Cesium reversibly suppresses HeLa cell proliferation by inhibiting cellular metabolism.

The aim of the present study was to investigate the influence of Cs+ on cultured human cells. We find that HeLa cell growth is suppressed by the addition of 10 mm CsCl into the culture media. In the Cs+ -treated cells, the intracellular Cs+ and K+ concentrations are increased and decreased, respectively. This leads to a decrease in activity of the glycolytic enzyme pyruvate kinase, which uses K+ as a cofactor. Cs+ -treated cells show an intracellular pH shift towards alkalization. Based on these results, CsCl presumably suppresses HeLa cell proliferation by inducing an intracellular cation imbalance that affects cell metabolism. Our findings may have implications for the use of Cs+ in cancer therapy.

Capillary isoelectric focusing after sample enrichment with immunoaffinity chromatography in a single capillary.

For accurate micro-scale quantification of a specific protein in biological fluids, immunoaffinity chromatography (IAC) and isoelectric focusing (IEF) were combined in a single fused-silica capillary. The inner wall of the capillary was coated with an anti-E-tag antibody at the inlet side to form an IAC column, and polydimethylacrylamide, a neutral polymer, at the outlet side to form the capillary for IEF. After loading a sample, the whole capillary was filled with a carrier ampholyte solution. An anode solution, an acid, was then introduced to fill only the IAC column segment. Focusing was started with a pressure that balances with the electroosmotic flow produced in the acidified IAC column. Fluorescence-labeled recombinant Fab with an E-tag spiked at 16 pM to 10 nM in 50% serum was separated and detected with high precision. The coupling principle allows rapid and high-resolution IEF analysis of a protein in a biological sample without any loss of the immunoaffinity captured protein.

Affinity probe capillary electrophoresis of insulin using a fluorescence-labeled recombinant Fab as an affinity probe.

Affinity probe CE (APCE) separates and detects a target molecule as a complex using a fluorescence-labeled affinity probe (AP) by CE. The electrophoretic separation of the complex ensures accurate identification of a specific signal among nonspecific ones, which often compromises the credibility of immunoassays. APCE of insulin using a recombinant Fab (rFab) as an AP was demonstrated as a model system in this report. Anti-insulin rFab was expressed in Escherichia coli and labeled at a cysteine residue in the hinge region with a thiol-reactive rhodamine dye. Electrophoretically pure labeled rFab was recovered from a focused band in slab-gel IEF and used as an AP. A mixture of standard insulin and the AP with carrier ampholyte was introduced into a neutral-polymer coated fused silica capillary (50 µm id, 120 mm long). IEF was carried out at 500 V/cm, and the capillary was scanned for laser-induced fluorescence under focusing conditions. The insulin-AP complex focused at pH 6.6 within 6 min along with the free AP at pH 7.6. The complex peak decayed according to the first-order reaction kinetics with a half life of 3.8 min. A linear calibration line was obtained for standard insulin at a concentration range of 20 pM to 5 nM using the AP at 50 nM. These results demonstrate that rFab is useful for the preparation of an AP for APCE.

Estimation of the deamidation rates of major deamidation sites in a Fab fragment of mouse IgG1-κ by capillary isoelectric focusing of mutated Fab fragments.

The deamidation of asparagine (Asn or N) residues in proteins is a common post-translational chemical modification. The identification of deamidation sites and determination of the degree of deamidation have been carried out by the combination of peptide mapping and mass spectrometry. However, when a peptide fragment contains multiple amides, such analysis becomes difficult and sometimes impossible. In this report, a quantitative method for estimating the deamidation rate of a specific amide in a protein is presented without using peptide mapping. Five Asn residues of a recombinant fragment antigen binding (rFab) (mouse IgG1, κ) were mutated to a serine (Ser) residue, one by one, through site-directed mutagenesis, and the single-residue deamidation rates of the original rFab and the mutants were determined using capillary isoelectric focusing. The difference of the rate between the original rFab and the mutant was assumed to be equal to the deamidation rate of the specific Asn residue, which had been mutated. Among five mutants established, three major deamidation sites-H chain Asn135, L chain Asn157, and L chain Asn161, using the Kabat numbering system-were identified, accounting for 66%, 29%, and 7% of the single-residue deamidation of the original rFab, respectively. Although the former two have been known by peptide mapping, the last one, which resides on the same tryptic peptide that carries one of the former two, previously has not been identified. For the first time, the deamidation rate constants of the three sites were estimated to be 10.5 × 10(-3) h(-1), 4.6 × 10(-3) h(-1), and 1.1 × 10(-3) h(-1) in 0.1 M phosphate buffer, pH 7.5 at 37 °C, respectively, with corresponding half-life of 2.8 days, 6.3 days, and 27 days. The method should be applicable to any recombinant proteins.

Quantitative evaluation of lectin-reactive glycoforms of α(1)-acid glycoprotein using lectin affinity capillary electrophoresis with fluorescence detection.

α(1)-Acid glycoprotein (AGP) was previously shown to be a marker candidate of disease progression and prognosis of patients with malignancies by analysis of its glycoforms via lectins. Herein, affinity capillary electrophoresis of fluorescein-labeled AGP using lectins with the aid of laser-induced fluorescence detection was developed for quantitative evaluation of the fractional ratios of concanavalin A-reactive or Aleuria aurantia lectin-reactive AGP. Labeled AGP was applied at the anodic end of a fused-silica capillary (50 µm id, 360 µm od, 27 cm long) coated with linear polyacryloyl-ß-alanyl-ß-alanine, and electrophoresis was carried out for about 10 min in 60 mM 3-morpholinopropane-1-sulfonic acid-NaOH buffer (pH 7.35). Addition of the lectins to the anode buffer resulted in the separation of lectin-reactive glycoform peaks from lectin-non-reactive glycoform peaks. Quantification of the peak area of each group revealed that the percent of lectin-reactive AGP is independent of a labeling ratio ranging from 0.4 to 1.5 mol fluorescein/mol AGP, i.e. the standard deviation of 0.5% for an average of 59.9% (n=3). In combination with a facile procedure for micro-purification of AGP from serum, the present procedure, marking the reactivity of AGP with lectins, should be useful in determining the prognosis for a large number of patients with malignancies.

Charge heterogeneity of a therapeutic monoclonal antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin.

A robust and highly reproducible capillary isoelectric focusing (cIEF) method for the evaluation of charge heterogeneity of monoclonal antibody (mAb) pharmaceutical which contains covalently bound antitumor compounds was developed using a combination of commercially available dimethylpolysiloxane-coated capillary and carrier ampholyte. In order to optimize major analytical parameters for robust mobilization, experimental responses from three pI markers were selected. The optimized method gave excellent repeatability and intermediate precision in estimated pI values of charge variants with relative standard deviations (RSDs) of not more than 0.06% and 0.95%, respectively, when using IgG(4) as a model. Furthermore, RSDs of charge variant compositions were less than 5.0%. These results suggest that the proposed method can be a powerful tool for reproducible evaluation of charge variants of both naked mAbs and their conjugates with high resolution, and it is applicable to quality testing and detailed characterization in the pharmaceutical industry. In addition, it should be noticed that the method provided non-linear pH gradient within the tested ranges, from pI 9.50 to 3.78, and the pH gradient caused the inconsistency of estimated pI ranges between cIEF and gel IEF. This result indicates that selecting appropriate pI markers based on the target pI ranges of charge variants for each mAb related pharmaceutical is highly recommended for the precise determination of pI values.

Two-step perpendicular free-solution isoelectric focusing in a microchamber array chip.

A free-solution microfluidic chip device was fabricated for small-scale protein fractionation based on the principle of two-step perpendicular isoelectric focusing (IEF) previously reported in a gel slab. The microchip was composed of two separate glass plates with a square array of 100 indentations etched on the facing side of each plate. By changing the relative position of the two plates, ten channels can be formed as ranges of the staggered indentations and can be switched to the perpendicular direction, or 100 chambers of 140 nL each can be produced by perfect overlapping of the indentations. The entire pH range of the carrier ampholyte was fractionated into 10 successive pH segments in the first IEF, and each of the segments was further fractionated into 10 fractions by the second IEF at a right angle to the first one. This chip enabled IEF fractionation at a lower voltage and in a shorter time, compared with a linear apparatus operated in one direction. Fluorescence-labeled peptides with a small isoelectric point (pI) difference (DeltapI = 0.3) were recovered in different chambers in a total separation time of 10 min at a maximum applied voltage of 500 V.

Micro OS-ELISA: Rapid noncompetitive detection of a small biomarker peptide by open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) integrated into microfluidic device.

A novel detection system that combines the merits of open-sandwich (OS) enzyme-linked immunoadsorbent assay (ELISA) and a microfluidic sensor chip system, and which enables rapid and noncompetitive immunodetection of small antigens of less than 1000 in molecular weight, has been proposed. Equipped with a sensitive thermal lens microscope, a minute amount of the carboxyl-terminal peptide of human osteocalcin (BGP), a biomarker for bone metabolism, was quantified utilizing antigen-dependent stabilization of an antibody variable region (OS principle). In a short analysis time (approximately 12 min), we could attain a detection limit comparable to that of the microplate-based OS ELISA (1 microg L(-1)). In addition, the effects of several pretreatments for serum-derived samples were investigated: an albumin absorption resin, addition of a protease inhibitor cocktail and heat treatment. Each pretreatment was found to be effective. Consequently, intrinsic BGP and its fragments could be detected in healthy human serum with a superior detection limit and working range compared to those of the conventional competitive ELISA method.

A capillary holder for scanning detection of capillary isoelectric focusing with laser-induced fluorescence.

A holder for a 12 cm long capillary was designed for scanning LIF detection of CIEF. The polyimide coat of a fused-silica capillary has been removed, and 1.5 mm diameter flanges have been attached near both ends. The holder is fixed on the stage of a fluorescence microscope via a translational stage, and a capillary guide is directly fixed on the microscope stage. The guide has a groove and a pressure plate for the capillary to slide in. The holder has two pulling plates with slits of 1 mm to accept the capillary just inside the flanges. The slits and the groove of the guide have been aligned. The motion of the translational stage brings the pulling plate into contact with the flange at the pulled side, and slides the capillary through the guide. The other end of the capillary is free and produces no strain on the capillary. When the motion of the stage is reversed, an unstrained contact is achieved at the other end. The baseline noise from scanning was only 50% larger than that without scanning. The fluorescence-signal variation during scanning was about 4% of the total signal, which was about twice that without scanning.

Recent advances in IEF in capillary tubes and microchips.

The methodological advancements in CIEF in tubes and microchips during the period between 2002 and early 2008 are reviewed as a continuation of previous review by the same author (Electrophoresis 2002, 23, 3847-3857). After a brief introduction, the following topics related to CIEF technologies are addressed: the materials used to form capillary tubes and chips; modes of CIEF related to the detection schemes; coatings of the capillary walls; additives to the separation media; sample-loading methods; development of pI markers and their use; focusing time in relation to the length of the pH gradient; resolution in terms of a peak capacity; and, the reproducibility and precision of CIEF. Three principal detection methods are examined, i.e. ultraviolet absorption, fluorescence and MS. The coupling of CIEF with other electrokinetic separation methods and chromatography is discussed, including many multidimensional systems constructed for proteome analysis using mass spectrometric identification. The developments in the separation of non-covalent complexes and microorganisms are examined. After a short conclusion, this review ends with 176 references.

Isoelectric focusing in a microfluidically defined electrophoresis channel.

A new form of microchip isoelectric focusing that allows efficient coupling with pretreatment processes is reported. The sample is conveyed in a carrier ampholyte solution to the separation channel that is connected at both ends by two V-shaped lead channels, which supply electrode solutions to the connection point and complete the electrical connection to off-chip electrodes. The relatively high electric conductivity of the electrode solutions compared with that of the pH gradient enables focusing with a 2% loss of applied voltage at the electrodes using the lead channels. A glass microchip was constructed specifically for this configuration. The channel wall was coated with polydimethylacrylamide, and the IEF chip was operated in a chip holder equipped with on-chip connector valves. A plug of fluorescence-labeled peptide p I markers with p I values ranging from 3.64 to 9.56 with carrier ampholyte solution (pH 3-10) was introduced into the separation channel. When the plug reached the channel segment (24 mm in length) between the connection points with the electrolyte lead channels, isoelectric focusing was started after filling the lead channels with electrolyte solutions. The peptide markers were observed using scanning fluorescence detection. The entire range of the pH gradient was established in the segment after approximately 2 min. Isoelectric focusing of three consecutively injected sample plugs containing different p I markers was demonstrated.

Pesticide analysis by MEKC on a microchip with hydrodynamic injection from organic extract.

An integrated microchip for monitoring carbamate pesticides in environmental water using continuous flow chemical processes is under development, i. e., the integration of hydrolysis, azo-derivatization, liquid-liquid extraction, electrophoretic separation, and quantification. The separation of the derivatives of four carbamate pesticides (carbaryl, carbofuran, propoxur, and bendiocarb) extracted in the continuous flow of a 1-butanol phase was studied in a silica microchip using micellar EKC. A baseline separation of four pesticide derivatives was achieved on a silica chip using hydrodynamic injection with electroosmotic gating. Detection using a thermal lens microscope showed good linearity in the concentration range of 10(-6 )-10(-5 )M with an LOD of 5 x 10(-7) M, which is superior to that of conventional CE with UV absorption detection at a level of 10(-4) M.

On-chip connector valve for immunoaffinity chromatography in a microfluidic chip.

A miniature valve that operates between a chip port and a tube fitting was developed. The valve functions by means of a rotor, 3 mm in diameter and 1.5 mm in height, made of Teflon, with a 0.2-mm diameter hole at its center that is co-axial with the tube fitting. It also has a radial groove, 0.85 mm long, 0.2 mm wide, and 0.2 mm deep, at the bottom surface, starting at its center. The chip port and the tube fitting have an offset of 0.75 mm, and, thus, the rotation of the rotor can make an on and off connection between the chip port and the groove, which is connected to the tubing. The valve had a pressure resistance of at least 1.0 MPa. The on-chip valve can be placed in position by adding only a single part, a valve rotor, and no changes in the fabrication of the glass microchip are required. Since the valve functions as a part of a connector, we refer to it as an on-chip connector valve. Immunoaffinity chromatography of a fluorescence-labeled recombinant antibody fragment was carried out in a glass microchip using the valves.

Application of a micro multiphase laminar flow on a microchip for extraction and determination of derivatized carbamate pesticides.

Determination of carbamate pesticides such as carbaryl, carbofuran, propoxur and bendiocarb was demonstrated on a microchip with newly designed microchannels developed for efficient solvent extraction. The pesticides were hydrolyzed to corresponding naphthols, coupled with p-nitrobenzenediazonium fluoroborate reagent, and then extracted into 1-butanol as colored azo derivatives and detected with a thermal lens microscope. Optimum flow rates for the aqueous and organic phases were evaluated in the continuous-flow chemical process established in the microchip. The calibration lines showed good linearity in the range of concentrations of 0.03 - 3 ppm (10(-7) - 10(-5) M) and a mass detection limit down to a nanogram level was achieved that is at least two orders of magnitude lower than the LODs for conventional spectrophotometric methods. Azo derivatives of the pesticides were successfully separated and identified by micellar electrokinetic chromatography (MEKC) using a sample prepared on a bulk scale.

Analysis of protein-protein interactions with a multi-capillary electrophoresis instrument.

Protein-protein interactions were analyzed by zone electrophoresis of premixed equilibrium mixtures of a fluorescence-labeled protein at a constant concentration and unlabeled protein at a variety of concentrations using a 96-CE instrument equipped with a LIF detector. The interactions between labeled-con A versus succinylated ovalbumin, labeled-trypsin versus four proteinaceous trypsin inhibitors and labeled-insulin versus seven anti-insulin monoclonal antibodies were analyzed using a dual buffer system, in which a 60 mM borate-Na buffer (pH 9.35) was used as electrophoresis buffer and 60 mM MOPS-Na (pH 7.35) containing 0.1% Tween 20 was used as a sample buffer. The dual buffer system allowed fast and reproducible analyses of interactions at a physiological pH using uncoated fused-silica capillaries. The change in the mobility moment, the first statistical moment of an electropherogram on the mobility axis (Shimura, K., Uchiyama, N., Enomoto, M., Matsumoto, H., Kasai, K., Anal. Chem. 2005, 77, 564-572), of the labeled proteins were analyzed as a function of the concentration of unlabeled proteins. The dissociation constants for seven antibodies ranging from sub nanomolar to micromolar was determined based on the results of one cycle of parallel electrophoresis runs, which completed in 30 min using 20 pmol (120 ng) of labeled insulin and 5 pmol (750 ng) each of the mAb.

Mobility moment analysis of molecular interactions by capillary electrophoresis.

A new method for the quantitative evaluation of molecular interactions that are observed in electrophoresis is described. One component taking part in the interaction is labeled with a fluorescent dye and is subjected to capillary zone electrophoresis with fluorescence detection in the presence or absence of an unlabeled interacting component. Fluorescence signals are collected at constant time intervals, and the electropherograms are converted to represent the fluorescence signal against mobility. After baseline subtraction, the first statistical moment of fluorescence signals on the mobility axis is calculated. This moment represents the average mobility of a labeled component. The change in the mobility moment in the presence and absence of the unlabeled component is used to evaluate the degree of saturation of the binding site of a labeled molecule with an unlabeled molecule. Mixtures of fluorescence-labeled protein (Fab' fragment of antibody or concanavalin A) and its unlabeled interacting partner (alpha(1)-antitrypsin or succinylated ovalbumin, respectively) at various concentrations were injected into a bare-silica capillary, and zone electrophoresis was carried out. The change in the mobility moment of the fluorescence-labeled molecules was used to determine the dissociation constants of the complexes. The determined constants are comparable to those obtained by a well-established method, that is, an analysis based on the peak height of the complex. Since the mobility moment analysis is not affected by the total intensity of the signals, it should be advantageous in analyses in which multiple capillaries are used, in which the injection volume and the sensitivity of detection might be more difficult to control at constant values. The mobility moment analysis also has advantages for the analysis of heterogeneous samples, since the identification of peaks is not necessarily required.

Recent advances in capillary isoelectric focusing: 1997-2001.

The methodological developments in the field of capillary isoelectric focusing (CIEF) published between 1997-2001 are reviewed as a continuation of the previous review by Rodriguez-Diaz et al. (Electrophoresis 1997, 18, 2134-2144). The applications are summarized and the progress in CIEF technologies, including experimental setup with coated and uncoated capillaries, remedies for the presence of salts in samples, additives to reduce precipitation of samples during the focusing process, calibration of the pH gradients, issues of reproducibility, carrier ampholyte-free CIEF, and a computer simulation of focusing process are discussed. Developments of IEF separations in fabricated microchannels and the advances in detection schemes, i.e., imaging, fluorescence and chemiluminescence, are summarized. The progress in micropreparation was noted, and the massive works for two-dimensional separations are described for the coupling with size-exclusion chromatography and mass spectrometry, in which the developments aimed at proteomics are discussed separately. The applications for the detection of noncovalent complexes and the separations of microorganisms are reviewed.

Determination of the affinity constants of recombinant human galectin-1 and -3 for simple saccharides by capillary affinophoresis.

The affinity constants of recombinant human galectin-1 and galectin-3 for sugars were determined by capillary affinophoresis. The monoliganded affinophore contains p-aminophenyl-beta-lactoside as an affinity ligand in the matrix of succinylglutathione and has three negative charges. An analysis of the mobility change of the lectins caused by the affinophore and its inhibition by neutral sugars allowed, for the first time, a determination of the affinity constants between the binding sites of the lectins and sugars. The relative magnitude of the affinity constants for each of the sugars in terms of dissociation constants found to be consistent with previously reported data on the concentrations of sugars that caused a 50% inhibition (I50) in the binding assay of the lectin to oligosaccharide-immobilized agarose beads but the absolute values of the dissociation constants were considerably smaller than the I50 values. Capillary affinophoresis indicated microheterogeneity of the lectin preparations and enabled the separate analysis of the affinity of each component simultaneously showing the advantage in using a separation method for analysis of bioaffinity.

Fluorescence-labeled peptide pI markers for capillary isoelectric focusing.

Nineteen fluorescent pH standards or pI markers ranging pH 3.64-10.12 were developed for use in capillary isoelectric focusing using laser-induced fluorescence detection. Tetra- to tridecapeptides containing one cysteine residue were designed to focus sharply at their respective isoelectric points by including amino acids that contain charged side chains, the pKa values of which are close to the corresponding pI values. An iodoacetylated derivative of tetramethylrhodamine was coupled to the thiol group of cysteine to yield fluorescent pI markers. The pI values of the labeled peptides were precisely determined after isoelectric focusing on polyacrylamide gel slabs by direct measurement of the pH of the focused bands. The markers were subjected to capillary isoelectric focusing for 10-15 min in coated capillaries under conditions of low electroosmosis and were detected by means of a scanning laser-induced fluorescence detector down to a level of subpicomolar range. The markers permitted the calibration of a wide-range pH gradient formed in a capillary by fluorescence detection for the first time and should facilitate the development of highly sensitive analytical methods based on a combination of capillary isoelectric focusing and laser-induced fluorescence detection.