Miniaturized capillary isoelectric focusing in plastic microfluidic devices

We report the demonstration of miniaturized capillary isoelectric focusing (CIEF) in plastic microfluidic devices. Conventional CIEF technique was adapted to the microfluidic devices to separate proteins and to detect protein-protein interactions. Both acidic and basic proteins with isoelectric points (pI) ranging from 5.4 to 11.0 were rapidly focused, mobilized, and detected in a 1.2 cm long channel (50 microm deep x 120 microm wide) with a total analysis time of 150 s. In a device with a focusing distance of 4.7 cm, the separation efficiency for a basic protein, lysozyme, was achieved as high as 1.5 x 10(5) plates, corresponding to 3.2 million plates per meter. We also experimentally confirmed that IEF resolution is essentially independent of focusing length when the applied voltage is kept the same and within a range that it does not cause Joule heating. Further, we demonstrated the use of miniaturized CIEF to study the interactions between two pairs of proteins, immunoglobulin G (IgG) with protein G and anti-six histidine (anti-6xHis) with 6xHis-tagged green fluorescent protein (GFP). Using this approach, protein-protein interactions can be detected for as little as 50 fmol of protein. We believe miniaturized CIEF is useful for studying protein-protein interactions when there is a difference in pI between a protein-protein complex and its constitutent proteins.     

Sub-second isoelectric focusing in free flow using a microfluidic device

Using a microfabricated chip with a bed volume of 0.2 microL we demonstrate the validity of the scaling laws for molecular mass transport of isoelectric focusing (IEF) in free flow. Nano- or microlitre sample volumes can be concentrated within 430 ms by a factor of up to 400. These very fast performances make the chip applicable to proteomic analysis and for continuous monitoring of biochemical processes.     

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.     

Effects of increased voltage on resolution in preparative isoelectric focusing of myoglobin varia

IEF is a powerful technique which separates proteins and other amphoteric solutes in a pH gradient according to their pI's. The current work evaluates the effect on resolution of increasing electric fields in a novel preparative, vortex-stabilized electrophoresis device. In shallow gradients spanning one pH unit, the variants of myoglobin were separated at applied voltages from 10 to 15 kV. Digital imaging of these separations indicated a 20% reduction in bandwidth and a 60% increase in resolution as the electric field strength is varied across this range. These results were confirmed by IEF-PAGE and ion-exchange chromatography.     

Micropreparative isoelectric focusing protein separation in a suspended drop

IEF protein binary separations were performed in a 12-μL drop suspended between two palladium electrodes, using pH gradients created by electrolysis of simple buffers at low voltages (1.5-5 V). The dynamics of pH gradient formation and protein separation were investigated by computer simulation and experimentally via digital video microscope imaging in the presence and absence of pH indicator solution. Albumin, ferritin, myoglobin, and cytochrome c were used as model proteins. A drop containing 2.4 μg of each protein was applied, electrophoresed, and allowed to evaporate until it splits to produce two fractions that were recovered by rinsing the electrodes with a few microliters of buffer. Analysis by gel electrophoresis revealed that anode and cathode fractions were depleted from high pI and low pI proteins, respectively, whereas proteins with intermediate pI values were recovered in both fractions. Comparable data were obtained with diluted bovine serum that was fortified with myoglobin and cytochrome c.     

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.     

Protein fractionation in a multicompartment device using Off-Gel isoelectric focusing

A new protein fractionation technique based on off-gel isoelectric focusing (IEF) is presented, where the proteins are separated according to their isoelectric point (pI) in a multiwell device with the advantage to be directly recovered in solution for further analysis. The protein fractions obtained with this technique have then been characterized with polymer nanoelectrospray for mass spectrometry (MS) analyses or with Bioanalyzer for mass identification. This methodology shows the possibility of developing alternatives to the classical two-dimensional (2-D) gel electrophoresis. One species numerical simulation of the electric field distribution during off-gel separation is also presented in order to demonstrate the principle of the purification. Experiments with pI protein markers have been carried out in order to highlight the kinetics and the efficiency of the technique. Moreover, the resolution of the fractionation was shown to be 0.1 pH unit for the separation of beta-lactoglobulin A and B. In addition, the isoelectric fractionation of an Escherichia coli extract was performed in standard solubilization buffer to demonstrate the performances of the technique, notably for proteomics applications.     

Preparative isoelectric focusing in a cellulose‐based separation medium

An improved preparative method based on isoelectric focusing of analytes in a cellulose‐based separation medium is described in this study. Cellulose is suspended in an aqueous solution of simple buffers, ethylene glycol, glycerol, nonionic surfactant, and colored pI markers. Water partially evaporates during focusing run and the separation takes place in an in situ generated layer of cellulose, which has a gel‐like appearance at the end of analysis. Final positions of analytes are indicated by the positions of zones of focused pI markers. Fractions, segments of the separation medium with analytes, can be simply collected by spatula and analyzed by downstream analytical methods. Good focusing ability of the new method and almost quantitative recovery of model proteins, cytochrome c and bovine serum albumin, was verified by gel electrophoresis and capillary isoelectric focusing of the collected fractions.     

In situ photo-immobilised pH gradient isoelectric focusing and zone electrophoresis integrated two-dimensional microfluidic chip electrophoresis for protein separation

A method is introduced for open-column photo-induced site-selective immobilization of pH gradients in a layer of PEG-methacrylate in a multi-dimensional microfluidic chip for use in electrophoresis. It has several attractive features: (a) mixtures of fluorescently labelled proteins carbonic anhydrase, catalase and myoglobin in their native state can be separated by pH-gradient isoelectric focusing (IEF) and zone electrophoresis (CZE) using integrated 2D chip electrophoresis; (b) compared to strip packing or monolithic photo-immobilization, it overcomes the shortcomings of free carrier ampholyte-based 2D chip electrophoresis in an easy way; (c) larger amount of sample can be loaded into the open column-mode electrophoresis (d) immobilized pH gradients can be re-used and the chip can be recycled; (e) a multilayer 3D pH gradient is established by a layer-by-layer assembly technique to further increase the separation capacity. In our perception, this strategy has a large potential in microfluidic chip-based separation schemes because of its simplicity, separation power, re-usability, and separation capacity.

An open-column layer-by-layer photo-immobilised pH gradient was introduced into two-dimensional chip electrophoresis with simplicity, reusability, improved separation performance and separation capacity.

     

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.     

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.     

Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

In the presence of methanol and n‐decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3–10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M‐IPG). In addition, an online self‐built platform for protein separation was established on account of the introduction of a cross‐shaped unit and two short‐off valves. In this platform, a cross‐shaped unit was not only used to join the M‐IPG column and a six‐way injection valve (1.5 μL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short‐off valve in the tee unit or cross‐shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7‐proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M‐IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A_1c have been successfully resolved with the newly developed M‐IPG column.     

Evaluation of protein loading techniques and improved separation in OFFGEL isoelectric focusing

2-DE proved to be a key technology in protein science since the two orthogonal separation dimensions are capable of protein isoform separation. Recently, Agilent introduced the OFFGEL 3100 fractionator for in solution IEF (off-gel) of proteins with the help of a 12- or 24-well frame. With this instrument also conventional focusing in IPG strips after passive in-tray rehydration can be performed. In this study, two novel IEF applications using the OFFGEL electrophoresis were developed. First, a sample cup was built and a cup-loading method for the OFFGEL device was implemented. Applying proteins via cup resulted in higher reproducibility and less protein loss compared with conventional in-tray rehydration loading. Especially, the recovery of basic and high-molecular-mass proteins seems to be favored by cup loading. These effects are more pronounced with low microgram sample amounts. Second, a 48-well OFFGEL frame was developed, which doubles the resolution of the commercially available 24-well frame. It is capable of separating proteins with small pI differences and shows potential for isoform/PTM separation.     

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.     

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum-protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and post-separation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum-protein bond stability under the conditions studied. SDS-PAGE in the absence of β-mercaptoethanol or dithiotreitol preserved the platinum-protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3-2.0 μg were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated.     

Dynamic analyte introduction and focusing in plastic microfluidic devices for proteomic analysis

Isoelectric focusing (IEF) separations, in general, involve the use of the entire channel filled with a solution mixture containing protein/peptide analytes and carrier ampholytes for the creation of a pH gradient. Thus, the preparative capabilities of IEF are inherently greater than most microfluidics-based electrokinetic separation techniques. To further increase sample loading and therefore the concentrations of focused analytes, a dynamic approach, which is based on electrokinetic injection of proteins/peptides from solution reservoirs, is demonstrated in this study. The proteins/peptides continuously migrate into the plastic microchannel and encounter a pH gradient established by carrier ampholytes originally present in the channel for focusing and separation. Dynamic sample introduction and analyte focusing in plastic microfluidic devices can be directly controlled by various electrokinetic conditions, including the injection time and the applied electric field strength. Differences in the sample loading are contributed by electrokinetic injection bias and are affected by the individual analyte's electrophoretic mobility. Under the influence of 30 min electrokinetic injection at constant electric field strength of 500 V/cm, the sample loading is enhanced by approximately 10-100 fold in comparison with conventional IEF.     

Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing

We have elaborated a protocol for the fractionation of both hydrophilic and hydrophobic proteins using as a model the matrix and membrane compartments of highly purified rat liver peroxisomes because of their distinct proteomes and characteristic composition with a high quota of basic proteins. To keep highly hydrophobic proteins in solution, an urea/thiourea/detergent mixture, as used in traditional gel-based isoelectric focusing (IEF), was added to the electrophoresis buffer. Electrophoresis was conducted in the ProTeam free-flow electrophoresis (FFE) apparatus of TECAN separating proteins into 96 fractions on a pH 3-12 gradient. Consecutive sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated that both matrix and the integral membrane proteins of peroxisomes could be successfully fractionated and then identified by mass spectrometry. This is documented by the detection of PMP22, which is the most hydrophobic and basic protein of the peroxisomal membrane with a pI > 10. The identification of 96 prominent spots corresponding to polypeptides with different physical and chemical properties, e.g., the most abundant integral membrane polypeptides of peroxisomes and specific ones of the mitochondrial and microsomal membrane, reflects the fractionation potential of free-flow (FF)-IEF, accentuating its value in proteomic research as an alternative perhaps superior to gel-based IEF.     

Effects of ampholyte concentration on protein behavior in on-chip isoelectric focusing

The effects of mobility corrections on carrier ampholytes are studied at various ampholyte concentrations to understand protein behavior during IEF. IEF simulations are conducted in the presence of 25 biprotic carrier ampholytes within a pH range of 6-9 after applying the Onsager-Debye-Hückel correction to the carrier ampholytes. Two model proteins with ten charge states but without ionic strength corrections are allowed to focus under an electric field of 300 V/cm in a 1 cm long channel. The IEF simulation results show that higher ionic strengths (50 - 100 mM) cause significant changes in the transient movement as well as the final focused profiles of both ampholytes and proteins. The time required for a single, well-defined peak to form increases with ionic strength when Onsager corrections are applied to the carrier ampholytes. For a particular ampholyte concentration, the space-averaged conductivity does not change during the final focusing stage, but the magnitude of space averaged conductivity is different for different ampholyte concentration. The simulation results also reveal that at steady-state ionic strength profiles remain flat throughout the channel except at the locations of proteins where a significant change in ampholyte concentration is obtained.     

Free-flow zone electrophoresis and isoelectric focusing using a microfabricated glass device with ion permeable membranes

This paper describes a microfabricated free-flow electrophoresis device with integrated ion permeable membranes. In order to obtain continuous lanes of separated components an electrical field is applied perpendicular to the sample flow direction. This sample stream is sandwiched between two sheath flow streams, by hydrodynamic focusing. The separation chamber has two open side beds with inserted electrodes to allow ventilation of gas generated during electrolysis. To hydrodynamically isolate the separation compartment from the side electrodes, a photo-polymerizable monomer solution is exposed to UV light through a slit mask for in situ membrane formation. These so-called salt-bridges resist the pressure driven fluid, but allow ion transport to enable electrical connection. In earlier devices the same was achieved by using open side channel arrays. However, only a small fraction of the applied voltage was effectively utilized across the separation chamber during free-flow electrophoresis and free-flow isoelectric focusing. Furthermore, the spreading of the carrier ampholytes into the side channels resulted in a very restricted pH gradient inside the separation chamber. The chip presented here allows at least 10 times more efficient use of the applied potential and a nearly linear pH gradient from pH 3 to 10 during free-flow isoelectric focusing could be established. Furthermore, the application of hydrodynamic focusing in combination with free-flow electrophoresis can be used for guiding the separated components to specific chip outlets. As a demonstration, several standard fluorescent markers were separated and focused by free-flow zone electrophoresis and by free-flow isoelectric focusing employing a transversal voltage of up to 150 V across the separation chamber.