Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.     

A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ～10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.     

Combining microscale solution-phase isoelectric focusing with Multiplexed Proteomics dye staining to analyze protein post-translational modifications

Previously, a strategy for rapidly identifying mitochondrial phosphoproteins was presented that involves prefractionating multisubunit complexes by sucrose gradient centrifugation, followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and selective staining of phosphoproteins and total protein with fluorescent dyes [1]. Though suitable for evaluating the mitochondrial proteome, which consists of numerous multisubunit complexes, the strategy is not generally applicable to other complex proteomes. We determined that prefractionating samples by solution-phase isoelectric focusing is an effective alternative to sucrose-gradient fractionation that can be applied equally well to the analysis of mitochondrial and plasma proteins. Fluorescence-based multiplexing dye technologies greatly extend the capacity of SDS-polyacrylamide gel electrophoresis with respect to the investigation of proteome-wide changes in protein expression and post-translational modification, such as phosphorylation and glycosylation [2]. Overall, the prefractionation/Multiplexed Proteomics staining technology permits rapid, higher throughput screening of specimens for the identification of potentially interesting fractions that can subsequently be evaluated more thoroughly by two-dimensional gel electrophoresis.     

Separation of the unique proteins of wheat protein fractions by capillary electrophoresis

Summary The wheat maturation process was monitored by high-performance capillary electrophoresis. The different protein components of the albumin, globulin, gliadin and glutenin fractions from the Osborne extraction procedure were analysed. The wheat sample was a Hungarian winter wheat, cultivar Martonvásári 23. The protein fractions were analysed by capillary zone electrophoresis using a low pH phosphate buffer containing a polymeric additive and organic modifiers. The albumins and gliadins as well as glutenins showed a characteristic pattern of development during the maturation process. For these fractions the development occurred at different stages of maturation. The formation of protein fractions of wheat at different stages of maturation—and thus the entire maturation process—could be well characterised by high-performance capillary electrophoresis. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September, 1–3, 1999     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage-like network obstructs an in-depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP-dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.     

Nonlinear electrophoresis of protein-detergent complexes

A comparatively new procedure is described for the nonlinear electrophoresis of proteins. Movement and separation of complexes formed by proteins and ionic detergents is first experimentally demonstrated for SDS rainbow colored protein molecular weight markers (Amersham). This result was revealed by SDS-PAGE in an asymmetric zero average pulsed electric field with a peak amplitude of up to 300 V cm(-1) and a frequency of 100 Hz. The highest molecular weight fractions were found to have the highest nonlinear drift velocity. A two-dimensional map of distribution of the protein complexes developed using nonlinear electrophoresis followed by SDS gel electrophoresis in an orthogonal direction, reveals nonuniform distribution of the fractions. Nonlinear electrophoresis can be run without electrode chambers, since the buffer electrolyte is not used up in alternating electric fields. Thus, this new type of electrophoresis can have advantages in microfluidic systems and biochips. Also possible uses are discussed of nonlinear electrophoresis via nonlinear focusing of protein-detergent complexes for further improvement of the SDS-PAGE technique for the separation and examination of these large hydrophobic complexes.     

Immune complexes in blood: a new strategy for the analysis of their antigen portion.

A method is described for the characterization of protein antigens from circulating immune complexes from plasma. Free immunoglobulins G were separated from larger immune complexes by gel filtration with a fast protein liquid chromatographic system. The collected immune complexes were dissociated with 4M urea into antigens and antibodies. With a second column run with 4M urea, antigens smaller than 120 kDa were separated from unloaded antibody fractions. After concentration, they were analyzed by two-dimensional gel electrophoresis.     

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.     

Gel electrophoresis as a means of detecting ternary complex formation of thymidylate synthetase

Polyacrylamide gel electrophoresis was used to resolve as many as three protein components from incubation mixtures containing the inhibitor, 5-fluoro-2′-deoxyuridylate, the cofactor, 5,10-methylene tetrahydrofolate, and thymidylate synthetase. In a series of mixtures containing excess 5,10-methylenetetrahydrofolate and constant levels of thymidylate synthetase, the relative amounts of the protein components were shown to be dependent on the concentration of the inhibitor. Evidence is presented which suggests that the three protein components correspond to (1) native enzyme, (2) an inhibitor-cofactor-enzyme complex in a 1:1:1 molar ratio, and (3) an inhibitorcofactorenzyme complex in a 2:2:1 molar ratio, respectively. Ternary complexes of thymidylate synthetase are stable to gel filtration and are shown to undergo a relatively slow rate of breakdown on storage at 25 °C.     

Influence of one- and two-dimensional gel electrophoresis procedure on metal-protein bindings examined by electrospray ionization mass spectrometry, inductively coupled plasma mass spectrometry, and ultrafiltration

Three independent methods, (i) electrospray ionization mass spectrometry (ESI-MS), (ii) carrying out the complete protein preparation procedure required for protein gel electrophoresis (GE) including extraction, precipitation, washing, and desalting with subsequent microwave digestion of the produced protein fractions for metal content quantification, and (iii) ultrafiltration for separating protein-bound and unbound metal fractions, were employed to elucidate the influences of protein sample preparation and GE running conditions on metal-protein bindings. A treatment of the protein solution with acetone instead of trichloroacetic acid or ammonium sulfate for precipitate formation led to a strongly enhanced metal binding capacity. The desalting step of the resolubilized protein sample caused a metal loss between 10 and 35%. The omission of some extraction buffer additives led to a diminished metal binding capacity of protein fractions obtained from the sample preparation procedure for GE, whereas a tenside addition to the protein solution inhibited metal-protein bindings. The binding stoichiometry of Cu and Zn-protein complexes determined by ESI-MS was influenced by the type of the metal salt which was applied to the protein solution. A higher pH value of the sample solution promoted the metal ion complexation by the proteins. Ultrafiltration experiments revealed a higher Cu- and Zn-binding capacity of the model protein lysozyme in both resolubilization buffers for 1D- and 2D-GE compared to the protein extraction buffer. Strongly diminished metal binding capacities of lysozyme were recorded in the running buffer of 1D-GE and in the gel staining solutions.     

Characterization of Protein Complexes Containing Nucleoside Diphosphate Kinase with Characteristics of Light Signal Transduction through Phytochrome in Etiolated Pea Seedlings

Abstract— At 5 days after sowing of pea seeds in darkness, intact seedlings were either irradiated with red light for 40 s at 50 μmol/m²/s at the third internode or with red light as above and then with far-red light for 180 s at 0.4 μmol/m²/s, and the stems were sectioned from below the hook (mainly the third internodes) and placed in liquid N₂ in a mortar. The samples were well ground, and after the addition of extraction buffer, homogenates were centrifuged to prepare the crude membrane and soluble fractions. Red-light irradiation increased the phosphorylation of an 18 kDa protein, while far-red-light irradiation decreased it. The 18 kDa protein (formerly 15 kDa protein) was identified as nucleoside diphosphate kinase (EC 2.4.6) (NDP kinase) by western blotting using an NDP kinase-specific antibody. The membrane and the soluble fractions of the red-light-irradiated samples were separated by native polyacrylamide gel electrophoresis. The protein complexes prepared from the membrane and soluble fractions differed in their mobilities, as determined by two-dimensional electrophoresis and nonequilibrium pH gradient electrophoresis. The major protein spots from both samples were cut out from the gel and tested for NDP kinase and protein kinase activity. Both protein preparations showed NDP kinase activity and changes from nucleoside diphosphates and deoxynucleoside diphosphates to nucleoside triphosphates and deoxynucleoside triphosphates in the presence of [γ-³²P]ATP. Both preparations showed protein kinase phosphorylation of myelin basic protein (MBP) rather than histone H1 as protein substrates, suggesting that NDP kinase possesses a function similar to that of MAP kinase.     

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

Native Protein Separation by Isoelectric Focusing and Blue Gel Electrophoresis-Coupled Two-Dimensional Microfluidic Chip Electrophoresis

The use of microfluidic chip-based two-dimensional separation holds great promise in the proteomics field, given its portability, simplicity, speed, efficiency, and throughput. However, inclusion of sodium dodecyl sulfate, reported to be necessary for increasing protein-resolving capability, was also accompanied by the loss of both protein conformation and biological function. Here, we describe separation of native proteins by introducing blue native gel electrophoresis into isoelectric focusing and gel electrophoresis (IEF/CGE)-coupled protein two-dimensional microfluidic chip electrophoresis. After assessing the influence of various experimental conditions, the best separation ability and reproducibility of blue native IEF/CGE (IEF/BN-CGE) chip electrophoresis achieved until now were demonstrated no matter whether with a simple simulated mixture or with a complex mixture of total Escherichia coli proteins. Finally, instead of theoretical calculations, the image analysis technique was also used for the first time to quantitatively evaluate the actual peak capacities of chip electrophoresis. According to the number of features abstracted in the electrophoresis patterns, the superiority of the IEF/BN-CGE two-dimensional microfluidic chip electrophoresis was then exhibited quantitatively. The high native protein separation performance makes this established chip electrophoresis method possible for further application in widely needed drug screening, analysis of bio-molecular function, and assays of protein–protein interactions.     

Some pitfalls in PAGE-LA-ICP-MS for quantitative elemental speciation of dissolved organic matter and metalomics

An experimental approach to evaluate the capabilities and limitations of polyacrylamide gel electrophoresis (PAGE) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for quantitative elemental speciation is presented. Two metalloproteins (superoxide dismutase, containing Cu and Zn, and thyroglobulin, containing I) with high binding affinity for metals, and metal-dissolved organic matter (DOM) complexes (from a compost leachate sample) which show different types of metal binding are studied. Iodine can be quantitatively detected in thyroglobulin after PAGE-LA-ICP-MS using either sodium dodecyl sulfate (SDS) PAGE or native PAGE. However, detection of Cu and Zn in superoxide dismutase after PAGE-LA-ICP-MS depends on the conditions of the PAGE method because possible metal losses can occur (either with SDS-PAGE or with native PAGE). The use of PAGE-LA-ICP-MS to study the contribution of DOM to the mobilization of metals from environmental samples is possible, but it depends also on the PAGE separation conditions owing to disequilibrium effects of metal-DOM complexes. Presented at the 4th International Conference on Trace Element Speciation in Biomedical, Nutritional and Environmental Sciences, 25–29 May 2008, Munich-Neuherberg, Germany.     

Simplified method for concentration of mitochondrial membrane protein complexes

Extracting and concentrating mitochondrial protein complexes from gel strips after blue native PAGE (BN‐PAGE) can be daunting tasks using the traditional methods, such as electroelution, passive diffusion and centrifugal concentration. We present a simplified gel electrophoresis method to concentrate mitochondrial protein complexes with excellent recovery rate. Mitochondrial complex I present in a long gel strip from BN‐PAGE can be easily concentrated into a 0.8 cm gel strip when a second BN‐PAGE is performed with a Y‐shaped gel and the addition of 0.01% n‐dodecyl β‐D ‐maltoside and 0.001% SDS in the cathode buffer. Once completed, the concentrated protein complex in the gel strip is ready for SDS‐PAGE or proteomic studies.     

Global mapping of rat plasma proteins with a native proteomic approach using nondenaturing micro 2DE and quantitative LC‐MS/MS

Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB‐stained spots were numbered and subjected to in‐gel digestion and quantitative LC‐MS/MS. The analysis provided the assignment of 1–25 (average eight) non‐redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC‐MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well‐known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis.     

Gel electrophoresis/electroelution sorting fractionator combined with filter-aided sample preparation for deep proteomic analysis

Sample preparation and protein fractionation are important issues for proteomic studies. Protein extraction procedures strongly affect the performance of fractionation methods by provoking protein dispersion in several fractions. The most notable exception is the gel-based electrophoretic protein fractionation due to its resolution and effectiveness of sodium dodecyl sulfate as a solubilizing agent, while its main limitation lies in the poor recovery of the gel-trapped proteins. We created a fractionator device to separate complex mixture of proteins and peptides that is based on the continuous gel electrophoresis/electroelution sorting of these molecules. In an unsupervised process, complex mixtures of proteins or peptides are fractionated into the gel while separated fractions are simultaneously and sequentially electroeluted to the solution containing wells. The performance of the device was studied for protein fractionation in terms of reproducibility, protein recovery, and loading capacity. In a setup free of sodium dodecyl sulfate, complex peptide mixtures can also be fractionated. More than 11,700 proteins were identified in the whole-cell lysate of the CaSki cell line by using the fractionator combined with the filter-aided sample preparation method and mass spectrometry analysis. Fractionator-based proteome characterization increased 1.7-fold the number of identified proteins compared to the unfractionated sample analysis.     

Capillary electrophoretic and mass spectrometric analysis of a polydisperse fluorosurfactant

A fluorosurfactant has been studied using capillary electrophoresis and mass spectrometry. The fluorosurfactant, FC134, can be used as a buffer additive in capillary electrophoresis in order to decrease wall adsorption of proteins and in micellar electrokinetic chromatography. However, it has been discovered that this fluorosurfactant is polydisperse, thus containing substances with different lengths and structures. In this work, the fluorosurfactant sample components were separated by capillary electrophoresis. An uncoated as well as a poly(vinyl alcohol)-coated capillary were used with running electrolytes containing methanol and acetic acid. Following the capillary electrophoretic separation, fractions were collected for further analysis by MALDI-MS. Non-fractionated samples were also analyzed both by MALDI-MS and by ESI-MS.     

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.