新型毛细管等电聚焦驱动方法的建立及其应用

毛细管等电聚焦(CIEF)在生物分离分析领域中的发展迅速．CIEF可分为两步法聚焦和一步法聚焦．电渗流(EOF)可作为一种新的驱动力．Rassi等根据串连体系中电渗流速率将被平均化的原理,将未涂层毛细管与聚乙二醇涂层的毛细管通过聚四氟乙烯管耦合起来,成功地利用毛细管区带电泳快速分离蛋白．此外,利用电渗流输液原理设计的电渗泵还可用作流动注射和微柱液相色谱的驱动系统。     

Use of quasi-isoelectric buffers as anolyte and catholyte to improve capillary isoelectric focusing performances

The use of quasi-isoelectric anolytes and catholytes has been investigated to improve CIEF performances. Narrow pH cuts of carrier ampholytes (NC) have been compared to more conventional couples of anolytes/catholytes (phosphoric acid/sodium hydroxide and glutamic acid/lysine). First, a CIEF setup that consists in a bare silica capillary and 70:30 water/glycerol separation medium has been used. The experiments have shown that when using NC instead of more classical anolytes and catholytes, an increase in the protein detection time was observed and the resolutions obtained for neutral and acidic proteins were doubled. Moreover, according to the NC fraction used, the resolution was modified. In order to investigate further the mechanisms involved, a second setup using a capillary coated with hydroxypropylcellulose was used. With this setup no difference has been observed when changing anolyte and catholyte nature. A simple methodology has then been developed to evaluate EOF during focusing and mobilization steps of CIEF experiments. It highlighted the crucial role played by EOF when using a bare silica capillary. EOF indeed decreased by 33% during mobilization step when using NC instead of classical anolytes and catholytes.     

DILUTE SOLUTION BEHAVIOR OF CHITOSAN IN DIFFERENT ACID SOLVENTS

Dilute solution behavior of chitosan was studied in formic acid, acetic acid, lactic acid andhydrochloric acid aqueous solution under different pH values. The reduced viscosities, η_(sp)/C,ofchitosan solutions were dependent on the properties of acid and pH value of solvents. For a givenchitosan concentration, η~(sp)/C decreased with the increase of acid concentration, or decreasing pHof solvent, indicating shielding effect of excessive acid similar to adding salt into solution. Thestabilities of dilute chitosan solution in formic acid and lactic acid were better than that in acetic acid and hvdrochloric acid.     

Analysis of formic and acetic acid in rain water by capillary electrophoresis

A simple technique is described for the routine capillary electrophoretic determination of formic and acetic acid in rain water. These acids were determined simultaneously in approximately 6 min using a carrier electrolyte containing lO mM phosphate and 0.5 mM myristyltrimethylammonium bromide (MTAB) as electroosmotic flow (EOF) modifier at pH 6.5 and direct UV detection at 185nm. The method is quantitative, with recoveries in the 99-101% range and linear up to 5mgL^-1. The precision is better than 2.1% and the procedure shows the appropriate sensitivity, with detection limits between 0.042 and 0.055mg L^-1. The proposed method was successfully employed for the determination of formic and acetic acid in 57 rain water samples, collected from October 2000 to February 2001 in four different sampling stations located in Galicia (NW Spain), by direct sample injection after filtration.     

毛细管等电聚焦方法及其应用

讨论了毛细管等电聚焦中所涉及的问题,如分离机理、电渗、迁移方法、检测器及其应用。由于毛细管等电聚焦操作方式的多样性,使其可适用于不同的仪器条件。非交联丙烯酰胺涂层能很好地消除电渗和蛋白吸附;而采用未处理的毛细管时,动态涂敷纤维素类亲水聚合物对碱性和中性蛋白亦能取得较好的分离效果。电荷耦合器件成像检测器尚待进一步发展才能成为常用的检测工具。对于复杂样品来说,仍需解决的问题是保证在较宽的ｐＨ范围内ｐＩ的线性。     

Comparison of different mobilization strategies for capillary isoelectric focusing of ovalbumin variants†

One pressure and three chemical mobilization strategies have been optimized and tested for two‐step capillary isoelectric focusing with ultraviolet detection with simultaneous refining of the composition of carrier ampholytes as well as of anodic and cathodic spacers. The comparison of individual mobilization strategies was performed on basis of model proteins and peptides covering a pI range of 4.1–10.0, finally targeting an acidic major food allergen, that is, ovalbumin. Resolution was improved by combining Pharmalyte 3–10 with Pharmalyte 5–6 with concentration adjustment of carrier ampholytes and the anodic and cathodic spacer, respectively. Analytes within pI 5–6 but not ovalbumin were prone to artificial peak duplication under selected capillary isoelectric focusing conditions due to retardation during focusing. l ‐Arginine and iminodiacetic acid were included as spacer to prevent drifts of the pH gradient and optionally block the distal capillary part. l ‐Arginine affected the baseline in the acidic regime in some instances by introducing irregularities that interfered with ovalbumin. Cathodic mobilization with an acidic zwitterion provided the best selectivity for ovalbumin and was successfully applied for the characterization of three commercial products of ovalbumin, revealing differences between the respective profiles. Up to 12 different fractions situated between pI 4.51 and 4.72 could be addressed.     

Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection

Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using a 3-10 pH gradient and detected in whole-column imaging mode. In other cases, a single point detector was used to detect zones during the focusing step of CIEF using a 2.5-5 pH gradient. In this case, analyte peaks passed the detector in approximately 5 min at an electric field of 350 V/cm. Detection during focusing allowed for more reproducible assays at shorter times but with a sacrifice in sensitivity compared to detection during mobilization. Resolution was adequate to separate BGTPgammaS-Ras and BGTPgammaS-Rab3A complexes. Formation of specific complexes was confirmed by adding GTPgammaS to samples containing BGTPgammaS-G protein. GTPgammaS competed with BGTPgammaS for G protein binding sites resulting in decreased BGTPgammaS-G protein peak heights. The concentrating effect of CIEF enabled detection limits of 30 pM.     

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.     

Recent applications of capillary isoelectric focusing

After the advent of capillary isoelectric focusing (CIEF) in the 80's several approaches have been developed in order to use the technique in routine analyses. The recent years showed an extensive increase in the applications of this technique employing its exceptionally high-resolution power. Methodological improvements, as well as hyphenation with other electrophoretic and chromatographic separation procedures, proved the versatility of CIEF in studies of clinically important proteins, recombinant product, cell lysates and other complex mixtures. The combination of CIEF with mass spectrometry detection is one of the major challenges for studying proteomics. This review collected the recent applications of CIEF including innovations in the experimental setup, remedies for the presence of salts in samples, calibration of the pH gradient, carrier ampholyte-free isoelectric focusing, the progress in micropreparation, two-dimensional separations, etc.     

Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing

Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.     

The effect of pH adjusted electrolytes on capillary isoelectric focusing assessed by high-resolution dynamic computer simulation

The effect of the composition of electrolytes on capillary IEF is assessed for systems with carrier ampholytes covering two pH units and with catholytes of decreased pH, anolytes of increased pH, and both electrode solutions with adjusted pH values. For electrolytes composed of formic acid as anolyte and ammonium hydroxide as catholyte, simulation is demonstrated to provide the expected IEF system in which analytes with pI values within the formed pH gradient are focused and become immobile. Addition of formic acid to the catholyte results in the formation of an isotachophoretic zone structure that migrates toward the cathode. With ammonium hydroxide added to the anolyte migration occurs toward the anode. In the two cases, all carrier components and amphoteric analytes migrate isotachophoretically as cations or anions, respectively. The data reveal that millimolar amounts of a counter ion are sufficient to convert an IEF pattern into an ITP system. With increasing amounts of the added counter ion, the overall length of the migrating zone structure shrinks, the range of the pH gradient changes, and the migration rate increases. The studied examples indicate that systems of this type reported in the literature should be classified as ITP and not IEF. When both electrolytes are titrated, a non-uniform background electrolyte composed of formic acid and ammonium hydroxide is established in which analytes migrate according to local pH and conductivity without forming IEF or ITP zone structures. Simulation data are in qualitative agreement with previously published experimental data.     

Modelling migration behavior of peptide hormones in capillary electrophoresis-electrospray mass spectrometry

The applicability in capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) of the classical semiempirical relationships between electrophoretic mobility and charge-to-mass ratio (me versus q/Malpha) has been investigated in order to describe the migration behavior of a series of bioactive peptide hormones. The influence upon the models of the separation electrolyte pH and the accuracy of the pK values of these compounds were studied first by capillary electrophoresis with ultraviolet detection (CE-UV). The classical polymer model, alpha = 1/2, resulted in slightly better correlations at any of the studied pH. Furthermore, a general linear equation can be adjusted combining all the experimental data pairs, which suggests that correlation in the whole pH range is independent of the ionic form of the studied peptide hormones. The plots of q/M1/2 against separation electrolyte pH were used to predict their electrophoretic separations, using the accurate pK values obtained in a previous work by CE-UV for charge calculations. A volatile separation electrolyte containing 50 mM of acetic acid and 50 mM of formic acid at pH 2.85 was selected for optimum CE-UV and CE-ESI-MS analysis of the peptide mixture. At this pH and taking into account the specific features of the coupling, the correlation using the classical polymer law was excellent and its parameters were similar to the ones of the general linear equation previously obtained by CE-UV. This confirmed the applicability in CE-ESI-MS of the semiempirical relationship originally established by CE-UV.     

Membrane-assisted capillary isoelectric focusing coupling with matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry for neuropeptide analysis

Herein we report a highly efficient and reliable membrane-assisted capillary isoelectric focusing (MA-CIEF) system being coupled with MALDI-FTMS for the analysis of complex neuropeptide mixtures. The new interface consists of two membrane-coated joints made near each end of the capillary for applying high voltage, while the capillary ends were placed in the two reservoirs which were filled with anolyte (acid) and catholyte (base) to provide pH difference. Optimizations of CIEF conditions and comparison with conventional CIEF were carried out by using bovine serum albumin (BSA) tryptic peptides. It was shown that the MA-CIEF could provide more efficient, reliable and faster separation with improved sequence coverage when coupled to MALDI-FTMS. Analyses of orcokinin family neuropeptides from crabs Cancer borealis and Callinectes sapidus brain extracts have been conducted using the established MA-CIEF/MALDI-FTMS platform. Increased number of neuropeptides was observed with significantly enhanced MS signal in comparison with direct analysis by MALDI-FTMS. The results highlighted the potential of MA-CIEF as an efficient fractionation tool for coupling to MALDI MS for neuropeptide analysis.     

Bis-indole derivatives for polysaccharide compositional analysis and chiral resolution of D-, L-monosaccharides by ligand exchange capillary electrophoresis using borate-cyclodextrin as a chiral selector

A series of aldo-bis-indole derivatives (aldo-BINs) was prepared by aromatic C-alkylation reactions of aldoses and indole in acetic acid solution. Common monosaccharides such as glucose, mannose, galactose, fucose, xylose, rhamnose, ribose, arabinose and N-acetylglucosamine were smoothly derivatized to form the UV absorbing aldo-BINs. The use of a capillary electrophoretic method to separate these novel aldo-BIN derivatives was established. The capillary electrophoresis conditions were set by using borate buffer (100 mM) at high pH (pH 9.0). The limit of determination was assessed to be 25 nM. The enantioseparation of D, L-pairs of aldo-BINs based on chiral ligand-exchange capillary electrophoresis technology was also achieved by using modified hydroxypropyl-β-cyclodextrin as the chiral selector in the presence of borate buffer. This aldose labeling method was applied successfully to the compositional and configurational analysis of saccharides, exemplified by a rapid and efficient method to simultaneously analyze the composition and configuration of saccharides from the medicinal herbs Cordyceps sinensis and Dendrobium huoshanense.     

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.     

Characterization of antithrombin III from human plasma by two-dimensional gel electrophoresis and capillary electrophoretic methods

The isoforms distribution of the glycoprotein antithrombin III (ATIII) derived from human plasma was investigated by means of isoelectric focusing (IEF) in polyacrylamide gels with immobilized pH gradients (IPG) and two-dimensional gel electrophoresis (2-DE) as well as capillary electrophoretic methods. It turned out that the presence of high concentrations of chaotropics (urea, thiourea) and zwitterionic detergents (3-[(3-cholamidepropyl)dimethylammonio]-1-propanesulfonate (CHAPS)) was decisive for attaining good resolution of the protein isoforms. Resolution by IPG-IEF was obtained with excellent reproducibility and pI differences down to 0.01 pH units could be distinguished. ATIII-alpha and ATIII-beta-fractions preseparated by heparin affinity chromatography showed an analogous but shifted spot pattern consisting each of one major and three minor isoforms. The main isoforms of ATIII-alpha and ATIII-beta exhibit pI values of 5.18 and 5.32, respectively, both values determined in the presence of high concentrations of urea. The pI difference of 0.14 pH units correspond to the effect of two sialic acids absent in ATIII-beta. The formation and occurrence of ATIII dimers and trimers turned out to be dependent on the sample preparation. The results obtained by 2-DE were compared with those of capillary zone electrophoresis (CZE) and capillary IEF (CIEF). Quantitative analysis regarding the CZE separated isoforms of plasma derived ATIII yielded a content of about 70% ATIII-alpha main isoform and about 6.6% of ATIII-beta. The pI values of ATIII determined by CIEF with internal calibration were in fair agreement with the pI values of the main isoforms achieved with 2-DE.     

Simplified capillary isoelectric focusing with chemical mobilization for intact protein analysis

We report a capillary isoelectric focusing system based on a sequential injection method for simplified chemical mobilization. This system was coupled to an ion trap mass spectrometer with an electrokinetically pumped nanoelectrospray interface. The nanoelectrospray emitter employed an acidic sheath electrolyte. To simplify focusing and mobilization, a plug of ammonium hydroxide was first injected into the capillary, followed by a section of mixed sample and ampholyte. During focusing, the NH₃H₂O section worked as catholyte. As focusing progressed, the NH₃H₂O section was titrated to lower pH by the acidic sheath electrolyte. Chemical mobilization started automatically once the ammonium hydroxide was consumed by the acidic sheath flow electrolyte, which then acted as the mobilization solution. In this report, the lengths of the NH₃H₂O section and sample were optimized. With a 1 m long capillary, a relative short plug of the NH₃H₂O section (3 cm) produced both fast migration and reasonable separation resolution. The simplified capillary isoelectric focusing mass spectrometry system produced base peak intensity relative standard deviation of 8.5% and migration time relative standard deviation ≤0.6% for myoglobin and cytochrome C in triplicate runs.     

Synthetic oligopeptides as isoelectric point markers for capillary isoelectric focusing with ultraviolet absorption detection

Sixteen peptides (trimers to hexamers) were designed for use as a set of pI markers for capillary isoelectric focusing (CIEF). Each peptide contains one tryptophan residue for detection by UV absorption and other amino acid residues having ionic side chains, which are responsible for focusing to its pI. The pIs of these peptides were determined by slab-gel IEF using commercial carrier ampholytes. The focused peptides in the gel were detected by absorption measurement at 280 nm using a scanning densitometer and the pH gradient was determined by measuring the pH of the gel using an oxidized metal membrane electrode. The pI values of the peptides ranged from 3.38 to 10.17. The obtained values agreed well with the predicted ones, which were calculated based on amino acid compositions, with root mean square differences of 0.15 pH unit. The peptides were detected at 280 nm as very sharp peaks when separated by CIEF. The pI values of some standard proteins were redetermined by CIEF by using this set of peptide pI markers and the values agreed closely with those reported previously. The sharp focusing, stability, high purity and high solubility of these synthetic pI markers should facilitate the profiling of a pH gradient in a capillary and the determination of the pI values of proteins.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

Gas-chromatographic determination of formic acid in the oxidation products of organic substances

Procedures for the gas-chromatographic determination of formic acid in oxidation produts of organic substances after its conversion to benzyl formate were developed. In determining formic acid in an organic phase, free formic acid was esterified with benzyl alcohol in a pyridine solution while adding acetic anhydride under mild conditions. The conversion of formic acid was complete even in the presence of other mono-and dicarboxylic acids in considerable amounts. It was found that the formation of benzyl formate occurred via a mixed aldehyde formed in situ from formic acid and acetic anhydride. The determination of formic acid in aqueous solutions involves the synthesis of its sodium or potassium salt and the successive treatment of this salt with acetyl chloride and benzyl alcohol in a pyridine solution.