High resolution two-dimensional gel electrophoresis of human erythrocyte membrane proteins.

Three different two-dimensional (2-D) gel electrophoretic techniques have been modified to provide high resolution of human erythrocyte membrane proteins. The resulting gels were referenced to the established one-dimensional (1-D) sodium dodecylsulfate (SDS) gel electrophoretic profile, and the effects of endogenous proteolysis and cytosolic contamination were studied. It is concluded that in vitro proteolysis and cytosolic contamination do not contribute significantly to the patterns observed on the 2-D gels, under the conditions used for erythrocyte ghost preparation. The procedures require only small quantities of blood; as many as twenty 2-D gel profiles can be obtained from 5 ml of blood. The combination of nonequilibrium isoelectric focusing (IEF) in the first dimension, SDS electrophoresis in the second dimension, and very sensitive silver staining techniques resolves more than 250 individual protein spots. This appears to be the most useful single procedure for the analysis of red cell membrane proteins. Membrane protein profiles from patients with Duchenne muscular dystrophy, Wernicke-Korsakoff syndrome, and acanthocytosis with degeneration of the basal ganglia were compared with normal controls. The patterns for Duchenne muscular dystrophy and Wernicke-Korsakoff syndrome were not different from normal patterns. The pattern for the patient with acanthocytosis and degeneration of the basal ganglia consistently showed a high level for one protein in the 100,000 mol. wt. range.     

Efficient solubilization buffers for two-dimensional gel electrophoresis of acidic and basic proteins extracted from wheat seeds

Plant tissues are made up of a broad range of proteins with a variety of properties. After extraction, solubilization of a diverse range of plant proteins for efficient proteomic analysis using two-dimensional electrophoresis is a challenging process. We tested the efficiency of 12 solubilization buffers in dissolving acidic and basic proteins extracted from mature seeds of wheat. The buffer containing two chaotropes (urea and thiourea), two detergents (3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate and N-decyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate), two reducing agents (dithiothreitol and tris (2-carboxyethyl) phosphine hydrochloride) and two types of carrier ampholytes (BioLyte pH 4-6 and pH 3-10) solubilized the most acidic proteins in the pH range between 4 and 7. The buffer made up of urea, thiourea, 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, DeStreak reagent (Amersham Biosciences, Uppsala, Sweden) and immobilized pH gradient buffer, pH 6-11 (Amersham Biosciences) solubilized the most basic proteins in the pH range between 6 and 11. These two buffers produced two-dimensional gels with high resolution, superior quality and maximum number of detectable protein (1425 acidic protein and 897 basic protein) spots.     

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes.

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuosus polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrance and of the whole platelet components established in this system.     

High resolution mini-two-dimensional gel electrophoresis of yeast ribosomal proteins

By applying two-dimensional gel electrophoresis (basic urea/acidic urea) in a mini-form yeast ribosomal proteins were separated with a high resolution. On basis of this separation pattern a standard nomenclature for the yeast ribosomal proteins is proposed.     

High resolution two-dimensional gel electrophoresis of the proteins and glycoproteins of human blood platelets and platelet membranes

The proteins and glycoproteins of human blood platelets and platelet membranes in both the reduced and the unreduced states have been analysed by isoelectric focusing and sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis in a two-dimensional technique. Gels which had been stained with periodic acid-Schiff's reagent could be counter-stained with Coomassie Brilliant Blue, simplifying the recognition of components which stain with both reagents. The major glycoproteins and some of the proteins have been identified and the characteristics of the membrane and of the whole platelet components established in this system.     

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.     

High-resolution two-dimensional electrophoretic analysis of acidic,basic and surface proteins of mouse neutrophils

The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 200 proteins detected) and with fluorography or autoradiography after [³⁵S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of ¹²⁵I associated with actin) yielded more than 20 major ¹²⁵I-labelled proteins on high-resolution electrophoretic maps. The major ¹²⁵I-labelled protein (M_r ≈ 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [³⁵S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.     

Acrylamide-agarose copolymers: improved resolution of high molecular mass proteins in two-dimensional gel electrophoresis

A method was developed in order to analyse high molecular mass proteins by two-dimensional (2-D) electrophoresis using a copolymer of acrylamide and allyl agarose instead of Bis cross-linked polyacrylamide (PA) gels in sodium dodecyl sulphate-electrophoresis. In this work, the matrix composition was optimised to improve the resolution of proteins larger than 200 kDa. The new gel type does not entrap large proteins and protein complexes at the application site. Mechanical properties were investigated through rheological measurements, which suggested the formation of a highly entangled elastomeric soft gel. A high 2-D resolution of proteins, extracted from membranes of red blood cells, was obtained in these gels. An example of tryptic digestion, peptide extraction and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry was reported. The results demonstrate that the new gel is fully compatible with mass spectrometry protein analysis.     

Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40, from a linear standard curve. A method for a quantitative determination of protein concentration by BCA in a sample containing 9 M urea and 4% Nonidet P-40 is also described. This method is effective for the determination of proteins in minute non-green and green plant tissue, and is especially designed for vegetative and floral shoot apices, and the primordia of inflorescences.     

Differentiation-related proteins of the broad bean rust fungus Uromyces viciae-fabae,as revealed by high resolution two-dimensional polyacrylamide gel electrophoresis

On artificial polyethylene membranes providing a thigmotropic signal, uredospores of the broad bean rust fungus Uromyces viciae-fabae differentiated a series of infection structures which in nature are necessary to invade the host tissue through the stomata. Within 24 h germ tubes, appressoria, substomatal vesicles, infection hyphae and haustorial mother cells were developed successively. Alterations in protein metabolism during infection structure differentiation of this obligate plant pathogen were analyzed in the absence of the host plant by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) and silver staining. The norm pattern representing the 2-DE protein patterns of the whole developmental sequence of infection structures of U. viciae-fabae showed 733 spots. During infection structure differentiation 55 proteins were newly formed, altered in quantity, or disappeared. Major alterations in the protein pattern occurred during uredospore germination and when infection hyphae were formed. Uredospore germination was characterized by a decrease of acidic proteins and an increase mainly of proteins with isoelectric points ranging from weakly acidic to basic.Abbreviations 2-DE two-dimensional polyacrylamide gel electrophoresis - DAPI 4,6-diamino-phenylindol - kDa kilo Dalton - pl isoelectric point - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis