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ABSTRACT: In 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.
Chromatographia 04/2012; 57(9):617-621. · 1.20 Impact Factor
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ABSTRACT: 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.
Journal of Pharmaceutical and Biomedical Analysis 10/2005; 39(3-4):848-52. · 2.97 Impact Factor
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ABSTRACT: 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.
Journal of Chromatography B 04/2005; 817(1):119-26. · 2.89 Impact Factor
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ABSTRACT: An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.
Analytical Chemistry 02/2003; 75(2):215-8. · 5.86 Impact Factor
