Multicompartment Electrolyzer Research Articles

Divergent-flow isoelectric focusing for separation and preparative analysis of peptides

A divergent-flow isoelectric focusing (DF IEF) technique has been applied for the separation and preparative analysis of peptides. The parameters of the developed DF IEF device such as dimension and shape of the separation bed, selection of nonwoven material of the channel, and separation conditions were optimized. The DF IEF device was tested by the separation of a peptide mixture originating from the tryptic digestion of BSA, cytochrome c, and myoglobin. The pH gradient of DF IEF was created by the autofocusing of tryptic peptides themselves without any addition of carrier ampholytes. The focusing process was monitored visually using colored pI markers, and the obtained fractions were analyzed by RP-HPLC and ESI/TOF-MS. DF IEF operating in the autofocusing mode provides an efficient preseparation of peptides, which is comparable with a commercially available MicroRotofor multicompartment electrolyzer and significantly improves sequence coverage of analyzed proteins. The potential of the DF IEF device as an efficient tool for the preparative scale separations was demonstrated by the isolation of caseinomacropeptide (CMP) from a crude whey solution.

SDS-PAGE Focusing: Preparative Aspects

As a followup of our previous report (Zilberstein, G.; Korol, L.; Antonioli, P.; Righetti, P. G.; Bukshpan, S. Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a novel method is here reported for small-scale prefractionation of complex protein mixtures, for subsequent proteome analysis, based on mass separation of SDS-protein micelles not in a gel matrix, but in liquid cationic polymers assembled in a multicompartment electrolyzer (MCE) in a stepwise fashion at discrete and increasing levels of positive charges (from 3 to 28 mM), the neighboring chambers being separated by neutral agarose membranes. Unlike conventional SDS-PAGE, in which separation by mass of SDS-laden polypeptide chains is obtained in constant concentration or porosity gradient gels, the present method of SDS-PAGE focusing exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path and trapped into different compartments of the MCE device via interaction (and subsequent charge neutralization) with cationic polymers of fixed (but increasing from chamber to chamber from cathode to anode) charge density. Minimization of migration of the liquid cationic polymers is obtained via use of low voltage and by arranging for a buffer conductivity gradient along the migration path. The present setup has the advantage of high protein recoveries (up to 90%) without any contamination from ungrafted monomers and catalysts, as occurring in proteins recovered by passive elution from gel matrixes. Additionally, resolution can be fine-tuned by selecting cationic polymers of varying charge density in microstep increments. The cationic polymers, of desired charge density and proper viscosity, are prepared by standard polymerization conditions, can be easily precipitated and washed free of monomeric contaminants, and stored in a dry form for subsequent use.

Autofocusing and ESI‐MS analysis of protein digests in a miniaturized multicompartment electrolyzer

Free-solution IEF of protein digests was studied in a newly introduced MicroRotofortrade mark multicompartment electrolyzer. The fractionation was performed in a cylindrical separation chamber divided into ten compartments with or without the addition of carrier ampholytes. In the case of autofocusing mode of operation, the tryptic digest itself served as the mixture of ampholytes leading to the separation of the peptides with well-defined pI's. The focusing process was monitored visually using colored pI markers. The resulting fractions from both modes of the separation were analyzed by CE and electrospray-TOF mass spectrometer using electrospray tips microfabricated in polyimide. Additional experiments, aiming at visualization of the mass flux within the focusing compartments were performed using isotachophoretic migration of color cationic tracers. The study considered the autofocusing of both the peptides with well-defined narrow pI's as well as those showing negligible net charge in a broader pH range. Although not all peptides in the protein digests have well-defined pI's the autofocusing process can preseparate many of them leading to higher S/N in the ESI-MS signals and improved protein sequence coverage.

Rapid isoelectric trapping in a micropreparative‐scale multicompartment electrolyzer

A multicompartmental electrolyzer called membrane-separated wells for isoelectric focusing and trapping (MSWIFT) has been developed and tested for micropreparative-scale isoelectric trapping separations. In the MSWIFT, the length of the heat conduction path from the center of the compartments to the wall is less than 1 mm. The compartments are made from 99.8% nonporous alumina that has a high heat conductivity and a high specific heat capacity, leading to adequate Joule heat dissipation even at power loads as high as 5 W. The length of any compartment parallel to the electric field (the intermembrane distance) can be selected to be multiples of 1.5 mm, leading to compartment volumes that are multiples of about 60 muL. A maximum of 20 (1.5 mm long) separation compartments can be readily assembled in the current version of MSWIFT. The MSWIFT has been used to desalt samples, isolate small ampholytic components (amino acids, peptides, and dyes), prefractionate complex protein mixtures and enrich minor components; these separations were achieved in 20-60 min.

Fractionation of Maize Embryo Proteins for Two-Dimensional Gel Electrophoresis Using a Multicompartment Electrolyzer

Fractionation of Maize Embryo Proteins for Two-Dimensional Gel Electrophoresis Using a Multicompartment Electrolyzer

Fractionation of Human Plasma Proteins for Two-Dimensional Gel Electrophoresis Using a Multicompartment Electrolyzer (MCE)

Fractionation of Human Plasma Proteins for Two-Dimensional Gel Electrophoresis Using a Multicompartment Electrolyzer (MCE)

Isoelectric beads for proteome pre‐fractionation. II: Experimental evaluation in a multicompartment electrolyzer

Proteome pre-fractionation in multicompartment electrolyzers is proposed here, with substantial modifications as compared to the standard technique. First of all, the classical isoelectric, buffering membranes, delimiting each compartment and acting, in pairs, as isoelectric traps, have been replaced by isoelectric buffering beads, operating on the same principle, but allowing unhindered migration of proteins (lack of sieving properties, contrary to typical continuous membrane barriers). Secondly, the isoelectric beads are not made in the conventional manner, with ionic acrylamide derivative monomers throughout their central core, but are composed of a hard, ceramic core, coated with an amphoteric buffering polymer. This minimizes mass transfer resistance of proteins that are transiently adsorbed onto the beads. As a result, significantly reduced separation times, of the order of ca. 3 h, are required for developing steady-state patterns, as compared to the lengthy times (overnight and much longer) in conventional multicompartment electrolyzers operating with isoelectric membranes. Examples of separation of standard marker proteins, as well as entire Escherichia coli lysates and human serum proteins, are given. The obtained fractions are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by surface enhanced laser desorption/ionization mass spectrometry.

Solution phase isoelectric fractionation in the multi-compartment electrolyser: A divide and conquer strategy for the analysis of complex proteomes

Sample complexity frequently interferes with the analysis of low-abundance proteins by two-dimensional gel electrophoresis (2DGE). Ideally, high abundance proteins should be removed, allowing low-abundance proteins to be applied at much higher concentrations than is possible with the unfractionated sample. One approach is to partition the sample in a manner that segregates the bulk of extraneous proteins from the protein(s) of interest. Solution phase isoelectric focusing in the multi-compartment electrolyser generates fractions of discrete isoelectric point (pI) intervals allowing isolated narrow segments of a proteome to be analysed individually by 2DGE. It is particularly useful for the isolation of low-abundance proteins of extremely basic or acidic pI.

Purification of an Enterobacter aerogenes plasmid DNA using MnCl 2 as compaction agent

Development of an efficient process for purification of DNA plasmids continues to be a technological challenge [1–3]. While earlier applications of DNA plasmids were mostly in cloning, the last few years have seen its increasing applications in gene therapy [1] and preparation of DNA vaccines [4]. A crucial step in the production process is the downstream processing stage which encompasses separation of plasmid DNA from cellular components of the host strain (including chromosomal DNA and its fragments). In the case where the intended applications are in therapeutics, it is also necessary to separate the plasmid from materials such as toxic solvents, mutagenic reagents, and animal-derived enzymes (e.g., bovine pancreatic RNase A, used for removal of abundant RNA present in cell lysate) which may have been used in the production process. The existing protocols for plasmid production are, in general, time consuming, expensive, and difficult to scale-up [5]. Almost all of these require at least one chromatographic step although recently a process using a multicompartment electrolyser separated by ultrafiltration has been described [6]. In this paper, we describe a mini-prep protocol that is a simple, inexpensive, and quick method for plasmid purification which in principle can be easily scaled-up. As the protocol described here does not require any aggressive chemicals and/or use any animal-origin enzymes it is potentially useful for therapeutic applications. The method is based upon two observations reported earlier by others: the possibility of plasmid DNA purification by selective precipitation [5,7] and condensation of DNA induced by MnCl2 [8]. The protocol described herein has been developed for purifica-

Prefractionation techniques in proteome analysis.

The present review deals with a number of prefractionation protocols in preparation for two-dimensional map analysis, both in the fields of chromatography and in the field of electrophoresis. In the first case, Fountoulaki's groups has reported just about any chromatographic procedure useful as a prefractionation step, including affinity, ion-exchange, and reversed-phase resins. As a result of the various enrichment steps, several hundred new species, previously undetected in unfractionated samples, could be revealed for the first time. Electrophoretic prefractionation protocols include all those electrokinetic methodologies which are performed in free solution, essentially all relying on isoelectric focusing steps. The devices here reviewed include multichamber apparatus, such as the multicompartment electrolyzer with Immobiline membranes, Off-Gel electrophoresis in a multicup device and the Rotofor, an instrument also based on a multichamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other instruments of interest are the Octopus, a continuous-flow device for isoelectric focusing in a upward flowing liquid curtain, and the Gradiflow, where different pI cuts are obtained by a multistep passage through two compartments buffered at different pH values. It is felt that this panoply of methods could offer a strong step forward in "mining below the tip of the iceberg" for detecting the "unseen proteome".

Separation of proteins in a multicompartment electrolyzer with chambers defined by a bed of gel beads.

Multicompartment electrolyzers (MEs) with isoelectric membranes were introduced in 1989 for purifying proteins in an electric field. At the basis of ME technology there are membranes consisting of cross-linked copolymers of acrylamide and acrylamido monomers bearing protolytic groups. The technology employed for casting the membranes is an extension of the isoelectric focusing in immobilized pH gradient technique for which specific acrylamido monomers, known with the trade name of Immobiline, have been developed. However, the use of continuous membranes presents several disadvantages. Due to the mechanical characteristics of polyacrylamide, the gel must physically adhere onto a rigid support, which prevents it from collapsing. The support must have a highly porous structure in order to be permeable to proteins. The mechanical fragility of the membranes is one of the main problems that hinders the industrial scale application of ME separators. In order to overcome this problem, we propose to substitute the continuous membranes with a bed of gel beads of identical comonomer composition, obtained by an inverse emulsion polymerization process.

Purification of plasmid DNA using a multicompartment electrolyser separated by ultrafilter membranes

A simple, scalable method for purification of plasmid DNA is described. Plasmid DNA was released from Escherichia coli JM109 by lysis (1% SDS, 0.2 M NaOH). Then a neutralization solution (3 M sodium acetate buffer, pH 4.8) was added to precipitate genomic DNA and protein. After the clarification of the lysate, the supernatant was placed in a multicompartment electrolyser separated by ultrafilter membranes to remove the remaining contamination (RNA, genomic DNA and protein). A recovery of 75%±2% of total plasmid DNA was obtained after 60 min electrophoresis with a field strength of 8 V cm−1 using cells at 30 g l−1 (quantified by dry cell weight). Genomic DNA, RNA and protein were undetectable in the purified plasmid DNA solution.

Stereoselective synthesis of ( S)-(+)-Naproxen catalyzed by carboxyl esterase in a multicompartment electrolyzer

The stereoselective hydrolysis of racemic 2-substituted propionates, catalyzed by carboxyl esterase, provides a cost-competitive route to produce the optically pure, anti-inflammatory drug Naproxen. In the present work, we describe the application of the multicompartment electrolyzer reactor (ME) for the stereoselective hydrolysis of a racemic Naproxen ester, ( R, S)-ethoxyethyl-[2-(6-methoxy-2-naphtyl)]propionate, catalyzed by a carboxyl esterase. The enzyme was trapped in a reactor chamber, delimited by two isoelectric membranes encompassing the p I value of the enzyme, together with the neutral substrate. After 90 min, a conversion of 45% was obtained with an enantiomeric excess of 84%. The reaction product, ( S)-(+)-Naproxen, was electrophoretically removed in continuous from the reaction chamber and collected in a contiguous, more acidic chamber, separated from the enzyme and from the unreacted substrate. Moreover, at the end of the reaction, it was possible to recover the enzyme from the reactor and use it again.

Mass transport characteristics and chromatographic behavior of preparative electrochromatography on hydroxyapatite

Introducing an electric field into chromatography on hydroxyapatite (HAP) was attempted in order to enhance mass transfer and separation performance. A membrane spaced multicompartment electrolyzer was developed for electrochromatography on HAP. The high performance of liquid transport by electroosmotic flux was identified and described in terms of dynamic electroosmotic pressure. The application of the electric field resulted in an improved adsorption of bovine serum albumin as shown by the breakthrough curve as function of the electric field. An improved elution was also obtained in the presence of the electric field. The results show that electroosmosis is a powerful tool of liquid transport and dispersion in a packed bed of fine particles and has potential in the large-scale chromatography of biological molecules.

Preparative-scale isoelectric focusing separation of enantiomers using a multicompartment electrolyzer with isoelectric membranes.

The IsoPrime multicompartment electrolyzer, equipped with a series of isoelectric membranes with closely spaced pI values, was used for the first time for the preparative-scale separation of the enantiomers of dansyl phenylalanine with hydroxypropyl beta-cyclodextrin as resolving agent. The final separation conditions could be established easily in three successive experiments by rationally narrowing the pH steps between the neighboring isoelectric membranes. The final separation yielded products with an enantiomeric excess greater than 99.9%, at production rates of about 0.1 mg/h. The greatest experimental difficulty was caused by the relatively high salt content of the hydroxypropyl beta-cyclodextrin used, which resulted in high conductivity and limited the maximum field strength one could use.

Preparative-scale isoelectric focusing separation of enantiomers using a multicompartment electrolyzer with isoelectric membranes

Preparative-scale isoelectric focusing separation of enantiomers using a multicompartment electrolyzer with isoelectric membranes

Experimental studies on affinity chromatography in an electric field

A multicompartment electrolyzer, which has been used for preparative electrophoresis [Z. Liu, Z. Huang, J.-Y. Cong, et al., Sep. Sci. Technol. 31 (1996) 427], is applied for carrying out affinity chromatography in an alternating electric field. The effect of electric field strength on the adsorption and desorption characteristics is experimentally examined with human serum albumin and Blue Sepharose Fast Flow as a model system. It is shown that the existence of an electric field leads to a significant change in the adsorption capacity of the blue dye, which may be used for establishing a preferential adsorption to achieve a high resolution. The adsorption speed increases slightly with respect to the increase of electric field strength, while adsorption capacity in the presence of an electric field is independent of the electric field strength. Different elution behavior is observed as function of adsorption condition and a high recovery of the adsorbed protein is obtained when the adsorption is carried out in the presence of an electric field.

Longitudinal Distribution of pH in Preparative Electrophoresis in a Multicompartment Electrolyzer

Generating an expected longitudinal pH distribution is of essential importance for achieving high resolution in an electrophoresis process conducted in a multicompartment electrolyzer. A simulation of the longitudinal distribution of pH in a multicompartment electrolyzer is proposed on the basis of mass transfer analysis and electrolysis equilibrium of the electrolytes. Selection of the operating conditions to establish an expected pH distribution with a conventional electrolyte rather than an ampholyte can thus be accomplished according to the simulation results. Numerical solution of this model is developed on the basis of the Gear method. The calculated results agree well with the experimental data in a wide range of operation conditions including applied potential, sample loading flow rate, and protein concentration in solution. This model may also be applied to other ampholyte-free electrophoresis processes conducted in a membrane-spaced multicompartment electrolyzer.

Electrophoretic affinity chromatography: method validation.

A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered.

Production of D-phenylglycine from racemic (D,L)-phenylglycine via isoelectrically-trapped penicillin G acylase.

Penicillin G acylase (PGA) is exploited for producing pure D-phenylglycine from a racemate mixture, via an acylation reaction onto a cosubstrate, the ester methyl-4-hydroxyphenyl acetate. The reaction, when carried in a batch, is severely hampered by the reverse process, by which the product, 4-hydroxyphenylacetyl-(L)phenyl glycine, upon consumption of L-phenylglycine, is converted by the enzyme back into free substrate and 4-hydroxyphenyl acetic acid via lysis of the amido bond. To prevent this noxious reaction, a multicompartment electrolyzer with isoelectric membranes (MIER) is used as enzyme reactor, operating in an electric field. PGA is trapped between pI 5.5 and pI 10.5 membranes, together with an amphoteric, isoelectric buffer (lysine). As the 4-hydroxyphenylacetyl-(L)phenyl glycine product is formed, it vacates the reaction chamber by electrophoretic transport and is collected close to the anode, in a chamber delimited by pI 2.5 and 4.0 membranes. The same fate occurs to the free acid 4-hydroxyphenyl acetic acid, formed upon spontaneous (and enzyme-driven) hydrolysis of the methyl ester in the reaction chamber. These combined processes leave behind, in the enzyme reaction chamber, the desired product, pure D-phenylglycine. The advantages of the MIER reactor over batch operations: the consumption of the L-form in the racemate is driven to completion and the enzyme is kept in a highly stable form, maintaining 100% activity after one day of operation, during which time the PGA enzyme, in the batch reactor, has already lost >75% catalytic activity.