Rapid imaging, using a cooled charge-coupled-device, of fluorescent two-dimensional polyacrylamide gels produced by labelling proteins in the first-dimensional isoelectric focusing gel with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone

A new method for visualising proteins in two-dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) while present in the first-dimensional gel after isoelectric focusing and subsequently electrophoresed into the second-dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge-coupled-device was used to view the two-dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two-dimensional gels at the end of electrophoresis with no further processing.     

中国小型猪心肌梗死模型的比较蛋白质组学研究

利用二维电泳(2DE)分离中国小型猪心肌梗死模型的正常与梗死心肌组织的蛋白提取液, 采用 PDQuest 软件对比分析了两种心肌组织在pH=5─8范围内的2DE谱图. 正常心肌组织检出851个蛋白点, 梗死组织检出1 032个蛋白点. 发现13个蛋白质点只在小型猪的正常心肌组织中表达, 而有14个蛋白质点只在梗死心肌组织中表达. 另外, 还有49个蛋白点在两种组织中表达量上有显著性变化(P<0.05), 选择进行质谱分析其中11个蛋白点, 成功地鉴定出7种蛋白, 蛋白功能分析结果表明, 这些蛋白的差异表达与心肌梗死过程相关.     

Selective purification of cardiac myosin by a high-performance salicylate affinity column

The cardiac muscle proteins, myosin and actin, were purified in one step using a salicylate-silica affinity column. The affinity columns were prepared by coupling sodium salicylate via its hydroxyl group to an Altex Ultraffinity-EP column. Crude detergent extracts from guinea pig hearts were passed through the column and the myosin-actin complex was then eluted with excess free salicylate or high salt. The affinity of cardiac myosin for immobilized salicylate was unique as myosin heavy chain from guinea pig leg muscle detergent extracts could not be purified by this procedure. Commercially purified rabbit leg muscle myosin also appeared to have no interaction with the salicylate affinity column, suggesting that the column is specific for cardiac myosin.     

A short history of electrophoretic methods

High resolution separation of proteins, based on charge differences, is possible with disc electrophoresis, displacement electrophoresis (isotachophoresis) and notably isoelectric focusing (IEF). Size separation is obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The combination of gel IEF, followed by SDS-PAGE in a second-dimensional slab gel, i.e. two-dimensional gel electrophoresis, affords the highest resolution with up to several thousand spots per gel. Staining of proteins gives high resolution patterns which can be scanned and stored in comprehensive databases. Over the last 10 years the electrophoretic separation in gels and subsequent visualization of nucleic acids (DNA, RNA) and even genes as well as nucleotides have been much improved, making possible efficient mapping of the genes in humans and all other organisms. This has led to the biggest concerted endeavor in the history of science, i.e. the mapping of the human genome, which will be of importance as long as mankind exists. In the last years electrophoresis in capillaries has attracted much interest because for numerous substances, such as proteins nucleic acids, pharmaceuticals, metabolites, and peptides, it offers high resolution on the analytical scale with over 1 million theoretical plates. Electrophoretic methods have unprecedented impact on life sciences, providing a basis for unique advances in biochemistry, molecular biology, genetics, gene technology and medicine.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

A high resolution two-dimensional gel electrophoresis and silver staining protocol demonstrated with nuclear matrix proteins.

An improved two-dimensional gel electrophoresis procedure has been developed utilizing isolated nuclear matrix proteins. The proteins of the cellular nuclear matrix are tissue specific. They are an example of a protein set whose two-dimensional electrophoretic patterns afford much information of clinical significance. However, current two-dimensional gel techniques were not completely satisfactory for the small amounts of protein present in tissue samples. There was a need for a two-dimensional gel procedure which was capable of increased sensitivity and resolution and at the same time was reliable and reproducible. This has been accomplished by implementing several modifications to the current two-dimensional gel procedures. In addition, changes were introduced in the silver staining process of the gels to increase the signal to background ratio. The overall procedure affects a dramatic increase in the resolution and clarity of the proteins visualized on two-dimensional gels and is no more laborious than current techniques.     

Two-dimensional map of the proteome of Haemophilus influenzae

We have constructed a two-dimensional database of the proteome of Haemophilus influenzae, a bacterium of medical interest of which the complete genome, comprising about 1742 open reading frames, has been sequenced. The soluble protein fraction of the microorganism was analyzed by two-dimensional electrophoresis, using immobilized pH gradient strips of various pH regions, gels with different acrylamide concentrations and buffers with different trailing ions. In order to visualize low-copy-number gene products, we employed a series of protein extraction and sample application approaches and several chromatographic steps, including heparin chromatography, chromatofocusing and hydrophobic interaction chromatography. We have also analyzed the cell envelope-bound protein fraction using either immobilized pH gradient strips or a two-detergent system with a cationic detergent in the first and an anionic detergent in the second-dimensional separation. Different proteins (502) were identified by matrix-assisted laser desorption/ionization mass spectrometry and amino acid composition analysis. This is at present one of the largest two-dimensional proteome databases.     

Electrophoretic variants of cardiac myosin heavy chain-alpha in Sprague Dawley rats

Analysis of cardiac myosin revealed differences in gel electrophoretic migration patterns of the alpha-isoform of myosin heavy chain, but not the beta-isoform, in Sprague Dawley rats. No differences in the migration patterns of the alpha-or beta-isoforms were observed in other rat strains. Three electrophoretic migration patterns of the alpha-isoforms were observed in individual rats: a slower migrating isoform alone (4% of all rats tested), a faster migrating isoform alone (55%), and both isoforms (41%). The isoform expression pattern was identical in all myocardial regions in each rat. Frequency of expression patterns suggests multiple gene sequences for alpha-cardiac myosin heavy chain in Sprague Dawley rats. Sequence analysis of amplified regions of the Sprague Dawley and Brown Norway rat alpha-myosin genes, specifically the 5'-untranslated region, exons 1-3, and associated introns, showed numerous single nucleotide polymorphisms in coding and noncoding regions, including putative regulatory sites in Sprague Dawley rats, but not in Brown Norway rats. All Sprague Dawley rats varied from Brown Norway rats and no heterogeneity was observed in Brown Norway rats. Several deletions and dimorphic positions were also observed. Dimorphic positions were evident on automated sequencing comparisons. The data indicate that at least two alpha-myosin heavy chain isoforms exist in Sprague Dawley rats and these rats exhibit sequence diversity within that portion of the alpha-myosin heavy chain gene reported in this study.     

Approach to stationary two-dimensional pattern: influence of focusing time and immobiline/carrier ampholytes concentrations

Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (G?rg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.     

Peptide mapping of polypeptides separated by two-dimensional electrophoresis: protease digestion directly on the two-dimensional gel followed by electrophoresis in reverse direction

A method is described which allows to reveal simultaneously the proteolytic patterns of numerous polypeptides separated by two-dimensional electrophoresis. After two-dimensional electrophoresis, the gels were dipped successively in buffers for preequilibration, protease digestion, and reequilibration. They were then returned to the electrophoresis tank, and electrophoresis was continued for a short time. After silver staining, digestion products appeared, lined up behind the original polypeptide spots. The method allows proteolytic patterns of numerous polypeptides to be visualized simply and quickly. Among proteins of wheat leaves, 31 groups of related polypeptides were found according to the similarity of their proteolytic patterns.     

Rapid high voltage isoelectric focusing of proteins in rod gels.

A rapid procedure of isoelectric focusing (IEF) of proteins in polyacrylamide rod gels (i.d., 1.1 mm; length, 7.5 cm) is described. The time required for IEF can be reduced to 0.5 h by using high voltages up to 3000 V in the presence or absence of urea in the gels. When used as the first dimension of a two-dimensional technique for IEF sodium dodecyl sulphate electrophoresis, high voltage IEF gives smaller protein spots on the second dimension gel, associated with an increase in resolution. The method has been tested by a two-dimensional separation of an eye sample of the goodeid fish Xenotoca eiseni.     

Cardiac sarcoplasmic reticulum and sarcolemmal proteins separated by two-dimensional electrophoresis: surfactant effects on membrane solubilization

We evaluated the use of the alkyaryl amidosulfobetaine zwitterionic detergent, designated as C8psi, to facilitate the solubilization of cardiac subcellular, membrane-associated proteins. Hearts from 7-week-old male Sprague-Dawley rats were isolated, and the left ventricles dissected and subsequently homogenized. The sarcolemma (SL) and the sarcoplasmic reticulum (SR) were isolated from different homogenate preparations originating from rat hearts by ultracentrifugation methods. The isolated membrane preparations were solubilized and the proteins precipitated. After resuspension, protein separation was achieved in first-dimensional IEF using an immobilized (pH 4-7) gradient and in the second dimension using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were then stained, images analyzed, and protein spots excised for subsequent identification. Protein identification from both SR and SL samples did not identify any of the known major membrane-associated proteins. Solubilization of whole tissue lysates with C8psi resulted in no increase in the total number of proteins detected relative to samples solubilized in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulforate (CHAPS). The data suggest the utility of newer surfactants such as C8psi to improve both the resolution of (2-D) protein profiles and increase the number of proteins extracted from subcellular organelle fractions.     

Differential Proteomic Analysis of Neonatal Cardiomyocytes in Response to β-Adrenergic Receptor Stimulation

β-Adrenoceptors(β-ARs) play a critical role in regulating cardiac functions under both physiological and pathological conditions. To further explore the mechanisms through whichβ-ARs perform its actions, proteomic approaches were adopted to study the global protein patterns in cultured neonatal rat cardiomyocytes exposed to isoproterenol(ISO). A modified method, "Mirror Images in One Gel", was used to improve the reproducibility and resolution power of two-dimensional electrophoresis. A 2-DE map with a good reproducibility was obtained in which 1281 ± 70spots were detected and about 1191 e 54 spots were matched, with an average matching rate of 92. 9%. Nine proteins with significant changes were identified by using peptide mass fingerprinting(PMF) data obtained via MALDI-MS.     

A simple method to remove contaminating salt from IPG strips prior to IEF

In this study, Saccharomyces cerevisiae yeast lysate is used to demonstrate how a simple wash procedure can improve IEF of IPG strips passively rehydrated in the presence of NaCl. By performing three 10 min washes after IPG strip rehydration and before IEF, corresponding second-dimensional gels from strips containing NaCl look similar to control strips while the second-dimensional gels of unwashed strips contains streaks and spaces devoid of protein. Up to 500 mM NaCl was added to the yeast lysate and successfully focused following this wash regime. Protein loss due to the washes was determined to be minimal by comparing replicates of washed and unwashed strips and analyzing the densities of their corresponding second-dimensional gel spots. In the event of unknown salt contamination, indicated by low voltage during focusing, it is possible to stop focusing, wash the strips, and then continue focusing with acceptable second dimension results.     

Two-dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins

An improved protocol for the two-dimensional analysis of proteins of the Corynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations (2-4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. More important, in contrast to a previously published method, both a sample tray and IPG-phor isoelectric focusing unit can be used for the in-gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, the preparation and analysis of C. glutamicum cell surface proteins is described. Proteins were extracted with lauroyl sarcosinate and 100-120 spots were separated on two-dimensional (2-D) gels in comparison to 18-20 spots observed previously by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). C. glutamicum proteins can now be separated into three distinct fractions resembling different functional units of the bacterial cell.     

Electrophoretic separation of protein kinases: altered mobility with different crosslinking agents in the presence of certain detergents

Nondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using an in situ phosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and with N,N'-methylenebisacrylamide as cross-linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent-treated preparations were electrophoresed through gels which contained either 0.1% Triton X-100 or 0.1% Nonidet P-40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP-dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with the detergent.     

A high-resolution reference map for cytoplasmic and membrane-associated proteins of Corynebacterium glutamicum

We present a high-resolution reference map for soluble proteins obtained from Corynebacterium glutamicum cells grown in glucose minimal medium. The analysis window covers the pl range from 4-6 and the molecular mass range from 5-100 kDa. Using overlapping narrow immobilized pH gradients for isoelectric focusing, 970 protein spots were detected after second-dimensional separation on SDS-polyacrylamide gels and colloidal Coomassie-staining. By tryptic peptide mass fingerprinting 169 protein spots were identified, representing 152 different proteins including many enzymes involved in central metabolism (18), amino acid biosynthesis (24) and nucleotide biosynthesis (11). Thirty-five of the identified proteins have no known function. A comparison of the observed and the expected physicochemical properties of the identified proteins indicated that nine proteins were covalently modified, since variants with apparently identical molecular mass, but differing pl were detected. The N-termini of eight proteins were determined by post-source decay (PSD) analysis of selected peptides. In addition to the soluble proteins, a map of the membrane-bound proteins within the pl range 4-7 is presented, which contains 660 protein spots, 22 of which were identified, representing 13 different proteins.     

Dual-label autoradiographic analysis of human skin fibroblast and myoblast proteins by two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradients in the first dimension

Horizontal two-dimensional polyacrylamide gel electrophoresis with immobilised pH gradients in the first dimension has been applied to the analysis of human skin fibroblast and muscle myoblast total cell proteins. Excellent two-dimensional separations of skin fibroblast proteins were obtained using pH 4-10 immobilised pH gradient gels with a long interelectrode distance (16 cm), but resolution was degraded, particularly of the more acidic proteins, by the use of shorter (10 cm) gels. Improved resolution of acidic and basic proteins was obtained using separate pH 4-7 and pH 7-10 immobilised pH gradient gels respectively in the first dimension. Two-dimensional protein maps of skin fibroblast proteins were visualised both by silver staining and by autoradiography of samples labelled synthetically with [35S]methionine. Horizontal two-dimensional electrophoresis, using pH 4-7 and pH 7-10 immobilised pH gradient gels in the first dimension, was applied to the analysis of protein samples from skin fibroblasts and muscle myoblasts dual-labelled synthetically with [35S]methionine and [75Se]selenomethionine in an attempt to identify sets of proteins specific to each cell type. In addition, two-dimensional maps or protein samples derived from normal individuals and patients with Duchenne muscular dystrophy were compared to search for protein changes associated with the disease state. Although sets of qualitative protein spot differences were observed by visual inspection of the two-dimensional gels, more rigorous qualitative and quantitative analysis of the patterns using a computerised analysis system will be required to obtain the maximum amount of information from these data.     

Determination of protein spots separated by two-dimensional polyacrylamide gel electrophoresis

A simple method for quantitating proteins in the spots on two-dimensional polyacrylamide gel electropherograms is described. The system consists in three steps: (1) O'Farrell's two-dimensional gel electrophoresis of the proteins to be analysed; (2) staining of the gels with Coomassie brilliant blue; and (3) determination of the area and integrated density of the stained spots by the Joyce Loebl Magiscan-1 image analysis system. The method can be used for the determination of proteins in the range 0.5-100 micrograms/cm2; the amount of protein involved in most spots detected by the staining method actually falls within this range. As the minimum spot diameter that can easily be handled by the method is about 2 mm, as much as 30 ng of protein in such a spot can be determined. The method can also be applied to autoradiograms.     

Dodecyl maltoside detergent improves resolution of hepatic membrane proteins in two-dimensional gels.

This report describes the incorporation of an alkyl maltoside detergent in two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) sample lysis buffer in order to improve resolution of protein patterns separated by nonequilibrium pH gradient electrophoresis. Membrane-associated proteins with alkaline isoelectric points form horizontal streaks on two-dimensional electrophoretograms when solubilized with conventional nonionic detergent. Dodecyl maltoside enhances protein delipidation during solubilization and improves pattern resolution and protein mobility.