and parallel hydrodynamic pressure allow the separation of two uncharged isothiazolone derivatives within 45 s.     

Affinity chromatography and capillary electrophoresis for analysis of the yeast ribosomal proteins

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.     

现代分离技术在抗生素提取中的应用

详细介绍了膜分离技术在抗生素分离提取中的应用进展,并简单阐明了高效毛细管电泳技术、双水相技术和反胶束萃取技术在抗生素提取中的应用情况。对这些现代分离技术的发展前景作了简要探讨。     

Octadecylated silica monolith capillary column with integrated nanoelectrospray ionization emitter for highly efficient proteome analysis

An improved strategy for the preparation of octadecylated silica monolith capillary column with high homogeneity was proposed. Column performance was evaluated by nanoscale HPLC. The design for constructing an integrated nanoelectrospray emitter on the octadecylated silica monolith capillary column was first introduced. In comparison with the separated configuration where the emitter is connected to monolithic capillary column by the aid of a zero dead volume union, the integrated capillary column has the inherent advantage of the minimized extracolumn volume thus providing improved separation quality. The performance of the integrated monolithic capillary column was evaluated by separation of BSA tryptic digest, and peak capacity of 313 with a 30-cm column was obtained. The high separation performance allowed highly confident identification of 662 distinct proteins through assignment of 1933 unique peptides by analysis of tryptic digest of 0.5 mug of Saccharomyces cerevisiae proteins. The higher separation efficiency by a 60-cm monolithic capillary column increased the proteome coverage with identification of 1323 proteins through assignment of 5501 unique peptides over 400-min gradient elution.     

二维液相色谱在中药分析的应用

中药的物质体系复杂,对其组分的分离、鉴定和制备需要更高的分离度,二维液相色谱能将分离机理不同而又相互独立的两支 色谱柱串联构成分离系统,应用于复杂基质的中药材及中药复方制剂的分析,可显著提高峰容量和色谱峰鉴定的可靠性,降低色谱峰重叠, 使分离效率与分析通量大为提高。综述二维液相色谱基本原理及其在中药分析中的应用研究新进展。     

手性药物分析方法研究进展

综述了近10 年来手性药物分离检测方法的发展,包括高效液相色谱法、气相色谱法、毛细管电泳法,以及超临界流体色谱法等,旨在为该领域的进一步发展提供参考。     

Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns

The high resolving power of the chromatographic separation of single- and double-stranded nucleic acids in 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns was utilized for mutation screening in polymerase chain reaction amplified polymorphic loci. Recognition of mutations is based on the separation of homo- and heteroduplex species by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions, resulting in characteristic peak patterns both for homozygous and heterozygous samples. Six different single nucleotide substitutions and combinations thereof were confidently identified in 413 bp amplicons from six heterozygous individuals each of which yielded a different unique chromatographic profile. Alternatively, mutations were identified in short, 62 bp PCR products upon their complete on-line denaturation at 75 degrees C taking advantage of the ability of IP-RP-HPLC to resolve single-stranded nucleic acids of identical length that differ in a single nucleotide. Separations in monolithic capillary columns can be readily hyphenated to electrospray ionization mass spectrometry and promise increased sample throughput by operating in arrays similar to those already used in capillary electrophoresis.     

Synergism of capillary isotachophoresis and capillary zone electrophoresis

The combination of capillary isotachophoresis and capillary zone electrophoresis may enhance greatly the performance of analytical capillary electrophoresis with respect to both separation power and the concentration sensitivity. The concentrating effects and the separation power of isotachophoresis allow the analysis of diluted samples and the elimination of interferences due to bulk components. The separation process of zone electrophoresis enables one to resolve the stack of trace analytes and detect the resulting individual zones with high sensitivity. The transition of isotachophoresis into zone electrophoresis plays the key role in the overall performance of this hyphenated technique. This article describes the dynamics of the conversion of isotachophoresis into zone electrophoretic mode and shows that the key role is played by the segments of the leading and terminating zones from the isotachophoretic stage. The magnitude of these segments directly effects the detection time as well as the separation width of the peaks of analytes. It is shown that these effects are also important in the analyses by capillary zone electrophoresis where isotachophoresis is induced by the sample itself. Finally, the paper presents a list of recommended, user-friendly, electrolyte systems which enable one to simply predict the performance of the combination isotachophoresis-zone electrophoresis.     

Recent advances in DNA sequencing by End-Labeled Free-Solution Electrophoresis (ELFSE)

End-Labeled Free-Solution Electrophoresis (ELFSE) is a new technique that is a promising bioconjugate method for DNA sequencing (or separation) and genotyping by both capillary and microfluidic device electrophoresis. Because ELFSE enables high-resolution electrophoretic separation in aqueous buffer alone (i.e., without a polymer matrix), it eliminates the need to load viscous polymer networks into electrophoresis microchannels. To achieve microchannel DNA separations with high performance, ELFSE requires monodisperse perturbing entities (i.e., drag-tags), which create a large amounts of frictional drag when pulled behind DNA during free-solution electrophoresis, and which have other properties suitable for microchannel electrophoresis. In this article, the theoretical concepts of ELFSE and the required characteristics of the drag-tag molecules for the ultimate performance of ELFSE are reviewed. Additionally, the merits and limitations of current drag-tags are also discussed in the context of recent experimental data of ELFSE separation (or sequencing).     

A rapid enantioseparation system of capillary electrochromatography modified by electrostatic adsorption with transfersomes

Transfersomes were a special kind of nanomaterials with higher deformability and flexibility. A rapid method for coated-column preparation using anionic transfersomes as a coating material by electrostatic adsorption was developed. With carboxymethyl-β-cyclodextrin added in running buffer as the chiral selector, the capillary electrochromatography enantioseparation system based on the transfersomes-coated column modified by electrostatic adsorption was established for the first time. Propranolol and metoprolol acted as model drugs to evaluate the enantioseparation performance, these two basic drugs achieved baseline separation with satisfactory resolution and selection factor in this transfersomes-electrochromatography system but only partial separation in bare column system. In order to get the optimal separation condition, concentration of chiral selector, buffer pH, and applied voltage were systematically investigated. A rapid and efficient enantioseparation electrochromatography system was established and showed that transfersomes as the stationary phase could efficiently improve chiral separation effect.     

Monoliths for microfluidic devices in proteomics

We report here on the preparation of monolithic capillary columns in view to their integration in a microsystem for on-chip sample preparation before their on-line analysis by electrospray and mass spectrometry (ESI-MS). These monolithic columns are based on polymer materials and consist of reverse phases for peptide separation and/or desalting. They were prepared using lauryl methacrylate (LMA), ethylene dimethacrylate (EDMA) as well as a suitable porogenic mixture composed of cyclohexanol and ethylene glycol. The resulting stationary phases present thus a C12-functionality. The LMA-based columns were first prepared in a capillary format using capillary tubing of 75 microm i.d. and tested in nanoLC-MS experiments for the separation of a commercial Cytochrome C digest composed of 12 peptidic fragments whose isoelectric point values and hydrophobic character cover a wide range. The LMA-based columns were capable of separating the peptidic fragments and their performances were seen to be similar as those of standard commercial columns dedicated to proteomic purposes with calculated separation efficiencies up to 145 x 10(3) plates/m. Monolithic LMA-based phases were then successfully polymerized in microchannels fabricated using the negative photoresist SU-8. After the polymerization, the systems were seen to withstand the pressures applied during the nanoLC-MS separation tests that were carried out in the same conditions as for the monolithic capillary columns. The pressure drop during these tests of the in-microchannel monoliths was as high as 50 bar; however, the separation was not as good as for a capillary format which could be accounted for by the monolith dimensions.     

Online multidimensional separation with biphasic monolithic capillary column for shotgun proteome analysis

A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 microm i.d. capillary. Separation performance of this column was evaluated by a five-cycle online multidimensional separation of 10 microg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only approximately 900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely attributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation.     

Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification

A resurgence of interest in the human plasma proteome has occurred in recent years because it holds great promise of revolution in disease diagnosis and therapeutic monitoring. As one of the most powerful separation techniques, multidimensional liquid chromatography has attracted extensive attention, but most published works have focused on the fractionation of tryptic peptides. In this study, proteins from human plasma were prefractionated by online sequential strong cation exchange chromatography and reversed-phase chromatography. The resulting 30 samples were individually digested by trypsin, and analyzed by capillary reversed-phase liquid chromatography coupled with linear ion trap mass spectrometry. After meeting stringent criteria, a total of 1292 distinct proteins were successfully identified in our work, among which, some proteins known to be present in serum in <10 ng/mL were detected. Compared with other works in published literatures, this analysis offered a more full-scale list of the plasma proteome. Considering our strategy allows high throughput of protein identification in serum, the prefractionation of proteins before MS analysis is a simple and effective method to facilitate human plasma proteome research.     

Simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and charge-coupled device, or photomultiplier for determining proteins in capillary electrophoresis: a critical comparison

The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices use commercially available, reasonably priced optical components that can be used by nonexperts. After fine optimization of several optical and separation parameters in both devices, a zone capillary electrophoresis methodology was achieved with 50mM borate buffer (pH 8.4) and 10mM phytic acid for the determination of two phycobiliproteins. Detection limits of 0.50 and 0.64microg/ml for R-phycoerythrin and B-phycoerythrin, respectively, were achieved by using the LED-induced fluorescence capillary electrophoresis (LED-IF-CE) system, and corresponding detection limits of 2.73 and 2.16microg/ml were achieved by using the Xe lamp-IF-CE system. Analytical performance and other parameters, such as cost and potential to miniaturization, are compared for both devices.     

Capillary electrochromatographic separation of aromatic amino acids possessing peptides using porphyrin derivatives as the inner wall modifiers

Two different porphyrin derivatives (H2TPP(m-OPh)4 and Rh(III)TPP(m-OPh)4) were investigated with respect to their capability to help resolution of five model aromatic peptides in capillary electrophoresis/open tubular capillary electrochromatography. Though the main separation mechanism was preferentially based on the ionic properties of the separated analytes, involvement of particularly H2TTPP(m-OPh)4-peptide interactions at alkaline pH (8.0) was clearly demonstrated. In combination with Tris-phosphate buffer, a speed up of the separation was observed at pH 2.25 (particularly if Rh(III)TPP(m-OPh)4 was used as capillary coating); in spite of the speed up of the separation the selectivity of the system was sufficient and resulted in a complete separation of the five model peptides. It can be expected that Rh(III)TPP(m-OPh)4 capillary coating in combination with Tris-phosphate buffer can be generally used for a considerable speeding up of lengthy separations of peptides in acidic media with some decrease in the separation power of the system.     

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis

The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.