Weibing Zhang

Recent advances in immobilized enzymatic reactors and their applications in proteome analysis.

Immobilized enzymatic reactors recently have drawn much attention because of the striking advantages, such as high substrate turnover rate and ease in coupling with the separation and detection systems. Carrier materials, which have great effects on the development of the immobilized enzymatic reactors, have always being the focus of study. In this paper, the contributions, mainly in the last 5 years, on the enzymatic reactors and their applications in proteome study are reviewed, with some newly developed inorganic and organic carriers for enzyme immobilization described in details. Moreover, the hyphenation of immobilized enzymatic reactors with the separation and identification systems is also summarized. By reviewing these achievements, it could be seen that enzymatic reactors have very bright future, especially in proteome analysis.

Determination of 1-phenyl-3-methyl-5-pyrazolone-labeled carbohydrates by liquid chromatography and micellar electrokinetic chromatography

In this paper, the method for the derivatization of carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) was simplified. One-third of the derivatization time was saved. Five monosaccharide derivatives have been well separated by MEKC and HPLC under optimized conditions. Good reproducibility could be obtained with relative standard deviation (RSD) values of the migration times within 5.0 and 2.3%, respectively. Furthermore, the developed methods have been successfully applied to the analysis of carbohydrates in Aloe powder and food. These methods are quite useful for routine analysis of monosaccharides and oligosaccharides in real samples.

Miniaturized two-dimensional capillary electrophoresis on a microchip for analysis of the tryptic digest of proteins

A two-dimensional capillary electrophoresis platform, combining isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), was established on a microchip with the channel width and depth as 100 mum and 40 mum, respectively. With polyacrylamide as permanent coating, EOF in the microchannel, which could impair the separation, was decreased to 3.4x10(-9)m(2).V(-1).s(-1), about 1/10 of that obtained in the uncoated set-up. During the separation, peptides were first focused by IEF in the first dimensional channel, and then directly driven into the perpendicular channel by controlling the applied voltages, and separated by CZE. Effects of various experimental parameters, including the electric field strength, channel length, and injection frequency from the first to the second dimensional separation channel, were studied. Under optimized condition, the digests of BSA and proteins extracted from E. coli were separated, and a peak capacity of 540 was obtained, which was far greater than that obtained by each single dimensional separation. All these results showed the promise of multidimensional separation on a microchip for the high-throughput and high-resolution analysis of complex samples.

Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60 degreesC with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C-1-C-20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C-18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (lambda (ex) 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n = 6) for each fatty acid derivative are <4%. The detection limits are at 45-68 fmol levels for C-14-C-20 fatty acids and even lower levels for

Preparation and application of monolithic columns with narrow immobilized pH gradients.

Monolith with immobilized pH gradient (M-IPG) is a novel separation matrix for amphoteric substances, such as peptides and proteins. To improve the properties of the newly designed column, efforts were made to optimize the preparation procedure, including the concentrations of the monomers, glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA), as well as the kinds of porogens, which led to a monolith with improved permeability, uniformity and continuity. In addition, different diamines, including ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane and 1,5-diaminopentane, were subjected to aminate the polymer, and the last two exhibited excellent reactivity with epoxy on the polymer surface, which could obviously make the immobilization of pH gradient facilitated. Under the optimal conditions, a simple method to prepare M-IPG column with narrow pH gradient was developed with commercial carrier ampholytes (CAs) solution. Such columns were applied into the analysis of proteins and peptides, and showed the improved resolution compared to the traditional one with a wide pH distribution.

Effects of Experimental Parameters on the Signal Intensity of Capillary Electrophoresis Electrospray Ionization Mass Spectrometry in Protein Analysis

SummaryIn recent years, the hyphenation of capillary electrophoresis (CE) and electrospray ionization mass spectrometry (ESI-MS) has been widely used for the analysis of biological molecules with high efficiency and high accuracy. However, the signal intensity of CE-ESI-MS is restricted by various parameters. This paper reports the effects on the CE-ESI-MS signal of the pH and the electrolyte concentration, the formic acid concentration of the sheath liquid, and the sheath liquid composition, using several proteins as samples. The study was performed systematically by experimental and theoretical analyses. The maximum signal intensity of three proteins (cytochrome c, insulin, bovine serum albumin) was attained with a pH 4.40 buffer containing 75 mM formic acid and 75 mM ammonium acetate. Investigation of the influence of the formic acid concentration in the sheath liquid (over the range of 0%–1%) on the ESI-MS signal of brovine serum albumin showed that the feasible amount of the formic acid in sheath liquid could improve the signal intensity of the sample ions. However, considerable band broadening was observed that should be attributed mainly to column overloading, band spreading at the interface, and scanning data acquisition.

Rapid separation of nucleosides by capillary electrochromatography with a methacrylate-based monolithic stationary phase

SummarySeparation of nucleosides by capillary electrochromatography with a methacrylate-based monolithic column is described. Because the nucleosides are relatively hydrophilic compounds, chromatographic parition is very weak on non-polar stationary phases. A small quantity of organic modifier is usually required to increase their retention factars but, because the wettability of the stationary phase by the mobile phase is rather poor, bubble formation can be a significant problem. This situation was improved by use of a polymeric monolithic column with higher level charged monomer in the polymerization mixlure, because of the presence of charged moieties on the surface of the monolithic stationary phase. The very high stability under conditions of very low organic modifier concentration used in the experiment showed that the wettability of the stationary phase by the mobile phase was satisfactory. The effect of pH, orgonic modifier content, and applied voltage on the separation was investigated. Baseline separction of six nucleosides was achieved within 4.5 min on this type of column. Because of the presence of charged moieties and butyl groups on the surface of the monolithic column, the mechanism of separation might involve both ion-exchange and hydrophobic interactions.

Study on the multiple sites binding of human serum albumin and porphyrin by affinity capillary electrophoresis

Equations to describe the two sites binding between proteins and ligands were deduced. According to these equations, not only the binding constants, but also the mole fraction of proteins in different forms could be obtained. Using the published data on the interaction between human serum albumin (HSA) and three kinds of porphyrin (coproporphyrin (CP), uroporphyrin I (UP) and protoporphyrin (PP)), a further study on their binding was carried out. It was concluded that there may exist two binding sites with the binding constants at the first site, proved to be the preferential one, being 6.50 x l0(5), 1.94 x 10(6) and 8.94 x 10(5), respectively. In addition, it was also demonstrated that the two binding sites of HSA with CP and UP might be of different kinds, though those of HSA and PP were of the same kind but at different positions.

Serially coupled microcolumn reversed phase liquid chromatography for shotgun proteomic analysis

Microcolumn RPLC (μRPLC) is one of the optimum separation modes for shotgun proteomic analysis. To identify as many proteins as possible by MS/MS, the improvement on separation efficiency and peak capacity of μRPLC is indispensable. Although the increase in column length is one of the effective solutions, the preparation of a long microcolumn is rather difficult due to the high backpressure generated during the packing procedure. In our recent work, through connecting microcolumns of 5, 10, and 15 cm length via unions with minimal dead volume, long microcolumns with length up to 30 cm were obtained, with which 318 proteins were identified from proteins extracted from Escherichia coli by μRPLC‐ESI MS/MS, and similar distributions of Mw and pI were found with single and various coupled microcolumns. Furthermore, by using MS/MS with improved sensitivity, with such a serially coupled 30 cm long microcolumn, 1692 proteins were identified within 7 h from rat brain tissue, with false positive rate (FPR) <1%. All these results demonstrated that serially couple microcolumns might be of great promising to improve the separation capacity of μRPLC in shotgun proteomic analysis.

Preparation and evaluation of a large-volume radial flow monolithic column

In this study, a 38 mL monolith with homogeneous porous structure was produced by a single polymerization from glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of porogens and an initiator. The uniform temperature distribution within the reaction system was achieved by adding reactant mixture continuously and enhancing the heat transfer ability of the polymerization system. Homogeneous porous structure in the monolith was proved by SEM and the pore size distribution profiles measured by mercury intrusion porosimetry. Experimental results from proteins separation indicated that the dynamic capacity and resolution of radial flow monolithic column were independent of flow rates. Furthermore, the pressure drop on the column was linearly dependent on the flow rate and did not exceed 1.7 MPa even at a flow rate of 50 mL/min, which proved that the prepared monolith could be used in the quick separation and preparation of biopolymers.