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Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins. [ABSTRACT FROM AUTHOR]

Abstract: A simple capillary array IEF device was developed for high resolution and micropreparative separation of trace amounts of proteins. Based on quasi-chip-scale manufacturing, the specific capillaries (600μm i.d., 1200μm o.d. and 20mm length) were integrated with the miniaturized polymethyl-methacrylate electrode trays. Electroosmotic flow was suppressed effectively by modified cross-linked polyacrylamide coating, and instability of IEF was addressed using the designed concentration of electrolytes via moving reaction boundary theory. As a prototyping, the resolution, reproducibility, throughput, speed and linearity of pH gradient were systemically evaluated with model proteins. The results revealed the following advantages: (i) the reproducibility of array was assessed as RSD values of 0.95% (intra-day) and 2.88% (inter-day); (ii) IEF could be completed in 20min with up to 400V/cm electric field; (iii) high resolution separation of model proteins achieved in 20mm length column; (iv) multi-units with 48 micro-columns can be easily integrated to obtain high throughput; and (v) good linearity of pH gradient (R=0.9989). More importantly, utility of the device was tested by using hemoglobins sample from human red blood cell. HbA0 and HbA1c with only ΔpI 0.03 have been successfully separated by the developed method. [Copyright &y& Elsevier]

Abstract: Hemoglobin A1c (HbA1c) has been proven to be a key biomarker for diabetes screening, and glutathiolation of HbA1c (viz., GSS-HbA1c) has been identified. However, the impact of GSS-HbA1c on the measurement of HbA1c for diabetes screening has not been quantitatively assessed yet. To address the issue, the micropreparative capillary isoelectric focusing (cIEF) developed in our previous work was used for the high resolution separation and purification of hemoglobin (Hb) species. The main fractions of HbA0, HbA3 and HbA1c extracted from the developed cIEF were identified by validated Mono S method. The proposed GSS-HbA1c fractions in the cIEF were pooled and identified by electrospray ionization mass spectrometry (ESI-MS). The HbA1c enzyme-linked immunosorbent assay (ELISA) kit was employed for further quantitative analysis of GSS-HbA1c. A total of 34 blood samples with HbA1c levels from 4.2% to 13.4% were assessed via the above comprehensive strategy of IEF-HPLC-MS-ELISA. It was demonstrated that the HbA1c levels detected by cation exchange LC were considerably influenced by the glutathiolation of Hb and the range of detected GSS-HbA1c values was between 0.23% and 0.74%. The results and developed cIEF methods have considerable significances for investigation of diabetes and clinical diagnosis. [Copyright &y& Elsevier]

Abstract: An immobilized pH gradient (IPG) has strong power against instability (e.g., drifting and plateau) existing in classic isoelectric focusing (IEF). However, the relevant mechanism against the instability of pH gradient is still unclear. In this work, the theories of diffusional current and water products in IEF were developed based on the Svensson–Tiselius's differential equation and concept of moving reaction boundary (MRB). Two novel methods of pH gradient mobilization in IPG–IEF and non-IPG–IEF (opposite to IPG–IEF) were developed to unveil stability mechanism of IPG–IEF. The theoretical and experimental results indicated that (i) the drifting of pH gradient in non-IPG–IEF could be effectively controlled by IPG technique due to the existence of equal-fluxes of hydroxyl and hydrogen ions in the IPG–IEF system, (ii) there existed high diffusional current in non-IPG–IEF because of the existence of free carrier ampholyte (CA), but weak current in the IPG–IEF due to the immobilization of CA species in gel matrix, and (iii) the high diffusional current resulted in a great amount of water formation in neutral zone of pH gradient that led to distinct plateau in non-IPG–IEF, conversely the weak diffusional current caused little of water formation and weak plateau of pH gradient in IPG–IEF. These studies have considerable significance to the understanding of mechanism and development of protein IEF separation technique. [Copyright &y& Elsevier]

Abstract: In this paper, a novel mathematical model of stump-like peak was constructed for alternative computer simulation of capillary zone electrophoresis (CZE) by using Haarhoff–Van der Linde (HVL) function. Unlike a classical model of Gaussian peak, the developed model contains both Gaussian and plateau concentration distributions of analytes. Based on the model, the relevant computer software was developed and implemented in Borland Delphi 7 environment. The relevant results revealed that (i) the software could freely simulate the plateau and Gaussian distribution peaks; (ii) the simulator could simulate a dynamic process of CZE properly; and (iii) the program could display the final electropherogram of numerous analytes in CZE. We further conducted the relevant experiments and compared them with the simulations. The comparisons demonstrated the high agreement between the simulation results and the experiments as well as those cited from references. In addition, the software could calculate effective mobility, diffusion coefficient and concentration of analytes based on the physico-chemical parameters input by users. The developed model and software have evident significance for understanding of electrophoretic separation, conditional optimization and basic computation on physico-chemical parameters of analytes in CZE. [Copyright &y& Elsevier]

Abstract: In this paper, a mathematical model and computer simulator were developed for offline sample pretreatment of heavy metal ion based on moving precipitate boundary (MPB) electrophoresis. The simulation indicates that (i) the program can easily accomplish numerical computations, such as the velocities of MPB and elution boundary (EB), and enrichment factor (EF) etc; (ii) the simulator can vividly imitate the dynamics of MPB, EB, precipitate zone, and precipitate-elution; and (iii) the software may simply optimized experimental conditions via the influence factors (e.g., voltage, hydroxyl, hydrogen and metal ions) on the EF. As a proof of concept, copper ion and its precipitate with definite blue color were, respectively chosen as mode heavy metal ion and alkaline precipitate for the relevant experiments of MPB-based sample preconcentration of heavy metal ion in large tube. All of the experimental results manifest the validity of developed mathematical model and the relevant simulation results. The model and simulator advanced herein are of clear significance to the optimization of experimental conditions and understanding of offline MPB- based sample condensation of heavy metal ion. [Copyright &y& Elsevier]

Abstract: The reason why a moving reaction boundary (MRB) can stack analyte in highly saline sample in capillary electrophoresis [C.X. Cao, Y.Z. He, M. Li, Y.T. Qian, S.L. Zhou, L. Yang, Q.S. Qu, Anal. Chem. 74 (2002) 4167] is still unclear. To illuminate the mechanism of such stacking, three MRBs formed by formic acid-NaOH buffer and sodium formate as well as 40, 80 and 120mmol/L sodium chloride in matrixes were studied. The computation with MRB theory shows that sodium chloride in matrix has weak effect on the stacking efficiency, whether the concentration of sodium chloride is set at 40, or 80, or 120mmol/L. The conclusion has been highly manifested by numerous experiments. Furthermore, the computer simulation and theoretical analyses depict that this kind of stacking is induced by the mechanism of MRB, rather than that of electrostacking or isotachophoresis (ITP) under the given electrolytic system. Finally, the application of the sample condensation was achieved for the stacking of analyte(s) in highly saline biological sample of skeletonema costarum’ culture with up to 527mmol/L total salt and health human urine with 150–320mmol/L inorganic ions (Cl−, Na+, K+, PO4 3−, etc.). The results herein have a clear significance to the design on stacking of analyte in highly saline biological sample. [Copyright &y& Elsevier]

Abstract: A simple but efficient capillary zone electrophoresis (CZE) method was developed for the fast separation and determination of two misused β2-agonists clenbuterol (CLB) and salbutamol (SAL) from complex background extractants existing in swine feed samples. The proper experimental conditions were achieved as 20.0mmol/l pH 10.5 Na2HPO4–NaOH buffer, 20kV applied voltage, fused-silica capillary of 60.5cm×75μm i.d. (50cm to detector). Under the proper conditions, the two abused drugs can be online isolated from the complex extractants and the separation between CLB and SAL is good, all of the target analytes can be detected within 4.5min. The linear response of CLB and SAL concentration ranges from 2.0 to 100.0μg/ml with high correlation coefficient (R 2 =0.9990) and (R 2 =0.9986), respectively. Limit of detection (LOD) and limit of quantification (LOQ) was calculated to be 0.95 and 3.17μg/ml for CLB, 1.07 and 3.57μg/ml for SAL. The precision values (expressed as R.S.D.) of intra- and inter-day were 1.24–2.36% and 0.90–3.85% for CLB, 0.47–1.64% and 0.91–3.46% for SAL. Recoveries spiked at levels 5.0, 25.0, 80.0μg/ml ranged between 93.30% and 104.33% with R.S.D. less than 5%. Finally, the developed method has been applied to the analysis of real swine feed samples and has achieved satisfactory results. [Copyright &y& Elsevier]

Abstract: Natural phenazines in secondary metabolites of bacteria have been receiving increasing attention in recent years due to their potential usage as antibiotics. In the present study, a rapid and reliable capillary zone electrophoresis (CZE) method was developed and validated for monitoring for the first time dynamic phenazine-1-carboxylic acid (PCA) and the 2-hydroxyphenazine (2-OH-PHZ) production of Pseudomonas chlororaphis GP72 during the entire fermentation cycle. The paper begins with the optimization of separate conditions for 2-OH-PHZ and PCA together with phenazine (PHZ), which is used as internal standard. The optimized conditions are: 10mM, pH 7.3 phosphate buffer, a fused-silica capillary with a total length of 49cm×75μmID, 375μmOD with an effective length of 40cm, 25kV, 13mbar 10s pressure sample injection and 25°C air-cooling. The three compounds could be separated within 2min under optimized conditions. The validation of the newly developed study shows the linear response of 2-OH-PHZ and PCA ranging from 10 to 250μgmL−1 with high correlation coefficient (r =0.9997 and 0.9993, n =7), low limits of detection (0.47 and 0.38μgmL−1) and quantification (1.56 and 1.28μgmL−1), respectively. Good precision values for intra- and inter-day detection and acceptable individual recovery ranges for 2-OH-PHZ and PCA are indicated. The newly developed method was also validated through monitoring dynamic PCA and 2-OH-PHZ production of P. chlororaphis GP72 during an 84h growth cycle. [Copyright &y& Elsevier]

Abstract: A simple and rapid method using micellar electrokinetic capillary chromatography (MEKC) was developed for the separation and determination of acrylamide in potato chips at low levels for the first time. The experimental conditions for the separation and quantification of acrylamide were optimized at first. The optimized conditions were: 50mmol/L Na2B4O7 and 40mmol/L SDS at pH 10.0, 12kV applied voltage, 76cm total length (67cm effective length) and 75μm i.d. capillary, 198nm wavelength, 15cm high 25s hydrodynamics sample injection, 20°C air-cooling. The linear response of acrylamide concentration ranges from 0.5 to 100μg/mL with high correlation coefficient (r =0.9986, n =9). The LOD and LOQ were estimated to be 0.1 and 0.33μg/mL based on S/N=3 and 10. The precision values (expressed as R.S.D.) of intra- and inter-day were 0.86–4.35% and 2.61–9.65%, respectively. Recoveries spiked at levels 2, 20, 60μg/mL ranged between 90.86% and 99.6% with R.S.D. less than 6.5%. Finally, the developed method has been applied to the analysis of real samples and has achieved satisfactory results. All of these indicated that it was a reliable method for the quantification of acrylamide in potato chips. [Copyright &y& Elsevier]