Comparison of ethanol-soluble proteins from different rye (Secale cereale) varieties by two-dimensional electrophoresis

The major storage proteins from six rye varieties, grown under the same conditions in 1997 and 1998 in Rønhave, Denmark, were analyzed by two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The proteins were extracted from ground rye kernels with 70% ethanol and separated by 2‐D electrophoresis. The gels were scanned, compared using ImageMaster® software and the data sets were analyzed by principal component analysis (PCA) using THE UNSCRAMBLER software. Afterwards MATLAB was used to make a cluster analysis of the varieties based on PCA. The analysis of the gels showed, that the protein patterns (number of different proteins and their isoelectric points and molecular weights) from the six rye varieties were different. Based on the presence of unique cultivar‐specific spots it was possible to differentiate between all six varieties if the two harvest years were investigated separately. When the results were combined from the two years five varieties could be differentiated. The results from the PCA confirmed the finding of the unique spots and cluster analysis was made in order to illustrate the results. The combination of the results from 2‐D electrophoresis and other grain characteristics showed that one protein spot was located close to the parameters bread volume and bread height.     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

Two-dimensional electrophoresis of human lymphocyte proteins: two-dimensional polymorphisms and paternity testing

Genetic polymorphisms of seven human lymphocyte proteins, analyzed by two-dimensional electrophoresis, were evaluated in respect to their suitability for paternity testing. Current data of an enlarged family and population study for five proteins (p23, p30, p40, p60, p66), already described for a smaller population sample of Southern Germany, are presented together with evidence for a new polymorphic protein (p42), recently observed in our survey. These six proteins occurred in isoelectric focusing as two different variants, acidic (a) and basic (b). The genetic basis of the protein variations was ascertained (i) by the presence of homozygous and heterozygous phenotypes, (ii) by the Mendelian mode of transmission of the variants as allelic gene products within 17 families and (iii) by the demonstration of a gene-dosage dependence comparing the spot intensities in homozygous and heterozygous phenotypes. For quantitative data, laser densitometric scanning of the protein spots followed by computer-assisted quantitative evaluation of the spot intensities was performed. The allele frequencies of the polymorphic protein were calculated from the phenotype distributions within a sample of 56 unrelated individuals from Southern Germany. Gene frequencies of the common alleles ranged between 0.991 and 0.518. To discuss the suitability of the two-dimensional polymorphisms for paternity testing the theoretical exclusion probabilities were assessed for seven polymorphic proteins observed in our population sample, the six polymorphisms with two alleles described here and a further polymorphism (p75) with six alleles. For five proteins (p23, p40, p42, p66 and p75) we found sufficiently high values for the theoretical exclusion probabilities, ranging from 10% to 34%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of two-dimensional crystalline aggregates of a protein studied by molecular dynamics

Two-dimensional protein (ferritin) aggregates with a square lattice symmetry, which were formed within a thin liquid layer on a mercury surface, were studied by molecular dynamics (MD) simulation. For the simulation, the ferritin molecule was modeled by an assembly of 49 spheres, and the intermolecular interactions were given by simple formulae. During the simulation, molecules were confined within a layer, which corresponds to the thin liquid layer. An annealing MD simulation was done starting from a random molecular configuration within the layer, and aggregates with the square lattice symmetry were also obtained. To study the stability of aggregates, dissociation processes of the aggregates were analyzed using MD simulations at room temperature. Interactions between the nearest-neighbor molecules were regarded as bonds. Mean bond energies and correlation coefficients between the bond energies were calculated from the MD trajectories. A decay profile according to the dissociation was obtained, yielding a dissociation rate constant. Buried bonds were stronger than peripheral bonds. The larger the aggregate size, the stronger the bond for each of the buried and peripheral bonds. A simple theoretical account, which is applicable to a general bonded network, was introduced to analyze the dynamics of the aggregates. © 1994 by John Wiley & Sons, Inc.     

Single-cell protein analysis of a single mouse embryo by two-dimensional capillary electrophoresis

The characterization of protein expression from a single-cell mouse embryo using two-dimensional capillary electrophoresis (2D-CE) is described. These zygotes were obtained from Hsf1 gene knockout mice. Single zygotes were lysed off-column and proteins were fluorescently labeled using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). After injection, analytes were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by micellar electrokinetic capillary chromatography (MEKC) to obtain protein expression fingerprints. Analytes were detected in a sheath flow cuvette using laser-induced fluorescence. In a 1-h 2D-CE separation, over 100 components were resolved with a spot capacity of 380.     

Precise quantitative results by capillary electrophoresis (CE): instrumental aspects

Summary The precision of different CE-instruments was tested by a standard procedure. These results and other interesting features are commented and presented in a table. The high technical standard of these instruments is demonstrated by obtainable day-to-day precisions of below 1%. Thermostating is very important to provide a reproducible dosage.     

A human myocardial two-dimensional electrophoresis database: protein characterisation by microsequencing and immunoblotting.

This communication briefly describes how a human heart two-dimensional electrophoresis (2-DE) protein database is being established in our laboratory. The database contains more than 1500 polypeptides and approximately fifty proteins from 2-DE gels of human myocardial tissue have been characterised. Information about the proteins has been compiled including molecular weight (M(r)), isoelectric point (pI), sample spot (SSP) number, protein name, partial sequence, and antibody reacting with the protein. The first stage of this project involves the investigation of protein with pIs in the range pH 4-7. Future studies will employ immobilised pH gradient (IPG) gels as the first dimension of the 2-DE to examine basic proteins. The ultimate goal of this project is to establish a global picture of human heart protein expression in both normal and disease conditions.     

Characterization of native multicomponent protein mixtures by one- and two-dimensional gradient electrophoresis

An arrangement of one- and two-dimensional polyacrylamide gradients has been applied to the analysis of multicomponent mixtures of soluble proteins: (1) two-dimensional 3-25% slabs; (2) 3-40% cylinders; and (3) 20-50% cylinders. The run in the first dimension is performed in non-restrictive agarose gel to ascertain a "pure" charge abscissa. Gradient electrophoresis is performed over prolonged periods in order to reach or closely approach the exclusion (pore) limit, which is taken as an operational parameter for the hydrodynamic size of globular molecules irrespective of their frictional ratios. For each of the three gradients one to four reference mixtures were formulated that allow size evaluations of non-denatured proteins. Human serum again proved its usefulness as a universal reference mixture for newly developed techniques. The number of stainable components is in accordance with expectation based on the broad knowledge of serum proteins. Two-dimensional agarose/gradient electrophoresis seems to fulfil the one band-one protein strategy of high-resolution techniques.     

Construction of two-dimensional (2D) H-bonded supramolecular nanostructures studied by STM

In this review,a group of two-dimensional（2D） hydrogen-bonded supramolecular networks developed in our laboratory are discussed.Our attention is mainly focused on:（1） recognition of Fe³⁺ through twocomponent molecular networks;（2） site-selective fabrication of 2D fullerene arrays;and（3） fabrication of the nanoporous structure regulated by photoisomerization reaction process.It is envisioned that special supramolecular nanostructures,through H-bonding interactions,can be constructed or reconstructed to be further investigated toward the research of multi-component systems,molecule recognition,single molecular switches,and host-guest supramolecular chemistry.     

Manipulation of protein fingerprints during on-column fluorescent labeling: protein fingerprinting of six Staphylococcus species by capillary electrophoresis

Bacterial proteomes were analyzed by use of electrophoretically mediated microanalysis (EMMA) and field-enhanced stacking. A water-soluble protein fraction was injected onto a capillary. Next, a fluorogenic reagent was injected and allowed to react with the protein mixture, producing fluorescent products that were separated by submicellar capillary electrophoresis and detected by laser-induced fluorescence. By use of a low-ionic strength sample buffer and a brief electrophoretic step, slow moving anionic proteins were stacked at the reagent-sample interface and were preferentially labeled. By reversing the order of sample injection and labeling reagent, fast moving cationic proteins were preferentially labeled. By adjustment of the sample buffer pH, proteins with different isoelectric points were selectively labeled. Electrophoresis fingerprints were generated for the water-soluble protein fraction from six Staphylococcus species. The protein patterns produced were species-specific and were used to construct a phylogenetic tree.     

Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study

Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities.     

Genetic variability of pepper (Capsicum annuum L.) seed proteins studied by 2-D electrophoresis with immobilized pH gradients.

Ten pepper (Capsicum annuum L.) inbred lines were successfully differentiated by two-dimensional electrophoresis with immobilized pH gradients. Qualitative polymorphism of water-soluble and urea/detergent-soluble seed proteins, respectively, was investigated by computer analysis and used for establishing a dendrogram derived from maximum-parsimony analysis. The dendrogram calculated from urea/detergent-soluble proteins shows four types of distance indices, whereas water-soluble proteins show two sets of inbred lines with similar intraset distance indices. The validity of the dendrograms with respect to quantitative inherited traits, such as cold tolerance and earliness, will be tested by field trials.     

Online mass spectrometry of CE (SDS)-separated proteins by two-dimensional capillary electrophoresis

Analytical and Bioanalytical Chemistry - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the fundamental technique for protein separation by size. Applying this technology...     

Composition analysis of poly(ethylene glycol)-poly(L-lactide) diblock copolymer studied by two-dimensional column chromatography

We used two-dimensional column chromatography to analyze the composition of a sample of presumably a diblock copolymer of poly(ethylene glycol) (PEG) and poly(L-lactide) synthesized from monomethoxy-terminated PEG. The first dimension of the separation is phase fluctuation chromatography to prepare fractions that contain various components of the copolymer in different ratios. The second dimension is size-exclusion chromatography, NMR, and HPLC at the critical condition of PEG. The PEG initiator has small amounts of diol-terminated dimeric components. We found that the copolymer sample contains a triblock copolymer and low-molecular-mass components in addition to the main part of the diblock copolymer. The SEC chromatograms show that the main part consists of two components with distinct peak lengths for the PLLA block. The low-molecular-mass components have a broad distribution in chemical composition. Phase fluctuation chromatography enriched the triblock copolymer and the diblock copolymer with the longer PLLA block in early fractions when the column was packed with carboxymethyl-modified porous silica. When the porous medium was PLLA-grafted silica, size exclusion dominated, but the low-molecular-mass components were separated according to their chemical composition.     

Determination of biotin on a protein by quantitative sodium dodecyl sulfate-capillary gel electrophoresis of monomeric avidin

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) is performed to quantify monomeric avidin and biotin on a protein. Under non-reducing SDS-CGE conditions, avidin migrates as monomers exhibiting apparent molecular mass 17,000. In the presence of a biotin-protein conjugate, monomeric avidin binds the conjugate and forms a larger complex that migrates later in the separation. The difference between the remaining monomeric avidin and the initial amount is the portion of monomeric avidin bound to the conjugate. Accordingly, the number of biotin on the protein can be calculated. The assay is linearly responsive to increasing biotin loading in a biotinylation reaction of a protein. Accuracy of the assay is also demonstrated by good sample dilution recovery. Excellent quantitative reproducibility < 2% (relative standard deviation) is obtained for both intra- and inter-day measurements. Main advantages of the method include the use of monomeric avidin that minimizes steric hindrance to capture biotin on a protein and assay automation on a capillary electrophoresis apparatus.     

Pressure-induced variation of cellulose tablet studied by two-dimensional (2D) near-infrared (NIR) correlation spectroscopy in conjunction with projection pretreatment

Projection two-dimensional (2D) correlation analysis, achieved by selectively eliminating specific portion of dynamic spectra synchronized with the projecting vector, was used to study compression-induced variation of cellulose tablet monitored by near-infrared (NIR) spectroscopy and water sorption test. The increased density of the tablet by compression provided apparent variation of spectral intensity much larger than those caused by the change in the cellulosic structure. The direct calculation of 2D NIR correlation spectra from the raw spectra generated a relatively uninformative synchronous correlation spectrum and a very noisy asynchronous correlation spectrum due to the predominant intensity variation arising from the light scattering. In contrast, significant correlation features were elucidated without being hampered by the baseline fluctuation when the projection-corrected NIR spectra were constructed by the projection onto the space spanned orthogonal to the baseline change. Fine features of the compression-induced variation of the system were also elucidated by 2D hetero-correlation analysis based on the NIR spectra and water sorption profiles. The 2D correlation analysis revealed that the compression produces a disordered amorphous component of cellulose. The development of mobile amorphous phase results in a more tightly packed matrix with less porosity, which in turn prevents the penetration of water into the tablet and delays the water sorption.     

Structural analysis of partially acetylated glycosides by refocused-decoupled insensitive nuclei enhanced by polarization transfer (INEPT) experiments: a qualitative and quantitative method

The application of the refocused-decoupled INEPT pulse sequence in 13C-NMR spectroscopy allows, in conjonction with computer simulations, a straightforward editing of the spectra of a raw mixture of glycosidic compounds.     

Spot overlapping in two-dimensional polyacrylamide gel electrophoresis separations: a statistical study of complex protein maps

A statistical approach able to extract the information contained in a two-dimenisional polyacrylamide gel electrophoresis (2-D PAGE) separation is here reported. The method is based on the quantitative theory of peak overlapping, a procedure previously developed by the authors and here extended to 2-D separations. The whole map is divided into many strips in order to obtain 1-D separations on which the statistic procedure is applied: the developed algorithms, on the basis of spot experimental data (intensity and spatial coordinates) permit to estimate the intrinsic number of components and to single out the specific order present in spot positions. The procedure was validated on computer-simulated maps. Its applicability to real samples was tested on maps obtained from literature sources. The following important information on protein mixtures can be extracted: (i) the number of proteins can be accurately estimated, on the basis of the spatial coordinates and intensities of spots detected in the 2-D PAGE map; (ii) the model describing distribution of interdistance between adjacent spots can be identified in both the separation dimensions; (iii) the presence of repeated interdistances in spot positions in the maps can be easily singled out: these regularities suggest specific protein modifications.