Biomedical subjects

Yukui Zhang

Publications and source records attributed to Yukui Zhang.

At least 19 recordsLinked to original sources

PubMed2006

Induction of tobacco genes in response to oligochitosan.

Oligochitosan has a variety of biological activities. To understand its mechanism, DDRT-PCR, reverse Northern blot and quantitative relative RT-PCR were used to identify and isolate genes whose transcription were altered in cultured Nicotiana tabacum (var. Samsun NN) plants that were treated with oligochitosan. Three genes whose mRNA levels significantly changed in response to oligochitosan were isolated and identified. One gene is up-regulated, and two genes are down-regulated. These genes encode a DNAJ heat shock N-terminal domain-containing protein, a histone H1 gene and a hypothetical protein, whose function is unknown. The results suggest that the usefulness of mRNA differential display technique for the detection of plant metabolic pathways affected by oligochitosan.

Blotting, Northern↗

PubMed2006

Separation and identification of compounds in Adinandra nitida by comprehensive two-dimensional liquid chromatography coupled to atmospheric pressure chemical ionization source ion trap tandem mass spectrometry.

A comprehensive two-dimensional liquid chromatographic (2D-LC) separation system based on the combination of a CN column and a Merck Chromolith Flash reversed-phase column was developed for the separation of components in Adinandra nitida, one type of traditional Chinese medicine (TCM). The two dimensions were connected by a ten-port, dual-position valve controlled automatically by software written in-house. The effluents were detected by both ultraviolet and atmospheric pressure chemical ionization source ion trap tandem mass spectrometry (MS). The calculated peak capacity of the 2D-LC-MS/MS system was above 1240. More than 57 components were resolved in the methanol extract from Adinandra nitida leaves, and five of these were identified based on their relative retention times, molecular weights and MS/MS spectra.

Chromatography, High Pressure Liquid↗

PubMed2006

Preparation and evaluation of C18-bonded 1-microm silica particles for pressurized capillary electrochromatography.

Nonporous silica spheres (1 microm) were synthesized and bonded with octadecylsilane functionality. These stationary phase particles were packed electrokinetically into fused-silica capillaries with 100 microm id for a length of 20 cm, which was evaluated by using pressurized CEC (pCEC). The efficiency of the C18 RP column was characterized through the theoretical plates of thiourea, benzyl alcohol, toluene, styrene, and naphthalene. The effects of experimental parameters such as the applied voltage, sample size, pump flow rate, pH value and the concentration of the buffer solution, and the content of methanol in the mobile phase, on-column efficiency were evaluated. Column efficiency as high as 200 000 theoretical plates per meter for naphthalene was obtained with the optimal condition of 70% v/v methanol and 30% v/v of 10 mmol/L phosphate buffer (pH 7.8) at an applied voltage of 10 kV and a supplementary pressure of 500 psi.

Benzyl Alcohol↗

PubMed2006

Macroporous polyacrylamide-based monolithic column with immobilized pH gradient for protein analysis.

Monolithic materials were prepared in capillaries by in situ polymerization of acrylamide, glycidyl methacrylate, and N,N'-methylenebisacrylamide in the presence of 1,4-butanediol, dodecanol, and DMSO as porogens. With Ampholine attached to the surface of the porous monolith via epoxide groups, a monolithic-IPG (M-IPG) was formed and showed good mechanical and chemical stability. With such a column immobilized by Ampholine 3.5-10, IEF-MIX 3.6-9.3 was separated and good linearity was obtained. The CIEF behavior of M-IPG was evinced by comparing the current with that in the open tubular capillary. In addition, the protein mixtures excreted from lung cancer cells of rats were analyzed with such a new M-IPG column.

Acrylic Resins↗

PubMed2006

On-line hyphenation of supercritical fluid extraction and two-dimensional high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometer for the analysis of Ganoderma lucidum.

A novel on-line system combining supercritical fluid extraction (SFE) and two-dimensional high performance liquid chromatography (2D-HPLC) was developed. A trap column and two three-port valves were employed to couple SFE and 2D-HPLC system, which was composed of a CN column and a monolithic silica column, connected by a 10-port dual-position valve. The analytes extracted by supercritical CO2 were completely transferred to the 2D-HPLC system. After separation in two orthogonal modes, the eluents were delivered to APCI-tandem-MS for identification of the samples. In this way, sample preparation, separation, detection, and identification were integrated into an on-line system permitting analysis of the fruiting bodies of Ganoderma lucidum, and at least 73 components in the extract were resolved with calculated peak capacity of up to 1643.

Acetonitriles↗

PubMed2006

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS.

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. In addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

Animals↗

PubMed2006

[Application of adjustable moving overlapping separation range map in multisegment stepwise gradient elution condition optimization of high performance liquid chromatography].

Computer-assisted optimization of high performance liquid chromatographic separation conditions can be used to obtain optimized experimental conditions in relatively short time and low cost, and has been widely applied to the separation of complex samples. Based on "moving overlapping separation range map" method, a novel method of optimizing multi-segment stepwise gradient elution conditions, "adjustable moving overlapping separation range map" method, was developed. By predicting the retention time, peak width and resolution of each solute under different mobile phase conditions, the overlapping separation range map of the solutes in the sample is plotted. When the mobile phase composition on the current step is optimized, the overlapping separation range map for solutes eluted in next one or two steps is replotted simultaneously by considering the influence of elution conditions for current step on the retention of uneluted solutes. It is helpful to consider the separation of each solute in the sample simultaneously and find optimized separation conditions close to the global optimum by investigating the effect of mobile phase conditions for current step on the separation of solutes eluted in the current and next one or two steps. By slightly adjusting the optimized separation conditions using grid search method, the separation performance can be further improved. The application of "adjustable moving overlapping separation range map" method was illustrated by using experimental data from literature. The advantage of this method in high performance liquid chromatography was verified in binary mobile phase system.

English Abstract↗

PubMed2006

[Separation and recognition of biomacromolecule by molecular imprinting technique].

Molecular imprinting technique is a novel technique based on mimicking specific action of antibody-antigen. The emergence and the development of the technique are reviewed in this article. The focuses of this article include the introductions of the synthesis conditions, the comparisons of the various approaches on preparation methods as well as the recognition mechanisms of the biomacromolecule imprinted polymers. The primary synthetic methods include the embed technique, the surface imprinting procedure and the epitope approach. The epitope approach is based on using a short peptide as a template that represents only part of a larger peptide or protein, which in turn can be recognized by the synthesized polymer. This approach for the development of the biomacromolecule imprinted polymers selective to proteins is attractive from an economic viewpoint: a small peptide is usually less expensive, and the quantity necessary for the polymer preparation is more readily available than that of the corresponding protein. In the end, the limitations and the prospective applications of this biomacromolecular imprinted technique are also discussed.

Epitope Mapping↗

PubMed2005

Rapid protein digestion and identification using monolithic enzymatic microreactor coupled with nano-liquid chromatography-electrospray ionization mass spectrometry.

A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-HPLC-MS) system. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at the fast flow rate of 1 microL/min, which afforded a residence time of 7s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Future more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture.

Bioreactors↗

PubMed2005

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography.

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing.

Animals↗

PubMed2005

On-line combination of capillary isoelectric focusing and capillary non-gel sieving electrophoresis using a hollow-fiber membrane interface: a novel two-dimensional separation system for proteins.

A novel two-dimensional (2D) separation system for proteins was reported. In the system, a piece of dialysis hollow-fiber membrane was employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) and capillary non-gel sieving electrophoresis (CNGSE). The system is similar equivalent to two-dimensional polyacrylamide gel electrophoresis (2D PAGE), by transferring the principal of 2D PAGE separation to the capillary format. Proteins were focused and separated in first dimension CIEF based on their differences in isoelectric points (pIs). Focused protein zones was transferred to the dialysis hollow-fiber interface, where proteins hydrophobically complexed with sodium dodecyl sulfate (SDS). The negatively charged proteins were electromigrated and further resolved by their differences in size in the second dimension CNGSE, in which dextran solution, a replaceable sieving matrix instead of cross-linked polyacrylamide gel was employed for size-dependent separation of proteins. The combination of the two techniques was attributed to high efficiency of the dialysis membrane interface. The feasibility and the orthogonality of the combined CIEF-CNGSE separation technique, an important factor for maximizing peak capacity or resolution elements, were demonstrated by examining each technique independently for the separation of hemoglobin and protein mixtures excreting from lung cancer cells of rat. The 2D separation strategy was found to greatly increase the resolving power and overall peak capacity over those obtained for either dimension alone.

Animals↗

PubMed2005

On-line concentration of proteins in pressurized capillary electrochromatography coupled with electrospray ionization-mass spectrometry.

Pressurized capillary electrochromatography (pCEC) and electrospray ionization-mass spectrometry (ESI-MS) have been hyphenated for protein analysis. Taken cytochrome c, lysozyme, and insulin as samples, the limits of detection (LODs) for absolute concentrations are 10(-11) mol (signal-to-noise ratio S/N = 3) with relative standard deviations (RSDs) of retention time and peak area, respectively, of less than 1.7% and 4.8%. In order to improve the detection sensitivity, on-line concentration by field-enhanced sample-stacking effect and chromatographic zone-sharpening effect has been developed, and parameters affecting separation and detection, such as pH and electrolyte concentration in the mobile phase, separation voltage, as well as enrichment voltage and time, have been studied systematically. Under the optimized conditions, the LODs of the three proteins could be decreased up to 100-fold. In addition, the feasibility of such techniques has been further demonstrated by the analysis of modified insulins at a concentration of 20 microg/mL.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2005

On-line concentration of peptides and proteins with the hyphenation of polymer monolithic immobilized metal affinity chromatography and capillary electrophoresis.

An iminodiacetic acid (IDA)-type adsorbent is prepared at the one end of a capillary by covalently bonding IDA to the monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate). Cu(II) is later introduced to the support via the interaction with IDA. By this means, polymer monolithic immobilized metal affinity chromatography (IMAC) materials are prepared. With such a column, IMAC for on-line concentration and capillary electrophoresis (CE) for the subsequent analysis are hyphenated for the analysis of peptides and proteins. The reproducibility of such a column has been proved good with relative standard deviations (RSDs) of dead time of less than 5% for injection-to-injection and 12% for column-to-column (n = 3). Through application on the analysis of standard peptides and real protein samples, such a technique has shown promising in proteome study.

Chromatography, Affinity↗

PubMed2005

Hybrid organic-inorganic phenyl monolithic column for capillary electrochromatography.

A novel hybrid organic-inorganic silica-based monolithic column possessing phenyl ligands for reversed-phase (RP) capillary electrochromatography (CEC) is described. The monolithic stationary phase was prepared by in situ co-condensation of tetraethoxysilane (TEOS) with phenyltriethoxysilane (PTES) via a two-step catalytic sol-gel procedure to introduce phenyl groups distributed throughout the silica matrix for chromatographic interaction. The hydrolysis and condensation reactions of precursors were chemically controlled through pH variation by adding hydrochloric acid and dodecylamine, respectively. The structural property of the monolithic column can be easily tailored through adjusting the composition of starting sol solution. The effect of PTES/TEOS ratios on the morphology of the created stationary phases was investigated. A variety of neutral and basic analytes were used to evaluate the column performance. The CEC columns exhibited typical RP chromatographic retention mechanism for neutral compounds and had improved peak shape for basic solutes.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed2005

Analysis of flavonoids in leaves of Adinandra nitida by capillary electrochromatography on monolithic columns with stepwise gradient elution.

Extracts of Adinandra nitida leaves, known as Shiyacha in China, were analyzed by monolithic columns of capillary electrochromatography (CEC). To obtain good resolution within a short time, stepwise gradient elution of CEC was employed, and the effects of experimental parameters, such as the buffer, the gradient conditions, and the mode of injection were studied systematically. Under optimized conditions, analysis could be accomplished in 25 min on a monolithic rod of macroporous poly(butyl methacrylate-co-ethylene dimethacrylate). With two identified flavonoids, epicatechin and apigenin, as markers, a quality control method for Shiyacha and its relevant products was established. The calibration curves exhibited good linear behavior over the concentration range of two orders of magnitude. On combination with an on-line concentration technique, the detection limit of flavonoids could be decreased to 25 ng for apigenin.

Apigenin↗

PubMed2005

Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins.

A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.

Animals↗

PubMed2005

[Application in capillary electrophoresis-based two-dimensional separation system].

An on-column, etched fused-silica porous junction interface was designed and fabricated, and an integrated two-dimensional separation platform involving on-line coupling of capillary isoelectric focusing (CIEF) with capillary electrophoresis (CE) was constructed by the interface. The primary advantages of this novel porous junction interface over previous designs are no dead volume, simplicity, ruggedness and convenience of column switching. This interface is particularly well suited for on-line coupling of CE-based multiple dimensional separation system. The feasibility and performance of the 2D CIEF-CE system constructed by such an etched porous junction were evaluated by the separation of soluble extracts of an antler. The results indicated that the total separation time was less than 1 h, and the 2D CE separation system was found to increase the resolving power and overall peak capacity over single dimension mode.

Animals↗

PubMed2005

[A study on peak compression effect in ion-exchange capillary electrochromatography with relaxation theory].

Due to the electric characteristics of the solute transport in capillary electrochromatography (CEC), some special phenomena might occur. An example is the peak compression effect in ion-exchange capillary electrochromatography, which cannot be explained with common chromatography theory. According to the basic model of relaxation theory, it is considered that there are "co-" and "counter-" solute flows in mobile phase and in stationary phase, the expressions of the first moment and the second center moment of profile, and column efficiency are derived by the method of Laplace transform. The relations of electrophoresis velocity, electroosmosis velocity and electric dispersion velocity are also investigated with these expressions. The results show that this new model offers a better explanation for peak compression effect on the migration behaviour of charged solute in CEC. The retention time will be shorter by peak compression effect, which is similar to a solvent gradients process. The peak compression effect is not steady, and doesn't appear until the conditions are fitted in some special situation.

Capillary Electrochromatography↗