A method for the two-dimensional electrophoresis of leaf proteins

Proteins extracted from green leaves of tobacco were subjected to analysis by two-dimensional electrophoresis. It was found that electrophoretic separations were unsatisfactory when leaf extracts were analyzed directly without prior extraction of pigments, phenols, and lipids by acetone treatment. The plant pigments and several phenolic compounds present in leaves presumably interacted with the proteins and created charge heterogeneity, streaking, and other artifacts. It was found that these problems could be overcome by treatment of leaf extracts with acetone followed by solubilization and electrophoresis of the acetone-treated proteins. Leaf extracts were prepared by grinding deribbed leaf disks in a buffer containing 5 mm potassium carbonate, 9.5 m urea, 0.5 dithiothreitol, 2% Nonidet P-40 detergent, 500 μg/ml l-lysine, and 2% Ampholines. The extracts, after centrifugation to remove cell debris and insoluble material, were treated several times with ice-cold acetone. The acetone-precipitated proteins were treated with nucleases, reprecipitated with cold acetone, and then resuspended in the above grinding buffer. The presence of l-lysine and Ampholines were required for good electrophoretic separations. The resuspended proteins were subjected to two-dimensional electrophoresis and proteins detected by staining and or fluorography. At least 300 distinct proteins could be recognized in radioactive samples. The method gives reproducible patterns even after repeated freezing and thawing of the samples.     

High-resolution two-dimensional electrophoresis of plant proteins

A technique for the analysis of plant proteins from seed, leaf, root, and coleoptile tissues by high resolution two-dimensional electrophoresis is described. This technique is based primarily on the procedure of P. O'Farrell (1975, J. Biol. Chem. 250, 4007-4021); however, a number of improvements and simplifications have been introduced. We have found that resolution of polypeptides from a range of plant tissues is improved if the concentrations of nonionic detergent and ampholytes used in the isoelectric focusing (IEF) step are increased to 4 and 5% (w/v), respectively. Further increase in the concentrations of these two components results in gels of decreased resolution and low mechanical strength. We have also found that substitution of n-octyl beta-D-glucopyranoside or 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate for Triton X-100 or Nonidet-P40 in the IEF dimension significantly increases the resolution of polypeptides in these gels. This technique also allows minor polypeptide differences between closely related cultivars of plants to be identified.     

A novel system for the two-dimensional electrophoresis of membrane proteins.

Membrane proteins were resolved in two dimensions by a novel technique that uses discontinuous electrophoresis in both directions. After electrophoresis in the first direction in chloral hydrate, the membrane proteins were further resolved by a novel system that used organic-base dodecyl sulphates to stack and then resolve them. This latter system has several advantages over conventional electrophoresis in sodium dodecyl sulphate, notably that it avoids the production of artifacts generated by other systems.     

Fast method for two-dimensional electrophoresis of proteins from biological samples

The method for two-dimensional gel electrophoresis of J. Klose and M. Feller [(1981) Electrophoresis 2, 12-24] has been simplified by reducing the thickness of the gels from 3.5 to 1.1 mm for isoelectric focusing gels and from 3.5 to 0.84 mm for sodium dodecyl sulfate slab gels. Thin gels need less reagents and smaller sample volumes. Cooling of the thin gels during electrophoresis is more effective, which allows the use of higher electric power. Therefore, less time is required for an electrophoretic run (approx 4 h). The resolution increases due to the smaller size of the spots. The time required for staining the gels is reduced from at least 3 days to about 1 h. The method has been tested with a protein sample from the filamentous fungus Fusarium solani.     

Triple-spot proteins in two-dimensional gel electrophoresis.

A triple-spot pattern of polypeptides occurring in two-dimensional gel electrophoresis of proteins is described. The presence of a mutant protein, Pc 1 Duarte, which results in a splitting of all three polypeptides, is evidence that they are produced by the same gene. This pattern is seen in about 1% of the proteins from a variety of sources. Typically, about 50% of the protein occurs as a single major spot, the remainder occurring as two polypeptides with an additional negative charge and slightly different molecular weight. The reproducibility of this pattern implies a functional significance which is presently unknown. The implication of this configuration for patterns seen by one-dimensional gel electrophoresis is discussed.     

Preparation of membrane proteins for analysis by two-dimensional gel electrophoresis

In order to separate hydrophobic membrane proteins, we have developed a novel two-dimensional electrophoresis system. For the iso-electric focusing, agarose was used as a supporting matrix and n-dodecyl-beta-D-maltopyranoside was used as a surfactant. In combination with a previously developed Tris/MES electrophoresis system in the second dimension, distinct spots were reproducibly detected from hydrophobic membrane proteins whose grand average hydropathicity (GRAVY) exceed 0.3. In contrast to the immobilized pH gradient system, c-type heme was also visualized in this system.     

A novel two-dimensional electrophoresis technique for the identification of intrinsically unstructured proteins

Intrinsically unstructured proteins (IUPs) lack a well defined three-dimensional structure under physiological conditions. They constitute a significant fraction of various proteomes, but only a handful of them have so far been identified. Here we report the development of a two-dimensional electrophoresis technique for their de novo recognition and characterization. This technique consists of the combination of native and 8 m urea electrophoresis of heat-treated proteins where IUPs are expected to run into the diagonal, whereas globular proteins either precipitate upon heat treatment or unfold and run off the diagonal in the second dimension. This behavior was born out by a collection of 10 known IUPs and four globular proteins. By running Escherichia coli and Saccharomyces cerevisiae extracts, several novel IUPs were also identified by mass spectrometric analysis of spots at or near the diagonal. By comparing this novel method to several other techniques, such as the PONDR(R) predictor, hydrophobicity-net charge plot, CD analysis, and gel filtration chromatography, it was shown to provide dependable global assessment of disorder even in dubious cases. Overall the reproducibility and ease of performance of this technique may promote the proteomic scale recognition and characterization of protein disorder.     

Micro-scale two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins

Summary A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 g of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours.     

A method for two-dimensional electrophoresis of proteins from green plant tissues

A method of extraction of proteins from green plant tissues for two-dimensional electrophoresis is presented. The method is demonstrated on barley and potato leaves and on spruce needles for use in isoelectric focusing as well as in nonequilibrium pH gradient electrophoresis. The following three-step procedure was used: (i) The tissue was ground in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenoloxidase and polyvinylpyrrolidone to bind phenolic compounds. (ii) The proteins were precipitated with acetone at -20 degrees C. (iii) The proteins were dialyzed. Protease activity was generally not a problem during this procedure.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis

Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore, the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. Efficient solubilization of proteins strictly depends on the chaotrope and detergent in the extraction buffer. Despite the large number of detergents that have been developed for the use in protein extraction and IEF, there is no single compound able to efficiently extract proteins from any source. Hence, optimization has to be performed for each type of sample. We tested several chaotrope/detergent combinations to achieve optimal solubilization and separation of proteins from Norway spruce [Picea abies (L.) H. Karst.] needles and European beech (Fagus sylvatica L.) leaves and roots. The same chaotrope mixture (7 M urea, 2 M thiourea) was found to be suitable for the extraction and separation of proteins from all samples. Nonetheless, the efficiency of the surfactants tested varied between samples so that optimal extraction and separation was achieved with different detergents or combination of detergents for each sample. The 2-DE separation of spruce needle proteins was optimal in a mixture of two zwitterionic detergents (2% CHAPS and 2% decyl dimethylammonio propanesulfonate). Beech proteins were best separated in buffers containing sugar-based detergents (2% n-octyl beta-D-glucopiranoside in the case of leaf samples and 2% dodecyl maltoside for the root samples). IEF was performed in buffers with the same composition as the extraction buffer except for the root proteins that were better focused in a buffer containing 2% CHAPS.     

Resolution of erythrocyte membrane proteins by two-dimensional electrophoresis.

A two-dimensional electrophoresis method has been developed which solubilizes erythrocyte membrane proteins, and which resolves the components of the band that migrates in detergent gels as if its molecular mass were 95,000 daltons. This method uses gel electrophoresis with sodium dodecyl sulfate in the first dimension and phenol, aqueous urea, and acetic acid in the second dimension. The 95,000 dalton band is known to contain several different membrane proteins, including those associated with anion transport, glucose transport, and (Na+,K+) transport. Two-dimensional electrophoresis resolved this band into one major spot and several minor ones. Pronase digestion of whole erythrocytes, followed by preparation of ghosts and two-dimensional electrophoresis, showed that only the major component of this band was digested by pronase.     

Solubilization of membrane proteins for two-dimensional gel electrophoresis: identification of sarcoplasmic reticulum membrane proteins

Solubilization of membrane proteins for two-dimensional electrophoresis (2DE) is very difficult. In this study, we report the use of 1,2-diheptanoyl-sn-glycero-3-phosphatdiyl choline (DHPC) as a detergent to solubilize integral membrane proteins for 2DE. Rat ventricular microsomal fractions enriched with sarco(endo)plasmic reticulum (SR) membrane proteins were used as a model system. Compatibility of DHPC with a high concentration of urea increases the solubility of proteins compared with sulphobetaines or ASB-14. Peptide mass analysis assisted in the identification of key SR membrane proteins including SR Ca(2+) ATPase and other membrane proteins, which have not previously been reported on 2DE. These results suggest that DHPC is a better detergent for solubilizing membrane proteins and may be useful in generating proteomic maps for most complex organelles including SR.     

Very-high-resolution two-dimensional gel electrophoresis of proteins using giant gels

An improved high-resolution two-dimensional gel system for separating complex protein mixtures is described that allows a threefold increase in the number of proteins detected. Like the original O'Farrell system, proteins are separated in the first dimension by isoelectric point and in the second dimension by size. The improved resolution results primarily from a 2.5-fold increase in the size of both dimensions. Although best resolution is obtained by application of <100 μg of protein containing >5 × 10⁶ cpm, as much as 150 μg of protein may be applied without appreciable loss of resolution. Useful separations may be made with up to 1.5 mg. By doubling the thickness of both dimensions, as much as 3 mg of protein can be separated into 300–400 separate peaks.     

Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDS-PAGE as two-dimensional gel electrophoresis

In the present study, an alternative procedure for two-dimensional (2D) electrophoretic analysis in proteomic investigation of the most represented basic muscle water-soluble proteins is suggested. Our method consists of Acetic acid-Urea-Triton polyacrylamide gel (AUT-PAGE) analysis in the first dimension and standard sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) in the second dimension. Although standard two-dimensional Immobilized pH Gradient-Sodium Dodecyl-Sulphate (2D IPG-SDS) gel electrophoresis has been successfully used to study these proteins, most of the water-soluble proteins are spread on the alkaline part of the 2D map and are poorly focused. Furthermore, the similarity in their molecular weights impairs resolution of the classical approach. The addition of Triton X-100, a non-ionic detergent, into the gel induces a differential electrophoretic mobility of proteins as a result of the formation of mixed micelles between the detergent and the hydrophobic moieties of polypeptides, separating basic proteins with a criterion similar to reversed phase chromatography based on their hydrophobicity. The acid pH induces positive net charges, increasing with the isoelectric point of proteins, thus allowing enhanced resolution in the separation. By using 2D AUT-PAGE/SDS electrophoresis approach to separate water-soluble proteins from fresh pork and from dry-cured products, we could spread proteins over a greater area, achieving a greater resolution than that obtained by IPG in the pH range 3-10 and 6-11. Sarcoplasmic proteins undergoing proteolysis during the ripening of products were identified by Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-ToF) mass spectrometry peptide mass fingerprinting in a easier and more effective way. Two-dimensional AUT-PAGE/SDS electrophoresis has allowed to simplify separation of sarcoplasmic protein mixtures making this technique suitable in the defining of quality of dry-cured pork products by immediate comparison of 2D maps to define the events occurring during their ripening and individuate candidate molecular markers of the characteristic proteolytic processes. Considering that, essentially, muscle endogenous enzymic activity, calpains and cathepsins, occur in the ripening process of dry-cured ham, whereas a combined action between endogenous and microbial enzymes takes place in the case of sausage ripening, these results provide deeper insight into the respective role of endogenous and microbial enzymes in performing proteolysis. Finally, image analysis and creation of data bank could be achieved to quickly identify and protect typical products.     

Human spermatogenic cell marker proteins detected by two-dimensional electrophoresis

Highly homogeneous populations of human pachytene spetmatocytes and round spermatids have been obtained from normal adult testis using unit gravity (STA-PUT) sedimentation. Contaminating Leydig cells have been removed by density centrifugation in discontinuous Percoll gradients to yield resultant germ cell purities of 90–95% for pachytene spermatocytes and 89–96% for round spermatids. The total cellular polypeptide composition of separated human germ cells has been analyzed by two-dimensional polyacrylamide gel electrophoresis to compare 1) human and mouse pachytene spermatocytes (species specificity), 2) samples of human spermatocytes obtained from different individuals (allo specificity), and 3) pachytene spermatocytes and round spermatids from the same patients (stage specificity). Mouse and human germ cells have been found to exhibit extensive homology, but identified marker proteins limited to human spermatocytes include a group of polypeptides at p45/5.9 as well as a protein at p67/5.2. Proteins unique to mouse germ cells include component p65/5.5. Comparisons between different preparations of human pachytene spermatocytes have revealed about 90% electrophoretic homology, but some striking allotypic variations have been noted including the proteins at p45/5.9. Finally, presumptive stage-specific spermatogenic cell markers have been identified including the p45/5.9 polypeptides that are present only in human spermatocytes. Although the physiological roles of particular marker proteins have not yet been determined, the present findings indicate that purified spermatogenic cell populations may be analyzed biochemically to identify constituents important in the regulation of sperm development in man.     

A tracking marker for the first dimension of the two-dimensional gel electrophoresis of ribosomal proteins

Protein content of different subcellular fractions from chick brain is compared by using Lowry, TCA—Lowry and Bradford assay methods. Caution is urged in application of Bradford's method to general assay for protein concentration in subcellular fractions.     

Chromatofocusing fractionation and two-dimensional difference gel electrophoresis for low abundance serum proteins

The technical challenge to analysis of the serum proteome is that the serum proteins are present at unequal concentrations. A few are so dominant, such as serum albumin and immunoglobulins, that they mask detection of other proteins. Because of these high abundance proteins, current technologies, while theoretically capable of analyzing protein amounts spanning four orders of magnitude, are only able to analyze proteins ranging over two orders of magnitude and cannot analyze the lower abundance proteins that may be the next biomarkers and drug targets. To facilitate the identification of low abundance proteins, we fractionated serum samples from patients with prostate cancer and patients with benign prostate hyperplasia using anion displacement liquid chromatofocusing chromatography, which separates proteins by a pH gradient and a positively charged column. Differential expression of proteins from fractions was then determined and identified by IEF gels and 2-D DIGE. Results demonstrate improved resolution of proteins within the chosen pH gradient when compared to the unfractionated samples. Several proteins that were differentially expressed in serum from patients with prostate cancer were identified in the fractionated serum. Three of these proteins, squamous cell carcinoma antigen 1 (SCCA1), calgranulin B, and haptoglobin-related protein, are present in the serum at levels below the classical protein level of mg/mL. SCCA1 is normally expressed in serum at ng/mL levels, and calgranulin B is an intracellular protein. Our results demonstrate that the use of anion displacement liquid chromatofocusing chromatography may reduce the complexity of the serum proteome by separating proteins into distinct pH ranges, and facilitate the identification of low abundance proteins.