On-line post-capillary affinity detection of immunoglobulin G for capillary zone electrophoresis

To address the quality issues of antibody manufacturing, post-capillary affinity detection of immunoglobulin G (IgG) is developed for capillary zone electrophoresis. In analogy to a two-dimensional separation system, capillary zone electrophoresis (CZE), as the first dimension, resolves IgG variants based on their differences in molecular structure. IgG variants separated by CZE are discriminated against other serum and cellular proteins by affinity complex formation with protein A binding fragment in a post-capillary reactor. The analytical power of post-capillary affinity detection is demonstrated for rapid and selective heterogeneity analysis of human IgG subclasses and monoclonal antibodies in complex sample matrices. By comparing with pre-capillary formation of affinity complexes between IgG and protein A, post-capillary affinity detection clearly exhibit greater resolving power for examining IgG microheterogeneity. Affinity complex formation prior to CZE analysis, however, has the advantage of lower detection limits. Detection limits suffer with post-capillary affinity detection because of the high fluorescence background contributed by the fluorescently labeled protein A in the post-capillary reactor, and the need to determine a small change in the background level upon complex formation.     

A Capillary Electrophoresis-Based Assay for Protein Kinases and Protein Phosphatases Using Peptide Substrates

A novel procedure for detection and assay of protein kinase and phosphatase activities in complex biological mixtures was developed. By means of capillary zone electrophoresis (CZE) methodology, the phosphorylated and dephosphorylated forms of the peptide Kemptide, a 46-amino-acid fragment from protein phosphatase inhibitor-1 and a peptide fragment corresponding to the R_II subunit of cAMP-dependent protein kinase (PKA), were rapidly resolved. This facilitated nonradioactive detection of PKA and protein phosphatase-2B (calcineurin) in rabbit skeletal muscle extracts. In addition, the CZE procedure enabled a site-specific assay of a 14-amino-acid peptide from the glycogen-binding subunit of protein phosphatase-1 monophosphorylated on distinct sites by PKA and casein kinase-II. These results suggest that CZE may prove to be extremely useful for the analysis of peptides that are phosphorylated at multiple sites in vivo.     

Carrier ampholytes as potential buffers in electrophoresis: physico-chemical study

Joule heating is a limiting factor when separating proteins in capillary zone electrophoresis (CZE). Low conductivity buffers, are required for high-speed separations. We investigated the use of carrier ampholytes (CA) as background electrolytes (BGE) in CZE. We prepared 25 "narrow pH cuts" of wide pH range (3-10) CA mixture in order to know if these fractions were suitable to be used as BGE in CZE. Each fraction was characterised by CZE analysis, giving an idea of its heterogeneity (number and relative abundance of molecular ampholytes). Conductivities and buffering capacities of each fraction have been also measured. Our conclusion is that "narrow pH cuts" of CA might be well suited buffers for electrophoretic separations.     

Determination of poorly separated monoclonal serum proteins by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) technique was developed for the determination of poorly separated monoclonal serum proteins by agarose gel electrophoresis (AGE). A P/ACE 5500 capillary instrument (Beckman) was used under the following conditions: 57 cm x 50 microm I.D. fused-silica capillary, pH 9.6 borate buffer, and 214 nm on-line detection. Sixty patients (61 +/- 13 years) with a well isolated (n=24, group A) or poorly separated monoclonal band(s) by AGE (n=36, group B) were included in this study. Within- and between-run precision for CZE was below 4% for albumin and 7% for gamma-globulin. A 100% (group A) or 61% agreement (group B, more bands detected by CZE in 10 cases) was obtained between CZE and AGE for the number of monoclonal bands. In group B, quantification was possible in 92% of samples by CZE vs. 64% by AGE (P<0.05, chi-square). The proposed CZE method appears as an additional helpful technique for the determination of poorly separated monoclonal serum proteins by AGE.     

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.     

Development of a capillary zone electrophoresis assay to examine the disposition of [d-pen]enkephalin in rats

A novel capillary zone electrophoresis (CZE) assay method was developed to evaluate the systemic disposition of [d-pen^2,5]enkephalin (DPDPE) in rats. DPDPE was recovered from serum samples (200 μl) by solid-phase extraction. Complete resolution of DPDPE and the internal standard ([d-ser²]leucine-enkephalin; DSLET) from other serum components was achieved within 15 min on a 50-μm I.D. capillary column with borate buffer (25 mM, pH 8.3). The peak-height ratio (DPDPE to DSLET) was linear through 100 μg/ml, with a detection limit of 250 ng/ml in serum, when absorbance of the column eluent was monitored at 210 nm. Serum samples obtained from rats after a 10 mg/kg intravenous bolus dose of DPDPE were analyzed with the present CZE method. The results suggest that CZE is a useful technique for quantitating therapeutic peptides in biological matrices.     

毛细管区带电泳／串联质谱联用法鉴定多肽和蛋白质

建立了毛细管区带电泳－串联质谱联用（ＣＺＥ／ＭＳ／ＭＳ）对多肽和蛋白质高灵敏度鉴定方法,对Ｍｅｔ－脑啡肽和Ｌｅｕ－脑啡肽的混合物进行了分析,用ＣＺＥ／ＭＳ／ＭＳ方法验证了各自的序列,同样对细胞色素ｃ的胰蛋白酶酶解产物用ＣＺＥ／ＭＳ／ＭＳ方法进行了肽质谱分析,几科所有肽段的序列及其与在分子中的位置都得到了确定,通过ＳＥＱＵＥＳＴ软件进行蛋白质序列数据库搜索得到准确的鉴定结果,所消耗的样品量均在低皮可     

Determination of amoxicillin in plasma samples by capillary electrophoresis

We have developed a capillary zone electrophoresis (CZE) method for determining amoxicillin in animal plasma samples. Sample clean-up involved solid-phase extraction onto Sep-Pak C₁₈ cartridges followed by elution with water–methanol (85:15). This paper describes two different techniques to increase the sensitivity of the CZE method: field-amplified sample injection (FASI) and electrokinetic injection. We have enhanced the detection limit to 280 μg l⁻¹ by the FASI technique.     

Determination of salicylic acid in human serum with capillary zone electrophoresis

The determination of salicylic acid (SA), a metabolite of aspirin, in human serum was developed using capillary zone electrophoresis (CZE) with diode array detection. The reproducibility of separation and quantification with CZE analysis of the extract of SA from human serum was appropriate for the intra- and inter-day assay coefficients. A high correlation was revealed between the serum SA levels in volunteers determined by CZE and those determined by a fluorescence polarization immunoassay (r=0.973, n=12), although the former values were slightly higher than the latter. There were no peaks interfering with the assay of SA by internal standard method. This CZE method could provide a simple and efficient method for monitoring SA in patients.     

Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine

A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm x 75 microm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 degrees C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C(18) cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0-6.0 mgL(-1) for MTX and 0.5-6.0 mgL(-1) for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mgL(-1). CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.     

Evaluation of microbial proteolysis in meat products by capillary electrophoresis

AIMS: The available methods for evaluating proteolysis in meat products, particularly the contribution of micro-organisms, are expensive, time-consuming and require an unacceptable sample size. To minimize these problems, two capillary electrophoresis-based methods have been developed. METHODS AND RESULTS: Six Gram-positive, catalase-positive cocci, four moulds and three yeasts, isolated from dry-cured ham, were tested on sterile pork slices. Using the Capillary Gel Electrophoresis (CGE) method, changes in sarcoplasmic and myofibrillar proteins due to endogenous and microbial enzymes were detected. The Capillary Zone Electrophoresis (CZE) analysis allowed evaluation of bulk changes by micro-organisms in soluble nitrogen compounds. CONCLUSION: CGE analysis of myofibrillar proteins and CZE determination of soluble nitrogen compounds have proved to be valuable tools for evaluating proteolytic activity of endogenous and microbial origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The CGE and CZE methods developed can be used for a rapid and sensitive analysis of proteolysis in meat products.     

Monitoring equilibria and kinetics of protein folding/unfolding reactions by capillary zone electrophoresis

A method is described here for studying conformational transitions of proteins due to denaturing agents: capillary zone electrophoresis (CZE) in acidic, isoelectric buffers. The sample is run in 50 mM isoelectric glutamic acid (pH = pI = 3.2) added with 1 mM oligoamine (tetraethylene pentamine) for quenching protein interaction to the capillary wall (final pH = 3.3). Muscle acylphosphatase (AcP), in this buffer, exhibited a free solution mobility of 2.63 x 10(-4) cm(2) V(-1) s(-1). By studying the unfolding kinetics, as a function of time of incubation in 7 M urea, it was possible to measure the rate constant of the unfolding reaction, estimated to be 0.00030+/-0.00006 s(-1). The same measurements, when repeated via spectroscopic monitoring of intrinsic fluorescence, gave a value of 0.00034+/-0.00002 s(-1), thus in excellent agreement with CZE data. By equilibrium unfolding CZE studies, it was possible to construct the typical sigmoidal transition of unfolding vs urea molarity: the midpoint of this transition, at which the folded and unfolded states should be equally populated, was estimated to be at 4.56 M urea. Similar experiments by fluorometric analysis gave a value of 4.60 M urea as midpoint of the unfolding curve.     

Comparison of high-performance liquid chromatography and capillary zone electrophoresis in penciclovir biodegradation kinetic studies

High-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) were used in biodegradation kinetic studies. This paper describes a rapid penciclovir separation using CZE with detection limits comparable to HPLC. The ionic-strength mediated stacking technique was employed while good resolution was maintained. With a shorter analysis time, comparable detection limits and no organic solvent consumption, CZE is a better method for penciclovir biodegradation studies than conventional reversed-phase HPLC (RP-HPLC).     

Heart-cut two-dimensional separation method via hyphenation of micellar electrokinetic capillary chromatography and capillary zone electrophoresis using analyte focusing by micelle collapse

A novel two-dimensional (2D) separation method, which hyphenated micellar electrokinetic capillary chromatography (MEKC) and capillary zone electrophoresis (CZE), was developed for analysis of flavonoids in Leonurus cardiaca. The Leonurus cardiaca sample was separated and purified in first dimension by MEKC. Then only a selected portion of the first dimension separation was transferred into the second dimension by pressure. Finally, the zone of flavonoids was separated by CZE. As the key to successful hyphenation of MEKC and CZE, an analyte focusing by micelle collapse (AFMC) concentration method was employed between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The whole heart-cut 2D separation process can be performed in a conventional CE analyzer. The relative standard deviation of peak height, peak area and migration time were in the range of 2.3-4.2%, 1.5-3.8% and 3.6-5.5%, respectively, and detection limits (S/N=3) were 15-55 ng/mL. The new methodology was applied with success for the flavonoids separation of Leonurus cardiaca.     

Simultaneous determination of phthalate di- and monoesters in poly(vinylchloride) products and human saliva by gas chromatography-mass spectrometry

This paper reports on the selectivity behaviour of tryptic peptides on a Cu(2+)-loaded immobilised metal ion affinity chromatography (IMAC) support. Ovalbumin was chosen as a model protein for investigation of the selection and separation of histidine-containing peptides by IMAC off-line coupled with capillary electrophoresis and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF). Two of five histidine-containing peptides in addition to some non-histidine-containing peptides from a tryptic digest of ovalbumin were captured by IMAC. To separate and purify the selected peptides, the IMAC sample was analysed by capillary zone electrophoresis (CZE). The sample was not separated by capillary zone electrophoresis, therefore, micellar electrokinetic chromatography (MEKC) using 10-75 mM SDS was used. Analysis of IMAC sample by MEKC, using low concentrations of SDS (10 mM) was characterised by MALDI-TOF. When using SDS at 75 mM, the migration times of reversed-phase fractions of the IMAC sample, were used to identify the peaks. One of the two selected histidine-containing peptides with two histidine residues was identified, analysing the sample by CZE or MEKC.     

Development and validation of a rapid capillary zone electrophoresis method for the determination of aconite alkaloids in aconite roots

Introduction – Aconites, with aconite alkaloids as the major therapeutic and toxic components, are used for the treatment of analgesic, antirheumatic and neurological symptoms. Quantification of the aconite alkaloids is important for the quality control of aconite‐containing drugs. Objective – To establish a validated capillary zone electrophoresis (CZE) method for the simultaneous determination of six major alkaloids, namely aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine, in crude and processed aconite roots. Methodology – The CZE method was optimised and validated using a stability‐indicating method. The optimised running buffer was a mixture of 200 mm Tris, 150 mm perchloric acid and 40% 1,4‐dioxane (pH 7.8) with the capillary thermostated at 25°C. Results – Using the optimised method, six aconite alkaloids were well separated. The established method showed good precision, accuracy and recovery. Contents of these alkaloids in crude and processed aconites were determined and it was observed that the levels of individual alkaloids varied between samples. Conclusion – The developed CZE method was reliable for the quality control of aconites contained in herbal medicines. The method could also be used as an approach for toxicological studies. Copyright © 2009 John Wiley & Sons, Ltd.     

High-performance separation methods in the analysis of a new peptide family: the galanins

An analytical investigation of a new peptide family, the human galanins and their fragments, was carried out by reversed-phase HPLC, capillary zone electrophoresis (CZE) at different pH values and micellar electrokinetic capillary chromatography (MECC) in phosphate-borate-sodium dodecyl sulphate buffer. None of the methods seems to be superior to the others. The complementary nature of the electrophoretic methods is obvious when the profiles of peptides are compared; impurities not separated by HPLC are separated by CZE or MECC and vice versa. With these three different separation methods, a more complex analytical control of the synthetic work can be achieved.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Determination of enantiomeric excess for 2,3-dihydroxy-3-phenylpropionate compounds by capillary electrophoresis using hydroxypropyl-beta-cyclodextrin as chiral selector

A new capillary zone electrophoresis (CZE) method was developed to separate three chiral 2,3-dihydroxy-3-phenylpropionate enantiomers using neutral hydroxypropyl-beta-CD (HP-beta-CD) as chiral selector and borate as background electrolyte. The results showed that HP-beta-CD exhibited good enantioselectivity and high resolution was achieved under the optimum condition of pH 10.3, 200 mM borate buffer containing 6% methanol and 50 mM HP-beta-CD at 15 kV and 20 degrees C within 16 min. The precision of the method was <0.9% for migration time and 4.5% for corrected peak area. In addition, the developed method was successfully applied to the determination of enantiomeric excess (ee) of synthetic 2,3-dihydroxy-3-phenylpropionate samples. With this method, low as 0.2% impurity of the undesirable enantiomer in the presence of high amount of target enantiomer was determined. The results demonstrated that the proposed CZE method is a simple and useful technique and is applicable to ee assay of 2,3-dihydroxy-3-phenylpropionate enantiomers.     

Determination of free radical reaction products and metabolites of salicylic acid using capillary electrophoresis and micellar electrokinetic chromatography

Hydroxylated radical products of salicylic acid are often used as a relative measurement in free radical research. Several analytical methods exist to determine the amount of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid. In this study we use capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) in order to determine these free radical products. The CZE experiment was optimized with a CZE simulation program in order to achieve an optimal pH. Calibration curves were recorded in the range 10⁻⁶–10⁻⁴ M and the detection limit was determined. For both CZE and MECC it was 2·10⁻⁷ M. Both methods resulted in a reproducible analysis of salicylate and its hydroxylated free radical products in 6 min.