Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

Two-dimensional gel analysis of soluble proteins. Charaterization of guinea pig exocrine pancreatic proteins.

A two-dimensional gel technique using slab gel isoelectric focusing in the first dimension and sodium dodecyl sulfate gradient gel electrophoresis in the second dimension has been developed for the separation of soluble proteins larger than 10,000 daltons. The technique is sensitive to 0.6 mug of protein and recovery of radiolabeled proteins averages 90%. Analysis of secretory protein from the guinea pig exocrine pancreas shows the presence of 19 distinct high molecular weight proteins. Each of these proteins has been characterized by isoelectric point, molecular weight, and proportionate mass. Thirteen of the 19 proteins have been identified by actual or potential enzymatic activity,accounting for 96% of the protein mass resolved by the two-dimensional gels.     

Characterization of the estrogen-induced uterine peroxidase by microelectrophoresis

Estrogen-dependent peroxidase from rat uterine fluid has been investigated by microelectrophoretic techniques. The molecular weight of the enzyme has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergent. The isoelectric points are located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, the enzyme was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000. The large subunit has slight enzymatic activiy, while the smaller subunit may be responsible for the charge difference in the holoenzyme pattern. The glycoprotein pattern of the uterine fluid peroxidase is further defined by its separation by affinity chromatography using a concanavalin A-Sepharose column and by its susceptibility to neuraminidase treatment.     

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.     

A multiple high-resolution mini two-dimensional polyacrylamide gel electrophoresis system: imaging two-dimensional gels using a cooled charge-coupled device after staining with silver or labeling with fluorophore.

A multiple mini two-dimensional electrophoretic method which results in three two-dimensional protein spot patterns being positioned side by side in an individual gel has been developed. Preparation time has been minimized by employing disposable capillary tubes for the isoelectric focusing gels and reducing the number of second-dimensional gels required. Commercially available vertical slab units were used for the second-dimensional electrophoresis. The protein spot patterns were visualized either by staining the second-dimensional gel with silver or fluorescently labeling the focused proteins while present in the isoelectric focusing gel and subsequently electrophoresing them into the second-dimensional gel. The fluorescently labeled second-dimensional gel was imaged while still present in the glass mold immediately following electrophoresis. Two fluorophores were compared: 2-methoxy-2,4-diphenyl-3(2H)-furanone and 5-(4,6-dichlorotriazin-2-yl)aminofluorescein hydrochloride. A rapid imaging system based on a cooled charge-coupled device was used to view both the silver-stained and fluorescently labeled two-dimensional spot patterns. The sensitivity of detection of protein spots in the mini two-dimensional gels was similar for the two types of fluorescently labeled gels and the silver-stained gels.     

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

Over two hundred polypeptides resolved from the human erythrocyte membrane

A modification of O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis has allowed for the resolution of erythrocyte membranes showing up to 200 individual components. Data is presented which indicates that this protein heterogeneity is not produced by artifactual protein-protein aggregation, ednogenous protease activity of secondary charge modification. Similar patterns are obtained when the samples are added to the unpolymerized isoelectric focusing gel, and isolated and stored in protease inhibitor. Individual spots could be eluted off of stained gels, resolubilized under extreme detergent solubilization conditions and run on one-dimensional gels; these run as sodium dodecyl sulfate in the solubilization procedure. The method chosen for solubilization prior to isoelectric focusing appears to cause selective aggregation of all or most of the spectrin and band 3 proteins. This further allows for excellent resolution of more components.     

Separation and identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels

A method is presented for the separation and detection of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase on isoelectric focusing gels and by immunoblotting. The gel staining procedure is a modification of a method used to demonstrate enzyme activity on blots after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis. The results show that immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase can be separated under equilibrium conditions on isoelectric focusing gels with an expanded alkaline pH range after solubilization in a mixture of nonionic/zwitterionic detergents and urea. Enzymatically active 2',3'-cyclic nucleotide 3'-phosphodiesterase focused as two closely spaced bands at pIapp 8.1 and 8.8, respectively, while 2',3'-cyclic nucleotide 3'-phosphodiesterase immunoreactivity was detected as four distinct bands at pIapp 4.2, 7.4, 8.8, and 9.3 and a diffuse band at pIapp 7.9-8.2. By two-dimensional separation these five bands showed molecular weights of about 43-47 kDa, i.e., corresponding to reported values for immunologically active 2',3'-cyclic nucleotide 3'-phosphodiesterase. Since enzyme activity is associated with only two of the bands, nonspecific and artifactual banding due to, e.g., detergent micelle formation, is unlikely.     

High resolution two-dimensional electrophoresis of basic as well as acidic proteins.

A previously described two-dimensional electrophoresis procedure (O'Farrell, 1975) combined isoelectric focusing and sodium dodecylsulfate slab gel electrophoresis to give high resolution of proteins with isoelectric points in the range of pH 4–7. This paper describes an alternate procedure for the first dimension which, unlike isoelectric focusing, resolves basic as well as acidic proteins. This method, referred to as nonequilibrium pH gradient electrophoresis (NEPHGE), involves a short time of electrophoresis toward the cathode and separates most proteins according to their isoelectric points. Ampholines of different pH ranges are used to optimize separation of proteins with different isoelectric points. The method is applied to the resolution of basic proteins with pH 7–10 Ampholines, and to the resolution of total cellular proteins with pH 3.5–10 Ampholines. Histones and ribosomal proteins can be readily resolved even though most have isoelectric points beyond the maximum pH attained in these gels. The separation obtained by NEPHGE with pH 3.5–10 Ampholines was compared to that obtained when isoelectric focusing was used in the first dimension. The protein spot size and resolution are similar (each method resolving more than 1000 proteins), but there is less resolution of acidic proteins in this NEPHGE gel due to compression of the pattern. On the other hand, NEPHGE gels extend the range of analysis to include the 15–30% of the proteins which are excluded from isoelectric focusing gels. The distribution of cell proteins according to isoelectric point and molecular weight for a procaryote (E. coli) was compared to that of a eucaryote (African green monkey kidney); the eucaryotic cell proteins are, on the average, larger and more basic.     

A nonurea electrophoretic gel system for resolution of polypeptides of Mr 2000 to Mr 200,000

A sodium dodecyl sulfate-polyacrylamide gel electrophoresis system which resolves proteins and peptides from Mr 2000 to Mr 200,000 is described. Gradients of polyacrylamide, crosslinker, and glycerol buffered in Tris-phosphate (pH 6.8) are employed. Neither urea nor a stacking gel is required. This system has been used to separate molecules below Mr 3000 which differed by only seven amino acid residues, yet has the capacity to survey masses up to Mr 200,000 on the same gel. Examples are given for separations of myoglobin cyanogen bromide fragments and adrenocorticotropin peptides. Utilizing the same gradient slab gel system in tandem with isoelectric focusing, a two-dimensional separation pattern of mammalian liver cell lysate is shown. A comparison of two different silver stain methods with this system is also given.     

A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension.

Two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension, initially applied for the separation of soluble and total cellular proteins, has been extended to the analysis of membrane proteins. We show that the usual procedures lead to artifacts and irreproducible results due to aggregation and precipitation of proteins and protein-phospholipid complexes during isoelectric focusing (first dimension) and sodium dodecyl sulfate (SDS) gel electrophoresis (second dimension). Optimized solubilization procedures for hydrophobic membrane proteins are presented and the use of dilute samples is shown to be essential to overcome the major problems in isoelectric focusing. Increased volumes of samples dissolved in rehydration buffer are applied by direct rehydration of dry immobilized pH gradient (IPG) gels. Isoelectric focusing in 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) without urea gives good results as does 2% Nonidet-P40 with 8 M urea. Heat denaturation should be avoided. An optimized equilibration procedure for IPG gel strips in SDS sample buffer prior to separation in the second dimension was developed that minimizes loss of proteins and results in high-resolution two-dimensional electropherographic maps with a minimum of streaking. The gel strips are partially dehydrated at 40 degrees C and shortly reswollen in situ on the SDS slab gel in SDS-sample buffer containing agarose.     

Preparative two-dimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients

A reproducible high-resolution protein separation method is the basis for a successful differential proteome analysis. Of the techniques currently available, two-dimensional gel electrophoresis is most widely used, because of its robustness under various experimental conditions. With the introduction of narrow range immobilized pH gradient (IPG) strips (also referred to as ultra-zoom gels) in the first dimension, the depth of analysis, i.e. the number of proteins that can be resolved, has increased substantially. However, for poorly understood reasons isoelectric focusing on ultra-zoom gels in the alkaline region above pH 7 has suffered from problems with resolution and reproducibility. To tackle these difficulties we have optimized the separation of semipreparative amounts of proteins on alkaline IPG strips by focusing on two important phenomena: counteracting water transport during isoelectric focusing and migration of dithiothreitol (DTT) in alkaline pH gradients. The first problem was alleviated by the addition of glycerol and isopropanol to the focusing medium, leading to a significant improvement in the resolution above pH 7. Even better results were obtained by the introduction of excess of the reducing agent DTT at the cathode. With these adaptations together with an optimized composition of the IPG strip, separation efficiency in the pH 6.2-8.2 range is now comparable to the widely used acidic ultra-zoom gels. We further demonstrated the usefulness of these modifications up to pH 9.5, although further improvements are still needed in that range. Thus, by extending the range covered by conventional ultra-zoom gels, the depth of analysis of two-dimensional gel electrophoresis can be significantly increased, underlining the importance of this method in differential proteomics.     

Analysis of cerebrospinal fluid proteins by electrophoresis

The cerebrospinal fluid (CSF) is a specific ultrafiltrate of plasma, which surrounds the brain and spinal cord. The study of its proteins and their alteration may yield useful information on several neurological diseases. By using various electrophoretic separation techniques, several CSF proteins have been identified derived from plasma or from brain. Different one-dimensional methods, such as agarose gel electrophoresis and isoelectric focusing, are of similar value in identifying the non-specific oligoclonal bands, which are mainly helpful in the diagnosis of multiple sclerosis and other inflammatory diseases. Isoelectric focusing has a greater resolution than other one-dimensional methods, and it yields additional data about disease-associated proteins occurring in Alzheimer's disease, Huntington's chorea and amyotrophic lateral sclerosis. Silver-stained two-dimensional gels provide more information about the complex protein composition of CSF, particularly about proteins produced in the brain, such as apolipoprotein E and neuron-specific enolase. For the detection of oligoclonal antibodies, the investigation of protein changes revealed by Parkinson's disease, schizophrenia and Creutzfeldt—Jakob disease, and the analysis of CSF immune complexes, two-dimensional electrophoresis has a greater sensitivity.     

Identification of Dictyostelium discoideum plasma membrane proteins by cell surface labeling and quantitative two-dimensional gel electrophoresis

Plasma membrane proteins of the cellular slime mold Dictyostelium discoideum were characterized by two-dimensional polyacrylamide gel electrophoresis using a variety of labeling techniques and a microcomputer-based videodensitometer. Algorithms for the determination of molecular weights and isoelectric points were developed to aid in the comparison of polypeptides from different autoradiographs, Coomassie blue-stained gels, and Western blots. Cell homogenates were compared to plasma membranes isolated by a silica density perturbation technique and to cytoskeletons obtained by nonionic detergent extraction. Plasma membrane proteins were distinguished from subcellular contaminants by lactoperoxidase-catalyzed radioiodination, by selective labeling with N-hydroxysuccinimidyl-2-iminobiotin, and by quantitatively determining the enrichments of individual polypeptides from gels of plasma membrane proteins relative to their counterparts in gels of total cell lysate proteins. In contrast to defining plasma membrane purity by measuring a representative marker enzyme activity, the quantitative two-dimensional gel analysis strategy presented allowed for a rigorous evaluation of the enrichments of all detectable polypeptides in the subcellular fraction. Quantitative two-dimensional gel analysis avoided problems encountered with marker enzyme activation or inhibition during subcellular fractionation as enrichments were based solely on polypeptide amounts. It was also capable of identifying a wider spectrum of plasma membrane proteins than any of the labeling techniques employed in this study. A high resolution two-dimensional gel catalog was generated containing information about plasma membrane protein orientation in the bilayer, association with the cytoskeleton, phosphorylation state, glycosylation state, copy number, isoelectric point, and molecular weight.     

A novel method for the identification and distinction of the beta-lactamases of the genus Acinetobacter

The characterization of the chromosomal beta-lactamases of Acinetobacter has proved difficult because of the poor focusing of these enzymes in conventional isoelectric focusing on polyacrylamide gels. We describe a novel isoelectric focusing method, which employs an agarose gel incorporating a detergent with sorbitol and urea, to examine the beta-lactamases produced by eight clinical strains of Acinetobacter calcoaceticus ; we have identified four different beta-lactamases. The molecular masses of each of the beta-lactamases was estimated and most of them ranged from 600000 to > 1000000. These are the largest beta-lactamases so far described and their size is likely to be one reason for their poor solubility in conventional polyacrylamide systems.     

A novel method for the identification and distinction of the beta-lactamases of the genus Acinetobacter

The characterization of the chromosomal beta-lactamases of Acinetobacter has proved difficult because of the poor focusing of these enzymes in conventional isoelectric focusing on polyacrylamide gels. We describe a novel isoelectric focusing method, which employs an agarose gel incorporating a detergent with sorbitol and urea, to examine the beta-lactamases produced by eight clinical strains of Acinetobacter calcoaceticus; we have identified four different beta-lactamases. The molecular masses of each of the beta-lactamases was estimated and most of them ranged from 600,000 to greater than 1,000,000. These are the largest beta-lactamases so far described and their size is likely to be one reason for their poor solubility in conventional polyacrylamide systems.     

Visualization of proteins after isoelectric focusing during two-dimensional gel electrophoresis.

A modification of P. H. O'Farrell's (1975, J. Biol. Chem.259, 4007–4021) two-dimensional gel electrophoresis is described. After isoelectric focusing, the cylindrical gels were fixed and stained with Coomassie brilliant blue R before sodium dodecyl sulfate-slab gel electrophoresis in the second dimension. The modification does not alter the protein patterns obtained, but provides sharper spots. In addition, bands are made visible before separation in the second dimension. Moreover, the modification helps to reduce the amount of ampholine in the dye front during electrophoresis.     

Solution isoelectric focusing for peptide analysis: comparative investigation of an insoluble nuclear protein fraction

In this study, a solution isoelectric focusing apparatus was modified and built into a two-dimensional separation method for peptides. Newly commercialized isoelectric membranes, which carry immobilized ampholytes, were integrated to establish the pH boundaries in this apparatus. High-performance liquid chromatography was employed as the second dimension, interfaced with mass spectrometry. An insoluble nuclear protein fraction was used to evaluate and optimize this method. This two-dimensional separation method dramatically improves peptide detection and identification compared with a single dimension LC-MS analysis. Off-line reversed-phase HPLC was used to ascertain reproducibility. The two-dimensional separation method was combined with (18)O labeling for comparative analysis of protein expression in two cell lines. Separation of peptides by solution isoelectric focusing (sIEF) offers the advantage that it can be accomplished after the (18)O labels are introduced. The labeled peptides can be mixed with unlabeled ones before fractionation by sIEF. The relative abundances of nuclear proteins from a drug resistant MCF-7 cancer cell line were compared to those from the drug susceptible parent cell line using this combined strategy. The abundances of several heterogeneous nuclear ribonucleoproteins were found to be increased in the mitoxantrone-resistant line.     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.