Identification of the immunoglobulin G receptor of human platelets

The binding site of IgG on human platelets was studied by the use of the cleavable heterobifunctional cross-linking agent N-succinimidyl (4-azidophenyldithio)propionate. Binding characteristics of the derivatized IgG were similar to normal IgG. Periodate-borohydride treatment of platelets also did not significantly alter their ability to bind IgG. N-Succinimidyl (4-azidophenyldithio)propionate was bound to IgG via a succinimidyl ester and then photolyzed in the presence of intact platelets. Their membrane glycoproteins were first tritiated by the periodate-borohydride method. The cross-linked product was analyzed by two dimensional sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The non-reduced first-dimension gels were subjected to 5% 2-mercaptoethanol prior to separation in the second dimension. Such gels were then evaluated by fluorography, silver staining, and counting the radioactivity of sequential gel strips in the area of cross-linking. The protein complexes at the interface between stacking and running gel were further resolved in isoelectric focusing gels. One IgG-containing band could be identified. After reduction, the constituent proteins of the cross-linked complex were analyzed by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and subsequent immunoblotting with an antiserum against platelet membrane glycoproteins. All of these studies gave evidence of glycoprotein IIIa as the receptor of IgG. Based on the results of the different experimental approaches, we conclude that glycoprotein IIIa is the IgG receptor in human platelets.     

Yet another improved silver staining method for the detection of proteins in polyacrylamide gels

Silver staining is very sensitive for detection of proteins in polyacrylamide gels and different procedures have been published. By combining and modifying some of the recipes, a very reproducible method, which is based upon staining with diamine complexes of silver, has been developed. The background staining is negligible and reduced silver does not precipitate on the gel surface. The technique works very well for sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both homogeneous and in gradient gels as well as for two-dimensional (2-D) PAGE. It was possible to detect 1-10 ng of protein corresponding to approximately 50 pg/mm2, provided that a discontinuous buffer system was used, which gives sharp bands.     

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.     

High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes

The recent upsurge in proteomics research has been facilitated largely by streamlining of two-dimensional (2-D) gel technology and the parallel development of facile mass spectrometry for analysis of peptides and proteins. However, application of these technologies to the mitochondrial proteome has been limited due to the considerable complement of hydrophobic membrane proteins in mitochondria, which precipitate during first dimension isoelectric focusing of standard 2-D gels. In addition, functional information regarding protein:protein interactions is lost during 2-D gel separation due to denaturing conditions in both gel dimensions. To resolve these issues, 2-D blue-native gel electrophoresis was applied to the mitochondrial proteome. In this technique, membrane protein complexes such as those of the respiratory chain are solubilized and resolved in native form in the first dimension. A second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel then denatures the complexes and resolves them into their component subunits. Refinements to this technique have yielded the levels of throughput and reproducibility required for proteomics. By coupling to tryptic peptide fingerprinting using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, a partial mitochondrial proteome map has been assembled. Applications of this functional mitochondrial proteomics method are discussed.     

Scrapie PrP 27-30 is a sialoglycoprotein.

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100 degrees C for 15 min in 0.1 N NaOH or 0.1 N HCl resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-beta-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.     

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.     

Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.

An Arabidopsis mitochondrial proteome project was started for a comprehensive investigation of mitochondrial functions in plants. Mitochondria were prepared from Arabidopsis stems and leaves or from Arabidopsis suspension cell cultures, and the purity of the generated fractions was tested by the resolution of organellar protein complexes applying two-dimensional blue-native/N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine (Tricine) sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Arabidopsis mitochondrial proteome was analyzed by two-dimensional isoelectric focusing/ Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 650 different proteins in a pI range of pH 3 to 10 were separated on single gels. Solubilization conditions, pH gradients for isoelectric focusing, and gel staining procedures were varied, and the number of separable proteins increased to about 800. Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. The characterized proteins cooperate in various processes, such as respiration, citric acid cycle, amino acid and nucleotide metabolism, protection against O(2), mitochondrial assembly, molecular transport, and protein biosynthesis. More than 20% of the identified proteins were not described previously for plant mitochondria, indicating novel mitochondrial functions. The map of the Arabidopsis mitochondrial proteome should be useful for the analysis of knockout mutants concerning nuclear-encoded mitochondrial genes. Considerations of the total complexity of the Arabidopsis mitochondrial proteome are discussed. The data from this investigation will be made available at http://www.gartenbau.uni-hannover.de/genetik/AMPP.     

Study of human erythrocyte membrane proteins by 2-dimensional electrophoresis

Fractionation of human erythrocyte membrane proteins was performed using a modification of two-dimensional gel electrophoresis described by P. O'Farrel with isoelectric point plotted against molecular mass. All major erythrocyte proteins, including high molecular weight proteins, such as spectrin and band 3 protein, identified by one-dimensional sodium dodecyl sulfate gel electrophoresis, were visualized by silver staining of two-dimensional gels. All in all about 50 polypeptides were distinguished on two-dimensional electrophoretic patterns. Preliminary protein map was developed.     

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.     

Detecting proteoglycans immobilized on positively charged nylon

Proteoglycans (PG) immobilized on positively charged Nylon 66 are detected readily by staining with Alcian blue. With the exception of hyaluronic acid, free glycosaminoglycans appear unreactive when treated similarly. Immobilization was performed by dot blotting or by electrophoretic transblotting from various gel supports. When transblotted to positively charged Nylon 66 from large-pore agarose-acrylamide gels, levels of 10-50 ng of PG could be detected by Alcian blue staining. This procedure appeared to be nearly 10(2) times more sensitive than staining of gels with toluidine blue. The transblot and staining procedure also appears to be effective with PG separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was applied to a preparation enriched in basement membrane components.     

Sequential two-dimensional and acetic acid/urea/Triton X-100 gel electrophoresis of proteins

A method is described which combines the resolving power of two-dimensional gel electrophoresis with that of acetic acid/urea/Triton X-100 gel electrophoresis, avoiding the necessity of eluting protein from the gels at any step of the procedure. The combination of electrophoretic separation on the basis of charge, mass, and hydrophobic properties of the proteins has the potential of resolving modified forms and isoforms present in very complex protein populations. The technique can be used for analytical purposes, or it may be scaled up to yield microgram amounts of highly purified proteins. The resolution obtained by tandem application of nonequilibrium pH gradient electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and polyacrylamide gel electrophoresis in the presence of nonionic detergent was evaluated using crude nuclear proteins of the nematode Caenorhabditis elegans.     

The basis for colored silver-protein complex formation in stained polyacrylamide gels

Using a modified silver stain of Merril et al. [(1981) Science 211, 1437-1438] for staining polypeptides in sodium dodecyl sulfate-polyacrylamide gels, protein bands reproducibly stain different shades of blue, yellow, red, and gray. The procedure is highly temperature dependent, with optimal color formation at 42 degrees C. The procedure may be completed within 2 h. Color formation is due to silver ion complexes with charged amino acid side chains. The color of the silver-protein complex can be predicted if the amino acid sequence is known, although some exceptions are discussed. This provides another dimension to the characterization of proteins by gel electrophoresis.     

Isolation and sequence analysis of proteins from mouse forebrain using two-dimensional gel electrophoresis coupled to high-pressure liquid extrusion

This report presents a technique for recovery of mouse forebrain proteins from two-dimensional sodium dodecyl sulfate-polyacrylamide gels for subsequent primary structure determination. Proteins were visualized by Coomassie staining or salt precipitation and manually cut out of the gel. Excised spots were minced and loaded into an empty precolumn of a reversed-phase high-performance liquid chromatography system. Purified protein was extruded from a gel matrix by pressurized liquid, then separated from gel contaminants by reversed-phase gradient elution, and finally collected in siliconized tubes or on polybrene-coated filter disks for gas-phase sequencing. Several mouse and rat forebrain proteins were purified by this method and sequenced. Three previously unidentified mouse brain proteins with molecular weights of 4,000, 12,000, and 18,500 were partially sequenced and three hemoglobin fragments were structurally identified and mapped. Ribonuclease A, myoglobin, adrenocorticotropin, and bovine somatotropin were also subjected to two-dimensional (2-D) analysis and partially sequenced. Recovery values of 27-95% were obtained for extruded 14C-labeled ribonuclease, carbonic anhydrase, and bovine serum albumin out of sodium dodecyl sulfate-polyacrylamide gel electrophoretic gels. Losses resulting from the multiple handling steps of a 2-D gel separation process were also investigated. Recoveries of 12-17%, as determined by sequencing signals, were achieved. These latter recovery values reflect overall losses incurred in gel-focusing, gel-sizing, staining, destaining, high-pressure liquid extrusion, and N-terminal blockage. This work demonstrates that an array of protein spots can be systematically identified or defined by partial sequencing after high-pressure liquid extrusion from a 2-D gel matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Defining the protein complex proteome of plant mitochondria

A classical approach, protein separation by two-dimensional blue native/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was combined with tandem mass spectrometry and up-to-date computer technology to characterize the mitochondrial "protein complex proteome" of Arabidopsis (Arabidopsis thaliana) in so far unrivaled depth. We further developed the novel GelMap software package to annotate and evaluate two-dimensional blue native/sodium dodecyl sulfate gels. The software allows (1) annotation of proteins according to functional and structural correlations (e.g. subunits of a distinct protein complex), (2) assignment of comprehensive protein identification lists to individual gel spots, and thereby (3) selective display of protein complexes of low abundance. In total, 471 distinct proteins were identified by mass spectrometry, several of which form part of at least 35 different mitochondrial protein complexes. To our knowledge, numerous protein complexes were described for the first time (e.g. complexes including pentatricopeptide repeat proteins involved in nucleic acid metabolism). Discovery of further protein complexes within our data set is open to everybody via the public GelMap portal at www.gelmap.de/arabidopsis_mito.     

The holm oak leaf proteome: analytical and biological variability in the protein expression level assessed by 2-DE and protein identification tandem mass spectrometry de novo sequencing and sequence similarity searching

As a first approach in establishing the holm oak leaf proteome, we have optimised a protocol for this plant and tissue which includes the following steps: trichloroacetic acid-acetone extraction, two-dimensional gel electrophoresis (2-DE) on pH 5 to 8 linear gradient immobilised pH gradient strips as the first dimension, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 13% polyacrylamide gels as the second one. Proteins were detected by Coomassie staining. Gel images were recorded and digitalized, and the protein spots quantified by using a linear regression equation of protein quantity on spot volume obtained against standard proteins. Analytical variance was calculated for one-hundred protein spots from three replicate 2-DE gels of the same protein extract. Biological variance was determined for the same protein spots from independent tissue extracts corresponding to leaves from different trees, or the same tree at different orientations or sampling times during a day. Values of 26% for the analytical variance and 58.6% for the biological variance among independent trees were obtained. These values provide a quantified and statistical basis for the evaluation of protein expression changes in comparative proteomic investigations with this species. A representative set of the major proteins, covering the isoelectric point range of 5 to 8 and the relative molecular mass(r) range of 14 to 78 kDa, were subjected to liquid chromatography-tandem mass spectrometry analysis. Due to the absence of Quercus DNA or protein sequence databases, a method based on the procedure reported by Liska and Shevchenko including de novo sequencing and BLAST similarity searching against other plant species databases was used for protein identification. Out of 43 analysed spots, 35 were positively identified. The identified proteins mainly corresponded to enzymes involved in photosynthesis and energetic metabolism, with a significant number corresponding to RubisCO.     

Electrophoretic identification of fusion proteins expressed in single recombinant lambda-bacteriophage plaques

Sufficient fusion protein is present in single plaques produced by lytic, recombinant lambda bacteriophage to be detected by Coomassie blue staining following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Agar plugs containing single plaques can be loaded directly onto the stacking gel thus avoiding the need for extensive sample preparation.     

Lipopolysaccharide-free Escherichia coli OmpF and Pseudomonas aeruginosa protein P porins are functionally active in lipid bilayer membranes.

Escherichia coli porin OmpF and Pseudomonas aeruginosa porin protein P were eluted from sodium dodecyl sulfate-polyacrylamide gels. The resultant porin preparations were found to be devoid of detectable lipopolysaccharide (LPS) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining for LPS, direct enzyme-linked immunosorbent assays with LPS-specific monoclonal antibodies, and 2-keto-3-deoxyoctulosonic acid assays. The average conductances, ionic selectivities and incorporation rates of the electroeluted porins were identical to those of their conventionally purified counterparts. These data suggest that LPS is not required per se for porin function.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Protein Components of the Purified Sodium Channel from Rat Skeletal Muscle Sarcolemma

Abstract: Sensitive detection systems have been used to study the protein components of the sodium channel purified from rat skeletal muscle sarcolemma. This functional, purified sodium channel contains at least three subunits on 7–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: a large glycoprotein which migrates anomalously in the high-molecular-weight range, a 45,000 molecular weight polypeptide, and a third protein often seen as a doublet at 38,000. The large glycoprotein runs as a diffuse band and stains very poorly with Coomassie blue, but is adequately visualized with silver staining or iodination followed by autoradiography. This glycoprotein exhibits anomalous electrophoretic behavior in SDS-polyacrylamide gels. The apparent molecular weight of the center of the band varies from ～230,000 on 13% acrylamide gels to ～130,000 on 5% gels; on 7–20% gradient gels a value of 160,000 is found. Plots of relative migration versus gel concentration suggest an unusually high apparent free solution mobility. Lectin binding to purified channel peptides separated by gel electrophoresis indicates that the large glycoprotein is the only subunit that binds either concanavalin A or wheat germ agglutinin, and this component has high binding capacity for both lectins. The smaller channel components run consistently at 45,000 and 38,000 molecular weight in a variety of gel systems and do not appear to be glycosylated.