Isoelectric Focusing Of Proteins Research Articles

Streamlined integrated protein isoelectric focusing using microfluidic paper-based device

An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device.

A novel reagent significantly improved assay robustness in imaged capillary isoelectric focusing

Imaged Capillary Isoelectric Focusing (icIEF) has been used as primary method for charge variants analysis of therapeutic antibodies and proteins [1], [9]. Proteins tend to precipitate around their pI values during focusing [14], which directly affects the reproducibility of their charge profiles. Protein concentration, focusing time and various supplementing additives are key parameters to minimize the protein precipitation and aggregation. Urea and sucrose are common additives to reduce protein aggregation, solubilize proteins in sample matrix and therefore improve assay repeatability [15]. However some proteins and antibodies are exceptions, we found urea and sucrose are not sufficient for a typical fusion protein (Fusion protein A) in icIEF assay and high variability is observed. We report a novel reagent, formamide, significantly improved reproducibility of protein charge profiles. Our results show formamide is a good supplementary reagent to reduce aggregation and stabilize proteins in isoelectric focusing. We further confirmed the method robustness, linearity, accuracy and precision after introducing the new reagent; extremely tight pI values, significantly improved method precision and sample on-board stability are achieved by formamide. Formamide is also proven to be equally functional to multiple antibodies as urea, which makes it an extra tool in icIEF method development.

Effect of Joule heating on isoelectric focusing of proteins in a microchannel.

Electric field-driven separation and purification techniques, such as isoelectric focusing (IEF) and isotachophoresis, generate heat in the system that can affect the performance of the separation process. In this study, a new mathematical model is presented for IEF that considers the temperature rise due to Joule heating. We used the model to study focusing phenomena and separation performance in a microchannel. A finite volume-based numerical technique is developed to study temperature-dependent IEF. Numerical simulation for narrow range IEF (6 < pH < 10) is performed in a straight microchannel for 100 ampholytes and two model proteins: staphylococcal nuclease and pancreatic ribonuclease. Separation results of the two proteins are obtained with and without considering the temperature rise due to Joule heating in the system for a nominal electric field of 100 V/cm. For the no Joule heating case, constant properties are used, while for the Joule heating case, temperature-dependent titration curves and thermo-physical properties are used. Our numerical results show that the temperature change due to Joule heating has a significant impact on the final focusing points of proteins, which can lower the separation performance considerably. In the absence of advection and any active cooling mechanism, the temperature increase is the highest at the mid-section of a microchannel. We also found that the maximum temperature in the system is a strong function of the [Formula: see text] value of the carrier ampholytes. Simulation results are also obtained for different values of applied electric fields in order to find the optimum working range considering the simulation time and buffer temperature. Moreover, the model is extended to study IEF in a straight microchip where pH is formed by supplying H(+) and OH(-), and the thermal analysis shows that the heat generation is negligible in ion supplied IEF.

On‐chip protein isoelectric focusing using a photo‐immobilized pH gradient

On‐chip protein isoelectric focusing using a photo‐immobilized pH gradient

Isoelectric focusing in a silica nanofluidic channel: effects of electromigration and electroosmosis.

Isoelectric focusing of proteins in a silica nanofluidic channel filled with citric acid and disodium phosphate buffers is investigated via numerical simulation. Ions in the channel migrate in response to (i) the electric field acting on their charge and (ii) the bulk electroosmotic flow (which is directed toward the cathode). Proteins are focused near the low pH (anode) end when the electromigration effect is more significant and closer to the high pH (cathode) end when the electroosmotic effect dominates. We simulate the focusing behavior of Dylight labeled streptavidin (Dyl-Strep) proteins in the channel, using a relationship between the protein's charge and pH measured in a previous experiment. Protein focusing results compare well to previous experimental measurements. The effect of some key parameters, such as applied voltage, isoelectric point (pI), bulk pH, and bulk conductivity, on the protein trapping behavior in a nanofluidic channel is examined.

Protein composition and micelle size of donkey milk with different protein patterns: Effects on digestibility

Qualitative and quantitative analysis of the protein profile of milk samples from 14 Ragusano donkeys was carried out. Isoelectric focusing (IEF) analysis revealed three IEF protein patterns: individual variability was related both to casein (one deviating sample, without αS1-casein) and whey proteins (two deviating samples, without β-lactoglobulin II). The observed polymorphism reflected the different distribution of the protein fractions (casein or whey protein content) but did not affect the size and zeta-potential of casein micelles. A simulated in vitro digestion, carried out in a two-step digestion assay with human gastric juice and duodenal juice, was also performed to provide a direct estimation of donkey milk protein digestibility. Individual IEF variability had an effect on digestibility only in the two samples lacking the β-lactoglobulin II, revealing a more rapid degradation of the β-lactoglobulin fraction when it consists of the single β-lactoglobulin I, particularly in the gastric digestion.

Study on stability mechanism of immobilized pH gradient in isoelectric focusing via the Svensson–Tiselius differential equation and moving reaction boundary

An immobilized pH gradient (IPG) has strong power against instability (e.g., drifting and plateau) existing in classic isoelectric focusing (IEF). However, the relevant mechanism against the instability of pH gradient is still unclear. In this work, the theories of diffusional current and water products in IEF were developed based on the Svensson–Tiselius's differential equation and concept of moving reaction boundary (MRB). Two novel methods of pH gradient mobilization in IPG–IEF and non-IPG–IEF (opposite to IPG–IEF) were developed to unveil stability mechanism of IPG–IEF. The theoretical and experimental results indicated that (i) the drifting of pH gradient in non-IPG–IEF could be effectively controlled by IPG technique due to the existence of equal-fluxes of hydroxyl and hydrogen ions in the IPG–IEF system, (ii) there existed high diffusional current in non-IPG–IEF because of the existence of free carrier ampholyte (CA), but weak current in the IPG–IEF due to the immobilization of CA species in gel matrix, and (iii) the high diffusional current resulted in a great amount of water formation in neutral zone of pH gradient that led to distinct plateau in non-IPG–IEF, conversely the weak diffusional current caused little of water formation and weak plateau of pH gradient in IPG–IEF. These studies have considerable significance to the understanding of mechanism and development of protein IEF separation technique.

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.

HSV-1 Cgal+ Infection Promotes Quaking RNA Binding Protein Production and Induces Nuclear-Cytoplasmic Shuttling of Quaking I-5 Isoform in Human Hepatoma Cells

Herpesvirus type 1 (HSV-1) based oncolytic vectors arise as a promising therapeutic alternative for neoplastic diseases including hepatocellular carcinoma. However, the mechanisms mediating the host cell response to such treatments are not completely known. It is well established that HSV-1 infection induces functional and structural alterations in the nucleus of the host cell. In the present work, we have used gel-based and shotgun proteomic strategies to elucidate the signaling pathways impaired in the nucleus of human hepatoma cells (Huh7) upon HSV-1 Cgal(+) infection. Both approaches allowed the identification of differential proteins suggesting impairment of cell functions involved in many aspects of host-virus interaction such as transcription regulation, mRNA processing, and mRNA splicing. Based on our proteomic data and additional functional studies, cellular protein quaking content (QKI) increases 4 hours postinfection (hpi), when viral immediate-early genes such as ICP4 and ICP27 could be also detected. Depletion of QKI expression by small interfering RNA results in reduction of viral immediate-early protein levels, subsequent decrease in early and late viral protein content, and a reduction in the viral yield indicating that QKI directly interferes with viral replication. In particular, HSV-1 Cgal(+) induces a transient increase in quaking I-5 isoform (QKI-5) levels, in parallel with an enhancement of p27(Kip1) protein content. Moreover, immunofluorescence microscopy showed an early nuclear redistribution of QKI-5, shuttling from the nucleus to the cytosol and colocalizing with nectin-1 in cell to cell contact regions at 16-24 hpi. This evidence sheds new light on mechanisms mediating hepatoma cell response to HSV-1 vectors highlighting QKI as a central molecular mediator.

Basic RNases of wild almond ( Prunus webbii): Cloning and characterization of six new S-RNase and one “non- S RNase” genes

In order to investigate the S-RNase allele structure of a Prunus webbii population from the Montenegrin region of the Balkans, we analyzed 10 Prunus webbii accessions. We detected 10 different S-RNase allelic variants and obtained the nucleotide sequences for six S-RNases. The BLAST analysis showed that these six sequences were new Prunus webbii S-RNase alleles. It also revealed that one of sequenced alleles, S(9)-RNase, coded for an amino acid sequence identical to that for Prunus dulcis S(14)-RNase, except for a single conservative amino acid replacement in the signal peptide region. Another, S(3)-RNase, was shown to differ by only three amino acid residues from Prunus salicina Se-RNase. The allele S(7)-RNase was found to be inactive by stylar protein isoelectric focusing followed by RNase-specific staining, but the reason for the inactivity was not at the coding sequence level. Further, in five of the 10 analyzed accessions, we detected the presence of one active basic RNase (marked PW(1)) that did not amplify with S-RNase-specific DNA primers. However, it was amplified with primers designed from the PA1 RNase nucleotide sequence (basic "non-S RNase" of Prunus avium) and the obtained sequence showed high homology (80%) with the PA1 allele. Although homologs of PA1 "non-S RNases" have been reported in four other Prunus species, this is the first recorded homolog in Prunus webbii. The evolutionary implications of the data are discussed.

Cover Picture: Electrophoresis 14/2008

AbstractIssue 14 is a regular issue including an Emphasis Section offering a series of 9 papers on „Microfluidics and Miniaturization”︁. These 9 research papers report on various topics including studying single DNA molecules, selective release of intracellular molecules on the single cell level, isoelectric focusing of proteins in an ordered micropillar array, sample stream focusing in a microchip, integrated microfluidic system for sensing infectious viral disease, EOF in annulus and rectangular channels, confinement effects on monolith morphology, accumulation and filtering of nanoparticles in microchannels, and carbon nanotubes disposable detectors.

Dynamic coating for protein separation in cyclic olefin copolymer microfluidic devices

We report our study on using hydroxyethyl cellulose (HEC) as a dynamic coating for protein separation in microfluidic devices made from cyclic olefin copolymer (COC). The coating significantly enhances hydrophilicity of COC surface, evident from the decrease in contact angle of water in a COC channel. Surface treatment of COC channels with HEC also results in a 72% drop in electroosmotic (EO) mobility and a significant reduction in protein adsorption on the channel wall. Using bovine serum albumin as a model protein, the number of theoretical plates of 1.1 × 104 was achieved in a separation distance of 3.3 cm using free solution electrophoresis. Hydroxyethyl cellulose dynamic coating is also found to have an effect on isoelectric focusing (IEF) of proteins. It not only prevents proteins from adsorption, but also reduces EO flow, both of which help achieve IEF of proteins with a difference of 0.1 pH values in isoelectric points (pI).

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: IEF of Proteins in an IEF-Dedicated Electrophoresis Unit

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels, providing the first dimension of the 2D separation. In this protocol, the IPG gels are focused using self-contained instruments for IEF. These high-voltage systems allow fewer manipulations of the IPG gels, resulting in less error, strip mix-up, contamination, air contact, or urea crystallization. Because rehydration and IEF can be performed consecutively within a single unit, these two steps can be performed unattended overnight. Finally, faster separations and sharper focusing are possible due to the higher voltage available in these instruments.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Isoelectric Focusing of Proteins in a Multipurpose Flatbed Electrophoresis Unit

INTRODUCTIONThis protocol describes a method for separating proteins based on their net charge using the technique of isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. This method serves as the first dimension of the 2D separation. The method described in this protocol utilizes a flatbed unit; however, self-contained instruments for IEF are also available.

Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Rehydration of IPG Strips for Isoelectric Focusing of Proteins

INTRODUCTIONThis protocol describes a method for rehydration of IPG gel strips in preparation for their use for isoelectric focusing (IEF) on immobilized pH gradient (IPG) gels. Following rehydration, IEF can be performed using either a flatbed unit or a self-contained instrument.

A Laser‐Induced Fluorescence Imaging System for Isoelectric Focusing*

This paper describes a laser‐induced fluorescence (LIF) imaging system for detecting proteins separated by isoelectric focusing (IEF). IEF was carried out in either microfabricated glass devices or in capillaries. Compared to the single‐point‐detection used in capillary IEF (CIEF), the imaging approach eliminates the need for mobilizing the focused proteins to the detector, thereby reducing proteins' de‐focusing during mobilization and improving separation resolution. The LIF imaging system consists of a laser; its diameter is first increased through a beam expander and subsequently the laser beam is focused into a line using a cylindrical lens. The resultant laser line is used to image an entire channel or capillary in which protein separation take place. The fluorescence emission is collected with a cooled, scientific grade charge‐coupled device (CCD) camera. The detection limit was determined using a series of concentrations of fluorescein solutions. The imaging system was demonstrated by isoelectric focusing of proteins, including Green Fluorescent Protein, B‐phycoerythrin, and R‐phycoerythrin. In addition, we can obtain temporal profiles of protein separation, providing useful information about the dynamic behavior of protein migration during IEF. *Portions of this work were presented in “Lab‐on‐a‐chip: platforms, devices, and applications” at the Optics East conference, Philadelphia, PA, Oct., 2004.

Comprehensive proteomics in yeast using chromatographic fractionation, gas phase fractionation, protein gel electrophoresis, and isoelectric focusing

We have investigated the use of a variety of different techniques to identify as many proteins as possible in a yeast lysate, with the aim of investigating the overlap and complementarity of data from different approaches. A standard lysate was prepared from log phase yeast (Saccharomyces cerevisiae). This was then subjected to analysis via five different approaches aimed at identifying as many proteins as possible using an ion trap mass spectrometer. The total number of non-redundant protein identifications from each experiment was: 524 proteins by 2-D (SCX/C18) nanoflow liquid chromatography-liquid chromatography tandem mass spectrometry (nanoLC-LC MS/MS (MudPIT)); 381 proteins by nanoLC-MS/MS with gas phase fractionation by mass range selection; 390 proteins by nanoLC-MS/MS with gas phase fractionation by ion abundance selection; 898 proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins, in-gel digestion, and nanoLC-MS/MS of gel slices; and 422 proteins by isoelectric focusing of proteins, in-gel digestion and nanoLC-MS/MS of gel slices. The total number of non-redundant protein identifications in the five experiments was 1204. Combining only the two best experiments, the SDS-PAGE gel slices and the Mudpit, produces 1024 proteins identified, more than 85% of the total. Clearly, combining a Mudpit analysis with an SDS-PAGE gel slice experiment gives the greatest amount of protein identification information from a limited amount of sample.

Rapid protein separations in ultra-short microchannels: microchip sodium dodecyl sulfate–polyacrylamide gel electrophoresis and isoelectric focusing

We have developed novel protein gel electrophoresis techniques, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and isoelectric focusing (IEF) in short microchannels (∼millimeters) that take less than a minute. A photopatterning technique was used to cast in situ crosslinked polyacrylamide gel in a microchannel to perform SDS–PAGE. A fluorescent protein marker sample (Mr range of 20 000–200 000) was separated in less than 30 s in less than 2 mm of channel length. Crosslinked polyacrylamide gel, patterned in channels using UV light, provides higher sieving power and sample stacking effect, therefore yielding faster and higher-resolution separation in a chip. IEF of proteins was also achieved in a microchannel, and several proteins were focussed within tens of seconds in mm-length channels. As resolution in IEF is independent of separation distance, focusing in ultra-short channels results in not only faster separation but also more concentrated bands potentially allowing detection of low-concentration species.

Rapid protein separations in ultra-short microchannels: microchip sodium dodecyl sulfate–polyacrylamide gel electrophoresis and isoelectric focusing

We have developed novel protein gel electrophoresis techniques, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and isoelectric focusing (IEF) in short microchannels (∼millimeters) that take less than a minute. A photopatterning technique was used to cast in situ crosslinked polyacrylamide gel in a microchannel to perform SDS–PAGE. A fluorescent protein marker sample ( M r range of 20 000–200 000) was separated in less than 30 s in less than 2 mm of channel length. Crosslinked polyacrylamide gel, patterned in channels using UV light, provides higher sieving power and sample stacking effect, therefore yielding faster and higher-resolution separation in a chip. IEF of proteins was also achieved in a microchannel, and several proteins were focussed within tens of seconds in mm-length channels. As resolution in IEF is independent of separation distance, focusing in ultra-short channels results in not only faster separation but also more concentrated bands potentially allowing detection of low-concentration species.

Metabolism of avenanthramide phytoalexins in oats.

Oat leaves produce phytoalexins, avenanthramides, in response to infection by pathogens or treatment with elicitors. The metabolism of avenanthramides was investigated using low molecular weight, partially deacetylated chitin as an elicitor. When oat leaf segments are floated on the elicitor solution, avenanthramides accumulate in the solution. The transfer of elicited oat leaves to solutions containing stable-isotope-labeled avenanthramides resulted in a rapid decrease in the labeled avenanthramides, suggesting the metabolism of avenanthramides. The rate of decrease was enhanced by elicitor treatment, and was dependent on the species of avenanthramides, with avenanthramide B decreasing most rapidly. The rates of biosynthesis and metabolism of avenanthramides A and B were measured using a model of isotope-labeling dynamics. Avenanthramide B was found to be more actively biosynthesized and metabolized than avenanthramide A. Radiolabeled avenanthramide B was incorporated into the cell wall fraction and 99% of incorporated activity was released by alkaline treatment. Gel filtration indicated that high-molecular-weight compounds derived from avenanthramide B were released by alkaline treatment. The decrease in stable-isotope-labeled avenanthramides was suppressed by catalase, salicylhydroxamic acid, and sodium ascorbate, suggesting the involvement of peroxidase in the metabolism. Consistent with this, peroxidase activity that accepts avenanthramide B as a substrate was induced in apoplastic fractions by elicitor treatment. The appearance of multiple basic isoperoxidases was observed by activity staining with 3-amino-9-ethylcarbazole coupled with isoelectric focusing of proteins from elicitor-treated leaves. These findings suggest that accumulated avenanthramides are further metabolized in apoplasts in oat leaves by inducible isoperoxidases.