Preparative-scale isoelectric trapping separations using a modified Gradiflow unit

The Gradiflow BF200 preparative electrophoretic unit (Gradipore), which has been developed for size-based and charge-sign-based protein separations and in which the hydraulic flow path of the recirculating sample stream in the separation cartridge is orthogonal to the electric field, has been modified to carry out binary protein separations using the principles of isoelectric trapping. The disposable separation cartridge contained three isoelectric membranes which, along with the cartridge holder, formed the anode and cathode compartments and the anodic and cathodic separation compartments. The utility of the modified instrument was demonstrated by effecting a binary separation of chicken egg white across an isoelectric point 5.5 isoelectric membrane. The desalting and subsequent binary separation steps proved to be remarkably rapid, due to the favorable combination of short electrophoretic path, high electric field strength and large effective isoelectric membrane surface area.     

Determination of the operational pH value of a buffering membrane by an isoelectric trapping separation of a carrier ampholyte mixture

The operational pH value of a buffering membrane used in an isoelectric trapping separation is determined by installing the membrane as the separation membrane into a multicompartmental electrolyzer operated in the two-separation compartment configuration. A 3相似文献   

Use of a preparative-scale, recirculating isoelectric trapping device for the isolation and enrichment of acidic proteins in bovine serum

A recirculating, preparative-scale isoelectric trapping device, developed for the binary isoelectric trapping separation of proteins has been used to desalt, isolate and enrich the pI<4 protein fraction from a 150 mL sample of bovine serum. Subsequent re-separation of the 2相似文献   

Preparative-scale isoelectric trapping enantiomer separations

The new Gradiflow BF200 IET unit, developed for isoelectric trapping protein separations has been modified and used to carry out preparative-scale enantiomer separations. Hydroxypropyl beta-cyclodextrin was used as the chiral resolving agent to induce an isoelectric point difference between the enantiomers. Three isoelectric membranes with isoelectric points below, in between and above the isoelectric points of the complexed enantiomers were used to trap the separated enantiomers in the anodic and cathodic separation compartments of the Gradiflow BF200 IET apparatus, respectively. The production rates were about 15 times higher than those previously obtained with another isoelectric trapping device and about 30% higher than those obtained in a continuous free-flow electrophoretic device operated in the isoelectric focusing mode. The remarkable separation speed observed in the modified Gradiflow BF200 IET unit is attributed to the favorable interplay of the short electrophoretic transfer distance, the high electric field strength and the large effective surface areas of the isoelectric membranes.     

Concentration and fractionation by isoelectric trapping in a micropreparative-scale multicompartmental electrolyzer having orthogonal pH gradients. Part 2

A micropreparative-scale multicompartmental electrolyzer called ConFrac has been developed and tested for isoelectric trapping separations. ConFrac can be operated in pass-by-pass mode or recirculating mode, using either asymmetrical feeding (feed enters only the anodic or the cathodic flow-through compartment) or symmetrical feeding (feed enters both the anodic and the cathodic flow-through compartment). Symmetrical feeding results in higher processing rates and is the preferred operation mode. Residence time in the flow-through compartments is set as a compromise between processing rate and temperature rise in the effluent. Ampholytic components have been isolated from 5 to 50 mL volumes of micromolar feed solutions and hundredfold concentrated into 100-μL collection compartments. Samples containing ampholytic analytes in highly conducting salt solutions were readily desalted and fractionated in ConFrac in one operation. pH transients formerly observed in other isoelectric trapping devices were observed in ConFrac as well. The pH transients were caused by the unequal ion transmission rates of the anodic- and cathodic-side buffering membranes.     

The Biflow: an instrument for transfer-loop mediated, continuous, preparative-scale isoelectric trapping separations

The Biflow, a new isoelectric trapping instrument was designed to obtain a narrow DeltapI fraction from a complex feed in one step. The Biflow contains two identical separation units, each unit houses: an anode and cathode compartment, an anodic and cathodic membrane, an anodic and cathodic separation compartment, and a separation membrane. The separation units are connected to independent power supplies. The anodic membranes in Units 1 and 2 typically buffer at the same pH value and so do the cathodic membranes. The separation membranes in Units 1 and 2 buffer at different pH values, these determine the pI range (DeltapI) of the product. The cathodic separation compartments in Units 1 and 2 contain the feed and harvest streams. The two anodic separation compartments, connected through an electrically insulating air gap, form the transfer loop through which the transfer stream is recirculated between Units 1 and 2. Ampholytic components in the feed, with pI values lower than the pH of the buffering membrane in Unit 1, pass into the transfer stream and are shuttled into Unit 2. In Unit 2, components in the transfer stream which have pI values higher than the pH of the buffering membrane in Unit 2, pass into the harvest stream. This double transfer of the target component, oppositely directed, guarantees the complete exclusion of products outside the desired DeltapI range from the harvest stream. The utility of the Biflow unit was demonstrated by isolating carnosine from a mixture of UV-absorbing ampholytes and ovalbumin isoforms as well as 4.4 相似文献   

Concentration and fractionation by isoelectric trapping in a micropreparative-scale multicompartmental electrolyzer having orthogonal pH gradients. Part 1

A multicompartmental electrolyzer called ConFrac has been developed and tested for micropreparative-scale isoelectric trapping separations. ConFrac contains n separate, minimalistic isoelectric trapping core units, each with a separate anode compartment, anodic flow-through compartment, collection compartment, cathodic flow-through compartment and a shared cathode compartment. The collection compartments are all isolated from each other and have volumes of 100 μL each. The liquid held in the collection compartments is stagnant. The respective anodic and cathodic flow-through compartments are hydraulically serially connected to each other by flexible, minimum-length, narrow internal diameter tubes. The respective feed solutions whose volumes are larger and variable are recirculated through the serially connected flow-through compartments. Poly(vinyl alcohol)-based buffering membranes are placed between the anode compartments, anodic flow-through compartments, collection compartments, cathodic flow-through compartments and cathode compartment. The membranes establish two orthogonal pH gradients in ConFrac. The primary pH gradient is parallel with the direction of the recirculating flows and orthogonal to that of the electric field. The secondary pH gradient is parallel with the direction of the electric field and orthogonal to that of the recirculating flows. Since the recirculating liquids are kept in thermostated reservoirs and the residence times in the flow-through compartments are shorter than 2 s, ConFrac can tolerate power loads as high as 2 W without overheating the solutions. The operation and performance of ConFrac has been quantitatively characterized: four 25 μM ampholytic components were isolated from 5 mL of feed solution in 20 min and their concentration increased approximately 50-fold.     

Synthesis of UV-absorbing and fluorescent carrier ampholyte mixtures and their application for the determination of the operational pH values of buffering membranes used in isoelectric trapping separations

Success in isoelectric trapping separations critically depends on the knowledge of the accurate operational pH value of the buffering membranes used. Currently, due to a lack of easy, rapid, accurate methods that can be used for the post-synthesis determination of the operational pH value of a buffering membrane, only nominal pH values calculated from the amounts of the reagents used in the synthesis of the membranes and their acid-base dissociation constants are available. To rectify this problem, UV-absorbing and fluorescent carrier ampholyte mixtures were prepared by alkylating pentaethylenehexamine with a chromophore and a fluorophore, followed by Michael addition of acrylic acid and itaconic acid to the resulting oligoamine. Carrier ampholyte mixtures, with evenly distributed absorbance values across the 3相似文献   

A family of high-buffering capacity diamino sulfate isoelectric buffers for pH-biased isoelectric trapping separations

pH-biased isoelectric trapping separations are hindered by the lack of suitable isoelectric buffers with pI values in the 5.8 < pI range. Two generic approaches are described here for the cost-effective synthesis of a family of diamino sulfate buffers that have high buffering capacities in their isoelectric state: the first approach relies on the sulfation of existing, commercially available diamino alcohol intermediates, the second approach calls for the synthesis of diamino alcohols from epichlorohydrin and widely available secondary amines, and subsequent sulfation of the new diamino alcohol. The diamino sulfate buffers are recovered in isoelectric state, in high purity. Four members of the family having pI values in the 5.8 < pI < 8.9 range have been synthesized, analytically characterized by capillary electrophoresis (CE), electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), 1-D and 2-D nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. All four diamino sulfates have been successfully used as pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping protein separations.     

Synthesis and characterization of quaternary ammonium dicarboxylic acid isoelectric buffers and their use in pH-biased isoelectric trapping separations

Two approaches are described in this paper for the synthesis of isoelectric buffers that have pI values in the 1.5 < pI < 4.3 range. The first synthesis relies on the alkylation of existing aminodicarboxylic acids and recovery of the ampholyte as an inner salt. The second synthesis method forms low-pI ampholytes by reacting a secondary amine with two equivalents of an alkylester of a haloalkanecarboxylic acid, followed by hydrolysis of the intermediate in an alkaline solution and recovery of the ampholyte as an inner salt. The new ampholytes have been analytically characterized by capillary electrophoresis, high-resolution electrospray ionization-mass spectrometry, one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. The isoionic solutions of the new ampholytes have high buffering capacity and conductivity, making them good pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping separations.     

High-buffering capacity, hydrolytically stable, low-pI isoelectric membranes for isoelectric trapping separations

Hydrolytically stable, low-pI isoelectric membranes have been synthesized from low-pI ampholytic components, poly(vinyl alcohol), and a bifunctional cross-linker, glycerol-1,3-diglycidyl ether. The low-pI ampholytic components used contain one amino group and at least two weakly acidic functional groups. The acidic functional groups are selected such that the pI value of the ampholytic component is determined by the pK(a) values of the acidic functional groups. When the concentration of the ampholytic component incorporated into the membrane is higher than a required minimum value, the pI of the membrane becomes independent of variations in the actual incorporation rate of the ampholytic compound. The new, low-pI isoelectric membranes have been successfully used as anodic membranes in isoelectric trapping separations with pH < 1.5 anolytes and replaced the hydrolytically less stable polyacrylamide-based isoelectric membranes. The new low-pI isoelectric membranes have excellent mechanical stability, low electric resistance, good buffering capacity, and long life time, even when used with as much as 50 W power and current densities as high as 33 mA/cm(2) during the isoelectric trapping separations.     

Preparative-scale isoelectric trapping by recursive electrophoresis in a compartmentalized system having orthogonal primary and secondary pH gradients. Part 2--operation in cascade mode

A parallel multicompartmental electrolyzer recently developed for preparative-scale isoelectric trapping separations, trapping by recursive electrophoresis in a compartmentalized system, was set up to operate as a cascade of binary separations to produce at least one pure target ampholyte (or more, with additional separation heads) without other ampholytes ever entering (even transiently) the harvest stream. This mode of operation avoids the need for exhaustive electrophoresis and the accompanying long separation times brought about by the exponentially decreasing concentrations over the course of batch separations. Continuous operation can be achieved in the cascade mode by continuously feeding the sample into the first separation head configured with three flow-through compartments and continuously harvesting one (or more) target components in additional separation heads configured with two flow-through compartments, attached to the respective branching points.     

Combining isoelectric point-based fractionation, liquid chromatography and mass spectrometry to improve peptide detection and protein identification

The off-line coupling of an isoelectric trapping device termed membrane separated wells for isoelectric focusing and trapping (MSWIFT) to mass spectrometry-based proteomic studies is described. The MSWIFT is a high capacity, high-throughput, mass spectrometry-compatible isoelectric trapping device that provides isoelectric point (pI)-based separations of complex mixtures of peptides. In MSWIFT, separation and analyte trapping are achieved by migrating the peptide ions through membranes having fixed pH values until the peptide pI is bracketed by the pH values of adjacent membranes. The pH values of the membranes can be tuned, thus affording a high degree of experimental flexibility. Specific advantages of using MSWIFT for sample prefractionation include: (1) small sample volumes (～200 μL), (2) customized membranes over a large pH range, (3) flexibility in the number of desired fractions, (4) membrane compatibility with a variety of solvents systems, and (5) resulting fractions do not require sample cleanup before MS analysis. Here, we demonstrate the utility of MSWIFT for mass spectrometry-based detection of peptides in improving dynamic range and the reduction of ion suppression effects for high-throughput separations of tryptic peptides.     

Use of full-column imaging capillary isoelectric focusing for the rapid determination of the operating conditions in the preparative-scale continuous free-flow isoelectric focusing separation of enantiomers

A rapid, simple method is proposed here for the identification of the experimental conditions that lead to satisfactory preparative-scale isoelectric focusing enantiomer separations in continuous free-flow electrophoretic units. The method first calls for the use of a commercially available, full-column imaging capillary electrophoretic system to find the background electrolyte composition that generates the largest pI difference between the bands of the enantiomers. The method then calls for the finding of the minimum residence time that permits full development of the pH gradient across the separation chamber of the continuous free-flow electrophoretic unit by measuring the pH in the sample-free carrier electrolyte fractions collected during these runs. Finally, the quality of the predicted preparative-scale separation is verified by analyzing the enantiomer-containing collected fractions by capillary electrophoresis using a 14-sulfated, single-isomer cyclodextrin as resolving agent. The pI difference values and production rate values observed in this work agree well with the literature values that were obtained by much more time-consuming methods.     

Hydrolytically stable, diaminocarboxylic acid-based membranes buffering in the pH range from 6 to 8.5 for isoelectric trapping separations

Diaminocarboxylic acid carrier ampholytes, such as L-histidine, 2,3-diaminopropionic acid, L-ornithine, and L-lysine, were reacted with glycerol-1,3-diglycidyl ether (GDGE) and poly(vinyl alcohol) (PVA) in the presence of sodium hydroxide to produce hydrolytically and mechanically stable hydrogels, supported on a PVA substrate, for use as buffering membranes in isoelectric trapping (IET) separations. The pH values of the DACAPVA membranes were determined with the help of small-molecule pI markers and proteins and were found to be in the 6 < pH < 8.5 range. The membranes were successfully used to isoelectrically trap small ampholytes, desalt ampholyte solutions in IET mode, and effect the binary separation of chicken egg white proteins.     

Estimation of isoelectric points of human plasma proteins employing capillary isoelectric focusing and peptide isoelectric point markers

Synthetic UV-detectable peptide pI markers were used to estimate isoelectric point (pI) values of proteins separated by capillary isoelectric focusing (CIEF) followed by cathodic mobilization in the absence of denaturing agents. The pI calculation and quantitative analysis of purified proteins showed the feasibility of these peptides as pI markers and internal standards in CIEF separation of proteins. Estimation of pI values of major proteins in human plasma was performed using the peptide pI markers, and the values were compared with those previously obtained by gel isoelectric focusing (IEF). Sera of immunoglobulin G (IgG) myeloma patients, which showed characteristic peaks of myeloma IgG in their CIEF patterns, were also subjected to the analysis and the pI values of the myeloma proteins have been estimated.     

Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organelles, and cells

In appropriate aqueous buffer solutions, biological particles usually exhibit a particular electric surface charge due to exposed charged or chargeable functional groups (amino acid residues, acidic carbohydrate moieties, etc.). Consequently, these bioparticles can migrate in solution under the influence of an electric field allowing separation according to their electrophoretic mobilities or their pI values. Based on these properties, electromigration methods are of eminent interest for the characterization, separation, and detection of such particles. The present review discusses the research papers published between 2008 and 2010 dealing with isoelectric focusing and zone electrophoresis of viruses, organelles and microorganisms (bacteria and yeast cells) in the capillary and the chip format.     

Chiral anion exchangers applied to capillary electrochromatography enantioseparation of oppositely charged chiral analytes: investigation of stationary and mobile phase parameters

Weak anion-exchange (WAX) type chiral stationary phases (CSPs) based on tert.-butyl carbamoyl quinine as chiral selector (SO) and different types of silica particles (porous and non-porous) as chromatographic support are evaluated in packed capillary electrochromatography (CEC). Their ability to resolve the enantiomers of negatively charged chiral analytes, e.g., N-derivatized amino acids, in the anion-exchange mode and their electrochromatographic characteristics are described in dependence of several mobile phase parameters (pH, buffer type and concentration, organic modifier type and concentration) and other experimental variables (electric field strength, capillary temperature). The inherent "zwitterionic" surface character of such silica-based WAX type CSPs (positively charged SO and negatively charged residual silanols) allows the reversal of the electroosmotic flow (EOF) towards the anode at pH values below the isoelectric point (pI) of the modified surface, whereas a cathodic EOF results at pH values above the pI. Since for negatively charged analytes also an electrophoretic transport increment has to be considered, which can be either in or against the EOF direction, several distinct modes of elution have been observed under different stationary phase and mobile phase conditions: (i) co-electrophoretic elution of the negatively charged solutes with the anodic EOF in the negative polarity mode, (ii) counter-electrophoretic elution with the cathodic EOF in the positive polarity mode, and (iii) electrophoretically dominated elution in the negative polarity mode with a cathodic EOF directed to the injection end of the capillary. Useful enantioseparations of chiral acids have been obtained with all three modes. Enantioselectivity values as high as under pressure-driven conditions and theoretical plate numbers up to 120000 per meter could be achieved under electrically driven conditions. A repeatability study yielded RSD values below 2% for retention times and RSD values in the range of 5-10% for theoretical plate numbers and resolution, thus clearly establishing the reliability of the investigated anion-exchange type CEC enantioseparation methods.     

General experimental aspects of the use of isoelectric buffers in capillary electrophoresis.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.     

Preparative-scale isoelectric trapping by recursive electrophoresis in a compartmentalized system having orthogonal primary and secondary pH gradients. Part 1--construction and standard operation

The performance of the current preparative-scale isoelectric trapping systems is limited by the serial arrangement of the separation compartments. A new system has been developed that achieves trapping by recursive electrophoresis in a compartmentalized system (T-RECS). T-RECS features (i) parallel-connected elementary separation heads with independent electrode compartments, feed compartments, and harvest compartments, (ii) orthogonal primary pH gradients and secondary pH gradients, (iii) directionally controlled convective analyte transport along the primary (resolving) pH gradients, and (iv) electrophoretic analyte transport along the secondary (harvesting) pH gradients. The operation of T-RECS has been quantitatively characterized via separation of small molecule ampholytes.