Electroelution of proteins from bands in gel electrophoresis without gel sectioning for the purpose of protein transfer into mass spectrometry: elements of a new procedure

Electroelution of protein bands from a gel has advantages over the competitive common technique requiring gel sectioning with respect to yield, speed and the potential for computer-controlled application to multicomponent two-dimensional (2-D) gels. The electroelution design for the commercial high-performance gel electrophoresis (HPGE) apparatus represented the most advanced technique to date until the recent discontinuation of its production. The present report serves to summarize the necessary design elements for the purpose of renewing and further developing the electroelution technique. A rudimentary technique is presented by which the electroeluate is collected in a glass tube superimposed on a reversibly stained gel band and connected to an anolyte reservoir. Although the stain used is insufficiently sensitive, the technique allowed for the qualitative verification of its usefulness in the transfer of the electroeluate into mass spectrometry.     

Electroelution without gel sectioning of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis: fluorescent detection, recovery, isoelectric focusing and matrix assisted laser desorption/ionization-time of flight of the electroeluate

A method of direct electroelution of intact proteins, without gel sectioning and orthogonal to the orientation of electrophoretic migration, was developed in application to Novex gels, using a simple home-made experimental setup. Six model proteins covering the molecular mass range of 14-120 kDa were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stained with an aqueous solution of the fluorescent dye, SYPRO-red, and electroeluted from the intact gel. The sensitivity of visual detection was 0.1-0.2 microg upon illumination by a green laser and 0.5-1 microg of protein on side-ways UV-illumination. Duration (for each protein) and field strength were optimized to render protein electroelution from the gel near-quantitative (above 80%) and relatively fast (1-12 min at 1 kV). At a given field strength, the optimal duration was found to be inversely proportional to the mobility of proteins in SDS-PAGE. Sequential ultrafiltration was evaluated as a simple approach to concentrate electroeluted proteins and deplete SDS to a level compatible with mass spectrometry of proteins: protein yields in the electroeluate were 25-33% (depending on the protein used) after three steps of ultrafiltration with water. The analysis of the electroeluate by isoelectric focusing in an immobilized pH gradient, to reveal protein heterogeneity under a single SDS-PAGE band (prior, e.g., to mass spectrometric analysis), was demonstrated.     

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies.

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.     

Isolation and recovery of acidic oligosaccharides from polyacrylamide gels by semi-dry electrotransfer.

Acidic oligosaccharides derived from glycosaminoglycan heparin were separated by polyacrylamide gradient gel electrophoresis (PAGE). The gel could be visualized using Alcian Blue dye to give a pattern of highly resolved, well defined bands. The particular banding pattern obtained was the result of a heparinase catalyzed depolymerization which afforded oligosaccharide products that differed in size by one disaccharide unit. The separated oligosaccharides could be recovered prior to staining by electroelution onto a positively charged nylon membrane by a semi-dry transfer procedure. Subsequent elution and quantitative recovery of individual oligosaccharides from the membrane was achieved. By using multiple membrane layers a second separation dimension was obtained, resulting in increased oligosaccharide purity proportional to transfer depth. Preparative gradient polyacrylamide gel electrophoresis followed by semi-dry electro-transfer and recovery represents a novel method for the preparation of homogeneous acidic oligosaccharides.     

Separation efficiency in protein zone electrophoresis performed in capillaries of different diameters

R-phycoerythrin (PHYCO, Mr 240 000), glucose-6-phosphate dehydrogenase (GPD, Mr 104 000) and two charge isomers of recombinant green fluorescent protein (GFP-1 and GFP-2, Mr 27 000) were subjected to capillary zone electrophoresis (CZE) in capillaries of 50, 100 and 150 microm inner diameter at various sample concentrations, electric field strengths, and lengths of the initial zone with the purpose of testing the hypothesis that protein - capillary wall interactions rather than thermal effects are predominantly responsible for the peak spreading of proteins in CZE. The efficiency of CZE was expressed in terms of the number of theoretical plates, N, or the plate height corrected by subtracting the contribution from initial zone length, H'. The latter has the advantage of solely reflecting contributions to the separation efficiency arising from intracolumn peak spreading in capillaries of different diameters. The separation efficiency measured varied widely, by two orders of magnitude, for these proteins under identical conditions, with GPD exhibiting the highest and PHYCO the lowest values of N. H' was found to be independent of sample concentrations within the concentration ranges studied, 1-100 microg/mL for PHYCO and 100-1000 microg/mL for GPD, while exhibiting a decrease with sample concentration for GFP, especially in 150 microm diameter capillaries, within the concentration range 1-100 microg/mL. H'was also found to be independent of electric field strength up to 300-400 V/cm for PHYCO and GFP. In all experiments, the CZE of proteins in 100 microm diameter capillaries provided a higher or, at least, equal efficiency, compared to that in 50 or 150 microm diameter capillaries. It may be concluded that the protein - capillary wall interactions and protein microheterogeneity are the dominant sources of peak spreading and their specific combinations are thought to be responsible for the wide variation in separation efficiency between proteins in CZE observed under identical conditions.     

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.     

Stacking of unlabeled sodium dodecyl sulfate-proteins within a fluorimetrically detected moving boundary, electroelution and mass spectrometric identification

The previously reported fluorimetric detection of sodium dodecyl sulfate (SDS)-protein in the presence of cascade blue in agarose gel electrophoresis using barbital buffer was found to be equally feasible in the absence of the fluorescent marker and using Tris-Tricinate buffer, provided that SDS was loaded with the sample but not contained in the catholyte. That fluorescent detection is thought to be due to the formation of a moving boundary between leading SDS and trailing barbital, or Tricinate buffer. This hypothesis is supported by the following evidence: (i) The fluorometrically detected band disappears with addition of SDS to the catholyte; (ii) band area is proportional to protein and/or SDS load; (iii) mobility of SDS-proteins differing in mass is the same at agarose concentrations up to 3%; (iv) lowering of protein mobility by increase in gel concentration and/or increase in the size of the SDS-protein leads to band disappearance. Fluorescent detection of the band is like to be nonspecific and due to the light scattering properties of a stack comprising moving boundaries of any analytes with net mobilities intermediate between SDS (or micellar SDS) and the trailing buffer constituent at their regulated very high concentrations. The steady-state stack of SDS-proteins in the size range of 14.4-45.0 kDa, and the transient stack of an SDS-protein of 66.2 kDa have lent themselves to electroelution and characterization by mass of the proteins after removal of SDS and buffer exchange using matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. The possibility to form a stack of protein between leading SDS and trailing buffer anions under conditions of weak molecular sieving (open-pore gel and small-sized protein) contributes to the understanding of moving boundaries in gel electrophoresis, but in view of the narrowly defined conditions, under which this stack forms, is of limited practical significance for the gel electrophoresis of SDS-proteins.     

Electroelution of nonfluorescent stacked proteins detected by fluorescence optics from gel electrophoretic bands for transfer into mass spectrometry

The extreme accuracy of spectrometrically determined masses of proteins has opened the possibility to identify proteins separated as gel electrophoretic bands in the absence of specific immunologic ways of identification. For the purpose of protein transfer from gel electrophoretic bands to mass spectrometer, electroelution from the intact gel has advantages, in particular when apparatus with capacity for fluorescent scanning allows one to direct the electroelution cell over the band under computer control. To avoid fluorescent labeling of the protein which is incompatible with mass spectrometric identification, it is proposed to selectively stack the unlabeled protein and detect it by comigrating tracking dye prior to electroelution. The feasibility of the approach is exemplified in case of a single protein, but still remains to be demonstrated in conjunction with the selective stacking or unstacking of a single protein from a mixture of several proteins.     

High-performance capillary electrophoresis of histones.

A high-performance capillary electrophoresis (HPCE) system was developed for the fractionation of histones. This system involves electroinjection of the sample and electrophoresis in 0.1 M phosphate buffer (pH 2.5) in a 35 cm x 50 micron I.D. coated capillary. Electrophoresis was accomplished in 9 min, separating a whole histone preparation into its components in the following order of decreasing mobility: (MHP) H3, H1 (major variant), H1 (minor variant), (LHP) H3, (MHP) H2A (major variant), (LHP) H2A, H4, H2B and (MHP) H2A (minor variant), where MHP is the more hydrophobic component and LHP is the less hydrophobic component. This order of separation is very different from that found in acid-urea polyacrylamide gel electrophoresis and in reversed-phase high-performance liquid chromatography and, thus, brings the histone biochemist a new dimension for the qualitative analysis of histone samples.     

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate-proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester

A gel electrophoresis apparatus capable of scanning the migration path fluorometrically and of computer-directed electroelution of bands was applied to the mass spectrometric identification of sequentially electroeluted 5(6)-carboxyfluorescein-N-hydrosuccinimide ester (FLUOS)-labeled sodium dodedyl sulfate (SDS)-proteins. The masses of four electroeluted SDS-proteins under study determined by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) spectrometry are changed by 1% due to their reaction with FLUOS in a 1:5 molar ratio of protein:label, allowing for the identification of the labeled intact proteins on the basis of mass. More importantly, the partial (10 or 50%) derivatization of proteins with FLUOS does not preclude their tryptic hydrolysis, and identification of the protein on the basis of the mass spectrometric analysis of its tryptic peptides. Potentially, the procedure allows for the automated mass spectrometric identification of SDS-proteins globally labeled with FLUOS and electrophoretically separated, without need for any gel sectioning.     

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.     

High resolution electrophoresis of soybean seed isoesterases

A modified discontinuous buffer system for vertical polyacrylamide electrophoresis was developed with large changes in component concentration from the original Tris-HCl/Tris-glycine system of the Ornstein-Davis type. The new buffer system is able to resolve the fast soybean seed isoesterases crucial for cultivar discrimination. A higher number of anodic bands is obtained in comparison with separations under standard conditions. A "gelating boundary", resulting from the aggregation of seed proteins, is retarded and does not migrate out of the upper stacking gel. The same buffer modification is also beneficial to the separation of native seed proteins.     

Online transient isotachophoresis concentration by the pseudo-terminating electrolyte buffer for the separation of DNA-aptamer and its thrombin complex in poly(methyl methacrylate) microchip

Online automatic transient isotachophoresis concentration of DNA-aptamer and its thrombin complex by using one kind of pseudo-terminating electrolyte buffer in a cross-channel poly(methyl methacrylate) microchip is reported. Sample injection, transient concentration and separation were done continuously and controlled by a sequential voltage switching program, time-consuming steps and complicated chip design were not required. Peak resolution between DNA-aptamer and its thrombin complex was influenced by this novel pseudo-terminating electrolyte buffer, which was prepared by the addition of chemical component with slow mobility into the same buffer as leading electrolyte buffer. 1100-fold signal enhancement of thrombin complex was achieved by this transient isotachophoresis on a standard cross-form microchip. The concentration effect or standing time of transient isotachophoresis was proved to be influenced by the concentration of leading electrolyte ion and the concentration of pseudo-terminating electrolyte buffer ion (glycine). The transient concentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution for the size-based separation. The detection limit, taken as the lowest thrombin concentration at threefold S/N, was determined to be 0.5 amol in mass by this method.     

Recovery of the contact site A glycoprotein of Dictyostelium discoideum from sodium dodecyl sulfate-polyacrylamide gel and characteristics of monoclonal antibodies against the recovered protein

Monoclonal antibodies were produced against a cell-cell adhesion (contact site A) glycoprotein of Dictyostelium discoideum, isolated by preparative gel electrophoresis. The glycoprotein was recovered by electroelution from a polyacrylamide gel strip and used for the production of monoclonal antibodies. Four of the five antibodies obtained bound specifically to the protein moiety of the contact site A glycoprotein. The specificities of the antibodies were in striking contrast to those of antibodies raised against the contact site A glycoprotein purified by Triton X-114 phase separation and DEAE chromatography. The majority of the latter antibodies recognized the carbohydrate moiety of the contact site A glycoprotein and cross-reacted heavily with other membrane glycoproteins.     

Modification of tricine–SDS–PAGE for online and offline analysis of phosphoproteins by ICP-MS

This study describes a modification to tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis to make it more effective for the separation of low molecular mass proteins and for coupling with inductively coupled plasma mass spectrometry (ICP-MS). The modified method employs low-percentage polyacrylamide gels (7–10%) (w/v) and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. Using phosphopeptides as a model system, and offline analysis, we obtained recoveries of 73% (w/v) in a 9% gel compared with 55% in a conventional 16% gel. Online coupling was achieved by modification of a standard commercially available gel electroelution apparatus and casting of the gel into a 7.3-cm-long tube. Online separation of a digest of β-casein was demonstrated with recovery of the mono- and tetraphosphopeptides, which were identified by comparison with peptide standards. A mass balance study with the standards yielded recoveries of 95% for tetraphosphopeptides and 48% for monophosphopeptides. The factors affecting the separations and recoveries are discussed in detail. The detection limits for 10-μL samples of the mono- and tetraphosphopeptides were 0.7 μM (7 pmol) and 0.2 μM (2 pmol) respectively.     

Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride

The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95 degrees C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84+/-0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94+/-0.60) was half that observed in phosphate buffer (pH 6.08+/-0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80 degrees C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65+/-0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80 degrees C. GFP pH- and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.     

Preparative separation of poliovirus structural polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, copper staining and electroelution, and induction of monospecific antisera

Viral polypeptides were prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by copper staining and electroelution from gel slices. Poliovirus capsid polypeptide VP 1 isolated by this procedure induced monospecific antibodies in rabbits, i.e., antisera reacting only with the homologous polypeptide. Our results demonstrate the applicability of the described copper staining method as a rapid visualization step for preparing viral proteins after SDS-PAGE.     

Electrophoretic immunodesorption of proteins according to molecular weight by use of a double-membrane system

Summary A simple method is described for electrophoretic desorption of proteins from antigen-antibody complexes, with more than 90% recovery and without denaturation, after immunosorbent affinity chromatography. Radiolabeled or unlabeled human serum albumin (HSA) and α-1-antitrypsin (AAT), conjugated to rabbit anti-HSA or anti-AAT polyclonal antisera, respectively, were electrophoretically desorbed from Sepharose 4B. In addition, purification and concentration of the major HSA protein band (monomer) of 68 kD from the other oligomeric protein bands were achieved by use of a two-membrane system in a simple electroelution apparatus. The system consisted of an upper cellulose acetate membrane, with pore size 20 nm and separation limit 70 kD, and a lower dialysis cellophane membrane with molecular weight cut-off from 1–50 kD that cnables separation according to size. Furthermore, purification of the monomer HSA or AAT from normal human serum was performed with 92% recovery. Homogeneity was implied by the presence of one band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, Western blot, and autoradiography.     

An improved sodium dodecyl sulfate-polyacrylamide gel electrophoresis system for the analysis of membrane protein complexes

Membrane protein complexes such as the reaction center complexes of oxygenic photosynthesis or the complex I of mitochondira are composed of many subunit polypeptides. To analyze their polypeptide compositions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a wide range of molecular sizes has to be resolved, especially in the low molecular mass range. We have improved the traditional Tris/HCI buffer systems adopting a Tris/2-(N-morpholino)ethanesulfonic acid (MES) buffer system containing 6 M urea. This gel system was used with an 18-24% acrylamide gradient for the separation of polypeptides with molecular masses from below 5 kDa to over 100 kDa. This buffer system can also be applied to the usual uniform concentration of acrylamide gel and also to minislab gels.     

Prevention of artifactual protein oxidation generated during sodium dodecyl sulfate-gel electrophoresis

Prevention of artifactual protein oxidation occurring during sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis is critical for identifying physiological protein oxidation implicated in human diseases due to the routine use of gel electrophoresis to separate the multiple proteins in proteomic studies. To develop a methodology that completely prevents artifactual protein oxidation in SDS acrylamide gel electrophoresis, cytochrome c was electrophoresed on polyacrylamide gels and subjected to trypsin in-gel digestion followed by tryptic peptide analysis by mass spectrometry. It was found that degassing the acrylamide solution to remove molecular oxygen prior to gel polymerization is a crucial process to protect the electrophoresed protein from reactive oxygen species generated during electrophoresis. However, significant artifactual protein oxidation remains that can only be eliminated entirely, if proteins are electrophoresed on an SDS gel photopolymerized with flavin as the photoinitiator and thioglycolate included in the cathode buffer as a reactive oxygen species scavenger. Using this combination of methodologies, cytochrome c isolated from adult rat heart mitochondria was purified and digested followed by mass spectrometric analysis, demonstrating the requisite high resolution of the polyacrylamide gel and the entire elimination of artifactual oxidation.