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Sample records for quantitative protein electrophoresis

  1. Free flow electrophoresis separation and AMS quantitation of C-naphthalene-protein adducts.

    PubMed

    Buchholz, Bruce A; Haack, Kurt W; Sporty, Jennifer L; Buckpitt, Alan R; Morin, Dexter

    2010-04-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of (14)C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with (14)C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts. PMID:20454606

  2. Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts

    PubMed Central

    Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter

    2009-01-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5–11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts. PMID:20454606

  3. Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts

    NASA Astrophysics Data System (ADS)

    Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter

    2010-04-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose-(concentration)dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 h post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

  4. Quantitation of yeast total proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer for uniform loading.

    PubMed

    Sheen, Hyukho

    2016-04-01

    Proteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer are difficult to quantitate due to SDS and reducing agents being in the buffer. Although acetone precipitation has long been used to clean up proteins from detergents and salts, previous studies showed that protein recovery from acetone precipitation varies from 50 to 100% depending on the samples tested. Here, this article shows that acetone precipitates proteins highly efficiently from SDS-PAGE sample buffer and that quantitative recovery is achieved in 5 min at room temperature. Moreover, precipitated proteins are resolubilized with urea/guanidine, rather than with SDS. Thus, the resolubilized samples are readily quantifiable with Bradford reagent without using SDS-compatible assays.

  5. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    PubMed Central

    2014-01-01

    Background Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated. Results Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction. Conclusions This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the

  6. Protein electrophoresis - serum

    MedlinePlus

    ... of protein and fat, called lipoproteins (such as LDL cholesterol). ... globulin proteins may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone ...

  7. Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication and further separated by size exclusion chromatography into monomeric and polymeric fractions. Proteins in each fraction were analyzed by quantitative two-dimensional gel...

  8. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    SciTech Connect

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  9. Quantitative analysis of electrophoresis data: novel curve fitting methodology and its application to the determination of a protein-DNA binding constant.

    PubMed

    Shadle, S E; Allen, D F; Guo, H; Pogozelski, W K; Bashkin, J S; Tullius, T D

    1997-02-15

    A computer program, GelExplorer, which uses a new methodology for obtaining quantitative information about electrophoresis has been developed. It provides a straightforward, easy-to-use graphical interface, and includes a number of features which offer significant advantages over existing methods for quantitative gel analysis. The method uses curve fitting with a nonlinear least-squares optimization to deconvolute overlapping bands. Unlike most curve fitting approaches, the data is treated in two dimensions, fitting all the data across the entire width of the lane. This allows for accurate determination of the intensities of individual, overlapping bands, and in particular allows imperfectly shaped bands to be accurately modeled. Experiments described in this paper demonstrate empirically that the Lorentzian lineshape reproduces the contours of an individual gel band and provides a better model than the Gaussian function for curve fitting of electrophoresis bands. Results from several fitting applications are presented and a discussion of the sources and magnitudes of uncertainties in the results is included. Finally, the method is applied to the quantitative analysis of a hydroxyl radical footprint titration experiment to obtain the free energy of binding of the lambda repressor protein to the OR1 operator DNA sequence.

  10. Human lymphocyte polymorphisms detected by quantitative two-dimensional electrophoresis

    SciTech Connect

    Goldman, D.; Merril, C.R.

    1983-09-01

    A survey of 186 soluble lymphocyte proteins for genetic polymorphism was carried out utilizing two-dimensional electrophoresis of /sup 14/C-labeled phytohemagglutinin (PHA)-stimulated human lymphocyte proteins. Nineteen of these proteins exhibited positional variation consistent with independent genetic polymorphism in a primary sample of 28 individuals. Each of these polymorphisms was characterized by quantitative gene-dosage dependence insofar as the heterozygous phenotype expressed approximately 50% of each allelic gene product as was seen in homozygotes. Patterns observed were also identical in monozygotic twins, replicate samples, and replicate gels. The three expected phenotypes (two homozygotes and a heterozygote) were observed in each of 10 of these polymorphisms while the remaining nine had one of the homozygous classes absent. The presence of the three phenotypes, the demonstration of gene-dosage dependence, and our own and previous pedigree analysis of certain of these polymorphisms supports the genetic basis of these variants. Based on this data, the frequency of polymorphic loci for man is: P . 19/186 . .102, and the average heterozygosity is .024. This estimate is approximately 1/3 to 1/2 the rate of polymorphism previously estimated for man in other studies using one-dimensional electrophoresis of isozyme loci. The newly described polymorphisms and others which should be detectable in larger protein surveys with two-dimensional electrophoresis hold promise as genetic markers of the human genome for use in gene mapping and pedigree analyses.

  11. Protein Cross-Linking Capillary Electrophoresis for Protein-Protein Interaction Analysis.

    PubMed

    Ouimet, Claire M; Shao, Hao; Rauch, Jennifer N; Dawod, Mohamed; Nordhues, Bryce; Dickey, Chad A; Gestwicki, Jason E; Kennedy, Robert T

    2016-08-16

    Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein-protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme-antibody interaction was found to have Kd = 24 ± 3 nM by PXCE and Kd = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have Kd = 25 ± 5 nM by PXCE which agrees with Kd values reported without cross-linking. Hsp70-Bag3 binding site mutants and small molecule inhibitors of Hsp70-Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC50 values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs. PMID:27434096

  12. A quantitative method for blood lipoproteins using cellulose acetate electrophoresis

    PubMed Central

    Magnani, H. N.; Howard, A. N.

    1971-01-01

    A rapid, inexpensive, and quantitative method is described for obtaining the levels of plasma very low, low, and high density lipoproteins using cellulose acetate electrophoresis and lipid assays without prior separation by ultracentrifuge or other techniques. It involves separation of the lipoproteins by cellulose acetate electrophoresis, followed by their identification with the ozone-Schiff reaction. The total lipoprotein concentration is estimated from the total plasma phospholipid, and the percentage of each component obtained by densitometric analysis of the stained electrophoretograms, using reflected light. For samples with a raised level of very low density lipoprotein, plasma triglyceride analysis is also required. The results obtained by the cellulose acetate electrophoresis method are in good agreement with those by the analytical ultracentrifuge and the preparative ultracentrifuge with refractometry. The theoretical assumptions on which the method is based have been shown to be valid. Images PMID:4110791

  13. Analysis of Protein Oligomerization by Electrophoresis.

    PubMed

    Cubillos-Rojas, Monica; Schneider, Taiane; Sánchez-Tena, Susana; Bartrons, Ramon; Ventura, Francesc; Rosa, Jose Luis

    2016-01-01

    A polypeptide chain can interact with other polypeptide chains and form stable and functional complexes called "oligomers." Frequently, biochemical analysis of these complexes is made difficult by their great size. Traditionally, size exclusion chromatography, immunoaffinity chromatography, or immunoprecipitation techniques have been used to isolate oligomers. Components of these oligomers are then further separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by immunoblotting with specific antibodies. Although they are sensitive, these techniques are not easy to perform and reproduce. The use of Tris-acetate polyacrylamide gradient gel electrophoresis allows the simultaneous analysis of proteins in the mass range of 10-500 kDa. We have used this characteristic together with cross-linking reagents to analyze the oligomerization of endogenous proteins with a single electrophoretic gel. We demonstrate how the oligomerization of p53, the pyruvate kinase isoform M2, or the heat shock protein 27 can be studied with this system. We also show how this system is useful for studying the oligomerization of large proteins such as clathrin heavy chain or the tuberous sclerosis complex. Oligomerization analysis is dependent on the cross-linker used and its concentration. All of these features make this system a very helpful tool for the analysis of protein oligomerization. PMID:27613048

  14. Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.

    ERIC Educational Resources Information Center

    Browning, Mark; Vanable, Joseph

    2002-01-01

    Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)

  15. Muscle protein analysis by two-dimensional gel electrophoresis

    SciTech Connect

    Giometti, C.S.

    1982-01-01

    Two-dimensional electrophoresis of muscle proteins has provided valuable new information concerning the heterogeneity of some of the major contractile proteins, alterations in the protein population of developing muscle fibers during various stages of myogenesis, and protein aberrations that correlate with muscle diseases. As with all electrophoretic techniques, careful attention must be paid to the preparation of samples and the selection of reagents to be used for the protein separations. Two-dimensional electrophoresis is the obvious method of choice when analysis of protein mixtures is required. The routine clinical application of two-dimensional electrophoresis to analysis of muscle tissue remains to be demonstrated. However, methods of sample preparation for two-dimensional electrophoresis compatible with existing clinical procedures have been described, and the equipment for multiple analyses is available. As protein abnormalities related to human myopathy are detected through the use of two-dimensional electrophoresis as a research tool, useful clinical markers of specific myopathic processes will be found. The preliminary work on muscle protein analysis by two-dimensional electrophoresis described in this review has begun a new approach to the enigma of human muscle disease.

  16. Scanning electrochemical microscopy as a readout tool for protein electrophoresis.

    PubMed

    Zhang, Meiqin; Wittstock, Gunther; Shao, Yuanhua; Girault, Hubert H

    2007-07-01

    Scanning electrochemical microscopy (SECM) was used to image silver-stained proteins on a poly(vinylidene difluoride) membrane. The method is based on measuring the current at a scanning microelectrode in the feedback mode. The electrochemical feedback is caused by the redox-mediated etching of the isolated 5 - 10-nm-diameter silver nanoparticles formed during the staining process. Several parameters, such as the redox mediator and the staining protocol, were optimized to ensure a high resolution and a low detection limit, i.e., 0.5 ng of bovine serum albumin (4 x 10(-14) mol) distributed on an area of 1 mm(2) (4 x 10(-16) mol x cm(-2)). Images of beta-lactoglobulin A and myoglobin bands after gel electrophoretic separation and electroblotting were obtained in order to demonstrate that SECM can be employed as a sensitive and quantitative readout method for detection of proteins after gel electrophoresis. An additional advantage is that the silver staining can be removed, allowing further downstream mass spectrometry analysis. PMID:17539601

  17. Analysis of protein therapeutics by capillary electrophoresis: applications and challenges.

    PubMed

    Ma, S

    2005-01-01

    Capillary electrophoresis (CE) has been increasingly used for the analysis of recombinant protein therapeutics in the biotechnology industry over the past several years. In this paper, an overview of the major applications implemented at Genentech Inc. is presented. The applications highlighted in this article are divided into the following three general areas: (i) CE as a replacement for slab gel electrophoresis, particularly capillary electrophoresis-sodium dodecylsulphate and capillary isoelectric focusing; (ii) CE to monitor protein charge heterogeneity as an orthogonal technique to the traditional on-exchange chromatographic methods; and (iii) CE for carbohydrate analysis, including both oligosaccharide and monosaccharide analysis. Overall, the advantages of these CE-based methodologies include automation, ease of quantification, rapid analysis time, enhanced resolution, and robustness when compared to the traditional methods. There are, however, still some challenges in applying CE for protein analysis, particularly in the area of characterization due to the miniaturization nature of CE.

  18. Capillary electrophoresis procedures for serum protein analysis: comparison with established techniques.

    PubMed

    Jenkins, M A; Guerin, M D

    1997-10-10

    Methods using automated capillary electrophoresis (CE) instrumentation are available for serum protein electrophoresis with monoclonal band quantitation, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis separations. The advantages of CE over previous gel methods relate to the time and labour saved by the automated instrumentation. High pI monoclonal bands and cryoglobulin specimens can be successfully analysed by CE. However, if the CE application uses a standard company supplied kit, then the cost savings are often negated by the high cost of the kit. Improvements such as the inclusion of both a UV-Vis as well as a fluorescence detector as standard within the one commercial instrument, the production of coated IEF capillaries with a useful life of at least 100 samples, and the introduction of a capillary array into all commercial instrumentation would ensure greater use of CE within both the clinical and other protein laboratories.

  19. Rapid identification and quantitation for oral bacteria based on short-end capillary electrophoresis.

    PubMed

    Chen, Jin; Ni, Yi; Liu, Chenchen; Yamaguchi, Yoshinori; Chen, Qinmiao; Sekine, Shinichi; Zhu, Xifang; Dou, Xiaoming

    2016-11-01

    High-speed capillary electrophoresis (HSCE) is a promising technology applied in ultra-rapid and high-performance analysis of biomolecules (such as nucleic acids, protein). In present study, the short-end capillary electrophoresis coupled with one novel space domain internal standard method (SDIS) was employed for the rapid and simultaneous analysis of specific genes from three oral bacteria (Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f)). The reliability, reproducibility and accuracy properties of above mentioned SDIS method were investigated in detail. The results showed the target gene fragments of P.g, T.d and T.f could be precisely, fast identified and quantitated within 95s via present short-end CE system. The analyte concentration and the ratio of space domain signals (between target sample and internal standard sample) featured a well linear relationship calculated via SDIS method. And the correlation coefficients R(2) and detection limits for P.g, T.d, T.f genes were 0.9855, 0.9896, 0.9969 and 0.077, 0.114 and 0.098ng/μl, respectively. PMID:27591633

  20. Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins.

    PubMed

    Kinghorn, N M; Norris, C S; Paterson, G R; Otter, D E

    1995-05-12

    The separation of the four major whey proteins by sodium dodecyl sulphate (SDS)-capillary gel electrophoresis (CGE) is described. Whilst commercially purified whey proteins could be analysed using the recommended protocol, the more complex nature of an acid whey and a reconstituted whey protein concentrate (WPC) powder necessitated considerable refinement of the CGE sample buffer. Individual whey proteins in the acid whey and WPC samples were then also separated and quantitated using capillary zone electrophoresis, polyacrylamide gel electrophoresis (PAGE) and HPLC methods and the results were compared. The values obtained for alpha-lactalbumin (alpha-Lac) and beta-lactoglobulin (beta-Lg) were consistent throughout the various methods, although size-exclusion HPLC, SDS-PAGE and SDS-CGE could not separate the two beta-Lg variants or the glycosylated form of alpha-Lac from the beta-Lg. There was considerable variation in the values for the bovine serum albumin and immunoglobulin determined by the different methods and it was concluded that none of the methods could satisfactorily quantitate all four whey proteins.

  1. [Determination of serum proteins by high performance capillary zone electrophoresis].

    PubMed

    Zhang, N; Tang, Y; Hao, D M; Zheng, L; Qiu, G B

    1999-11-01

    The separation method of serum proteins was established with an untreared 50 microns i.d. x 47 cm (40 cm to detector) capillary and detection of absorbance at 200 nm. Analysis was performed by pressure injectction 17.23 kPa.s and by applying 23 kV in the constant voltage mode. Serum samples were diluted 40-folds with assay buffer (12.5 mmol/L sodium borate, 1 mmol/L calcium lactate, 0.7 mmol/L magnesium sulfate, 1 mmol/L EDTA were mixed). A normal control serum protein was separated into 6 fractions. In pregnant serum, the alpha 0 was an additionally unknown fraction. Comparison of capillary electrophoresis with conventional cellulose acetate electrophoresis for analysis of serum proteins from normal control, pregnant women multiple myeloma and tonic rachitis patients indicates that capillary clectrophoresis is a new technique for the analysis of serum proteins because of its high efficiency, on-line data processing and automation. Capillary electrophoresis is the reliable technique for clinical diagnosis of serum protein abnormalities.

  2. Human muscle proteins: analysis by two-dimensional electrophoresis

    SciTech Connect

    Giometti, C.S.; Danon, M.J.; Anderson, N.G.

    1983-09-01

    Proteins from single frozen sections of human muscle were separated by two-dimensional gel electrophoresis and detected by fluorography or Coomassie Blue staining. The major proteins were identical in different normal muscles obtained from either sex at different ages, and in Duchenne and myotonic dystrophy samples. Congenital myopathy denervation atrophy, polymyositis, and Becker's muscular dystrophy samples, however, showed abnormal myosin light chain compositions, some with a decrease of fast-fiber myosin light chains and others with a decrease of slow-fiber light chains. These protein alterations did not correlate with any specific disease, and may be cause by generalized muscle-fiber damage.

  3. Procedures for two-dimensional electrophoresis of proteins

    SciTech Connect

    Tollaksen, S.L.; Giometti, C.S.

    1996-10-01

    High-resolution two-dimensional gel electrophoresis (2DE) of proteins, using isoelectric focusing in the first dimension and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) in the second, was first described in 1975. In the 20 years since those publications, numerous modifications of the original method have evolved. The ISO-DALT system of 2DE is a high-throughput approach that has stood the test of time. The problem of casting many isoelectric focusing gels and SDS-PAGE slab gels (up to 20) in a reproducible manner has been solved by the use of the techniques and equipment described in this manual. The ISO-DALT system of two-dimensional gel electrophoresis originated in the late 1970s and has been modified many times to improve its high-resolution, high-throughput capabilities. This report provides the detailed procedures used with the current ISO-DALT system to prepare, run, stain, and photograph two-dimensional gels for protein analysis.

  4. Two-dimensional electrophoresis and computer imaging: quantitation of human milk casein.

    PubMed

    Goldfarb, M

    1999-01-01

    Because human casein does not precipitate from milk at its isoelectric point as does bovine casein, there is no easy method of quantitation. Casein represents only approximately 30% of the protein fraction in human milk, and the complex methods necessary for isolation cannot be used easily with small samples in a survey of a large number of mothers. Two-dimensional electrophoresis coupled with computer imaging has the potential to compare and quantitate proteins expeditiously using a small sample size. IsoDalt, a denaturing methodology, separates the casein micelle into its component parts, beta-casein, kappa-casein, parakappa-casein and casomorphins. Identification of these spots was made by immunoassay of a Western blot with monoclonal anti-human casein. Two spots at 24 kDa and 26 kDa, thought to be phosphorylated isomers of beta casein, were selected for quantitation. Milk samples from 20 mothers, 8 weeks post partum, were run on two-dimensional (2-D) gels; a slide was taken of each silverstained gel with a Kodak control strip; the slide was scanned into powerMac Photoshop 3 with a Polaroid-Sprintscan; spots were isolated using "threshold", "mask" with IPTK (Imaging Processing Tool Kit, Reindeer Games) a Photoshop plug-in, and transferred to the NIH-Image program. Using an NIH-Image gel macro (Thomas Seebacher), the area and integrated density of the spots were measured. The Kodak control scale provided calibration and conversion to OD units. Visual scanning of the gels and computer units indicated a wide range of concentrations. To understand the range in units of weight, a standard was generated using bovine alpha casein (Sigma). Measurements will be used in a statistical program, Statview (Abacus), in an attempt to correlate information from a questionaire with casein concentration.

  5. Capillary zone electrophoresis and capillary electrophoresis-mass spectrometry for analyzing qualitative and quantitative variations in therapeutic albumin.

    PubMed

    Marie, Anne-Lise; Przybylski, Cédric; Gonnet, Florence; Daniel, Régis; Urbain, Rémi; Chevreux, Guillaume; Jorieux, Sylvie; Taverna, Myriam

    2013-10-24

    The present study describes a reproducible and quantitative capillary zone electrophoresis (CZE) method, which leads to the separation of nine forms (native, oxidized and glycated) of human serum albumin (HSA). In an attempt to identify the different species separated by this CZE method, the capillary electrophoresis was coupled to mass spectrometry using a sheath liquid interface, an optimized capillary coating and a suitable CE running buffer. CE-MS analyses confirmed the heterogeneity of albumin preparation and revealed new truncated and modified forms such as Advanced Glycation End products (AGEs). Assignment of the CZE peaks was carried out using specific antibodies, carboxypeptidase A or sample reduction before or during the CE separation. Thus, five HSA forms were unambiguously identified. Using this CZE method several albumin batches produced by slightly different fractionation ways could be discriminated. Furthermore, analyses of HSA preparations marketed by five pharmaceutical industries revealed that two therapeutic albumins, including that marketed by LFB, contained the highest proportion of native form and lower levels of oxidized forms. PMID:24120174

  6. Capillary zone electrophoresis-mass spectrometry of peptides and proteins

    SciTech Connect

    Loo, J.A.; Udseth, H.R.; Smith, R.D.

    1989-05-01

    Capillary zone electrophoresis (CZE) is attracting extensive attention as a fast, high resolution analytical and micro-preparative separations technique for systems of biological interest. In zone electrophoresis, a column is filled with a single electrolyte having a specific conductivity. The mixture of substances to be separated is applied as a narrow band to the head of a buffer filled column in a band whose width is much less than the length of the column and at a concentration too low to affect the buffer conductivity. An electric field is then applied across the length of the column and the individual substances migrate and separate according to their net electrophoretic velocities. Zone electrophoresis carried out in small diameter (<100 ..mu..m) fused silica capillaries is a relatively new approach to the high resolution separation of aqueous samples. Very small volume samples (picoliter range) with separation efficiencies on the order of 10/sup 6/ theoretical plates for amino acids have been achieved. The method can be further enhanced by the dynamic combination of detection sensitivity and selectivity offered by mass spectrometry (MS). The on-line marriage of mass spectrometry to CZE is accomplished by an atmospheric pressure electrospray ionization source interface. Our research efforts have demonstrated that proteins with MW's greater than 100 kDa can be analyzed using a conventional quadrupole mass spectrometer with an upper m/z limit of only 1700. 6 refs.

  7. High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.

    PubMed

    Thiede, Bernd; Koehler, Christian J; Strozynski, Margarita; Treumann, Achim; Stein, Robert; Zimny-Arndt, Ursula; Schmid, Monika; Jungblut, Peter R

    2013-02-01

    The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows.

  8. High Resolution Quantitative Proteomics of HeLa Cells Protein Species Using Stable Isotope Labeling with Amino Acids in Cell Culture(SILAC), Two-Dimensional Gel Electrophoresis(2DE) and Nano-Liquid Chromatograpohy Coupled to an LTQ-OrbitrapMass Spectrometer*

    PubMed Central

    Thiede, Bernd; Koehler, Christian J.; Strozynski, Margarita; Treumann, Achim; Stein, Robert; Zimny-Arndt, Ursula; Schmid, Monika; Jungblut, Peter R.

    2013-01-01

    The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows. PMID:23033477

  9. Capillary Zone Electrophoresis-Mass Spectrometry of Intact Proteins.

    PubMed

    Domínguez-Vega, Elena; Haselberg, Rob; Somsen, Govert W

    2016-01-01

    Capillary electrophoresis (CE) coupled with mass spectrometry (MS) has proven to be a powerful analytical tool for the characterization of intact proteins. It combines the high separation efficiency, short analysis time, and versatility of CE with the mass selectivity and sensitivity offered by MS detection. This chapter focuses on important practical considerations when applying CE-MS for the analysis of intact proteins. Technological aspects with respect to the use of CE-MS interfaces and application of noncovalent capillary coatings preventing protein adsorption are treated. Critical factors for successful protein analysis are discussed and four typical CE-MS systems are described demonstrating the characterization of different types of intact proteins by CE-MS. These methodologies comprise the use of sheath-liquid and sheathless CE-MS interfaces, and various types of noncovalent capillary coatings allowing efficient and reproducible protein separations. The discussion includes the analysis of lysozyme-drug conjugates and the therapeutic proteins human growth hormone, human interferon-β-1a, and human erythropoietin. PMID:27473479

  10. Data analysis methods for detection of differential protein expression in two-dimensional gel electrophoresis.

    PubMed

    Meunier, Bruno; Bouley, Julien; Piec, Isabelle; Bernard, Carine; Picard, Brigitte; Hocquette, Jean-François

    2005-05-15

    The recent development of microarray technology has led statisticians and bioinformaticians to develop new statistical methodologies for comparing different biological samples. The objective is to identify a small number of differentially expressed genes from among thousands. In quantitative proteomics, analysis of protein expression using two-dimensional gel electrophoresis shows some similarities with transcriptomic studies. Thus, the goal of this study was to evaluate different data analysis methodologies widely used in array analysis using different proteomic data sets of hundreds of proteins. Even with few replications, the significance analysis of microarrays method appeared to be more powerful than the Student's t test in truly declaring differentially expressed proteins. This procedure will avoid wasting time due to false positives and losing information with false negatives.

  11. Leverage principle of retardation signal in titration of double protein via chip moving reaction boundary electrophoresis.

    PubMed

    Zhang, Liu-Xia; Cao, Yi-Ren; Xiao, Hua; Liu, Xiao-Ping; Liu, Shao-Rong; Meng, Qing-Hua; Fan, Liu-Yin; Cao, Cheng-Xi

    2016-03-15

    In the present work we address a simple, rapid and quantitative analytical method for detection of different proteins present in biological samples. For this, we proposed the model of titration of double protein (TDP) and its relevant leverage theory relied on the retardation signal of chip moving reaction boundary electrophoresis (MRBE). The leverage principle showed that the product of the first protein content and its absolute retardation signal is equal to that of the second protein content and its absolute one. To manifest the model, we achieved theoretical self-evidence for the demonstration of the leverage principle at first. Then relevant experiments were conducted on the TDP-MRBE chip. The results revealed that (i) there was a leverage principle of retardation signal within the TDP of two pure proteins, and (ii) a lever also existed within these two complex protein samples, evidently demonstrating the validity of TDP model and leverage theory in MRBE chip. It was also showed that the proposed technique could provide a rapid and simple quantitative analysis of two protein samples in a mixture. Finally, we successfully applied the developed technique for the quantification of soymilk in adulterated infant formula. The TDP-MRBE opens up a new window for the detection of adulteration ratio of the poor food (milk) in blended high quality one.

  12. Protein electrophoresis, immunoelectrophoresis and immunofixation electrophoresis as predictors for high-risk phenotype in familial Waldenström macroglobulinemia.

    PubMed

    McMaster, Mary L; Csako, Gyorgy

    2008-03-01

    Protein electrophoresis is used for the detection, evaluation and follow-up of monoclonal gammopathy (MG) conditions such as Waldenström macroglobulinemia (WM). Immunofixation electrophoresis (IFE) is currently the most common method for isotyping of monoclonal gammopathy because of its superior sensitivity relative to immunoelectrophoresis (IEP). We designed a study to evaluate the clinicobiological relevance of small monoclonal bands detected by serum protein electrophoresis, IEP, and IFE. Serum protein electrophoresis, IEP, and IFE were used to evaluate possible monoclonal gammopathy in 46 members (29 relatives and 17 nonbloodline spouses) from 3 families with multiple cases of WM. IFE identified small monoclonal bands initially missed by IEP in 5 individuals (2 blood relatives, 3 spouses) among 46 study participants. All bands were IgM type. Twenty-three individuals, including the 2 blood relatives and 2 of 3 spouses with monoclonal gammopathy, were then followed for a median of 17 years (range, 13-25). The monoclonal gammopathy progressed in the 2 relatives but disappeared in the spouses, and new IgM MG developed in 2 additional relatives with a prior history of IgM polyclonal gammopathy. Small monoclonal bands detected by IFE in a familial context may be biologically meaningful, both as phenotypic biomarkers and possibly as predictors of high risk for WM. Polyclonal IgM may also be a marker of genetic susceptibility in WM families. Larger studies are needed to confirm these observations.

  13. Development of fully automated quantitative capillary electrophoresis with high accuracy and repeatability.

    PubMed

    Xu, Yuan; Ling, Bang-Zan; Zhu, Wen-Jun; Yao, Dong; Zhang, Lin; Wang, Yan; Yan, Chao

    2016-03-01

    A quantitative capillary electrophoresis (qCE) was developed by utilizing a rotary type of nano-volume injector, an autosampler, and a thermostat with cooling capacity. The accuracy and precision were greatly improved compared with conventional capillary electrophoresis. The 10 nL volume accuracy was guaranteed by the carefully designed nano-injector with an accurate internal loop. The system repeatability (precision) in terms of RSD <0.5% for migration time and 1% for peak area were achieved by using DMSO as a test sample. We believe that this fully automated qCE system has the potential to be employed broadly in quality control and quality assurance in the pharmaceutical industry.

  14. Calibration-free quantitation in microchip zone electrophoresis with conductivity detection.

    PubMed

    Noblitt, Scott D; Henry, Charles S

    2015-08-01

    The relationship between electrophoretic mobility and molar conductivity has previously led to speculation on achieving quantitation in zone electrophoresis without calibration curves when using conductivity detection. However, little work in this area has been pursued, possibly because of the breakdown of simple sensitivity-mobility relationships when working with partially protonated species. This topic is revisited with the aid of electrophoretic simulation software that produces facile predictions of analyte sensitivity relative to an internal standard. Calibration curve slopes for over 50 analyte/internal standard/BGE combinations were measured with both unbiased and electrokinetically biased injections using microchip electrophoresis with conductivity detection. The results were compared to theoretical expectations as computed with PeakMaster software. Good agreement was observed, with some systems being predicted with quantitative accuracy while others showed significant deviations. Some mechanisms that can lead to deviations from theory are demonstrated, but the causes for some discrepancies are still not understood. Overall, this work exhibits another useful application for simulation software, particularly for disposable devices where device-specific calibration curves cannot be collected. It also serves as quantitative validation for some outputs of PeakMaster simulation software.

  15. Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum.

    PubMed

    Corthals, G L; Collins, B M; Mabbutt, B C; Williams, K L; Gooley, A A

    1997-06-27

    Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not

  16. ACUTE PHASE PROTEIN AND ELECTROPHORESIS PROTEIN FRACTION VALUES FOR CAPTIVE AMERICAN FLAMINGOS (PHOENICOPTERUS RUBER).

    PubMed

    Delk, Katie W; Wack, Raymund F; Burgdorf-Moisuk, Anne; Kass, Philip H; Cray, Carolyn

    2015-12-01

    Protein electrophoresis has recognized applications in determining the health status of various species. While reference intervals for electrophoresis have been determined for psittacine and raptor species, there are none reported for Phoenicopteriformes species. Reference intervals for haptoglobin and protein fractions obtained by electrophoresis were determined for the American flamingo (Phoenicopterus ruber) based on plasma samples from 39 captive birds. The reference intervals were as follows: haptoglobin, 0.17-0.8 mg/ml; total protein, 3.65-6.38 g/dl; prealbumin, 0.26-1.9 g/dl; albumin, 1.51-3.12 g/dl; α-1 globulin, 0.06-0.38 g/dl; α-2 globulin, 0.17-0.67 g/dl; β globulin, 0.38-1.33 g/dl; γ globulin, 0.26-0.68 g/dl; albumin : globulin ratio, 0.93-2.17. As captive flamingos often suffer from pododermatitis, feet of all flamingos were scored to determine if pododermatitis would be reflected in the acute phase proteins. Spearman rank correlation was performed on each of the protein fractions and pododermatitis scores, and only albumin had a significant correlation. This indicates that albumin, as a negative acute phase protein, may be a marker for this disease process.

  17. Quantitative detection of protein arrays.

    PubMed

    Levit-Binnun, Nava; Lindner, Ariel B; Zik, Ory; Eshhar, Zelig; Moses, Elisha

    2003-03-15

    We introduce a quantitative method that utilizes scanning electron microscopy for the analysis of protein chips (SEMPC). SEMPC is based upon counting target-coated gold particles interacting specifically with ligands or proteins arrayed on a derivative microscope glass slide by utilizing backscattering electron detection. As model systems, we quantified the interactions of biotin and streptavidin and of an antibody with its cognate hapten. Our method gives quantitative molecule-counting capabilities with an excellent signal-to-noise ratio and demonstrates a broad dynamic range while retaining easy sample preparation and realistic automation capability. Increased sensitivity and dynamic range are achieved in comparison to currently used array detection methods such as fluorescence, with no signal bleaching, affording high reproducibility and compatibility with miniaturization. Thus, our approach facilitates the determination of the absolute number of molecules bound to the chip rather than their relative amounts, as well as the use of smaller samples.

  18. Quantitative enantiomeric analysis of chlorcyclizine, hydroxyzine, and meclizine by capillary electrophoresis.

    PubMed

    Ho, Yu- Hsiang; Wu, Hsin- Lung; Wu, Shou- Mei; Chen, Su- Hwei; Kou, Hwang- Shang

    2003-07-01

    A simple capillary zone electrophoresis method was developed for the quantitative enantiomeric analysis of piperazine antihistamines with teratogenic suspicion in animals. Enantioseparation of chlorcyclizine, hydroxyzine, and meclizine was performed in glycine buffer (0.6 mol L(-1); pH 3.00) with sulfated beta-cyclodextrin (5 mg mL(-1)) as a chiral selector; and the separated drugs were monitored by ultra-violet detector. The lower quantitation of the individual enantiomer is attainable at 10 micro mol L(-1), using an achiral piperazine drug (cyclizine) as internal standard. The method is simple and rapid with a short run time (<5 min) for the analysis of chlorcyclizine, hydroxyzine or meclizine enantiomers. PMID:12830360

  19. Assay of multiplex proteins from cell metabolism based on tunable aptamer and microchip electrophoresis.

    PubMed

    Lin, Xuexia; Chen, Qiushui; Liu, Wu; Yi, Linglu; Li, Haifang; Wang, Zhihua; Lin, Jin-Ming

    2015-01-15

    A simple and rapid method for multiplex protein assay based on tunable aptamer by microchip electrophoresis has been developed. Different lengths of aptamers can modulate the electrophoretic mobility of proteins, allowing the protein molecules to be effectively separated in hydroxyethyl cellulose buffer with 1.00 mM magnesium ion. A non-specific DNA was exploited as an internal standard to achieve the quantitative assay and to reduce the interference. A fluorescence dye SYBR gold was exploited to improve the sensitivity and to suppress the interference from sample matrix. Under optimum conditions, quantitative assay of PDGF-BB (R(2)=0.9986), VEGF165 (R(2)=0.9909), and thrombin (R(2)=0.9947) were achieved with a dynamic range in the 5.00-150.0 nM and RSDs in the 5.87-16.3% range. The recoveries were varied from 83.6% to 113.1%. Finally, the proposed method was successfully applied to analyze cell secretions, and then the concentration of PDGF-BB and VEGF165 were detected from 5.15 nM to 2.03 nM, and 3.14 to 2.53 nM, respectively, indicating the established method can be used to analyze cell secretions.

  20. Attomole quantitation of protein separations with accelerator mass spectrometry

    SciTech Connect

    Vogel, J S; Grant, P G; Buccholz, B A; Dingley, K; Turteltaub, K W

    2000-12-15

    Quantification of specific proteins depends on separation by chromatography or electrophoresis followed by chemical detection schemes such as staining and fluorophore adhesion. Chemical exchange of short-lived isotopes, particularly sulfur, is also prevalent despite the inconveniences of counting radioactivity. Physical methods based on isotopic and elemental analyses offer highly sensitive protein quantitation that has linear response over wide dynamic ranges and is independent of protein conformation. Accelerator mass spectrometry quantifies long-lived isotopes such as 14C to sub-attomole sensitivity. We quantified protein interactions with small molecules such as toxins, vitamins, and natural biochemicals at precisions of 1-5% . Micro-proton-induced-xray-emission quantifies elemental abundances in separated metalloprotein samples to nanogram amounts and is capable of quantifying phosphorylated loci in gels. Accelerator-based quantitation is a possible tool for quantifying the genome translation into proteome.

  1. THERMAL DETECTION OF DNA AND PROTEINS DURING GEL ELECTROPHORESIS

    SciTech Connect

    R. JOHNSTON

    2000-08-01

    This is the final report of a three-year, Laboratory-Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The goal of this project was to try to detect unstained, untagged, unlabeled DNA bands in real-time during gel electrophoresis using simple thermal measurements. The technical and ES&H advantages to this approach could potentially be quite significant, especially given the extreme importance of gel electrophoresis to a wide variety of practical and research fields. The project was unable to demonstrate sufficient thermal sensitivity to detect DNA bands. It is clear that we still do not understand the gel electrophoresis phenomenon very well. The temperature control techniques developed during the course of this project have other useful applications.

  2. Minimizing adsorption of histidine-tagged proteins for the study of protein-deoxyribonucleic acid interactions by kinetic capillary electrophoresis.

    PubMed

    Liyanage, Ruchi; Krylova, Svetlana M; Krylov, Sergey N

    2013-12-27

    Affinity interactions between DNA and proteins play a crucial role in many cellular processes. Kinetic Capillary Electrophoresis is a highly efficient tool for kinetic and equilibrium studies of protein-DNA interactions. Recombinant proteins, which are typically used for in vitro studies of protein-DNA interactions, are often expressed with a His tag to aid in their purification. In this work, we study how His tags affect Kinetic Capillary Electrophoresis analysis of protein-DNA interactions. We found that the addition of a His tag can increase or decrease protein adsorption to a bare-silica capillary wall, dependent on the protein. For Kinetic Capillary Electrophoresis measurements, it is essential to have as little protein adsorption as possible. We screened a number of capillary coatings to reduce adsorption of the His-tagged DNA mismatch repair protein MutS to the capillary wall and found that UltraTrol LN was the most effective coating. The effectiveness of the coating was confirmed with the prevention of adsorption of His-tagged fat mass and obesity-associated protein. Under typical conditions, the coating reduced protein adsorption to a level at which accurate Kinetic Capillary Electrophoresis analysis of protein-DNA interactions was possible. We further used Kinetic Capillary Electrophoresis to study how the His tag affected Kd of protein-DNA interactions for the MutS protein. Using UltraTrol LN, we found that the effect of the His tag was insignificant.

  3. Two-dimensional electrophoresis of liver proteins: characterization of a drug-induced hepatomegaly in rats.

    PubMed

    Newsholme, S J; Maleeff, B F; Steiner, S; Anderson, N L; Schwartz, L W

    2000-06-01

    Two-dimensional electrophoresis (2-DE) of liver proteins was applied to further characterize an unusual drug-induced increase in hepatocellular rough endoplasmic reticulum (RER) in Sprague-Dawley rats given a substituted pyrimidine derivative. Absolute liver weights of drug-treated rats (9.9 +/- 0.4 g) increased above vehicle-treated controls (7.2 +/- 0.2 g) by 37%. Light microscopy revealed diffuse granular basophilia of the hepatocellular cytoplasm, uncharacteristic of hepatocytes and suggested cells rich in ribosomes, which was confirmed by electron microscopy. Immunostaining for cell proliferation, viz., 5-bromo-2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA), indicated marked hepatocellular proliferative activity. 2-DE of solubilized liver using an ISO-DALT gel system indicated significant (p<0.001) quantitative changes in at least 17 liver proteins (12 increased, 5 decreased) compared to controls. The protein with the largest increase was homologous to acute-phase reactant, contrapsin-like protein inhibitor-6. Other markedly upregulated proteins were methionine adenosyltransferase, a catalyst in methionine/ATP metabolism and mitochondrial HMG-CoA synthase, involved in cholesterol synthesis. The complementary strategies of 2-DE coupled either with database spot mapping or protein isolation and amino acid sequencing successfully identified a subset of proteins from xenobiotic-damaged rodent livers, the expression of which differed from controls. However, the current bioinformatics platform for rodent hepatic proteins and limited knowledge of specific protein functionality restricted application of this proteomics profile to further define a mechanistic basis for this unusual hepatotoxicity.

  4. [Quantitative determination of morphine in opium powder by addition and correlation method using capillary electrophoresis].

    PubMed

    Sun, Guo-xiang; Miao, Ju-ru; Wang, Yu; Sun, Yu-qing

    2002-01-01

    The morphine in opium powder has been quantitatively determined by addition and correlation method (ACM), in which capillary zone electrophoresis was applied, and the average recovery was 100.6%. The relative standard deviation (RSD) of migration time was not more than 2.4%, the RSD of relative migration time was not more than 1.1%, and the RSD of the relative area was not more than 0.51%. Meanwhile, the contrast test has been done by the calibration curve method with an internal standard correlation. The content of morphine in opium powder determined by ACM was the same as that by using the calibration curve method with an internal standard correlated. The study shows that ACM is simple, quick and accurate.

  5. Capillary electrophoresis to quantitate gossypol enantiomers in cotton flower petals and seed.

    PubMed

    Vshivkov, Sergey; Pshenichnov, Egor; Golubenko, Zamira; Akhunov, Alik; Namazov, Shadman; Stipanovic, Robert D

    2012-11-01

    Gossypol is a toxic compound that occurs as a mixture of enantiomers in cotton plant tissues including seed and flower petals. The (-)-enantiomer is more toxic to non-ruminant animals. Efforts to breed cottonseed with a low percentage of (-)-gossypol requires determination of the (+)- to (-)-gossypol ratio in seed and flower petals. We report a method to quantitatively determine the total gossypol and percent of its enantiomers in cotton tissues using high performance capillary electrophoresis (HPCE). The method utilizes a borate buffer at pH 9.3 using a capillary with internal diameter of 50μm, effective length of 24.5cm, 15kV and cassette temperature of 15°C. This method provides high accuracy and reproducible results with a limit of detection of the individual enantiomers of less than 36ng/mL providing base line separation in less than 6min. PMID:23122406

  6. Analysis of soybean embryonic axis proteins by two-dimensional gel electrophoresis and mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and subsequent mass spectrometry (MS) for protein identification was applied to establish a proteomic reference map for the soybean embryonic axis. Proteins were extracted from dissecte...

  7. Quantitative assessment of fluorescent proteins.

    PubMed

    Cranfill, Paula J; Sell, Brittney R; Baird, Michelle A; Allen, John R; Lavagnino, Zeno; de Gruiter, H Martijn; Kremers, Gert-Jan; Davidson, Michael W; Ustione, Alessandro; Piston, David W

    2016-07-01

    The advent of fluorescent proteins (FPs) for genetic labeling of molecules and cells has revolutionized fluorescence microscopy. Genetic manipulations have created a vast array of bright and stable FPs spanning blue to red spectral regions. Common to autofluorescent FPs is their tight β-barrel structure, which provides the rigidity and chemical environment needed for effectual fluorescence. Despite the common structure, each FP has unique properties. Thus, there is no single 'best' FP for every circumstance, and each FP has advantages and disadvantages. To guide decisions about which FP is right for a given application, we have quantitatively characterized the brightness, photostability, pH stability and monomeric properties of more than 40 FPs to enable straightforward and direct comparison between them. We focus on popular and/or top-performing FPs in each spectral region. PMID:27240257

  8. Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

    PubMed Central

    Birdsall, Robert E.; Koshel, Brooke M.; Hua, Yimin; Ratnayaka, Saliya N.; Wirth, Mary J.

    2013-01-01

    Sieving of proteins in silica colloidal crystals of mm dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 μm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization. PMID:23229163

  9. Separation and quantitation of phycobiliproteins using phytic acid in capillary electrophoresis with laser-induced fluorescence detection.

    PubMed

    Viskari, Pertti J; Colyer, Christa L

    2002-10-01

    The similar electrophoretic mobilities and sizes of several of the phycobiliproteins, which are derived from the photosynthetic apparatus of cyanobacteria and eukaryotic algae, render their separation and quantitation a challenging problem. However, we have developed a suitable capillary electrophoresis (CE) method that employs a phytic acid-boric acid buffer and laser-induced fluorescence (LIF) detection with a single 594 nm He-Ne laser. This method takes advantage of the remarkably high quantum yields of these naturally fluorescent proteins, which can be attributed to their linear tetrapyrrole chromophores covalently bound to cysteinyl residues. As such, limits of detection of 1.18 x 10(-14), 5.26 x 10(-15), and 2.38 x 10(-15) mol/l were obtained for R-phycoerythrin, C-phycocyanin, and allophycocyanin proteins, respectively, with a linear dynamic range of eight orders of magnitude in each case. Unlike previously published CE-LIF methods, this work describes the separation of all three major classes of phycobiliproteins in under 5 min. Very good recoveries, ranging from 93.2 to 105.5%, were obtained for a standard mixture of the phycobiliproteins, based on seven-point calibration curves for both peak height and peak area. It is believed that this development will prove useful for the determination of phycobiliprotein content in naturally occurring cyanobacteria populations, thus providing a useful tool for understanding biological and chemical oceanographic processes.

  10. Separation of Recombinant Therapeutic Proteins Using Capillary Gel Electrophoresis and Capillary Isoelectric Focusing.

    PubMed

    De Jong, Caitlyn A G; Risley, Jessica; Lee, Alexis K; Zhao, Shuai Sherry; Chen, David D Y

    2016-01-01

    Detailed step-by-step methods for protein separation techniques based on capillary electrophoresis (CE) are described in this chapter. Focus is placed on two techniques, capillary gel electrophoresis (CGE) and capillary isoelectric focusing (cIEF). CGE is essentially gel electrophoresis, performed in a capillary, where a hydrogel is used as a sieving matrix to separate proteins or peptides based on size. cIEF separates proteins or peptides based on their isoelectric point (pI), the pH at which the protein or peptide bears no charges. Detailed protocols and steps (including capillary preparation, sample preparation, CE separation conditions, and detection) for both CGE and cIEF presented so that readers can follow the described methods in their own labs. PMID:27473487

  11. Bence-Jones protein - quantitative

    MedlinePlus

    Immunoglobulin light chains - urine; Urine Bence-Jones protein ... Bence-Jones proteins are a part of regular antibodies called light chains. These proteins are not normally in urine. Sometimes, when ...

  12. Fully automated two-dimensional electrophoresis system for high-throughput protein analysis.

    PubMed

    Hiratsuka, Atsunori; Yokoyama, Kenji

    2009-01-01

    A fully automated two-dimensional electrophoresis (2DE) system for rapid and reproducible protein analysis is described. 2DE that is a combination of isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein expression analysis. Here, all the operations are achieved in a shorter time and all the transferring procedures are performed automatically. The system completed the entire process within 1.5 h. A device configuration, operational procedure, and data analysis are described using this system.

  13. Multidimensional microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

    PubMed

    Wu, Ruige; Wang, Zhiping; Fung, Ying Sing

    2015-01-01

    Functional proteins have been found in infant milk formula as supplements, added by an increasing number of manufacturers. Their supplementations are expected to be controlled and monitored. Here, we describe a microchip-integrated CE method for the determination of these low levels of functional proteins in a protein-rich sample matrix. On-chip isoelectric focusing (IEF) is used to separate high-abundance proteins from low-abundance proteins instead of using some complicated time-consuming protein purification process. After that, transient isotachophoresis hyphenated capillary zone electrophoresis (t-ITP-CZE) can preconcentrate, separate, and analyze transferred functional proteins in the embedded capillary under UV detection. PMID:25673487

  14. Multidimensional microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

    PubMed

    Wu, Ruige; Wang, Zhiping; Fung, Ying Sing

    2015-01-01

    Functional proteins have been found in infant milk formula as supplements, added by an increasing number of manufacturers. Their supplementations are expected to be controlled and monitored. Here, we describe a microchip-integrated CE method for the determination of these low levels of functional proteins in a protein-rich sample matrix. On-chip isoelectric focusing (IEF) is used to separate high-abundance proteins from low-abundance proteins instead of using some complicated time-consuming protein purification process. After that, transient isotachophoresis hyphenated capillary zone electrophoresis (t-ITP-CZE) can preconcentrate, separate, and analyze transferred functional proteins in the embedded capillary under UV detection.

  15. Buffer additives other than the surfactant sodium dodecyl sulfate for protein separations by capillary electrophoresis.

    PubMed

    Corradini, D

    1997-10-10

    The different compounds utilized as additives to the electrolyte solutions employed in protein capillary zone electrophoresis (CZE) for minimizing protein-capillary wall interactions, for improving selectivity and resolution and for controlling the electroosmotic flow are reviewed. The dependence of the electroosmotic flow on the different variables that can be affected by the incorporation of an additive into the electrolytic solution is discussed. A list of the most effective additives employed for protein separations by CZE is reported in Appendix A.

  16. Chemical modification of proteins to improve the accuracy of their relative molecular mass determination by electrophoresis.

    PubMed

    Dolnik, Vladislav; Gurske, William A

    2011-10-01

    We studied the electrophoretic behavior of basic proteins (cytochrome c and histone III) and developed a carbamylation method that normalizes their electrophoretic size separation and improves the accuracy of their relative molecular mass determined electrophoretically. In capillary zone electrophoresis with cationic hitchhiking, native cytochrome c does not sufficiently bind cationic surfactants due to electrostatic repulsion between the basic protein and cationic surfactant. Carbamylation suppresses the strong positive charge of the basic proteins and results in more accurate relative molecular masses.

  17. Protein electrophoresis as a diagnostic and prognostic tool in raptor medicine.

    PubMed

    Tatum, L M; Zaias, J; Mealey, B K; Cray, C; Bossart, G D

    2000-12-01

    Plasma proteins of 139 healthy adult birds of prey from 10 species were separated by electrophoresis to characterize and document normal reference ranges and species-specific electrophoretic patternsand to evaluate the value of this technique for health screening, disease diagnosis, and prognostic indication. Species studied included bald eagle (Haliaeetus leucocephalus), red-tailed hawk (Buteo jamaicensis), barn owl (Tyto alba), great horned owl (Bubo virginianus), turkey vulture (Cathartes aura), Harris' hawk (Parabuteo unicinctus), Stellar's sea eagle (Haliaeetus pelagicus), barred owl (Strix varia), screech owl (Otus asio), and black vulture (Coragyps atratus). Several clinical cases show the diagnostic/therapeutic value of protein electrophoresis in raptors. This study establishes species-specific reference ranges for several birds of prey and discusses the benefit of electrophoresis as a diagnostic technique in health screens, as a diagnostic aid in conjunction with other tests, and as a prognostic indicator in clinical evaluation of raptors. PMID:11428396

  18. Determination of free L- and D-alanine in hydrolysed protein fertilisers by capillary electrophoresis.

    PubMed

    Cavani, Luciano; Ciavatta, Claudio; Gessa, Carlo

    2003-01-24

    of racemisation of hydrolysed protein fertilisers (HPFs) using an The objective of this study was to determine the degree inexpensive and easy to handle analytical method for qualitative control of the products. Using a polyacrylamide coated capillary and a run buffer containing 0.1 M Tris-borate+2.5 mM EDTA-Na2+0.1% sodium dodecylsulfate+10 mM beta-cyclodextrin a quantitative separation of D- and L-alanine (Ala) was made from an not treated HPF sample derivatised with dansyl chlorine by capillary electrophoresis. The D-Ala:[D-Ala+L-Ala] ratio, called degree of racemisation (RD), was calculated. The analysis of ten commercial HPFs has shown that more than 60% of HPFs have an RD > or = 40%. while only one product has shown an RD <5%. These results showed that most of the HPFs on the market are obtained with strong hydrolytic processes and high contents of D-amino acids are probably less effective as plant nutrients or even potentially dangerous to plants.

  19. [Total protein analysis by two-dimensional electrophoresis in cysticerci of Taenia solium and Taenia asiatica].

    PubMed

    Fang, Wen; Xiao, Liang-Liang; Bao, Huai-En; Mu, Rong

    2011-06-01

    Two 20-day-old three-way crossed hybrid pigs were infected with 80000 Taenia solium or T. asiatica eggs, respectively. Immature cysticerci of the two species in liver were collected at 40 days after infection. The total proteins were separated by two-dimensional electrophoresis, and differentially expressed proteins were analyzed by Image-Master 2D Platinum 6.0 software. The results showed that there were (236 +/- 12) and (231 +/- 14) protein spots in 2D electrophoresis gel images of T. solium and T. asiatica, respectively, with 3 proteins up-regulated and 7 proteins down-regulated in T. solium cysticercus by 2-fold or more compared with those in T. asiatica cysticercus.

  20. Pneumatic Microvalve-Based Hydrodynamic Sample Injection for High-Throughput, Quantitative Zone Electrophoresis in Capillaries

    PubMed Central

    2015-01-01

    A hybrid microchip/capillary electrophoresis (CE) system was developed to allow unbiased and lossless sample loading and high-throughput repeated injections. This new hybrid CE system consists of a poly(dimethylsiloxane) (PDMS) microchip sample injector featuring a pneumatic microvalve that separates a sample introduction channel from a short sample loading channel, and a fused-silica capillary separation column that connects seamlessly to the sample loading channel. The sample introduction channel is pressurized such that when the pneumatic microvalve opens briefly, a variable-volume sample plug is introduced into the loading channel. A high voltage for CE separation is continuously applied across the loading channel and the fused-silica capillary separation column. Analytes are rapidly separated in the fused-silica capillary, and following separation, high-sensitivity MS detection is accomplished via a sheathless CE/ESI-MS interface. The performance evaluation of the complete CE/ESI-MS platform demonstrated that reproducible sample injection with well controlled sample plug volumes could be achieved by using the PDMS microchip injector. The absence of band broadening from microchip to capillary indicated a minimum dead volume at the junction. The capabilities of the new CE/ESI-MS platform in performing high-throughput and quantitative sample analyses were demonstrated by the repeated sample injection without interrupting an ongoing separation and a linear dependence of the total analyte ion abundance on the sample plug volume using a mixture of peptide standards. The separation efficiency of the new platform was also evaluated systematically at different sample injection times, flow rates, and CE separation voltages. PMID:24865952

  1. Screening of Small-Molecule Inhibitors of Protein-Protein Interaction with Capillary Electrophoresis Frontal Analysis.

    PubMed

    Xu, Mei; Liu, Chao; Zhou, Mi; Li, Qing; Wang, Renxiao; Kang, Jingwu

    2016-08-16

    A simple and effective method for identifying inhibitors of protein-protein interactions (PPIs) was developed by using capillary electrophoresis frontal analysis (CE-FA). Antiapoptotic B-cell-2 (Bcl-2) family member Bcl-XL protein, a 5-carboxyfluorescein labeled peptide truncated from the BH3 domain of Bid (F-Bid) as the ligand, and a known Bcl-XL-Bid interaction inhibitor ABT-263 were employed as an experimental model for the proof of concept. In CE-FA, the free ligand is separated from the protein and protein-ligand complex to permit the measurement of the equilibrium concentration of the ligand, hence the dissociation constant of the protein-ligand complex. In the presence of inhibitors, formation of the protein-ligand complex is hindered, thereby the inhibition can be easily identified by the raised plateau height of the ligand and the decayed plateau of the complex. Further, we proposed an equation used to convert the IC50 value into the inhibition constant Ki value, which is more useful than the former for comparison. In addition, the sample pooling strategy was employed to improve the screening throughput more than 10 times. A small chemical library composed of synthetic compounds and natural extracts were screened with the method, two natural products, namely, demethylzeylasteral and celastrol, were identified as new inhibitors to block the Bcl-XL-Bid interaction. Cell-based assay was performed to validate the activity of the identified compounds. The result demonstrated that CE-FA represents a straightforward and robust technique for screening of PPI inhibitors. PMID:27425825

  2. Can You Solve the Crime? Using Agarose Electrophoresis To Identify an Unknown Colored Protein.

    ERIC Educational Resources Information Center

    Wiltfong, Cynthia L.; Chester, Emily; Albertin, Faith; Smith, Julia; Hall, Judith C.; Arth, Emily C.; Martin, Stephanie

    2003-01-01

    Describes a lab that introduces agarose electrophoresis techniques and basic information on proteins to middle school and high school students. Insists that, built around a scenario in which students must solve a crime, the lab has real-world applications that should spark student interest. (KHR)

  3. Introducing Proteomics in the Undergraduate Curriculum: A Simple 2D Gel Electrophoresis Exercise with Serum Proteins

    ERIC Educational Resources Information Center

    Kim, Thomas D.; Craig, Paul A.

    2010-01-01

    Two-dimensional gel electrophoresis (2DGE) remains an important tool in the study of biological systems by proteomics. While the use of 2DGE is commonplace in research publications, there are few instructional laboratories that address the use of 2DGE for analyzing complex protein samples. One reason for this lack is the fact that the preparation…

  4. Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins.

    PubMed

    Bjellqvist, B; Sanchez, J C; Pasquali, C; Ravier, F; Paquet, N; Frutiger, S; Hughes, G J; Hochstrasser, D

    1993-12-01

    We describe some simple modifications to the micropreparative two-dimensional (2-D) electrophoresis procedure using immobilized pH gradients in the first dimension and sodium dodecyl sulfate-electrophoresis in the second dimension. The geometry of the immobilized pH gradient strips has been changed to allow the use of large sample application cups that can accommodate greater sample volumes. The use of narrow range immobilized pH gradients with a large sample loading volume allows an efficient resolubilization of polypeptides after the first dimension. As a result, the vertical streaking caused by too high a protein concentration is eliminated in the second dimension. Protein identification by N-terminal sequencing is facilitated by the large protein load (1-15 mg) which can be employed using this modification. Spots not normally detectable on conventional analytical 2-D maps, even with sensitive silver staining, are observed. Results for plasma and liver proteins are shown. PMID:8137805

  5. Identification of Methanococcus Jannaschii Proteins in 2-D Gel Electrophoresis Patterns by Mass Spectrometry

    DOE R&D Accomplishments Database

    Liang, X.

    1998-06-10

    The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.

  6. Database of two-dimensional polyacrylamide gel electrophoresis of proteins labeled with CyDye DIGE Fluor saturation dye.

    PubMed

    Fujii, Kazuyasu; Kondo, Tadashi; Yokoo, Hideki; Okano, Tetsuya; Yamada, Masayo; Yamada, Tesshi; Iwatsuki, Keiji; Hirohashi, Setsuo

    2006-03-01

    CyDye DIGE Fluor saturation dye (saturation dye, GE Healthcare Amersham Biosciences) enables highly sensitive 2-D PAGE. As the dye reacts with all reduced cysteine thiols, 2-D PAGE can be performed with a lower amount of protein, compared with CyDye DIGE Fluor minimal dye (GE Healthcare Amersham Biosciences), the sensitivity of which is equivalent to that of silver staining. We constructed a 2-D map of the saturation dye-labeled proteins of a liver cancer cell line (HepG2) and identified by MS 92 proteins corresponding to 123 protein spots. Functional classification revealed that the identified proteins had chaperone, protein binding, nucleotide binding, metal ion binding, isomerase activity, and motor activity. The functional distribution and the cysteine contents of the proteins were similar to those in the most comprehensive 2-D database of hepatoma cells (Seow et al.., Electrophoresis 2000, 21, 1787-1813), where silver staining was used for protein visualization. Hierarchical clustering on the basis of the quantitative expression profiles of the 123 characterized spots labeled with two charge- and mass-matched saturation dyes (Cy3 and Cy5) discriminated between nine hepatocellular carcinoma cell lines and primary cultured hepatocytes from five individuals, suggesting the utility of saturation dye and our database for proteomic studies of liver cancer.

  7. Speciation of protein-bound trace elements by gel electrophoresis and atomic spectrometry.

    PubMed

    Ma, Renli; McLeod, Cameron W; Tomlinson, Kerry; Poole, Robert K

    2004-08-01

    The metabolism of trace elements, in particular their binding to proteins in biological systems is of great importance in biochemical, toxicological, and pharmacological studies. As a result there has been a sustained interest over the last two decades in the speciation of protein-bound metals. Various analytical approaches have been employed, combining efficient separation of metalloproteins by liquid chromatography or electrophoresis with high-sensitivity elemental detection. Slab-gel electrophoresis (GE) is a key platform for high-resolution protein separation, and has been combined with autoradiography and various atomic spectrometric techniques for in-gel determination of protein-bound metals. Recently, the combination of GE with state-of-the-art inductively coupled plasma-mass spectrometry (ICP-MS), particularly when linked to laser ablation (LA) for direct gel interrogation, has opened up new opportunities for rapid characterization of metalloproteins. The use of GE and atomic spectrometry for the speciation of protein-bound trace elements is reviewed in this paper. Technical requirements for gel electrophoresis/atomic spectrometric measurement are considered in terms of method compatibilities, detection capability and potential usefulness. The literature is also surveyed to illustrate current status and future trends. PMID:15300764

  8. Urine protein electrophoresis and immunoelectrophoresis using unconcentrated or minimally concentrated urine samples.

    PubMed

    Roden, Anja C; Lockington, Karen S; Tostrud, Linda J; Katzmann, Jerry A

    2008-07-01

    Our objective was to evaluate a gel system that uses unconcentrated urine specimens for protein electrophoresis (PEL) and immunofixation electrophoresis (IFE) in patients with monoclonal gammopathies. For the study, 222 urine specimens were analyzed by our current PEL method (Helena Laboratories, Beaumont, TX) and by a system that recommends use of unconcentrated urine (Sebia, Norcross, GA). M protein concentrations were compared in the 43 cases with a measurable M spike. IFE was performed on 111 of the samples using both methods. There was a 97% concordance for detection of PEL abnormalities. The concordance for IFE was 98%. M protein concentrations by the 2 methods correlated well (r2=0.99; slope, 1.04). Cases with insufficient urine volumes for concentration (PEL, 7; IFE, 20) were analyzed in the Sebia gel system, and in 11 cases (PEL, 2; IFE, 9) an M protein was identified.High-resolution gel electrophoresis of urine using the Sebia system offers similar performance for detection, characterization, and quantification of M proteins when compared with our current gel system. Testing unconcentrated urine specimens will mean fewer sample rejections owing to insufficient sample volume.

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

    PubMed

    Tsai, Irene Y; Tomczyk, Nancy; Eckmann, Joshua I; Composto, Russell J; Eckmann, David M

    2011-05-01

    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. PMID:21277175

  10. Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry.

    PubMed

    Singh, Pramod Kumar; Shrivastava, Nidhi; Chaturvedi, Krishna; Sharma, Bechan; Bhagyawant, Sameer S

    2016-01-01

    Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE) across a broad range 3.0-10.0 immobilized pH gradient (IPG) strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected in these accessions. In-gel protein expression patterns revealed three protein spots as upregulated and three other as downregulated. Using trypsin in-gel digestion, these differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) which showed 45% amino acid homology of chickpea seed storage proteins with Arabidopsis thaliana. PMID:27144024

  11. Fully automated two-dimensional electrophoresis system for high-throughput protein analysis.

    PubMed

    Hiratsuka, Atsunori; Kinoshita, Hideki; Maruo, Yuji; Takahashi, Katsuyoshi; Akutsu, Satonari; Hayashida, Chie; Sakairi, Koji; Usui, Keisuke; Shiseki, Kisho; Inamochi, Hajime; Nakada, Yoshiko; Yodoya, Kouhei; Namatame, Ichiji; Unuma, Yutaka; Nakamura, Makoto; Ueyama, Kosuke; Ishii, Yoshinori; Yano, Kazuyoshi; Yokoyama, Kenji

    2007-08-01

    We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.

  12. Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis.

    PubMed

    Sørensen, Birgitte Kjaer; Højrup, Peter; Østergård, Erik; Jørgensen, Charlotte Svaerke; Enghild, Jan; Ryder, Lisa Rebekka; Houen, Gunnar

    2002-05-01

    A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry. PMID:11969186

  13. [Blood serum proteins studied by a method of polyacrylamide gel disc electrophoresis in children with phenylketonuria].

    PubMed

    Lupinovich, V L; Kopylova, N V

    1980-01-01

    Protein spectra of blood serum were studied in 25 children suffering from phenlyketonuria (PKU) by disc electrophoresis in polyacrylamide gel. 14 children were examined in the course of being given a specialized diet on the basis of berlafen and 11 children were examined after they were switched over to common diet. PKU patients had an increased protein content in the zone of transferrins. After switching over to common diet the patients demonstrated the reduced protein content, increased amount of postalbumins of medium and low electrophoretic mobility, and transferrins as well.

  14. Indirect tissue electrophoresis: a new method for analyzing solid tissue protein.

    PubMed

    Smith, A C

    1988-01-01

    1. The eye lens core (nucleus) has been a valuable source of molecular biologic information. 2. In these studies, lens nuclei are usually homogenized so that any protein information related to anatomical subdivisions, or layers, of the nucleus is lost. 3. The present report is of a new method, indirect tissue electrophoresis (ITE), which, when applied to fish lens nuclei, permitted (a) automatic correlation of protein information with anatomic layer, (b) production of large, clear electrophoretic patterns even from small tissue samples and (c) detection of more proteins than in liquid extracts of homogenized tissues. 4. ITE seems potentially applicable to a variety of solid tissues.

  15. Capillary electrophoresis determination of non-protein amino acids as quality markers in foods.

    PubMed

    Pérez-Míguez, Raquel; Marina, María Luisa; Castro-Puyana, María

    2016-01-01

    Non-protein amino acids mainly exist in food as products formed during food processing, as metabolic intermediates or as additives to increase nutritional and functional properties of food. This fact makes their analysis and determination an attractive field in food science since they can give interesting information on the quality and safety of foods. This article presents a comprehensive review devoted to describe the latest advances in the development of (achiral and chiral) analytical methodologies by capillary electrophoresis and microchip capillary electrophoresis for the analysis of non-protein amino acids in a variety of food samples. Most relevant information related to sample treatment, experimental separation and detection conditions, preconcentration strategies and limits of detection will be provided.

  16. Green fluorescent protein as a quantitative tool.

    PubMed

    Hack, N J; Billups, B; Guthrie, P B; Rogers, J H; Muir, E M; Parks, T N; Kater, S B

    2000-02-15

    Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.

  17. Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis.

    PubMed

    Creamer, Jessica S; Oborny, Nathan J; Lunte, Susan M

    2014-07-01

    The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis.

  18. Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

    PubMed Central

    Creamer, Jessica S.; Oborny, Nathan J.; Lunte, Susan M.

    2014-01-01

    The development of therapeutic proteins and peptides is an expensive and time-intensive process. Biologics, which have become a multi-billion dollar industry, are chemically complex products that require constant observation during each stage of development and production. Post-translational modifications along with chemical and physical degradation from oxidation, deamidation, and aggregation, lead to high levels of heterogeneity that affect drug quality and efficacy. The various separation modes of capillary electrophoresis (CE) are commonly utilized to perform quality control and assess protein heterogeneity. This review attempts to highlight the most recent developments and applications of CE separation techniques for the characterization of protein and peptide therapeutics by focusing on papers accepted for publication in the in the two-year period between January 2012 and December 2013. The separation principles and technological advances of CE, capillary gel electrophoresis, capillary isoelectric focusing, capillary electrochromatography and CE-mass spectrometry are discussed, along with exciting new applications of these techniques to relevant pharmaceutical issues. Also included is a small selection of papers on microchip electrophoresis to show the direction this field is moving with regards to the development of inexpensive and portable analysis systems for on-site, high-throughput analysis. PMID:25126117

  19. Two-dimensional electrophoresis analysis of proteins extracted from Alexandrium sp. LC3

    NASA Astrophysics Data System (ADS)

    Li, Hao; Miao, Jinlai; Cui, Fengxia; Li, Guangyou

    2007-10-01

    Two-dimensional electrophoresis(2-DE) of protein extracted and purified from Alexandrium sp. LC3 was conducted. In the SDS-PAGE study, the relative molecular weights of the proteins were mainly in the range of 14kDa-31kDa and 43kDa-66kDa, and more proteins were detected between 14kDa and 31kDa. With the improved protein preparation, the two-dimensional electrophoresis patterns indicated that the relative molecular weights of the proteins were between 14kDa and 100kDa, and most of them ranged from 14kDa to 31kDa. This was consistent with the result of the SDS-PAGE analysis. The isoelectric points were found to lie between 3.0 and 8.0, and most of them were in the range of 3.0 6.0. Better separation effect was acquired with pre-prepared immobilized gradient (IPG) strip (pH3 5.6), and about 320 protein spots could be visualized on the 2-DE map by staining. Within pH3 10 and pH3 5.6 strips, the protein samples of Alexandrium sp. LC3 could be separated well.

  20. Completely monodisperse, highly repetitive proteins for bioconjugate capillary electrophoresis: Development and characterization

    PubMed Central

    Lin, Jennifer S.; Albrecht, Jennifer Coyne; Meagher, Robert J.; Wang, Xiaoxiao; Barron, Annelise E.

    2011-01-01

    Protein-based polymers are increasingly being used in biomaterial applications due to their ease of customization and potential monodispersity. These advantages make protein polymers excellent candidates for bioanalytical applications. Here we describe improved methods for producing drag-tags for Free-Solution Conjugate Electrophoresis (FSCE). FSCE utilizes a pure, monodisperse recombinant protein, tethered end-on to a ssDNA molecule, to enable DNA size separation in aqueous buffer. FSCE also provides a highly sensitive method to evaluate the polydispersity of a protein drag-tag and thus its suitability for bioanalytical uses. This method is able to detect slight differences in drag-tag charge or mass. We have devised an improved cloning, expression, and purification strategy that enables us to generate, for the first time, a truly monodisperse 20 kDa protein polymer and a nearly monodisperse 38 kDa protein. These newly produced proteins can be used as drag-tags to enable longer read DNA sequencing by free-solution microchannel electrophoresis. PMID:21553840

  1. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut (Juglans regia L.) proteins and protein fractionations.

    PubMed

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-27

    As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8-6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa.

  2. Amino Acid Composition, Molecular Weight Distribution and Gel Electrophoresis of Walnut (Juglans regia L.) Proteins and Protein Fractionations

    PubMed Central

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-01

    As a by-product of oil production, walnut proteins are considered as an additional source of plant protein for human food. To make full use of the protein resource, a comprehensive understanding of composition and characteristics of walnut proteins are required. Walnut proteins have been fractionated and characterized in this study. Amino acid composition, molecular weight distribution and gel electrophoresis of walnut proteins and protein fractionations were analyzed. The proteins were sequentially separated into four fractions according to their solubility. Glutelin was the main component of the protein extract. The content of glutelin, albumin, globulin and prolamin was about 72.06%, 7.54%, 15.67% and 4.73% respectively. Glutelin, albumin and globulin have a balanced content of essential amino acids, except for methionine, with respect to the FAO pattern recommended for adults. SDS-PAGE patterns of albumin, globulin and glutelin showed several polypeptides with molecular weights 14.4 to 66.2 kDa. The pattern of walnut proteins in two-dimension electrophoresis (2-DE) showed that the isoelectric point was mainly in the range of 4.8–6.8. The results of size exclusion chromatogram indicated molecular weight of the major components of walnut proteins were between 3.54 and 81.76 kDa. PMID:24473146

  3. Capillary zone electrophoresis for separation and quantitative determination of mexiletine and its main phase I metabolites.

    PubMed

    Bruno, Claudio; Cavalluzzi, Maria Maddalena; Carocci, Alessia; Catalano, Alessia; Franchini, Carlo; Lentini, Giovanni

    2013-03-01

    The simultaneous separation and quantification of the analytes within the minimum analysis time and the maximum resolution and efficiency are the main objectives in the development of a capillary electrophoretic method for the determination of solutes. In this paper we describe a specific, sensitive and robust method, using capillary zone electrophoresis with internal standard and UV detection, for the separation and quantification of the anti-arrhythmic drug mexiletine, its main phase I metabolites, and its main nitrogenous degradation product. PMID:23826880

  4. Capillary Electrophoresis in Wine Science.

    PubMed

    Coelho, Christian; Bagala, Franck; Gougeon, Régis D; Schmitt-Kopplin, Philippe

    2016-01-01

    Capillary electrophoresis appeared to be a powerful and reliable technique to analyze the diversity of wine compounds. Wine presents a great variety of natural chemicals coming from the grape berry extraction and the fermentation processes. The first and more abundant after water, ethanol has been quantified in wines via capillary electrophoresis. Other families like organic acids, neutral and acid sugars, polyphenols, amines, thiols, vitamins, and soluble proteins are electrophoretically separated from the complex matrix.Here, we will focus on the different methodologies that have been employed to conduct properly capillary electrophoresis in wine analysis.Two examples informing on wine chemistry obtained by capillary electrophoresis will be detailed. They concern polyphenol analysis and protein profiling. The first category is a well-developed quantitative approach important for the quality and the antioxidant properties conferred to wine. The second aspect involves more research aspects dealing with microbiota infections in the vineyard or in the grape as well as enological practices. PMID:27645750

  5. Comparison of Serum Protein Electrophoresis Values in Wild and Captive Whooping Cranes ( Grus americana ).

    PubMed

    Hausmann, Jennifer C; Cray, Carolyn; Hartup, Barry K

    2015-09-01

    Protein electrophoresis of serum samples from endangered, wild whooping cranes ( Grus americana ) was performed to help assess the health of the only self-sustaining, migratory population in North America. Serum samples from wild adult cranes (n = 22) were taken at Aransas National Wildlife Refuge, Texas, USA during winter. Wild juvenile cranes (n = 26) were sampled at Wood Buffalo National Park, Northwest Territories, Canada, in midsummer. All captive crane samples were acquired from the International Crane Foundation, Baraboo, WI, USA. Captive adult cranes (n = 30) were sampled during annual examinations, and archived serum samples from captive juvenile cranes (n = 19) were selected to match the estimated age of wild juveniles. Wild juveniles had significantly lower concentrations of all protein fractions than wild adults, except for prealbumin and γ globulins. All protein fraction concentrations for wild juveniles were significantly lower compared with captive juveniles, except for prealbumin and γ globulins, which were higher. Wild adults had significantly greater γ globulin concentrations than captive adults. Captive juveniles had significantly lower prealbumin and albumin concentrations and albumin : globulin ratios than captive adults. The higher γ globulin concentrations in wild versus captive cranes are likely because of increased antigenic exposure and immune stimulation. Protein fraction concentrations vary significantly with age and natural history in this species. Reference intervals for serum protein electrophoresis results from captive adult whooping cranes are provided in this study. PMID:26378665

  6. Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus).

    PubMed

    Isaza, Ramiro; Wiedner, Ellen; Hiser, Sarah; Cray, Carolyn

    2014-09-01

    Acute phase protein (APP) immunoassays and serum protein electrophoresis (SPEP) are assays for evaluating the inflammatory response and have use as diagnostic tools in a variety of species. Acute phase proteins are markers of inflammation that are highly conserved across different species while SPEP separates and quantifies serum protein fractions based on their physical properties. In the current study, serum samples from 35 clinically healthy Asian elephants (Elephas maximus) were analyzed using automated assays for C-reactive protein, serum amyloid A, and haptoglobin and SPEP. Robust methods were used to generate reference intervals for the APPs: C-reactive protein (1.3-12.8 mg/l), serum amyloid A (0-47.5 mg/l), and haptoglobin (0-1.10 mg/ml). In addition, SPEP was performed on these samples to establish reference intervals for each protein fraction. A combination of APPs and SPEP measurements are valuable adjunctive diagnostic tools in elephant health care.

  7. Reference intervals for acute phase protein and serum protein electrophoresis values in captive Asian elephants (Elephas maximus).

    PubMed

    Isaza, Ramiro; Wiedner, Ellen; Hiser, Sarah; Cray, Carolyn

    2014-09-01

    Acute phase protein (APP) immunoassays and serum protein electrophoresis (SPEP) are assays for evaluating the inflammatory response and have use as diagnostic tools in a variety of species. Acute phase proteins are markers of inflammation that are highly conserved across different species while SPEP separates and quantifies serum protein fractions based on their physical properties. In the current study, serum samples from 35 clinically healthy Asian elephants (Elephas maximus) were analyzed using automated assays for C-reactive protein, serum amyloid A, and haptoglobin and SPEP. Robust methods were used to generate reference intervals for the APPs: C-reactive protein (1.3-12.8 mg/l), serum amyloid A (0-47.5 mg/l), and haptoglobin (0-1.10 mg/ml). In addition, SPEP was performed on these samples to establish reference intervals for each protein fraction. A combination of APPs and SPEP measurements are valuable adjunctive diagnostic tools in elephant health care. PMID:25057161

  8. Purification of glycosylphosphatidylinositol-anchored proteins by modified triton X-114 partitioning and preparative gel electrophoresis.

    PubMed

    Ko, Y G; Thompson, G A

    1995-01-01

    Triton X-114 phase partitioning has frequently been used to obtain preparations enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and other hydrophobic proteins from crude cellular homogenates. We have developed a new modification of this phase-partitioning technique which allows two distinct GPI-anchored proteins of Tetrahymena mimbres to be separated from other hydrophobic as well as hydrophilic proteins and recovered in approximately 90% yield. The unique feature of the new method is a 24-h incubation of the first Triton X-114 extract at -20 degrees C. This improves the partitioning of GPI-anchored proteins into the detergent phase while promoting the aggregation of other hydrophobic proteins. Individual GPI-anchored proteins in the detergent phase are then purified to near homogeneity by one-step preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By substituting Triton X-114/water mixtures for the conventional SDS buffer used to collect proteins eluting from the preparative gel, the purified GPI-anchored protein can be rapidly concentrated from relatively large volumes of eluate by phase partitioning at 32 degrees C. The method is also effective in separating mammalian GPI-anchored alkaline phosphatase from other proteins. It is likely to be of general utility in characterizing the GPI anchor structures associated with nonabundant and abundant GPI-anchored proteins coexisting within the same cell type.

  9. Effect of bile salts stress on protein synthesis of Lactobacillus casei Zhang revealed by 2-dimensional gel electrophoresis.

    PubMed

    Wu, R; Sun, Z; Wu, J; Meng, H; Zhang, H

    2010-08-01

    Lactobacillus casei Zhang, isolated from koumiss in Inner Mongolia of China, is known from previous findings to be tolerant to bile salts. Bile salts secreted by mammals act as a natural antibacterial barrier and may serve as a component of innate immunity, as they have limited antagonistic effect against resident microflora. In this work, we compared the growth and protein expression patterns of L. casei Zhang with and without bile salts. Twenty-six proteins were found to be differentially expressed using 2-dimensional gel electrophoresis. Peptide mass fingerprinting was used to identify these proteins. Further verification by using real-time, quantitative reverse transcription-PCR and bioinformatics analysis showed that the implicated pathways are involved with a complex physiological response under bile salts stress, particularly including cell protection (DnaK and GroEL), modifications in cell membranes (NagA, GalU, and PyrD), and key components of central metabolism (PFK, PGM, CysK, LuxS, PepC, and EF-Tu). These results provide insight on the protein expression pattern of L. casei under bile salts stress and offer a new perspective for the molecular mechanisms involved in stress tolerance and adaptation of bacteria. PMID:20655455

  10. Fabrication of anti-protein-fouling poly(ethylene glycol) microfluidic chip electrophoresis by sandwich photolithography.

    PubMed

    Cong, Hailin; Xu, Xiaodan; Yu, Bing; Liu, Huwei; Yuan, Hua

    2016-07-01

    Microfluidic chip electrophoresis (MCE) is a powerful separation tool for biomacromolecule analysis. However, adsorption of biomacromolecules, particularly proteins onto microfluidic channels severely degrades the separation performance of MCE. In this paper, an anti-protein-fouling MCE was fabricated using a novel sandwich photolithography of poly(ethylene glycol) (PEG) prepolymers. Photopatterned microchannel with a minimum resolution of 10 μm was achieved. After equipped with a conventional online electrochemical detector, the device enabled baseline separation of bovine serum albumin, lysozyme (Lys), and cytochrome c (Cyt-c) in 53 s under a voltage of 200 V. Compared with a traditional polydimethylsiloxane MCE made by soft lithography, the PEG MCE made by the sandwich photolithography not only eliminated the need of a master mold and the additional modification process of the microchannel but also showed excellent anti-protein-fouling properties for protein separation.

  11. Quantitative interaction proteomics of neurodegenerative disease proteins.

    PubMed

    Hosp, Fabian; Vossfeldt, Hannes; Heinig, Matthias; Vasiljevic, Djordje; Arumughan, Anup; Wyler, Emanuel; Landthaler, Markus; Hubner, Norbert; Wanker, Erich E; Lannfelt, Lars; Ingelsson, Martin; Lalowski, Maciej; Voigt, Aaron; Selbach, Matthias

    2015-05-19

    Several proteins have been linked to neurodegenerative disorders (NDDs), but their molecular function is not completely understood. Here, we used quantitative interaction proteomics to identify binding partners of Amyloid beta precursor protein (APP) and Presenilin-1 (PSEN1) for Alzheimer's disease (AD), Huntingtin (HTT) for Huntington's disease, Parkin (PARK2) for Parkinson's disease, and Ataxin-1 (ATXN1) for spinocerebellar ataxia type 1. Our network reveals common signatures of protein degradation and misfolding and recapitulates known biology. Toxicity modifier screens and comparison to genome-wide association studies show that interaction partners are significantly linked to disease phenotypes in vivo. Direct comparison of wild-type proteins and disease-associated variants identified binders involved in pathogenesis, highlighting the value of differential interactome mapping. Finally, we show that the mitochondrial protein LRPPRC interacts preferentially with an early-onset AD variant of APP. This interaction appears to induce mitochondrial dysfunction, which is an early phenotype of AD.

  12. Comparative analysis of the tear protein expression in blepharitis patients using two-dimensional electrophoresis.

    PubMed

    Koo, Bon-Suk; Lee, Do-Yeon; Ha, Hyo-Shin; Kim, Jae-Chan; Kim, Chan-Wha

    2005-01-01

    Change in the expression of body fluid proteins is caused by many diseases or environmental disturbances. The changes in tear proteins are also associated with various pathological eye conditions. Especially, chronic blepharitis is one of the most common conditions seen in the ophthalmologist's office. However, there are no specific clinical diagnostic tests for blepharitis, and it is difficult to treat effectively. Therefore, the aim of this study was to screen prognostic or diagnostic marker tear proteins for blepharitis and investigate pathogenesis of this disease using proteomics techniques. The tear proteins expressed in patients suffering from blepharitis (patient, n=19) and healthy volunteers (control, n=27) were analyzed using the two-dimensional electrophoresis (2-DE) technique. The differentially expressed proteins in patients were identified with ESI-Q-TOF (electrospray-quadrupole-time-of-flight) mass spectrometry and confirmed with western blotting. Nine proteins in patient were down regulated about 50% compared to those of the control: serum albumin precursor, alpha-1 antitrypsin, lacritin precursor, lysozyme, Ig-kappa chain VIII, prolactin inducible protein (PIP/GCDFP-15), cystatin-SA III, pyruvate kinase, and an unnamed protein. The use of the two-dimensional eletrophoretic technique could give more insight into the disease-related protein expression changes in tear fluids. Our findings reveal that the composition of tear proteins in blepharitis patients is different from that of healthy subjects and may provide further insights into the pathogenesis of blepharitis.

  13. Qualitative and quantitative metabolomic investigation of single neurons by capillary electrophoresis electrospray ionization mass spectrometry

    PubMed Central

    Nemes, Peter; Rubakhin, Stanislav S.; Aerts, Jordan T.; Sweedler, Jonathan V.

    2013-01-01

    Single-cell mass spectrometry (MS) empowers metabolomic investigations by decreasing analytical dimensions to the size of individual cells and subcellular structures. We describe a protocol for investigating and quantifying metabolites in individual isolated neurons using single-cell capillary electrophoresis hyphenated to electrospray ionization time-of-flight MS. The protocol requires ~2 h for sample preparation, neuron isolation, and metabolite extraction, and 1 h for metabolic measurement. The approach was used to detect more than 300 distinct compounds in the mass range of typical metabolites in various individual neurons (25–500-µm in diameter) isolated from the sea slug (Aplysia californica) central and rat (Rattus norvegicus) peripheral nervous systems. A subset of identified compounds was sufficient to reveal metabolic differences among freshly isolated neurons of different types and changes in the metabolite profiles of cultured neurons. The protocol can be applied to the characterization of the metabolome in a variety of smaller cells and/or subcellular domains. PMID:23538882

  14. Quantitative analysis of protein turnover in plants.

    PubMed

    Nelson, Clark J; Li, Lei; Millar, A Harvey

    2014-03-01

    Proteins are constantly being synthesised and degraded as plant cells age and as plants grow, develop and adapt the proteome. Given that plants develop through a series of events from germination to fruiting and even undertake whole organ senescence, an understanding of protein turnover as a fundamental part of this process in plants is essential. Both synthesis and degradation processes are spatially separated in a cell across its compartmented structure. The majority of protein synthesis occurs in the cytosol, while synthesis of specific components occurs inside plastids and mitochondria. Degradation of proteins occurs in both the cytosol, through the action of the plant proteasome, and in organelles and lytic structures through different protease classes. Tracking the specific synthesis and degradation rate of individual proteins can be undertaken using stable isotope feeding and the ability of peptide MS to track labelled peptide fractions over time. Mathematical modelling can be used to follow the isotope signature of newly synthesised protein as it accumulates and natural abundance proteins as they are lost through degradation. Different technical and biological constraints govern the potential for the use of (13)C, (15)N, (2)H and (18)O for these experiments in complete labelling and partial labelling strategies. Future development of quantitative protein turnover analysis will involve analysis of protein populations in complexes and subcellular compartments, assessing the effect of PTMs and integrating turnover studies into wider system biology study of plants.

  15. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

    Zhang, Jian-Shi; Giometti, Carol S.; Tollaksen, Sandra L.

    1989-01-01

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a DC power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. A high percentage extraction of proteins is achieved. The extracted proteins can be removed and subjected to partial digestion by trypsin or the like, followed by two-dimensional electrophoresis, resulting in a gel slab having a pattern of peptide gel spots which can be cored out and subjected to electrophoretic extraction to extract individual peptides.

  16. Re-use of commercial microfluidics chips for DNA, RNA, and protein electrophoresis.

    PubMed

    Nguyen, Thi; Kwak, Sukyoung; Karpowicz, Steven J

    2014-11-01

    Microfluidics chip technology is a powerful and convenient alternative to agarose gels and PAGE, but costs can be high due to certain chips being non-reusable. Here we describe a method to regenerate, re-use, and store Agilent DNA, RNA, and protein electrophoresis chips designed for use in the Bioanalyzer 2100. By washing the sample wells and displacing the old gel matrix with new gel-dye mix, we have run samples on the same chip up to ten times with negligible loss of signal quality. Chips whose wells were loaded with buffer or water were stored successfully for one week before re-use.

  17. High-performance capillary electrophoresis for characterization of hapten-protein conjugates used for production of antibodies against soyasaponin I.

    PubMed

    Frøkiaer, H; Møller, P; Sørensen, H; Sørensen, S

    1994-10-01

    Micellar electrokinetic capillary chromatography using sodium cholate as the micellar phase has been investigated for characterization of hapten-protein conjugates. Special focus has been placed on the hapten soyasaponin I which is a quantitatively dominating glycoside in seeds of several legumes including pea (Pisum sativum L.) and soybean [Glycine max (L.) Merr.]. Soyasaponin I has been isolated from pea and used as hapten for production of anti-saponin specific polyclonal antibodies. Soyasaponin I was coupled to Kunitz soybean trypsin inhibitor (KSTI) and bovine serum albumin. The degree of coupling was determined by high-performance capillary electrophoresis (HPCE). Capillaries dynamically coated with zwitterions were found to be efficient for reduction of interaction between the silica capillary surface and the proteins. The applicability of HPCE for determination of coupling density was confirmed by investigation of a model hapten (p-nitrophenyl-alpha-D-galactoside; PNPG) coupled to KSTI. The PNPG-KSTI conjugates were examined by both HPCE and by spectrophotometric determination of the PNPG density on KSTI. The HPCE method was shown to be efficient in studies of the formation of hapten-protein conjugates and to be more specific than alternative techniques applied for determination of coupling densities.

  18. Quantitative study of protein-protein interactions by quartz nanopipettes.

    PubMed

    Tiwari, Purushottam Babu; Astudillo, Luisana; Miksovska, Jaroslava; Wang, Xuewen; Li, Wenzhi; Darici, Yesim; He, Jin

    2014-09-01

    In this report, protein-modified quartz nanopipettes were used to quantitatively study protein-protein interactions in attoliter sensing volumes. As shown by numerical simulations, the ionic current through the conical-shaped nanopipette is very sensitive to the surface charge variation near the pore mouth. With the appropriate modification of negatively charged human neuroglobin (hNgb) onto the inner surface of a nanopipette, we were able to detect concentration-dependent current change when the hNgb-modified nanopipette tip was exposed to positively charged cytochrome c (Cyt c) with a series of concentrations in the bath solution. Such current change is due to the adsorption of Cyt c to the inner surface of the nanopipette through specific interactions with hNgb. In contrast, a smaller current change with weak concentration dependence was observed when Cyt c was replaced with lysozyme, which does not specifically bind to hNgb. The equilibrium dissociation constant (KD) for the Cyt c-hNgb complex formation was derived and the value matched very well with the result from surface plasmon resonance measurement. This is the first quantitative study of protein-protein interactions by a conical-shaped nanopore based on charge sensing. Our results demonstrate that nanopipettes can potentially be used as a label-free analytical tool to quantitatively characterize protein-protein interactions.

  19. Semi-quantitative digital analysis of polymerase chain reaction-electrophoresis gel: Potential applications in low-income veterinary laboratories

    PubMed Central

    Antiabong, John F.; Ngoepe, Mafora G.; Abechi, Adakole S.

    2016-01-01

    Aim: The interpretation of conventional polymerase chain reaction (PCR) assay results is often limited to either positive or negative (non-detectable). The more robust quantitative PCR (qPCR) method is mostly reserved for quantitation studies and not a readily accessible technology in laboratories across developing nations. The aim of this study was to evaluate a semi-quantitative method for conventional PCR amplicons using digital image analysis of electrophoretic gel. The potential applications are also discussed. Materials and Methods: This study describes standard conditions for the digital image analysis of PCR amplicons using the freely available ImageJ software and confirmed using the qPCR assay. Results and Conclusion: Comparison of ImageJ analysis of PCR-electrophoresis gel and qPCR methods showed similar trends in the Fusobacterium necrophorum DNA concentration associated with healthy and periodontal disease infected wallabies (p≤0.03). Based on these empirical data, this study adds descriptive attributes (“more” or “less”) to the interpretation of conventional PCR results. The potential applications in low-income veterinary laboratories are suggested, and guidelines for the adoption of the method are also highlighted. PMID:27733792

  20. Quantitative analysis of pungent and anti-inflammatory phenolic compounds in olive oil by capillary electrophoresis.

    PubMed

    Vulcano, Isabella; Halabalaki, Maria; Skaltsounis, Leandros; Ganzera, Markus

    2015-02-15

    The first CE procedure for the quantitative determination of pharmacologically relevant secoiridoids in olive oil, oleocanthal and oleacein, is described. Together with their precursors tyrosol and hydroxytyrosol they could be baseline separated in less than 15min using a borax buffer with pH 9.5, at 25kV and 30°C. Method validation confirmed that the procedure is selective, accurate (recovery rates from 94.0 to 104.6%), reproducible (σmax⩽6.8%) and precise (inter-day precision⩽6.4%), and that the compounds do not degrade quickly if non-aqueous acetonitrile is used as solvent. Quantitative results indicated a low occurrence of oleocanthal (0.004-0.021%) and oleacein (0.002-0.048%) in olive oil samples, which is in agreement to published HPLC data. The CE method impresses with its simple instrumental and methodological design, combined with reproducible and valid quantitative results. PMID:25236241

  1. Quantitative Determination of Lercanidipine Enantiomers in Commercial Formulations by Capillary Electrophoresis

    PubMed Central

    Lourenço, Luciana Pereira; Aguiar, Fernando Armani; de Oliveira, Anderson Rodrigo Moraes; de Gaitani, Cristiane Masetto

    2015-01-01

    An enantioselective method based on capillary electrophoresis (CE) using cyclodextrin (CD) as chiral selector was developed and validated for determination of lercanidipine (LER) enantiomers, a drug calcium channel blocker which exerts antihypertensive effects of long duration, in a pharmaceutical formulation. Optimum separation of LER enantiomers was obtained on a 50 cm × 50 μm id capillary using a sodium acetate buffer solution 200 mmol/L pH 4.0 containing 10 mmol/L of 2,3,6-o-methyl-β-cyclodextrin (TM-β-CD) as background electrolyte. The capillary temperature and voltage were 15°C and 25 kV, respectively, hydrodynamic injection and detection at 237 nm. Linearity was obtained in the range 12.5–100 μg/mL for both enantiomers (r ≥ 0.995). The RSD (%) and relative errors (E, %) obtained in precision and accuracy studies (intraday and interday) were lower than 5%. After validation, the method was applied to quantify the enantiomers of LER in commercial tablets and the results were satisfactory in terms of accuracy and precision, both less than 5%. Therefore, this method was found to be appropriate for enantioselective quality control of LER enantiomers in pharmaceutical formulations. PMID:25821632

  2. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

    PubMed

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J

    2014-09-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P < 0.001) serum amyloid A, alpha2-globulin, and haptoglobin concentrations compared with the healthy subgroup. Moreover, serum amyloid A (P = 0.020), alpha2-globulin (P < 0.001) and haptoglobin (P = 0.001) concentrations in cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

  3. Optimization of an HP Scanjet for quantification of protein electrophoresis gels.

    PubMed

    Kendrick, N C; Johansen, J J; Lee, P R; Santek, D A

    1994-06-01

    An inexpensive desktop scanner, the Hewlett-Packard Scanjet IIp (HP), has been optimized for analysis of protein electrophoresis gels by comparison with a calibrated laser densitometer (Laser). Images from both densitometers were transferred to a personal computer and analyzed with QGEL software. Without correction the HP response was often in poor agreement with the Laser. However, when the HP response to Coomassie blue stained gels and x-ray films was linearized using a HP software option called Emphasis, the HP results agreed with results from the Laser. For 2D gels scanned with appropriate Emphasis applied, spot integrated density values were a constant multiple of 1.8 +/- 0.3 times the corresponding Laser value for x-ray films (CV = 17%) and 2.1 +/- 0.5 for Coomassie blue stained gels (CV = 24%). The highest error was observed for density extremes. For proteins quantified relative to standards using sodium dodecyl sulfate-slab gel electrophoresis, the HP values were within 15% of the Laser values. Data is shown concerning linearity and reproducibility of response, optical density range (about 0 to 1.8 OD units), variability of the imaging field, and resolution of the HP.

  4. Urine Proteins Identified by Two-Dimensional Differential Gel Electrophoresis Facilitate the Differential Diagnoses of Scrapie

    PubMed Central

    Lamoureux, Lise; Simon, Sharon L. R.; Plews, Margot; Ruddat, Viola; Brunet, Simone; Graham, Catherine; Czub, Stefanie; Knox, J. David

    2013-01-01

    The difficulty in developing a diagnostic assay for Creutzfeldt - Jakob disease (CJD) and other transmissible spongiform encephalopathies (TSEs) stems in part from the fact that the infectious agent is an aberrantly folded form of an endogenous cellular protein. This precludes the use of the powerful gene based technologies currently applied to the direct detection of other infectious agents. To circumvent this problem our research objective has been to identify a set of proteins exhibiting characteristic differential abundance in response to TSE infection. The objective of the present study was to assess the disease specificity of differentially abundant urine proteins able to identify scrapie infected mice. Two-dimensional differential gel electrophoresis was used to analyze longitudinal collections of urine samples from both prion-infected mice and a transgenic mouse model of Alzheimer's disease. The introduction of fluorescent dyes, that allow multiple samples to be co-resolved and visualized on one two dimensional gel, have increased the accuracy of this methodology for the discovery of robust protein biomarkers for disease. The accuracy of a small panel of differentially abundant proteins to correctly classify an independent naïve sample set was determined. The results demonstrated that at the time of clinical presentation the differential abundance of urine proteins were capable of identifying the prion infected mice with 87% sensitivity and 93% specificity. The identity of the diagnostic differentially abundant proteins was investigated by mass spectrometry. PMID:23704971

  5. Optimisation of a simple and reliable label-free methodology for the relative quantitation of raw pork meat proteins.

    PubMed

    Gallego, Marta; Mora, Leticia; Aristoy, M Concepción; Toldrá, Fidel

    2015-09-01

    Recent advances in proteomics have become an indispensable tool for a fast, precise and sensitive analysis of proteins in complex biological samples at both, qualitative and quantitative level. In this study, a label-free quantitative proteomic methodology has been optimised for the relative quantitation of proteins extracted from raw pork meat. So, after the separation of proteins by one-dimensional gel electrophoresis and trypsin digestion, their identification and quantitation have been done using nanoliquid chromatography coupled to a quadrupole/time-of-flight (Q/ToF) mass spectrometer. Relative quantitation has been based on the measurement of mass spectral peak intensities, which have been described that are correlated with protein abundances. The results obtained regarding linearity, robustness, repeatability and accuracy show that this procedure could be used as a fast, simple, and reliable method to quantify changes in protein abundance in meat samples.

  6. Comparison of protein expression profiles between three Perkinsus spp., protozoan parasites of molluscs, through 2D electrophoresis and mass spectrometry.

    PubMed

    Fernández-Boo, S; Chicano-Gálvez, E; Alhama, J; Barea, J L; Villalba, A; Cao, A

    2014-05-01

    The genus Perkinsus includes protozoan parasites of a wide range of marine molluscs worldwide, some of which have been responsible for heavy mollusc mortalities and dramatic economic losses. This study was performed with the aim of increasing the knowledge of Perkinsus spp. proteome. Proteins extracted from in vitro cultured cells of three species of this genus, P. marinus, P. olseni and P. chesapeaki, were analysed using 2D electrophoresis. Four gels from each species were produced. Qualitative and quantitative comparisons among gels were performed with Proteamweaver software. Cluster analysis grouped the four gels of each Perkinsus sp.; furthermore, P. marinus and P. olseni gels were grouped in a cluster different from P. chesapeaki. Around 2000 spots of each species were considered, from which 213 spots were common to the 3 species; P. chesapeaki and P. marinus shared 310 spots, P. chesapeaki and P. olseni shared 315 spots and P. marinus and P. olseni shared 242 spots. A number of spots were exclusive of each Perkinsus species: 1161 spots were exclusive of P. chesapeaki, 1124 of P. olseni and 895 of P. marinus. A total of 84 spots, including common and species-specific ones, were excised from the gels and analysed using MALDI-TOF and nESI-IT (MS/MS) techniques. Forty-two spots were successfully sequenced, from which 28 were annotated, most of them clustered into electron transport, oxidative stress and detoxification, protein synthesis, carbohydrate metabolism, signal transduction, metabolic process and proteolysis.

  7. Seasonal influence on biochemical profile and serum protein electrophoresis for Boa constrictor amarali in captivity.

    PubMed

    Silva, L F N; Riani-Costa, C C M; Ramos, P R R; Takahira, R K

    2011-05-01

    Similarly to other reptiles, snakes are ectothermic animals and depend exclusively on the environment for the maintenance of their physiological, biochemical and immunological processes. Thus, changes in biochemical values can be expected due to seasonal influence. Twenty-two adult specimens of Boa constrictor amarali kept in captivity were used. Blood collections were done in two different seasons: winter (July 2004) and summer (January 2005) for the following assays: uric acid, aspartate aminotransferase (AST), glucose, cholesterol, total protein, and serum protein electrophoresis. The mean biochemical results found in summer and winter, respectively, were: 6.3 ± 3.4 and 11.3 ± 6.2 mg/dL for uric acid; 28.7 ± 12.4 and 20.7 ± 16.2 UI/L for AST; 26.3 ± 17 and 17.4 ± 6.8 mg/dL for glucose; 67.3 ± 30.2 and 69.7 ± 38.5 mg/dL for cholesterol; and 5.9 ± 1.6 and 5.9 ± 1.4 g/dL for total protein. Results regarding electrophoresis in summer and winter, respectively, were: 1.9 ± 0.7 and 2.4 ± 0.6 g/dL for albumin; 0.7 ± 0.2 and 0.5 ± 0.2 g/dL for α-globulin; 1.5 ± 0.5 and 1.7 ± 0.6 g/dL for β-globulin; and 1.8 ± 0.5 and 1.5 ± 0.5 g/dL for γ-globulin. In the summer, there was a significant increase in AST and a decrease in uric acid (p < 0.05). Serum protein electrophoresis showed a significant increase in α-globulin fraction (p < 0.05) in the same season. There were not significant differences between seasons for the remaining variables. Based on these results, the period of the year must be considered in the interpretation of some biochemical values for these animals.

  8. PIXE-electrophoresis shows starving collembolan reallocates protein-bound metals.

    PubMed

    Bengtsson, Göran; Pallon, Jan; Nilsson, Christina; Triebskorn, Rita; Köhler, Heinz-R

    2016-01-01

    One of multiple functions of metalloproteins is to provide detoxification to excess metal levels in organisms. Here we address the induction and persistence of a range of low to high molecular weight copper- and zinc binding proteins in the collembolan species Tetrodontophora bielanensis exposed to copper- and zinc-enriched food, followed by a period of recovery from metal exposure, in absence and presence of food. After 10 days of feeding copper and zinc contaminated yeast, specimens were either moved to ample of leaf litter material from their woodland stand of origin or starved (no food offered). The molecular weight distribution of metal binding proteins was determined by native polyacryl gel electrophoresis. One gel was stained with Comassie brilliant blue and a duplicate gel dried and scanned for the amount of copper and zinc by particle-induced X-ray emission. Specimens exposed to copper and recovered from it with ample of food had copper bound to two groups of rather low molecular weight proteins (40-50 kDa) and two of intermediate size (70-80 kDa). Most zinc in specimens from the woodland stand was bound to two large proteins of about 104 and 106 kDa. The same proteins were holding some zinc in metal-exposed specimens, but most zinc was found in proteins <40 kDa in size. Specimens recovered from metal exposure in presence of ample of food had the same distribution pattern of zinc binding proteins, whereas starved specimens had zinc as well as copper mainly bound to two proteins of 8 and 10 kDa in size. Thus, the induction and distribution of copper- and zinc-binding proteins depend on exposure conditions, and the presence of low molecular weight binding proteins, characteristic of metallothioneins, was mainly limited to starving conditions. PMID:26507895

  9. Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

    PubMed Central

    Gauci, Victoria J.; Wright, Elise P.

    2010-01-01

    Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist. PMID:21686332

  10. Serum protein electrophoresis by using high-resolution agarose gel in clinically healthy and Aspergillus species-infected falcons.

    PubMed

    Kummrow, Maya; Silvanose, Christudas; Di Somma, Antonio; Bailey, Thomas A; Vorbrüggen, Susanne

    2012-12-01

    Serum protein electrophoresis has gained importance in avian medicine during the past decade. Interpretation of electrophoretic patterns should be based on species-specific reference intervals and the electrophoresis gel system. In this study, serum protein electrophoresis by using high-resolution agarose gels was performed on blood samples collected from 105 falcons, including peregrine falcons (Falco peregrinus), gyrfalcons (Falco rusticolus), saker falcons (Falco cherrug), red-naped shaheens (Falco pelegrinoides babylonicus), and hybrid falcons, that were submitted to the Dubai Falcon Hospital (Dubai, United Arab Emirates) between 2003 and 2006. Reference values were established in clinically healthy birds and compared with values from falcons infected with Aspergillus species (n = 32). Falcons with confirmed aspergillosis showed significantly lower prealbumin values, which is a novel finding. Prealbumin has been documented in many avian species, but further investigation is required to illuminate the diagnostic significance of this negative acute-phase protein.

  11. Electrophoretic extraction of proteins from two-dimensional electrophoresis gel spots

    DOEpatents

    Zhang, Jian-Shi; Giometti, C.S.; Tollaksen, S.L.

    1987-09-04

    After two-dimensional electrophoresis of proteins or the like, resulting in a polyacrylamide gel slab having a pattern of protein gel spots thereon, an individual protein gel spot is cored out from the slab, to form a gel spot core which is placed in an extraction tube, with a dialysis membrane across the lower end of the tube. Replicate gel spots can be cored out from replicate gel slabs and placed in the extraction tube. Molten agarose gel is poured into the extraction tube where the agarose gel hardens to form an immobilizing gel, covering the gel spot cores. The upper end portion of the extraction tube is filled with a volume of buffer solution, and the upper end is closed by another dialysis membrane. Upper and lower bodies of a buffer solution are brought into contact with the upper and lower membranes and are provided with electrodes connected to the positive and negative terminals of a dc power supply, thereby producing an electrical current which flows through the upper membrane, the volume of buffer solution, the agarose, the gel spot cores and the lower membrane. The current causes the proteins to be extracted electrophoretically from the gel spot cores, so that the extracted proteins accumulate and are contained in the space between the agarose gel and the upper membrane. 8 figs.

  12. Muscle protein analysis. II. Two-dimensional electrophoresis of normal and diseased human skeletal muscle

    SciTech Connect

    Giometti, C.S.; Barany, M.; Danon, M.J.; Anderson, N.G.

    1980-07-01

    High-resolution two-dimensional electrophoresis was used to analyze the major proteins of normal and pathological human-muscle samples. The normal human-muscle pattern contains four myosin light chains: three that co-migrate with the myosin light chains from rabbit fast muscle (extensor digitorum longus), and one that co-migrates with the light chain 2 from rabbit slow muscle (soleus). Of seven Duchenne muscular dystrophy samples, four yielded patterns with decreased amounts of actin and myosin relative to normal muscle, while three samples gave patterns comparable to that for normal muscle. Six samples from patients with myotonic dystrophy also gave normal patterns. In nemaline rod myopathy, in contrast, the pattern was deficient in two of the fast-type myosin light chains.

  13. Sensitive detection of C-reactive protein in serum by immunoprecipitation-microchip capillary gel electrophoresis.

    PubMed

    Herwig, Ela; Marchetti-Deschmann, Martina; Wenz, Christian; Rüfer, Andreas; Redl, Heinz; Bahrami, Soheyl; Allmaier, Günter

    2015-06-01

    Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP-MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R(2) = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP-MCGE bears potential for point-of-care diagnosis. PMID:25778394

  14. Electrophoresis characterisation of protein as a method to establish the entomological origin of stingless bee honeys.

    PubMed

    Ramón-Sierra, Jesús Manuel; Ruiz-Ruiz, Jorge Carlos; de la Luz Ortiz-Vázquez, Elizabeth

    2015-09-15

    Increasing production of stingless-bee honey and the prospect of broader marker for natural and organic products indicate the need to establish parameters to determinate the entomological origin and authenticity of honey. In this research, honeys of Apis mellifera, Melipona beecheii and Trigona spp. were collected in Yucatan, Mexico. Stingless-bee honeys contained more water and less total sugars and reducing sugars. SDS-PAGE patterns show distinctive bands for each kind of honey. The SDS-PAGE pattern of A. mellifera proteins honey showed three bands with molecular weights between 10.2 and 74.8kDa, there were five proteins bands in M. beecheii honey with molecular weights between 6.1 and 97.0kDa and nine for Trigona spp. proteins between 9.3 and 86.7kDa. Conventional physicochemical parameters along with electrophoresis profiles of stingless-bee honeys proteins could be an alternative for determination of entomological origin.

  15. Sensitive detection of C-reactive protein in serum by immunoprecipitation-microchip capillary gel electrophoresis.

    PubMed

    Herwig, Ela; Marchetti-Deschmann, Martina; Wenz, Christian; Rüfer, Andreas; Redl, Heinz; Bahrami, Soheyl; Allmaier, Günter

    2015-06-01

    Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP-MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R(2) = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP-MCGE bears potential for point-of-care diagnosis.

  16. Microchip capillary electrophoresis-electrospray ionization-mass spectrometry of intact proteins using uncoated Ormocomp microchips.

    PubMed

    Sikanen, Tiina; Aura, Susanna; Franssila, Sami; Kotiaho, Tapio; Kostiainen, Risto

    2012-01-20

    We present rapid (<5 min) and efficient intact protein analysis by mass spectrometry (MS) using fully microfabricated and monolithically integrated capillary electrophoresis-electrospray ionization (CE-ESI) microchips. The microchips are fabricated fully of commercial inorganic-organic hybrid material, Ormocomp, by UV-embossing and adhesive Ormocomp-Ormocomp bonding (CE microchannels). A sheath-flow ESI interface is monolithically integrated with the UV-embossed separation channels by cutting a rectangular emitter tip in the end with a dicing saw. As a result, electrospray was produced from the corner of chip with good reproducibility between parallel tips (stability within 3.8-9.2% RSD). Thanks to its inherent biocompatibility and stable (negative) surface charge, Ormocomp microchips enable efficient intact protein analysis with up to ∼10(4) theoretical separation plates per meter without any chemical or physical surface modification before analysis. The same microchip setup is also feasible for rapid peptide sequencing and mass fingerprinting and shows excellent migration time repeatability from run to run for both peptides (5.6-5.9% RSD, n=4) and intact proteins (1.3-7.5% RSD, n=3). Thus, the Ormocomp microchips provide a versatile new tool for MS-based proteomics. Particularly, the feasibility of the Ormocomp chips for rapid analysis of intact proteins with such a simple setup is a valuable increment to the current technology.

  17. Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry.

    PubMed

    Tengattini, Sara; Domínguez-Vega, Elena; Temporini, Caterina; Terreni, Marco; Somsen, Govert W

    2016-09-01

    A capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the characterization and integrity assessment of the Mycobacterium tuberculosis (MTB) antigens TB10.4 and Ag85B and their chemically produced glycoconjugates, which are glycovaccine candidates against tuberculosis (TB). In order to prevent protein adsorption to the inner capillary wall and to achieve efficient separation of the antigen proteoforms, a polyionic multilayer coating of polybrene-dextran sulfate-polybrene (PB-DS-PB) was used in combination with 1.5 M acetic acid as background electrolyte (BGE). Coupling of CE to high-resolution time-of-flight MS was achieved by a coaxial interface employing a sheath liquid of isopropanol-water (50:50, v/v) containing 0.1 % formic acid. The MTB antigens were exposed to experimental conditions used for chemical glycosylation (but no activated saccharide was added) in order to investigate their stability during glycovaccine production. CE-MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants, which were assigned by accurate mass. Analysis of synthesized mannose conjugates of TB10.4 and Ag85B allowed the determination of the glycoform composition of the neo-glycoproteins next to the characterization of degradation products which were shown to be partly glycoconjugated. Moreover, the selectivity of CE-MS allowed specific detection of deamidated species (protein mass change of 1.0 Da only), indicating that chemical glycosylation increased susceptibility to deamidation. Overall, the results show that CE-MS represents a useful analytical tool for the detailed characterization and optimization of neo-glycoconjugate products. Graphical Abstract Flowchart illustrating Mycobacterium tuberculosis (MTB) antigen glycosylation, glycoconjugate variant and degradation product separation by capillary electrophoresis (CE) and their characterization by intact mass

  18. [Evaluation of the relations between serum proteins electrophoresis and other laboratory tests in monoclonal gammopathies (author's transl)].

    PubMed

    Ramacciotti, P G; Lazzari, L; Minardi, P

    1976-03-01

    We have considered interesting to determine monoclonal gammopathies incidence, in 2191 serum proteins electrophoresis performed in our laboratory from January to December 1974. We have found 15 cases of monoclonal gammopathies, some cases combined with Mieloma (3 cases), some other with other with non specific diseases. We have considered the relations between type of gammopathy and other laboratory tests useful for any other diagnose: they are: immunochemical analysis, E.S.R., red and white count, total proteins, Bence Jones protein. PMID:65779

  19. Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations

    PubMed Central

    Volpi, Nicola; Maccari, Francesca; Linhardt, Robert J.

    2014-01-01

    A simple, accurate and robust quantitative CE method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced, i.e., GlcN from Hep and GalN from OSCS, were derivatized with anthranilic acid (AA) and separated by means of CE in approx. 10 min with high sensitivity detection at 214 nm (limit of detection (LOD) of approx. 200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity allowing for direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination, since chondroitin ABC lyase is unable to act on this semi-synthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS while a formulated contaminated Hep was calculated to have 39.7% OSCS. PMID:19232311

  20. Laser ablation inductively coupled plasma-mass spectrometry in combination with gel electrophoresis: a new strategy for speciation of metal binding serum proteins

    NASA Astrophysics Data System (ADS)

    Neilsen, J. L.; Abildtrup, A.; Christensen, J.; Watson, P.; Cox, A.; McLeod, C. W.

    1998-02-01

    A new hyphenated technique-crossed immunoelectrophoresis in combination with laser ablation inductively coupled plasma (ICP)-mass spectrometry—for the identification and quantitation of metal binding proteins in blood serum is described. Human serum enriched with Co was subjected to electrophoresis and the agarose gels corresponding to the first and second dimensions were interrogated and analysed using a Nd Yag laser (1064 nm) interfaced to ICP-mass spectrometry. Comparison of the distribution map for Co with the protein distribution map obtained via Coommassie Brilliant Blue staining allowed identification of main Co binding serum proteins. Signals for Co (single ion monitoring, mle 59) were transient in nature and for gels enriched with increasing concentrations of Co, peak area response was linear with concentration. Precision for replicate analyses was 6% R.S.D. and the limit of detection was - 0.29 ng.

  1. Pneumatic Microvalve-Based Hydrodynamic Sample Injection for High-Throughput, Quantitative Zone Electrophoresis in Capillaries

    SciTech Connect

    Kelly, Ryan T.; Wang, Chenchen; Rausch, Sarah J.; Lee, Cheng S.; Tang, Keqi

    2014-07-01

    A hybrid microchip/capillary CE system was developed to allow unbiased and lossless sample loading and high throughput repeated injections. This new hybrid CE system consists of a polydimethylsiloxane (PDMS) microchip sample injector featuring a pneumatic microvalve that separates a sample introduction channel from a short sample loading channel and a fused silica capillary separation column that connects seamlessly to the sample loading channel. The sample introduction channel is pressurized such that when the pneumatic microvalve opens briefly, a variable-volume sample plug is introduced into the loading channel. A high voltage for CE separation is continuously applied across the loading channel and the fused silica capillary separation column. Analytes are rapidly separated in the fused silica capillary with high resolution. High sensitivity MS detection after CE separation is accomplished via a sheathless CE/ESI-MS interface. The performance evaluation of the complete CE/ESI-MS platform demonstrated that reproducible sample injection with well controlled sample plug volumes could be achieved by using the PDMS microchip injector. The absence of band broadening from microchip to capillary indicated a minimum dead volume at the junction. The capabilities of the new CE/ESI-MS platform in performing high throughput and quantitative sample analyses were demonstrated by the repeated sample injection without interrupting an ongoing separation and a good linear dependence of the total analyte ion abundance on the sample plug volume using a mixture of peptide standards. The separation efficiency of the new platform was also evaluated systematically at different sample injection times, flow rates and CE separation voltages.

  2. Optimization of Protein Extraction and Two-Dimensional Electrophoresis Protocols for Oil Palm Leaf.

    PubMed

    Daim, Leona Daniela Jeffery; Ooi, Tony Eng Keong; Yusof, Hirzun Mohd; Majid, Nazia Abdul; Karsani, Saiful Anuar Bin

    2015-08-01

    Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work.

  3. Photosensitive diazotized poly(ethylene glycol) covalent capillary coatings for analysis of proteins by capillary electrophoresis.

    PubMed

    Yu, Bing; Chen, Xin; Cong, Hailin; Shu, Xi; Peng, Qiaohong

    2016-09-01

    A new method for the fabrication of covalently cross-linked capillary coatings of poly(ethylene glycol) (PEG) is described using diazotized PEG (diazo-PEG) as a new photosensitive coating agent. The film of diazo-PEG depends on ionic bonding and was first prepared on the inner surface of capillary by self-assembly, and ionic bonding was converted into covalent bonding after reaction of ultraviolet light with diazo groups through unique photochemical reaction. The covalently bonded coating impedance adsorption of protein on the central surface of capillary and hence the four proteins ribonuclease A, cytochrome c, bovine serum albumin, and lysosome can be baseline separated by using capillary electrophoresis (CE). The covalently cross-linked diazo-PEG capillary column coatings not only improved the CE separation performance for proteins compared to non-covalently cross-linked coatings or bare capillary but also showed a remarkable chemical solidity and repeatability. Because photosensitive diazo-PEG took the place of the highly noxious and silane moisture-sensitive coating reagents in the fabrication of covalent coating, this technique shows the advantage of being environment-friendly and having a high efficiency for CE to make the covalently bonded capillaries. PMID:27475442

  4. Optimization of Protein Extraction and Two-Dimensional Electrophoresis Protocols for Oil Palm Leaf.

    PubMed

    Daim, Leona Daniela Jeffery; Ooi, Tony Eng Keong; Yusof, Hirzun Mohd; Majid, Nazia Abdul; Karsani, Saiful Anuar Bin

    2015-08-01

    Oil palm (Elaeis guineensis) is an important economic crop cultivated for its nutritional palm oil. A significant amount of effort has been undertaken to understand oil palm growth and physiology at the molecular level, particularly in genomics and transcriptomics. Recently, proteomics studies have begun to garner interest. However, this effort is impeded by technical challenges. Plant sample preparation for proteomics analysis is plagued with technical challenges due to the presence of polysaccharides, secondary metabolites and other interfering compounds. Although protein extraction methods for plant tissues exist, none work universally on all sample types. Therefore, this study aims to compare and optimize different protein extraction protocols for use with two-dimensional gel electrophoresis of young and mature leaves from the oil palm. Four protein extraction methods were evaluated: phenol-guanidine isothiocyanate, trichloroacetic acid-acetone precipitation, sucrose and trichloroacetic acid-acetone-phenol. Of these four protocols, the trichloroacetic acid-acetone-phenol method was found to give the highest resolution and most reproducible gel. The results from this study can be used in sample preparations of oil palm tissue for proteomics work. PMID:26263918

  5. Detection of the end point temperature of thermal denatured protein in fish and chicken meat through SDS-PAGE electrophoresis

    NASA Astrophysics Data System (ADS)

    Gao, Hongwei; Mao, Mao; Liang, Chengzhu; Lin, Chao; Xiang, Jianhai

    2009-03-01

    Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was applied in the detection of the end point temperature (EPT) of thermal denatured protein in fish and meat in this study. It was also used in studying the thermal denatured temperature range of proteins in salmon and chicken meat. The results show that the temperature ranges of denatured proteins were from 65°C to 75°C, and these temperature ranges were influenced by the processing methods. Through SDS-PAGE, the features of repeated heating thermal denatured proteins under the same temperature and processing time were studied. The electrophoresis patterns of thermal denatured proteins determined through repeated heating at the same temperature did not exhibit any change. For the detection of cooked fish and meat samples, they were subjected to applying the SDS-PAGE method, which revealed an EPT ranging from 60°C to 80°C.

  6. Quantitative identification of protein nitration sites.

    PubMed

    Chiappetta, Giovanni; Corbo, Claudia; Palmese, Angelo; Galli, Francesco; Piroddi, Marta; Marino, Gennaro; Amoresano, Angela

    2009-03-01

    Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs have emerged. Many research groups have focused their attention towards o-nitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o-nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems. PMID:19242934

  7. A comprehensive platform to investigate protein-metal ion interactions by affinity capillary electrophoresis.

    PubMed

    Alhazmi, Hassan A; Nachbar, Markus; Albishri, Hassan M; Abd El-Hady, Deia; Redweik, Sabine; El Deeb, Sami; Wätzig, Hermann

    2015-03-25

    In this work, the behavior of several metal ions with different globular proteins was investigated by affinity capillary electrophoresis. Screening was conducted by applying a proper rinsing protocol developed by our group. The use of 0.1M EDTA in the rinsing solution successfully desorbs metal ions from the capillary wall. The mobility ratio was used to evaluate the precision of the method. Excellent precision for repeated runs was achieved for different protein metal ion interactions (RSD% of 0.05-1.0%). Run times were less than 6 min for all of the investigated interactions. The method has been successfully applied for the interaction study of Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Al(3+), Ga(3+), La(3+), Pd(2+), Ir(3+), Ru(3+), Rh(3+), Pt(2+), Pt(4+), Os(3+), Au(3+), Au(+), Ag(+), Cu(1+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cr(3+), V(3+), MoO4(2-) and SeO3(2-) with bovine serum albumin, ovalbumin, β-lactoglobulin and myoglobin. Different interaction values were obtained for most of the tested metal ions even for that in the same metal group. Results were discussed and compared in view of metal and semimetal group's interaction behavior with the tested proteins. The calculated normalized difference of mobility ratios for each protein-metal ion interaction and its sign (positive and negative) has been successfully used to detect the interaction and estimate further coordination of the bound metal ion, respectively. The comprehensive platform summarizes all the obtained interaction results, and is valuable for any future protein-metal ion investigation.

  8. Studies on proteinograms in dermatorphytes by disc electrophoresis. Part 2: Protein bands of keratinophilic fungi

    NASA Technical Reports Server (NTRS)

    Danev, P.; Balabanov, V.; Friedrich, E.

    1983-01-01

    Disc electrophoresis studies on keratinophili fungi demonstrated corresponding proteinograms in morphologically homogeneous strains of the same species, but different in different species of one and the same genus.

  9. Migration behaviour of discontinuous buffers in capillary electrophoresis during protein enrichment.

    PubMed

    Li, Ting; Booker, Christina J; Yeung, Ken K-C

    2012-10-21

    Capillary electrophoresis (CE) is not only an effective separation technique, but can also serve as a sample preparation tool for enrichment and purification at sub-microliter sample volumes. Our approach is based on the use of a discontinuous buffer system consisting of an acid and a base (acetate and ammonium). Proteins and/or peptides with isoelectric points between the pH values of these two buffers will become stacked at the neutralization reaction boundary (NRB). To understand the mechanism of the NRB formation and the electrophoretic migration of various ions during the enrichment, we performed experiments using myoglobin and mesityl oxide to reveal the ion migration patterns at the buffer junction, and utilized Simul 5 to computer simulate the process. The simulated results closely resembled the experimental data, and together, they effectively revealed the characteristics of the discontinuous buffers. Importantly, the discovery allowed the manipulation of NRB behaviours by controlling the discontinuous buffer composition. To illustrate this, the removal of urea as an unwanted background molecule from the enriched protein sample was achieved based on the acquired information.

  10. Hematological, protein electrophoresis and cholinesterase values of free-living nestling peregrine falcons in Spain.

    PubMed

    Lanzarot, M P; Montesinos, A; San Andrés, M I; Rodríguez, C; Barahona, M V

    2001-01-01

    Protein electrophoresis, hematological and cholinesterase values were determined in 32 nestling free-living peregrine falcons (Falco peregrinus) (15- to 27-days-old) in order to establish normal reference values for this population. The following values (mean +/- SD) were observed: prealbumin 0.31 +/- 0.04 g/dl, albumin 1.25 +/- 0.06 g/dl, alpha1 and alpha2-globulin 0.23 +/- 0.02 and 0.16 +/- 0.02 g/dl respectively, beta-globulin 1.02 +/- 0.05 g/dl, gamma-globulin 0.060 +/- 0.08 g/dl, total protein 3.79 +/- 0.18 g/dl, 21.26 +/- 1.30 white blood cells/microl (1 x 10(3)), 2.17 +/- 0.07 red blood cells/microl (1 x 10(6)), packed cell volume 37.58 +/- 0.82%, hemoglobin 20.96 +/- 0.29 g/dl, heterophils 61.14 +/- 2.50% and cholinesterase 1,184 +/- 75 IU/L. There were no difference in any of these parameters among males and females. The hematological values obtained could be considered as representative values in free-living nestling peregrine falcons.

  11. High-throughput viscosity measurement using capillary electrophoresis instrumentation and its application to protein formulation.

    PubMed

    Allmendinger, Andrea; Dieu, Le-Ha; Fischer, Stefan; Mueller, Robert; Mahler, Hanns-Christian; Huwyler, Jörg

    2014-10-01

    Viscosity characterization of protein formulations is of utmost importance for the development of subcutaneously administered formulations. However, viscosity determinations are time-consuming and require large sample volumes in the range of hundreds of microliters to a few milliliters, depending on the method used. In this article, an automated, high-throughput method is described to determine dynamic viscosity of Newtonian fluids using standard capillary electrophoresis (CE) equipment. CE is an analytical method routinely used for the separation and characterization of proteins. In our set-up, the capillary is filled with the test sample, and a constant pressure is applied. A small aliquot of riboflavin is subsequently loaded into the capillary and used as a dye to monitor movement of protein samples. Migration time of the riboflavin peak moving through the filled capillary is converted to the viscosity by applying the Hagen-Poiseuille's law. The instrument is operated without using an electrical field. Repeatability, robustness, linearity, and reproducibility were demonstrated for different capillary lots and instruments, as well as for different capillary lengths and diameters. Accuracy was verified by comparing the viscosity data obtained by CE instrumentation with those obtained by plate/cone rheometry. The suitability of the method for protein formulations was demonstrated, and limitations were discussed. Typical viscosities in the range of 5-40mPas were reliably measured with this method. Advantages of the CE instrumentation-based method included short measurement times (1-15min), small sample volumes (few microliters) for a capillary with a diameter of 50μm and a length of 20.5cm as well as potential to be suitable for high-throughput measurements.

  12. Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of (/sup 35/S)methionine-labeled proteins

    SciTech Connect

    Tabaqchali, S.; O'Farrell, S.; Holland, D.; Silman, R.

    1986-01-01

    A typing method for Clostridium difficile based on the incorporation of (/sup 35/S)methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described. On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z). The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C. difficile.

  13. Detection of C-reactive protein based on magnetic nanoparticles and capillary zone electrophoresis with laser-induced fluorescence detection.

    PubMed

    Lin, Yi-Jyun; Yang, Jian-Ying; Shu, Ting-Yu; Lin, Ting-Yu; Chen, Yen-Yi; Su, Mei-Yu; Li, Wen-Jie; Liu, Mine-Yine

    2013-11-01

    A simple and fast method based on magnetic nanoparticles (MNPs) and capillary zone electrophoresis (CZE) with laser-induced fluorescence (LIF) detection was developed for the detection of C-reactive protein (CRP). To optimize the CZE conditions, several factors including buffer compositions, buffer ionic strength, buffer pH, applied voltage and capillary temperature have been examined. The optimal separation buffer selected was a 30 mM sodium phosphate (PB) buffer, pH 8.0. The optimal CE applied voltage and temperature selected were 20 kV and 35°C, respectively. The CZE profile of the MNP-1°Ab-CRP-2°Ab/FITC bioconjugates showed good reproducibility. One major peak was observed for the MNP bioconjugates. The quantitative analysis also showed good results. The coefficient of variation (CV%) for the major peak area was 8.7%, and the CV% for the major peak migration time was 2.5%. The linear range for CRP analysis was 10-150 μg/mL, and the concentration limit of detection (LOD) was 9.2 μg/mL. Non-specific interactions between bovine serum albumin (BSA) and the system can be prevented by including 10% (v/v) of human plasma in the binding buffers. The CE/LIF method might be helpful for analyzing high concentrations of CRP in a patient's plasma after an acute-phase inflammation. This new method demonstrated the possibility of using MNPs and CE/LIF for the detection of proteins, and provided information for the establishment of appropriate CE conditions.

  14. A multi-channel gel electrophoresis and continuous fraction collection apparatus for high throughput protein separation and characterization

    SciTech Connect

    Choi, Megan; Nordmeyer, Robert A.; Cornell, Earl; Dong, Ming; Biggin, Mark D.; Jin, Jian

    2009-10-02

    To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A Counter Free-Flow elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high-resolution separation of a complex protein mixture can be achieved on this system using SDS-PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48-96 fractions over a mass range of 10-150 kDa; sample recovery rates were 50percent or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 L/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 g per channel and reduced resolution.

  15. Quantitative determination of dopamine in single rat pheochromocytoma cells by microchip electrophoresis with only one high-voltage power supply.

    PubMed

    Sha, Cuicui; Fan, Yuejuan; Cheng, Jieke; Cheng, Han

    2015-07-01

    We developed a method for the direct identification of dopamine in single cultured rat pheochromocytoma cells by capillary electrophoresis using an end-channel carbon fiber nanoelectrode amperometric detector. The operation mode was designed to achieve single-cell injection and lysis in microfluidic chip electrophoresis with only one high-voltage power supply. The separation and detection conditions were optimized. Four catecholamines were baseline-separated and determined with this system, and the cell density and liquid height of the reservoirs were accommodated for single cell loading, docking and analysis. The microchip capillary electrophoresis system was successfully applied to determine dopamine in single cultured rat pheochromocytoma cells. PMID:25893961

  16. A quantitative measure for protein conformational heterogeneity

    PubMed Central

    Lyle, Nicholas; Das, Rahul K.; Pappu, Rohit V.

    2013-01-01

    Conformational heterogeneity is a defining characteristic of proteins. Intrinsically disordered proteins (IDPs) and denatured state ensembles are extreme manifestations of this heterogeneity. Inferences regarding globule versus coil formation can be drawn from analysis of polymeric properties such as average size, shape, and density fluctuations. Here we introduce a new parameter to quantify the degree of conformational heterogeneity within an ensemble to complement polymeric descriptors. The design of this parameter is guided by the need to distinguish between systems that couple their unfolding-folding transitions with coil-to-globule transitions and those systems that undergo coil-to-globule transitions with no evidence of acquiring a homogeneous ensemble of conformations upon collapse. The approach is as follows: Each conformation in an ensemble is converted into a conformational vector where the elements are inter-residue distances. Similarity between pairs of conformations is quantified using the projection between the corresponding conformational vectors. An ensemble of conformations yields a distribution of pairwise projections, which is converted into a distribution of pairwise conformational dissimilarities. The first moment of this dissimilarity distribution is normalized against the first moment of the distribution obtained by comparing conformations from the ensemble of interest to conformations drawn from a Flory random coil model. The latter sets an upper bound on conformational heterogeneity thus ensuring that the proposed measure for intra-ensemble heterogeneity is properly calibrated and can be used to compare ensembles for different sequences and across different temperatures. The new measure of conformational heterogeneity will be useful in quantitative studies of coupled folding and binding of IDPs and in de novo sequence design efforts that are geared toward controlling the degree of heterogeneity in unbound forms of IDPs. PMID:24089719

  17. Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas.

    PubMed

    Flint, Mark; Matthews, Beren J; Limpus, Colin J; Mills, Paul C

    2015-01-01

    Biochemical and haematological parameters are increasingly used to diagnose disease in green sea turtles. Specific clinical pathology tools, such as plasma protein electrophoresis analysis, are now being used more frequently to improve our ability to diagnose disease in the live animal. Plasma protein reference intervals were calculated from 55 clinically healthy green sea turtles using pulsed field electrophoresis to determine pre-albumin, albumin, α-, β- and γ-globulin concentrations. The estimated reference intervals were then compared with data profiles from clinically unhealthy turtles admitted to a local wildlife hospital to assess the validity of the derived intervals and identify the clinically useful plasma protein fractions. Eighty-six per cent {19 of 22 [95% confidence interval (CI) 65-97]} of clinically unhealthy turtles had values outside the derived reference intervals, including the following: total protein [six of 22 turtles or 27% (95% CI 11-50%)], pre-albumin [two of five, 40% (95% CI 5-85%)], albumin [13 of 22, 59% (95% CI 36-79%)], total albumin [13 of 22, 59% (95% CI 36-79%)], α- [10 of 22, 45% (95% CI 24-68%)], β- [two of 10, 20% (95% CI 3-56%)], γ- [one of 10, 10% (95% CI 0.3-45%)] and β-γ-globulin [one of 12, 8% (95% CI 0.2-38%)] and total globulin [five of 22, 23% (8-45%)]. Plasma protein electrophoresis shows promise as an accurate adjunct tool to identify a disease state in marine turtles. This study presents the first reference interval for plasma protein electrophoresis in the Indo-Pacific green sea turtle.

  18. Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas.

    PubMed

    Flint, Mark; Matthews, Beren J; Limpus, Colin J; Mills, Paul C

    2015-01-01

    Biochemical and haematological parameters are increasingly used to diagnose disease in green sea turtles. Specific clinical pathology tools, such as plasma protein electrophoresis analysis, are now being used more frequently to improve our ability to diagnose disease in the live animal. Plasma protein reference intervals were calculated from 55 clinically healthy green sea turtles using pulsed field electrophoresis to determine pre-albumin, albumin, α-, β- and γ-globulin concentrations. The estimated reference intervals were then compared with data profiles from clinically unhealthy turtles admitted to a local wildlife hospital to assess the validity of the derived intervals and identify the clinically useful plasma protein fractions. Eighty-six per cent {19 of 22 [95% confidence interval (CI) 65-97]} of clinically unhealthy turtles had values outside the derived reference intervals, including the following: total protein [six of 22 turtles or 27% (95% CI 11-50%)], pre-albumin [two of five, 40% (95% CI 5-85%)], albumin [13 of 22, 59% (95% CI 36-79%)], total albumin [13 of 22, 59% (95% CI 36-79%)], α- [10 of 22, 45% (95% CI 24-68%)], β- [two of 10, 20% (95% CI 3-56%)], γ- [one of 10, 10% (95% CI 0.3-45%)] and β-γ-globulin [one of 12, 8% (95% CI 0.2-38%)] and total globulin [five of 22, 23% (8-45%)]. Plasma protein electrophoresis shows promise as an accurate adjunct tool to identify a disease state in marine turtles. This study presents the first reference interval for plasma protein electrophoresis in the Indo-Pacific green sea turtle. PMID:27293722

  19. Establishment of reference intervals for plasma protein electrophoresis in Indo-Pacific green sea turtles, Chelonia mydas

    PubMed Central

    Flint, Mark; Matthews, Beren J.; Limpus, Colin J.; Mills, Paul C.

    2015-01-01

    Biochemical and haematological parameters are increasingly used to diagnose disease in green sea turtles. Specific clinical pathology tools, such as plasma protein electrophoresis analysis, are now being used more frequently to improve our ability to diagnose disease in the live animal. Plasma protein reference intervals were calculated from 55 clinically healthy green sea turtles using pulsed field electrophoresis to determine pre-albumin, albumin, α-, β- and γ-globulin concentrations. The estimated reference intervals were then compared with data profiles from clinically unhealthy turtles admitted to a local wildlife hospital to assess the validity of the derived intervals and identify the clinically useful plasma protein fractions. Eighty-six per cent {19 of 22 [95% confidence interval (CI) 65–97]} of clinically unhealthy turtles had values outside the derived reference intervals, including the following: total protein [six of 22 turtles or 27% (95% CI 11–50%)], pre-albumin [two of five, 40% (95% CI 5–85%)], albumin [13 of 22, 59% (95% CI 36–79%)], total albumin [13 of 22, 59% (95% CI 36–79%)], α- [10 of 22, 45% (95% CI 24–68%)], β- [two of 10, 20% (95% CI 3–56%)], γ- [one of 10, 10% (95% CI 0.3–45%)] and β–γ-globulin [one of 12, 8% (95% CI 0.2–38%)] and total globulin [five of 22, 23% (8–45%)]. Plasma protein electrophoresis shows promise as an accurate adjunct tool to identify a disease state in marine turtles. This study presents the first reference interval for plasma protein electrophoresis in the Indo-Pacific green sea turtle. PMID:27293722

  20. Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain.

    PubMed

    Nishihara, Julie C; Champion, Kathleen M

    2002-07-01

    The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.

  1. Protein Electrophoresis/Immunofixation Electrophoresis

    MedlinePlus

    ... be decreased in: May be increased in: Albumin Malnutrition and malabsorption Pregnancy Kidney disease (especially nephrotic syndrome ) ... Acute or chronic inflammatory diseases Alpha 2 globulin Malnutrition Severe liver disease Hemolysis Kidney disease (nephrotic syndrome) ...

  2. [Application of capillary zone electrophoresis in the interaction analysis of protein C with protein C activator from Agkistrodon acutus venom].

    PubMed

    Sun, Yao; Bao, Pengju; Zhang, Genbao

    2013-01-01

    A new capillary zone electrophoresis method (CZE) has been established for the interaction analysis of protein C (PC) with a protein C activator (PCA) from Agkistrodon acutus venom. The analysis was performed on an uncoated fused-silica capillary with 75 microm i.d. and a total length of 60.2 cm (50 cm to the detector) with a buffer solution of 50 mmol/L Tris-HCl (pH 7.4) and 198 nm of wavelength. The factors which influence the separation of the PCA, such as buffer solution and ion concentration, and the interaction between the PCA and PC incubated for different times at 37.5 degrees C were studied. The linear range was from 10 to 300 mg/L. The limit of detection was 3 mg/L (S/N = 3). The relative standard deviation (RSD) for the migration time of the PCA was 0.56%. The RSD for the peak area was 3.8% (n = 6). The equal volumes of the PCA (200 mg/L) and PC (60 mg/L) were incubated for five minutes, at which their binding rate reached the maximum. And no hydrolyzed peptide chain from PC was found in the electropherogram. The PCA from Agkistrodon acutus venom could activate PC directly through changing the space conformation of PC. The method is simple, and highly sensitive with high resolution, and will provide important theoretical basis for the rapid detection of venom proteins and their activities in the future. PMID:23667991

  3. Speciation of iodine-containing proteins in Nori seaweed by gel electrophoresis laser ablation ICP-MS.

    PubMed

    Romarís-Hortas, V; Bianga, J; Moreda-Piñeiro, A; Bermejo-Barrera, P; Szpunar, J

    2014-09-01

    An analytical approach providing an insight into speciation of iodine in water insoluble fraction of edible seaweed (Nori) was developed. The seaweed, harvested in the Galician coast (Northwestern Spain), contained 67.7±1.3 μg g(-1) iodine of which 25% was water soluble and could be identifies as iodide. Extraction conditions of water insoluble residue using urea, NaOH, SDS and Triton X-100 were investigated. The protein pellets obtained in optimized conditions (after precipitation of urea extracts with acetone), were digested with trypsin and protease XIV. Size exclusion chromatography-ICP-MS of both enzymatic digests demonstrated the occurrence of iodoaminoacids putatively present in proteins. Intact proteins could be separated by gel electrophoresis after an additional extraction of the protein extract with phenol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) with laser ablation ICP-MS detection of (127)I indicated the presence of iodine in protein bands corresponding to molecular masses of 110 kDa, 40 kDa, 27 kDa, 20 kDa and 10 kDa. 2D IEF-SDS PAGE with laser ablation ICP-MS (127)I imaging allowed the detection of 5 iodine containing protein spots in the alkaline pI range.

  4. Deamidation as a widespread phenomenon in two-dimensional polyacrylamide gel electrophoresis of human blood plasma proteins.

    PubMed

    Sarioglu, H; Lottspeich, F; Walk, T; Jung, G; Eckerskorn, C

    2000-06-01

    The human plasma protein patterns obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a good model system for post-translational modifications because of the existence of several "ladders" of protein spots [Anderson, N. L., Anderson, N. G., Electrophoresis 1991, 12, 883-906], so-called "trains" of spots. Our investigation of several proteins, amongst others beta2-microglobulin and the haptoglobin chains, found the differences in isoelectric points (p/) to be due to deamidation of asparagines. After enzymatic cleavage with endopeptidases in the 2-D polyacrylamide gel, the asparagine and deamidated asparagine containing peptides were separated and quantified by reversed-phase HPLC. In order to separate these peptides, a neutral pH system was established and, as a result, the differences in hydrophobicity of asparagine-containing and deamidated asparagine-containing peptides increased. But how do deamidated asparagines contribute to the observed spot pattern? One spot in the 2-D gel consists of a mixture of protein species with the same number of deamidated asparagines but on different sequence position sites. The difference between the spots in the "ladder" is a growing number of negative charges introduced in the protein by an increasing number of deamidated asparagines. As a consequence, the mass difference between two spots is exactly 1 Da, which is shown in this paper for intact protein masses and the corresponding deamidated peptides.

  5. Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state

    PubMed Central

    Migliore, Luciana; Rotini, Alice; Randazzo, Davide; Albanese, Nadia N; Giallongo, Agata

    2007-01-01

    Background The endemic seagrass Posidonia oceanica (L.) Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. Results The overall expression pattern from Posidonia rhizome was determined using a preliminary proteomic approach, 437 protein spots were characterized by pI and molecular weight. We found that protein expression differs in samples belonging to sites with high or low phenols: 22 unique protein spots are peculiar of "low phenols" and 27 other spots characterize "high phenols" samples. Conclusion Posidonia showed phenols variations within the meadow, that probably reflect the heterogeneity of environmental pressures. In addition, comparison of the 2-D electrophoresis patterns allowed to highlight qualitative protein expression differences in response to these pressures. These differences may account for changes in metabolic/physiological pathways as adaptation to stress. A combined approach, based on phenols content determination and 2-D electrophoresis protein pattern, seems a promising tool to monitor Posidonia meadows health state. PMID:17663776

  6. Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Lu, Y; Qi, Y X; Zhang, H; Zhang, H Q; Pu, J J; Xie, Y X

    2013-12-19

    To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

  7. Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis.

    PubMed

    Urzainqui, A; Tabarés, E; Carrasco, L

    1987-09-01

    At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins a1, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32.

  8. ICSH recommendations for assessing automated high-performance liquid chromatography and capillary electrophoresis equipment for the quantitation of HbA2.

    PubMed

    Stephens, A D; Colah, R; Fucharoen, S; Hoyer, J; Keren, D; McFarlane, A; Perrett, D; Wild, B J

    2015-10-01

    Automated high performance liquid chromatography and Capillary electrophoresis are used to quantitate the proportion of Hemoglobin A2 (HbA2 ) in blood samples order to enable screening and diagnosis of carriers of β-thalassemia. Since there is only a very small difference in HbA2 levels between people who are carriers and people who are not carriers such analyses need to be both precise and accurate. This paper examines the different parameters of such equipment and discusses how they should be assessed.

  9. Determination of free acidic and alkaline residues of protein via moving reaction boundary titration in microdevice electrophoresis.

    PubMed

    Wang, Hou-yu; Li, Si; Tang, Yun-yun; Dong, Jing-yu; Fan, Liu-yin; Cao, Cheng-xi

    2013-06-21

    As two important physico-chemical parameters, the acidic and alkaline residues of protein are of evident significance for the evaluation of protein properties and the design of relevant separation and analysis. However, there is still no electrophoretic method used for the direct detection of free acidic and alkaline residues of protein. Herein, we developed the concepts of moving reaction boundary (MRB) and MRB titration, relevant MRB titration theory, and the method of microdevice electrophoresis for the determination of free acidic and alkaline residues of protein. In the MRB titration, the boundary was created with acid or alkali and target protein immobilized via highly cross-linked polyacrylamide gel (PAG). It was theoretically revealed that the number of free acidic or alkaline residues of protein was as a function of MRB displacement in the electrophoretic titration system. As a proof of concept, seven model proteins were chosen for the determination of acidic or alkaline residues of protein via MRB titration. The results showed that the numbers of free acidic and alkaline residues of proteins detected were in good agreement with those obtained from the relevant amino sequences in the NCBI database, demonstrating the feasibility of the developed concept, theory and technique. The general methodology of MRB titration has potential application for inexpensive, facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.

  10. Characterization of lactosylated proteins of infant formula powders using two-dimensional gel electrophoresis and nanoelectrospray mass spectrometry.

    PubMed

    Marvin, Laure F; Parisod, Véronique; Fay, Laurent B; Guy, Philippe A

    2002-08-01

    Infant formula powders were analyzed by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to assess the whey proteins quality, which may be altered by the heat treatment used during the processing conditions. Lactosylation was found to be the major chemical modification occurring in whey proteins. In parallel, a two-dimensional (2-D) gel electrophoresis was performed on the milk sample and the entire protein patterns were analyzed by nano-ESI-MS after cutting the different gel spots and in-gel trypsin digestion. A highly selective and specific tandem MS technique has been developed to characterize and localize up to ten lactosylation sites in beta-lactoglobulin (beta-Lg) and alpha(S2)-casein. alpha-Lactalbumin (alpha-La), with five lactosylated peptides, was found to be an interesting protein marker in the milk powder sample to detect chemical modification induced by the processing/storage conditions.

  11. High resolution two-dimensional polyacrylamide gel electrophoresis using Immobilines. Application to the study of brain proteins.

    PubMed

    Vocanson, C; Honnorat, J; Aguera, M; Antoine, J C; Caudie, C; Belin, M F

    1994-02-01

    Cytoplasmic soluble proteins of unknown origin but which could be associated with the development and maturation of the central nervous system are recognized by antibodies found in serum of patients with a paraneoplastic neurological syndrome. The associated antigens are of great interest in understanding these neurological pathologies. To study these antigens we describe and criticize a two dimensional polyacrylamide gel electrophoresis (2D-PAGE), using immobilized pH gradient (IPG) in first dimension. Proteins from brain complex mixtures were separated by this technique in order to identify and characterize (molecular weight, p-isoelectric point, sequencing) a protein of interest. Results prove the high reproducibility and the good resolution of the technique. Without applying enrichment method prior to 2D-PAGE, any proteins are not sequenceable after transblotting to PVDF (polyvinylidene difluoride) membrane and Coomassie blue staining. In these cases the meaning to reach the wanted aim, i.e. sequencing, was discussed. PMID:8003939

  12. Quantitative assessment of protein function prediction programs.

    PubMed

    Rodrigues, B N; Steffens, M B R; Raittz, R T; Santos-Weiss, I C R; Marchaukoski, J N

    2015-12-21

    Fast prediction of protein function is essential for high-throughput sequencing analysis. Bioinformatic resources provide cheaper and faster techniques for function prediction and have helped to accelerate the process of protein sequence characterization. In this study, we assessed protein function prediction programs that accept amino acid sequences as input. We analyzed the classification, equality, and similarity between programs, and, additionally, compared program performance. The following programs were selected for our assessment: Blast2GO, InterProScan, PANTHER, Pfam, and ScanProsite. This selection was based on the high number of citations (over 500), fully automatic analysis, and the possibility of returning a single best classification per sequence. We tested these programs using 12 gold standard datasets from four different sources. The gold standard classification of the databases was based on expert analysis, the Protein Data Bank, or the Structure-Function Linkage Database. We found that the miss rate among the programs is globally over 50%. Furthermore, we observed little overlap in the correct predictions from each program. Therefore, a combination of multiple types of sources and methods, including experimental data, protein-protein interaction, and data mining, may be the best way to generate more reliable predictions and decrease the miss rate.

  13. Quantitative assessment of protein function prediction programs.

    PubMed

    Rodrigues, B N; Steffens, M B R; Raittz, R T; Santos-Weiss, I C R; Marchaukoski, J N

    2015-01-01

    Fast prediction of protein function is essential for high-throughput sequencing analysis. Bioinformatic resources provide cheaper and faster techniques for function prediction and have helped to accelerate the process of protein sequence characterization. In this study, we assessed protein function prediction programs that accept amino acid sequences as input. We analyzed the classification, equality, and similarity between programs, and, additionally, compared program performance. The following programs were selected for our assessment: Blast2GO, InterProScan, PANTHER, Pfam, and ScanProsite. This selection was based on the high number of citations (over 500), fully automatic analysis, and the possibility of returning a single best classification per sequence. We tested these programs using 12 gold standard datasets from four different sources. The gold standard classification of the databases was based on expert analysis, the Protein Data Bank, or the Structure-Function Linkage Database. We found that the miss rate among the programs is globally over 50%. Furthermore, we observed little overlap in the correct predictions from each program. Therefore, a combination of multiple types of sources and methods, including experimental data, protein-protein interaction, and data mining, may be the best way to generate more reliable predictions and decrease the miss rate. PMID:26782400

  14. Investigating the fate of activated sludge extracellular proteins in sludge digestion using sodium dodecyl sulfate polyacrylamide gel electrophoresis.

    PubMed

    Park, Chul; Helm, Richard F; Novak, John T

    2008-12-01

    The fate of activated sludge extracellular proteins in sludge digestion was investigated using three different cation-associated extraction methods and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Extraction methods used were the cation exchange resin (CER) method for extracting calcium (Ca2+) and magnesium (Mg2+), sulfide extraction for removing iron, and base treatment (pH 10.5) for dissolving aluminum. Extracellular polymeric substances extracted were then subjected to SDS-PAGE, and the resultant protein profiles were examined before and after sludge digestion. The SDS-PAGE results showed that three methods led to different SDS-PAGE profiles for both undigested and digested sludges. The results further revealed that CER-extracted proteins remained mainly undegraded in anaerobic digestion, but were degraded in aerobic digestion. While the fate of sulfide- and base-extracted proteins was not clear for aerobic digestion, their changes in anaerobic digestion were elucidated. Most sulfide-extracted proteins were removed by anaerobic digestion, while the increase in protein band intensity and diversity was observed for base-extracted proteins. These results suggest that activated sludge flocs contain different fractions of proteins that are distinguishable by their association with certain cations and that each fraction undergoes different fates in anaerobic and aerobic digestion. The proteins that were resistant to degradation and generated during anaerobic digestion were identified by liquid chromatography tandem mass spectrometry. Protein identification results and their putative roles in activated sludge and anaerobic digestion are discussed in this study. PMID:19146099

  15. Evaluation of different protein extraction methods for banana (Musa spp.) root proteome analysis by two-dimensional electrophoresis.

    PubMed

    Vaganan, M Mayil; Sarumathi, S; Nandakumar, A; Ravi, I; Mustaffa, M M

    2015-02-01

    Four protocols viz., the trichloroacetic acid-acetone (TCA), phenol-ammonium acetate (PAA), phenol/SDS-ammonium acetate (PSA) and trisbase-acetone (TBA) were evaluated with modifications for protein extraction from banana (Grand Naine) roots, considered as recalcitrant tissues for proteomic analysis. The two-dimensional electrophoresis (2-DE) separated proteins were compared based on protein yield, number of resolved proteins, sum of spot quantity, average spot intensity and proteins resolved in 4-7 pI range. The PAA protocol yielded more proteins (0.89 mg/g of tissues) and protein spots (584) in 2-DE gel than TCA and other protocols. Also, the PAA protocol was superior in terms of sum of total spot quantity and average spot intensity than TCA and other protocols, suggesting phenol as extractant and ammonium acetate as precipitant of proteins were the most suitable for banana rooteomics analysis by 2-DE. In addition, 1:3 ratios of root tissue to extraction buffer and overnight protein precipitation were most efficient to obtain maximum protein yield.

  16. Decoding protein networks during virus entry by quantitative proteomics.

    PubMed

    Gerold, Gisa; Bruening, Janina; Pietschmann, Thomas

    2016-06-15

    Virus entry into host cells relies on interactions between viral and host structures including lipids, carbohydrates and proteins. Particularly, protein-protein interactions between viral surface proteins and host proteins as well as secondary host protein-protein interactions play a pivotal role in coordinating virus binding and uptake. These interactions are dynamic and frequently involve multiprotein complexes. In the past decade mass spectrometry based proteomics methods have reached sensitivities and high throughput compatibilities of genomics methods and now allow the reliable quantitation of proteins in complex samples from limited material. As proteomics provides essential information on the biologically active entity namely the protein, including its posttranslational modifications and its interactions with other proteins, it is an indispensable method in the virologist's toolbox. Here we review protein interactions during virus entry and compare classical biochemical methods to study entry with novel technically advanced quantitative proteomics techniques. We highlight the value of quantitative proteomics in mapping functional virus entry networks, discuss the benefits and limitations and illustrate how the methodology will help resolve unsettled questions in virus entry research in the future. PMID:26365680

  17. Prototype integration of protein electrophoresis laboratory results in an information warehouse to improve workflow and data analysis.

    PubMed

    Liu, Jianhua; Silvey, Scott A; Bissell, Michael G; Saltz, Joel H; Kamal, Jyoti

    2006-01-01

    This poster demonstrates our efforts to enhance workflow and clinical analysis of protein electrophoresis (PEP) data through integration with the Information Warehouse (IW) at The Ohio State University Medical Center (OSUMC). A new desktop application has been developed with the aim of enabling more efficient and accurate gel analysis by clinical pathologists. This tool gives the pathologists the ability to perform their analysis conveniently from anywhere on the OSUMC network along with the aid of numerical analysis algorithms, image enhancement techniques, and access to historical PEP results for the given patient.

  18. Measurement of Protein Sulfhydryls in Response to Cellular Oxidative Stress Using Gel Electrophoresis and Multiplexed Fluorescent Imaging Analysis

    PubMed Central

    Spiess, Page C.; Morin, Dexter; Jewell, William T.; Buckpitt, Alan R.

    2008-01-01

    The significance of free radicals in biology has been established by numerous investigations spanning a period of over 40 years. Whereas there are many intracellular targets for these radical species, the importance of cysteine thiol posttranslational modification has received considerable attention. The current studies present a highly sensitive method for measurement of the posttranslational modification of protein thiols. This method is based on labeling of proteins with monofunctional maleimide dyes followed by 2D gel electrophoresis to separate proteins and multiplexed fluorescent imaging analysis. The method correctly interrogates the thiol/disulfide ratio present in commercially available proteins. Exposure of pulmonary airway epithelial cells to high concentrations of menadione or t-butyl hydroperoxide resulted in the modification of cysteines in more than 141 proteins of which 60 were subsequently identified by MALDI-TOF/TOF MS. Although some proteins were modified similarly by these two oxidants, several showed detectably different maleimide ratios in response to these two agents. Proteins that were modified by one or both oxidants include those involved in transcription, protein synthesis and folding, and cell death/growth. In conclusion, these studies provide a novel procedure for measuring the redox status of cysteine thiols on individual proteins with a clearly demonstrated applicability to interactions of chemicals with pulmonary epithelial cells. PMID:18416539

  19. Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome

    PubMed Central

    2009-01-01

    The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics. PMID:19563668

  20. Quantitative Proteomic Approaches for Analysis of Protein S-Nitrosylation.

    PubMed

    Qu, Zhe; Greenlief, C Michael; Gu, Zezong

    2016-01-01

    S-Nitrosylation is a redox-based post-translational modification of a protein in response to nitric oxide (NO) signaling, and it participates in a variety of processes in diverse biological systems. The significance of this type of protein modification in health and diseases is increasingly recognized. In the central nervous system, aberrant S-nitrosylation, due to excessive NO production, is known to cause protein misfolding, mitochondrial dysfunction, transcriptional dysregulation, and neuronal death. This leads to an altered physiological state and consequently contributes to pathogenesis of neurodegenerative disorders. To date, much effort has been made to understand the mechanisms underlying protein S-nitrosylation, and several approaches have been developed to unveil S-nitrosylated proteins from different organisms. Interest in determining the dynamic changes of protein S-nitrosylation under different physiological and pathophysiological conditions has underscored the need for the development of quantitative proteomic approaches. Currently, both gel-based and gel-free mass spectrometry-based quantitative methods are widely used, and they each have advantages and disadvantages but may also be used together to produce complementary data. This review evaluates current available quantitative proteomic techniques for the analysis of protein S-nitrosylation and highlights recent advances, with emphasis on applications in neurodegenerative diseases. An important goal is to provide a comprehensive guide of feasible quantitative proteomic methodologies for examining protein S-nitrosylation in research to yield insights into disease mechanisms, diagnostic biomarkers, and drug discovery.

  1. Quantitative peptide and protein profiling by mass spectrometry.

    PubMed

    Schmidt, Alexander; Bisle, Birgit; Kislinger, Thomas

    2009-01-01

    Proteomics may be defined as the systematic analysis of proteins expressed in a given organism (Electrophoresis 16:1090-1094, 1995). Important technical innovations in mass spectrometry (MS), protein identification methods, and database annotation, over the past decade, now make it possible to routinely identify thousands of proteins in complex biological samples (Nature 422:198-207, 2003). However, to gain new insights regarding fundamental biological questions, accurate protein quantification is also required. In this chapter, we present methods for the biochemical separation of different cellular compartments, two-dimensional chromatographic separation of the constituent peptide populations, and the recently published Spectral Counting Strategy, a label-free MS-based protein quantification technology (Cell 125:173-186, 2006; Anal Chem 76:4193-4201, 2004; Mol Cell Proteomics 4:1487-1502, 2005; Cell 125:1003-1013, 2006; Methods 40:135-142, 2006; Anal Chem 77:6218-6224, 2005; J Proteome Res 5:2339-2347, 2006). Additionally, highly accurate protein quantification based on isotope dilution, describing the isotope coded protein label (ICPL) -- method shall be explained in detail (Mol Cell Proteomics 5:1543-1558, 2006; Proteomics 5:4-15, 2005).

  2. The detection and quantitation of protein oligomerization.

    PubMed

    Gell, David A; Grant, Richard P; Mackay, Joel P

    2012-01-01

    There are many different techniques available to biologists and biochemists that can be used to detect and characterize the self-association of proteins. Each technique has strengths and weaknesses and it is often useful to combine several approaches to maximize the former and minimize the latter. Here we review a range of methodologies that identify protein self-association and/or allow the stoichiometry and affinity of the interaction to be determined, placing an emphasis on what type of information can be obtained and outlining the advantages and disadvantages involved. In general, in vitro biophysical techniques, such as size exclusion chromatography, analytical ultracentrifugation, scattering techniques, NMR spectroscopy, isothermal titration calorimetry, fluorescence anisotropy and mass spectrometry, provide information on stoichiometry and/or binding affinities. Other approaches such as cross-linking, fluorescence methods (e.g., fluorescence correlation spectroscopy, FCS; Förster resonance energy transfer, FRET; fluorescence recovery after photobleaching, FRAP; and proximity imaging, PRIM) and complementation approaches (e.g., yeast two hybrid assays and bimolecular fluorescence complementation, BiFC) can be used to detect protein self-association in a cellular context.

  3. An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies.

    PubMed

    Schulenberg, Birte; Arnold, Brad; Patton, Wayne F

    2003-07-01

    Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.

  4. Differences in serum protein 2D gel electrophoresis patterns of Przewalski's (Mongolian wild horse) and thoroughbred horses.

    PubMed

    Barsuren, Enkhbolor; Namkhai, Bandi; Kong, Hong Sik

    2015-04-01

    The objective of this study was to assess differences in serum protein expression profiles of Przewalski's (Mongolian wild horse) and thoroughbred horses using proteome analysis. The serum proteins were separated by two-dimensional electrophoresis (2-DE) and five different gene products were identified. Proteins represented by the five spots were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS technology. The identities of all proteins were deduced based on their similarity to proteins in the human plasma protein database. Three proteins (a haptoglobin-2 alpha glycoprotein and two haptoglobin-2beta glycoproteins with different accession numbers) were downregulated in Przewalski's horse sera compared to thoroughbred horse sera. Moreover, two proteins (tetraspanin-18 and pM5) were upregulated in Przewalski's horses compared to thoroughbred horses. Haptoglobin-2 alpha and haptoglobin-2beta may serve as candidate molecules in future studies of inflammation, coagulation, immune modulation and pro-oxidant and antioxidant activity with consequential effects on the entire metabolism of the horse.

  5. Characterization of proteins in latex of the opium poppy (Papaver somniferum) using two-dimensional gel electrophoresis and microsequencing.

    PubMed

    Decker, G; Wanner, G; Zenk, M H; Lottspeich, F

    2000-10-01

    The opium poppy (Papaver somniferum) belongs to the group of latex-containing plants. Latex is the milky-like fluid within laticifer cells. In this study, poppy latex was analyzed with respect to ultrastructure, alkaloid, and protein content. The main goal of this project was the examination of the proteins by two-dimensional gel electrophoresis. In a proteomics approach, we investigated two main fractions of the latex, namely the cytosolic serum and the sedimented fraction containing the alkaloid-accumulating vesicles. Of the serum, representing the protein-rich part of the latex, 75 spots were analyzed by internal peptide microsequencing, followed by a database searching. For 69 proteins a function could be assigned due to homology to known proteins, whereas six spots could not be identified. Furthermore, codeinone reductase, a representative of the specific enzyme system in morphine biosynthesis, could be detected within the cytosolic serum fraction. In the vesicle-containing pellet, 23 protein spots were analyzed. An attempt was also made to separate the vesicle pellet by density centrifugation, followed by investigation of the alkaloid content, ultrastructure, and protein pattern. This study describes the first database of soluble proteins present in the latex of P. somniferum

  6. Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis.

    PubMed

    Jessen, Flemming; Wulff, Tune

    2015-09-15

    A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications.

  7. An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies.

    PubMed

    Schulenberg, Birte; Arnold, Brad; Patton, Wayne F

    2003-07-01

    Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively. PMID:12872220

  8. Endoplasmic reticulium protein profiling of heat-stressed Jurkat cells by one dimensional electrophoresis and liquid chromatography tandem mass spectrometry.

    PubMed

    Zhang, Xiulian; Kuramitsu, Yasuhiro; Ma, Aiguo; Zhang, Hui; Nakamura, Kazuyuki

    2016-08-01

    Proteomic study on membrane-integrated proteins in endoplasmic reticulum (ER) fractions was performed. In this study, we examined the effects of heat stress on Jurkat cells. The ER fractions were highly purified by differential centrifugation with sodium carbonate washing and acetone methanol precipitations. The ER membrane proteins were separated by one dimensional electrophoresis (1-DE), and some of the protein bands changed their abundance by heat stress, 12 of the 14 bands containing 40 and 60 ribosomal proteins whose expression level were decreased, on the contrary, 2 of the 14 bands containing ubiquitin and eukaryotic translation initiation factor 3 were increased. Heat treatment of human Jurkat cells led to an increase in the phosphorylation of PERK and eIF2α within 30 min of exposure. This was followed by an increase in the expression of the GRP78. Protein ubiquitination and subsequent degradation by the proteasome are important mechanisms regulating cell cycle, growth and differentiation, the result showed that heat stress enhanced ubiquitination modification of the microsomal proteins. The data of this study strongly suggest that heat treatment led to a significant reduction in protein expression and activated UPR, concomitant with protein hyperubiqutination in ER.

  9. On-Line Solid-Phase Extraction Capillary Electrophoresis Mass Spectrometry for Preconcentration and Clean-Up of Peptides and Proteins.

    PubMed

    Benavente, Fernando; Medina-Casanellas, Silvia; Giménez, Estela; Sanz-Nebot, Victoria

    2016-01-01

    One of the major drawbacks of capillary electrophoresis (CE) and other microscale separation techniques, for the analysis of low abundant peptides and proteins in complex samples, are the poor concentration limits of detection. Several strategies have been developed to improve CE sensitivity. Here, we describe an on-line solid-phase extraction capillary electrophoresis mass spectrometry method with a commercial C18 sorbent for clean-up and preconcentration of neuropeptides from highly diluted biological samples.

  10. New algorithmic approaches to protein spot detection and pattern matching in two-dimensional electrophoresis gel databases.

    PubMed

    Pleissner, K P; Hoffmann, F; Kriegel, K; Wenk, C; Wegner, S; Sahlström, A; Oswald, H; Alt, H; Fleck, E

    1999-01-01

    Protein spot identification in two-dimensional electrophoresis gels can be supported by the comparison of gel images accessible in different World Wide Web two-dimensional electrophoresis (2-DE) gel protein databases. The comparison may be performed either by visual cross-matching between gel images or by automatic recognition of similar protein spot patterns. A prerequisite for the automatic point pattern matching approach is the detection of protein spots yielding the x(s),y(s) coordinates and integrated spot intensities i(s). For this purpose an algorithm is developed based on a combination of hierarchical watershed transformation and feature extraction methods. This approach reduces the strong over-segmentation of spot regions normally produced by watershed transformation. Measures for the ellipticity and curvature are determined as features of spot regions. The resulting spot lists containing x(s),y(s),i(s)-triplets are calculated for a source as well as for a target gel image accessible in 2-DE gel protein databases. After spot detection a matching procedure is applied. Both the matching of a local pattern vs. a full 2-DE gel image and the global matching between full images are discussed. Preset slope and length tolerances of pattern edges serve as matching criteria. The local matching algorithm relies on a data structure derived from the incremental Delaunay triangulation of a point set and a two-step hashing technique. For the incremental construction of triangles the spot intensities are considered in decreasing order. The algorithm needs neither landmarks nor an a priori image alignment. A graphical user interface for spot detection and gel matching is written in the Java programming language for the Internet. The software package called CAROL (http://gelmatching.inf.fu-berlin.de) is realized in a client-server architecture.

  11. A comparative method for protein extraction and 2-D gel electrophoresis from different tissues of Cajanus cajan.

    PubMed

    Singh, Nisha; Jain, Neha; Kumar, Ram; Jain, Ajay; Singh, Nagendra K; Rai, Vandna

    2015-01-01

    Pigeonpea is an important legume crop with high protein content. However, it is often subjected to various abiotic and biotic stresses. Proteomics is a state-of-the-art technique used to analyze the protein profiling of a tissue for deciphering the molecular entities that could be manipulated for developing crops resistant to these stresses. In this context, developing a comprehensive proteome profile from different vegetative and reproductive tissues has become mandatory. Although several protein extraction protocols from different tissues of diverse plant species have been reported, there is no report for pigeonpea. Here, we report tissue-specific protein extraction protocols representing vegetative (young leaves), and reproductive (flowers and seeds) organs and their subsequent analysis on 2-dimensional gel electrophoresis. The study explicitly demonstrated that the efficacy of a particular protein extraction protocol is dependent on the different tissues, such as leaves, flowers and seeds that differ in their structure and metabolic constituents. For instance, phenol-based protocol showed an efficacy toward higher protein yield, better spot resolution and a minimal streaking on 2-DE gel for both leaves and flowers. Protein extraction from seeds was best achieved by employing phosphate-TCA-acetone protocol. PMID:26300903

  12. Electrophoretic mobilities of the complexes between sodium dodecyl sulfate and various peptides or proteins determined by free solution electrophoresis using coated capillaries.

    PubMed

    Karim, M R; Shinagawa, S; Takagi, T

    1994-01-01

    Electrophoretic mobilities of various proteins or peptides complexed with sodium dodecyl sulfate (SDS) were determined by free solution capillary electrophoresis. The complexes formed between SDS and protein polypeptide showed electrophoretic mobilities virtually insensitive to the protein molecular weight. On the other hand, scattered and larger negative electrophoretic mobilities were observed for peptides of molecular weights less than 10000. Mobility differences as small as 0.3-3% among the three differently modified derivatives of bovine serum albumin could also be successfully determined due to the high resolving power of capillary electrophoresis.

  13. Specific proteins synthesized during the viral lytic cycle in vaccinia virus-infected HeLa cells: analysis by high-resolution, two-dimensional gel electrophoresis

    SciTech Connect

    Carrasco, L.; Bravo, R.

    1986-05-01

    The proteins synthesized in vaccinia-infected HeLa cells have been analyzed at different times after infection by using two-dimensional gel electrophoresis. Vaccinia-infected cells present up to 198 polypeptides (138 acidic, isoelectric focusing; 60 basic, nonequilibrium pH gradient electrophoresis) not detected in control cells. Cells infected in the presence of cycloheximide show 81 additional polypeptides after cycloheximide removal, resulting in a total estimate of 279 proteins induced after vaccinia infection. The glycoproteins made at various time postinfection were also analyzed. At least 13 proteins labeled with (/sup 3/H)glucosamine were detected in vaccinia-infected HeLa cells.

  14. Multiplex and quantitative pathogen detection with high-resolution capillary electrophoresis-based single-strand conformation polymorphism.

    PubMed

    Hwang, Hee Sung; Shin, Gi Won; Chung, Boram; Na, Jeongkyeong; Jung, Gyoo Yeol

    2013-01-01

    Among the molecular diagnostic methods for bacteria-induced diseases, capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) combined with 16S rRNA gene-specific PCR has enormous potential because it can separate sequence variants using a simple procedure. However, conventional CE-SSCP systems have limited resolution and cannot separate most 16S rRNA gene-specific markers into separate peaks. A high-resolution CE-SSCP system that uses a poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) triblock copolymer matrix was recently developed and shown to effectively separate highly similar PCR products. In this report, a protocol for the detection of 12 pathogenic bacteria is provided. Pathogen markers were amplified by PCR using universal primers and separated by CE-SSCP; each marker peak was well separated at baseline and showed a characteristic mobility, allowing the easy identification of the pathogens.

  15. Multilayer polymer microchip capillary array electrophoresis devices with integrated on-chip labeling for high-throughput protein analysis

    PubMed Central

    Yu, Ming; Wang, Qingsong; Patterson, James E.; Woolley, Adam T.

    2011-01-01

    It is desirable to have inexpensive, high-throughput systems that integrate multiple sample analysis processes and procedures, for applications in biology, chemical analysis, drug discovery, and disease screening. In this paper, we demonstrate multilayer polymer microfluidic devices with integrated on-chip labeling and parallel electrophoretic separation of up to 8 samples. Microchannels were distributed in two different layers and connected through interlayer through-holes in the middle layer. A single set of electrophoresis reservoirs and one fluorescent label reservoir address parallel analysis units for up to 8 samples. Individual proteins and a mixture of cancer biomarkers have been successfully labeled on-chip and separated in parallel with this system. A detection limit of 600 ng/mL was obtained for heat shock protein 90. Our integrated on-chip labeling microdevices show great potential for low-cost, simplified, rapid and high-throughput analysis. PMID:21449615

  16. Proteomics analysis in mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritive, and allergenic proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano electrospray ionization liquid chromatography tandem mass ...

  17. Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis.

    PubMed

    Guo, Cheng-ye; Wang, Hou-yu; Liu, Xiao-ping; Fan, Liu-yin; Zhang, Lei; Cao, Cheng-xi

    2013-05-01

    In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross-linked polyacrylamide gel (PAG), an acid-base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.

  18. Protein expression profile related to cisplatin resistance in bladder cancer cell lines detected by two-dimensional gel electrophoresis.

    PubMed

    Taoka, Yoshinori; Matsumoto, Kazumasa; Ohashi, Kazuya; Minamida, Satoru; Hagiwara, Masahiro; Nagi, Shoji; Saito, Tatsuya; Kodera, Yoshio; Iwamura, Masatsugu

    2015-01-01

    We used a proteomic approach to compare the differentially regulated protein expression profiles of cisplatin-naïve and cisplatin-resistant bladder cancer cell lines to screen candidate molecules related to cisplatin resistance. The cisplatin-resistant cell line T24 was established by the stepwise exposure of T24 cells to up to 40 μM of cisplatin. We performed a comprehensive study of protein expression in bladder cancer cell lines that included cisplatin-naïve (T24) and cisplatin-resistant cells (T24CDDPR) by means of agarose two-dimensional gel electrophoresis followed by analysis of liquid chromatography tandem mass spectroscopy. We identified 25 obviously different spots for T24 and T24 CDDPR. Seven spots had increased expression and 18 spots had decreased expression in T24CDDPR compared to those in T24. Cytoskeletal proteins and enzyme modulators were prominent among differential proteins. Of the 25 proteins, we selected HNRNPA3, PCK2, PPL, PGK1, TKT, SERPINB2, GOT2, and EIF3A for further validation by Western blot. HNRNPA3, PGK1, TKT, and SERPINB2 had more than 1.5-times incremental expression in T24CDDPR compared to that in T24. PCK2 and PPL expressions were decreased less than 20% in T24CDDPR compared to that in T24. The results of 25 new proteins in this study could be valuable and could lead to the development of a new molecular marker. PMID:26299484

  19. Two-Dimensional Differential Gel Electrophoresis to Identify Protein Biomarkers in Amniotic Fluid of Edwards Syndrome (Trisomy 18) Pregnancies

    PubMed Central

    Hsu, Te-Yao; Lin, Hao; Hung, Hsuan-Ning; Yang, Kuender D.; Ou, Chia-Yu; Tsai, Ching-Chang; Cheng, Hsin-Hsin; Chung, Su-Hai; Cheng, Bi-Hua; Wong, Yi-Hsun; Chou, An Kuo; Hsiao, Chang-Chun

    2016-01-01

    Background Edwards syndrome (ES) is a severe chromosomal abnormality with a prevalence of about 0.8 in 10,000 infants born alive. The aims of this study were to identify candidate proteins associated with ES pregnancies from amniotic fluid supernatant (AFS) using proteomics, and to explore the role of biological networks in the pathophysiology of ES. Methods AFS from six second trimester pregnancies with ES fetuses and six normal cases were included in this study. Fluorescence-based two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) were used for comparative proteomic analysis. The identified proteins were further validated by Western blotting and the role of biological networks was analyzed. Results Twelve protein spots were differentially expressed by more than 1.5-fold in the AFS of the ES pregnancies. MALDI-TOF/MS identified one up-regulated protein: apolipoprotein A1 (ApoA1), and four under-regulated proteins: vitamin D binding protein (VDBP), alpha-1-antitrypsin (A1AT), insulin-like growth factor-binding protein 1 (IGFBP-1), and transthyretin (TTR). Western blot and densitometric analysis of ApoA1, A1AT, IGFBP-1, and TTR confirmed the alteration of these proteins in the amniotic fluid samples. Biological network analysis revealed that the proteins of the ES AFS were involved mainly in lipid and hormone metabolism, immune response, and cardiovascular disease. Conclusions These five proteins may be involved in the pathogenesis of ES. Further studies are needed to explore. PMID:26752631

  20. HP-Lattice QSAR for dynein proteins: experimental proteomics (2D-electrophoresis, mass spectrometry) and theoretic study of a Leishmania infantum sequence.

    PubMed

    Dea-Ayuela, María Auxiliadora; Pérez-Castillo, Yunierkis; Meneses-Marcel, Alfredo; Ubeira, Florencio M; Bolas-Fernández, Francisco; Chou, Kuo-Chen; González-Díaz, Humberto

    2008-08-15

    The toxicity and inefficacy of actual organic drugs against Leishmaniosis justify research projects to find new molecular targets in Leishmania species including Leishmania infantum (L. infantum) and Leishmaniamajor (L. major), both important pathogens. In this sense, quantitative structure-activity relationship (QSAR) methods, which are very useful in Bioorganic and Medicinal Chemistry to discover small-sized drugs, may help to identify not only new drugs but also new drug targets, if we apply them to proteins. Dyneins are important proteins of these parasites governing fundamental processes such as cilia and flagella motion, nuclear migration, organization of the mitotic splinde, and chromosome separation during mitosis. However, despite the interest for them as potential drug targets, so far there has been no report whatsoever on dyneins with QSAR techniques. To the best of our knowledge, we report here the first QSAR for dynein proteins. We used as input the Spectral Moments of a Markov matrix associated to the HP-Lattice Network of the protein sequence. The data contain 411 protein sequences of different species selected by ClustalX to develop a QSAR that correctly discriminates on average between 92.75% and 92.51% of dyneins and other proteins in four different train and cross-validation datasets. We also report a combined experimental and theoretic study of a new dynein sequence in order to illustrate the utility of the model to search for potential drug targets with a practical example. First, we carried out a 2D-electrophoresis analysis of L. infantum biological samples. Next, we excised from 2D-E gels one spot of interest belonging to an unknown protein or protein fragment in the region M<20,200 and pI<4. We used MASCOT search engine to find proteins in the L. major data base with the highest similarity score to the MS of the protein isolated from L. infantum. We used the QSAR model to predict the new sequence as dynein with probability of 99.99% without

  1. Molecular phylogeny of the hominoid primates as indicated by two-dimensional protein electrophoresis

    SciTech Connect

    Goldman, D.; Giri, P.R.; O'Brien, J.O.

    1987-05-01

    A molecular phylogeny for the hominoid primates was constructed by using genetic distances from a survey of 383 radiolabeled fibroblast polypeptides resolved by two-dimensional electrophoresis (2DE). An internally consistent matrix of Nei genetic distances was generated on the basis of variants in electrophoretic position. The derived phylogenetic tree indicated a branching sequence, from oldest to most recent, of cercopithecoids (Macaca fascicularis), gibbon-siamang, orangutan, gorilla, and human-chimpanzee. A cladistic analysis of 240 electrophoretic characters that varied between ape species produced an identical tree. Genetic distance measures obtained by 2DE are largely consistent with those generated by other molecular procedures. In addition, the 2DE data set appears to resolve the human-chimpanzee-gorilla trichotomy in favor of a more recent association of chimpanzees and humans.

  2. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  3. Comparison of structural and nonstructural proteins of virulent and less virulent Theiler's virus isolates using two-dimensional gel electrophoresis.

    PubMed

    Rozhon, E J; Kratochvil, J D; Lipton, H L

    1985-02-01

    The Theiler's murine encephalomyelitis viruses (TMEV) are important neurotropic picornaviruses because they persist in the central nervous system (CNS) and produce an inflammatory demyelinating disease in the mouse, their natural host. Insight into the pathogenesis of this disease may come from studying the genetic and biochemical compositions of these viruses; therefore, in this report, the structural and nonstructural proteins specified by both highly and less virulent TMEV were examined. Using two-dimensional gel electrophoresis, structural and nonstructural proteins, originating from each of the three regions of the picornavirus genome (Kitamura et al., 1981; Rueckert and Wimmer, 1984), from nine TMEV isolates were compared on the basis of isoelectric points (pI). Proteins of two virulent TMEV (GDVII and FA viruses) had almost indistinguishable pI values, whereas two of the three major capsid proteins of the less virulent TMEV varied considerably. For example, the structural proteins VP1 and VP3 from seven less virulent viruses ranged from pI 6.3 to 6.9 and 6.5 to 8.3, respectively. On the other hand, the pI values of VP2 and nonstructural proteins from the less virulent TMEV varied relatively little. In general, structural proteins of each TMEV group had pI ranges unique to their respective biological group, while most nonstructural proteins were similar for all TMEV. The virus-specified proteins of Vilyuisk virus, which is serologically related to the TMEV and a possible cause of encephalomyelitis in man, had pI values similar to the less virulent TMEV. Finally, VP3 not only showed the greatest variation in pI among the less virulent TMEV, but it also was preferentially radioiodinated in intact virus from each of the two biological groups using the lactoperoxidase technique.

  4. Efficient method of protein extraction from Theobroma cacao L. roots for two-dimensional gel electrophoresis and mass spectrometry analyses.

    PubMed

    Bertolde, F Z; Almeida, A-A F; Silva, F A C; Oliveira, T M; Pirovani, C P

    2014-07-04

    Theobroma cacao is a woody and recalcitrant plant with a very high level of interfering compounds. Standard protocols for protein extraction were proposed for various types of samples, but the presence of interfering compounds in many samples prevented the isolation of proteins suitable for two-dimensional gel electrophoresis (2-DE). An efficient method to extract root proteins for 2-DE was established to overcome these problems. The main features of this protocol are: i) precipitation with trichloroacetic acid/acetone overnight to prepare the acetone dry powder (ADP), ii) several additional steps of sonication in the ADP preparation and extractions with dense sodium dodecyl sulfate and phenol, and iii) adding two stages of phenol extractions. Proteins were extracted from roots using this new protocol (Method B) and a protocol described in the literature for T. cacao leaves and meristems (Method A). Using these methods, we obtained a protein yield of about 0.7 and 2.5 mg per 1.0 g lyophilized root, and a total of 60 and 400 spots could be separated, respectively. Through Method B, it was possible to isolate high-quality protein and a high yield of roots from T. cacao for high-quality 2-DE gels. To demonstrate the quality of the extracted proteins from roots of T. cacao using Method B, several protein spots were cut from the 2-DE gels, analyzed by tandem mass spectrometry, and identified. Method B was further tested on Citrus roots, with a protein yield of about 2.7 mg per 1.0 g lyophilized root and 800 detected spots.

  5. Binary Oscillatory Crossflow Electrophoresis

    NASA Technical Reports Server (NTRS)

    Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.

    1996-01-01

    We present preliminary results of our implementation of a novel electrophoresis separation technique: Binary Oscillatory Cross flow Electrophoresis (BOCE). The technique utilizes the interaction of two driving forces, an oscillatory electric field and an oscillatory shear flow, to create an active binary filter for the separation of charged species. Analytical and numerical studies have indicated that this technique is capable of separating proteins with electrophoretic mobilities differing by less than 10%. With an experimental device containing a separation chamber 20 cm long, 5 cm wide, and 1 mm thick, an order of magnitude increase in throughput over commercially available electrophoresis devices is theoretically possible.

  6. Differentially regulated proteins in Prevotella intermedia after oxidative stress analyzed by 2D electrophoresis and mass spectrometry.

    PubMed

    Santos, Simone G; Diniz, Cláudio G; Silva, Vânia L; Lima, Francisca L; Andrade, Hélida M; Chapeaurouge, Donat A; Perales, Jonas; Serufo, José Carlos; Carvalho, Maria Auxiliadora R; Farias, Luiz M

    2012-02-01

    Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells. PMID:22193554

  7. Quantitative capillary electrophoresis and its application in analysis of alkaloids in tea, coffee, coca cola, and theophylline tablets.

    PubMed

    Li, Mengjia; Zhou, Junyi; Gu, Xue; Wang, Yan; Huang, Xiaojing; Yan, Chao

    2009-01-01

    A quantitative CE (qCE) system with high precision has been developed, in which a 4-port nano-valve was isolated from the electric field and served as sample injector. The accurate amount of sample was introduced into the CE system with high reproducibility. Based on this system, consecutive injections and separations were performed without voltage interruption. Reproducibilities in terms of RSD lower than 0.8% for retention time and 1.7% for peak area were achieved. The effectiveness of the system was demonstrated by the quantitative analysis of caffeine, theobromine, and theophylline in real samples, such as tea leaf, roasted coffee, coca cola, and theophylline tablets.

  8. Field-amplified sample stacking β-cyclodextrin modified capillary electrophoresis for quantitative determination of diastereomeric saponins.

    PubMed

    Emara, Samy; Masujima, Tsutomu; Zarad, Walaa; Mohamed, Khaled; Kamal, Maha; Fouad, Marwa; EL-Bagary, Ramzia

    2014-01-01

    Successful simultaneous diastereomeric separation and sensitive determination of two pairs of triterpenoidal saponins have been achieved by capillary electrophoresis (CE) using β-cyclodextrin (β-CD) as a stereoselective agent to cooperate with borate complexation. A usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to the determination of individual saponins by CE. Soyasaponin I ( S1: ), azukisaponin V ( S2: ), bersimoside I ( S3: ) and bersimoside II ( S4: ) could be well separated within 14 min in a fused-silica capillary (60 cm long to the detector with an additional 10 cm to the cathode; 75 µm i.d.). The background electrolyte was borate buffer (80 mM, pH 10), containing 24 mM β-CD. The separation voltage was 14 kV with a detection wavelength of 195 nm. The sample was electrokinetically injected using a voltage of 16 kV for 12 s. Methanol (70%) was used as the diluent for field-amplified sample stacking after hydrodynamic injection of short water plug (5 cm, 4 s). The method was partially validated for linearity, repeatability, reproducibility, limits of detection and limits of quantification. The correlation coefficients of the calibration curves were all >0.998, and the recoveries were from 98.23 to 96.21%.

  9. Improved Solubilization of Surface Proteins from Listeria monocytogenes for Two-dimensional Gel Electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Solubilization of bacterial surface (cell wall and membrane-associated) proteins for 2-DE is challenging, particularly in the case of Gram-positive bacteria. This is primarily due to strong protein association with the cell wall peptidoglycan and protein hydrophobicity. We solubilized surface protei...

  10. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat flour is one of the world's major food ingredients, but it is difficult to distinguish and identify the many proteins in a flour sample. The abundant glutamine and proline rich gluten proteins are responsible for many of the unique end-use qualities of wheat flour but it is challenging to dis...

  11. Two-dimensional isoelectric focusing OFFGEL and microfluidic lab-on-chip electrophoresis for assessing dissolved proteins in seawater.

    PubMed

    García-Otero, Natalia; Peña-Vázquez, Elena; Barciela-Alonso, María Carmen; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

    2013-06-18

    Dissolved proteins were assessed in surface and deep seawater by two-dimensional isoelectric focusing (IEF) OFFGEL-lab-on-chip (LOC) electrophoresis after tangential flow ultrafiltration followed by centrifugal ultrafiltration (preconcentration factor of 3000). Dissolved protein isolation was performed by treating the ultrafiltrated retentate with cold acetone and also with chloroform as precipitating reagents. The best electrophoretic behavior of the isolated proteins was obtained after protein precipitation with chloroform before different rinsing stages for removing methanol and water interferences. Metals bound to proteins in the different OFFGEL fractions were assessed by inductively coupled plasma-optical emission spectrometry and electrothermal atomic absorption spectrometry, under optimized operating conditions. Experiments regarding stability of the metal-binding proteins [superoxide dismutase (SOD) and alcohol dehydrogenase (ADH) as protein models] showed the integrity of the Zn-binding SOD/ADH under the OFFGEL electrophoretic conditions. However, stability of Cu bound to SOD is not guaranteed. The first electrophoretic dimension (IEF OFFGEL) showed that dissolved proteins in surface seawater exhibit alkaline isoelectric points (pIs of 8.10 and 8.37) and also acid Ips (4.82, 5.13, 5.43, and 5.73), while LOC showed that the isolated proteins exhibit a spread molecular weight range (within 15 - 63 kDa); although, high molecular weights were the most commonly found. Regarding deep seawater, isolated proteins were of acid Ips (from 3.30 to 4.22) and low molecular weight (within the 21-24 kDa range). Elements such as Cd, Cu, Mn, and Ni were mainly associated with dissolved proteins of alkaline pIs in surface seawater, while Zn was mainly associated to proteins of acid pIs. However, only Cu and Mn were found to be bound to dissolved proteins of higher Ips in deep seawater, and the amount of Mn (from 68 to 84 μg L(-1)) was higher than that found in dissolved

  12. Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry

    PubMed Central

    2011-01-01

    Background Wheat flour is one of the world's major food ingredients, in part because of the unique end-use qualities conferred by the abundant glutamine- and proline-rich gluten proteins. Many wheat flour proteins also present dietary problems for consumers with celiac disease or wheat allergies. Despite the importance of these proteins it has been particularly challenging to use MS/MS to distinguish the many proteins in a flour sample and relate them to gene sequences. Results Grain from the extensively characterized spring wheat cultivar Triticum aestivum 'Butte 86' was milled to white flour from which proteins were extracted, then separated and quantified by 2-DE. Protein spots were identified by separate digestions with three proteases, followed by tandem mass spectrometry analysis of the peptides. The spectra were used to interrogate an improved protein sequence database and results were integrated using the Scaffold program. Inclusion of cultivar specific sequences in the database greatly improved the results, and 233 spots were identified, accounting for 93.1% of normalized spot volume. Identified proteins were assigned to 157 wheat sequences, many for proteins unique to wheat and nearly 40% from Butte 86. Alpha-gliadins accounted for 20.4% of flour protein, low molecular weight glutenin subunits 18.0%, high molecular weight glutenin subunits 17.1%, gamma-gliadins 12.2%, omega-gliadins 10.5%, amylase/protease inhibitors 4.1%, triticins 1.6%, serpins 1.6%, purinins 0.9%, farinins 0.8%, beta-amylase 0.5%, globulins 0.4%, other enzymes and factors 1.9%, and all other 3%. Conclusions This is the first successful effort to identify the majority of abundant flour proteins for a single wheat cultivar, relate them to individual gene sequences and estimate their relative levels. Many genes for wheat flour proteins are not expressed, so this study represents further progress in describing the expressed wheat genome. Use of cultivar-specific contigs helped to overcome

  13. Whole genome scan to detect quantitative trait loci for bovine milk protein composition.

    PubMed

    Schopen, G C B; Koks, P D; van Arendonk, J A M; Bovenhuis, H; Visker, M H P W

    2009-08-01

    The objective of this study was to perform a whole genome scan to detect quantitative trait loci (QTL) for milk protein composition in 849 Holstein-Friesian cows originating from seven sires. One morning milk sample was analysed for the major milk proteins using capillary zone electrophoresis. A genetic map was constructed with 1341 single nucleotide polymorphisms, covering 2829 centimorgans (cM) and 95% of the cattle genome. The chromosomal regions most significantly related to milk protein composition (P(genome) < 0.05) were found on Bos taurus autosomes (BTA) 6, 11 and 14. The QTL on BTA6 was found at about 80 cM, and affected alpha(S1)-casein, alpha(S2)-casein, beta-casein and kappa-casein. The QTL on BTA11 was found at 124 cM, and affected beta-lactoglobulin, and the QTL on BTA14 was found at 0 cM, and affected protein percentage. The proportion of phenotypic variance explained by the QTL was 3.6% for beta-casein and 7.9% for kappa-casein on BTA6, 28.3% for beta-lactoglobulin on BTA11, and 8.6% for protein percentage on BTA14. The QTL affecting alpha(S2)-casein on BTA6 and 17 showed a significant interaction. We investigated the extent to which the detected QTL affecting milk protein composition could be explained by known polymorphisms in beta-casein, kappa-casein, beta-lactoglobulin and DGAT1 genes. Correction for these polymorphisms decreased the proportion of phenotypic variance explained by the QTL previously found on BTA6, 11 and 14. Thus, several significant QTL affecting milk protein composition were found, of which some QTL could partially be explained by polymorphisms in milk protein genes.

  14. A general method for the rapid characterization of tyrosine-phosphorylated proteins by mini two-dimensional gel electrophoresis.

    PubMed

    Ducret, A; Desponts, C; Desmarais, S; Gresser, M J; Ramachandran, C

    2000-06-01

    Our preliminary results are reported in the investigation of the tyrosine phosphorylation cascade triggered by the stimulation of the insulin receptor in the adipocyte cell line 3T3-L1 using a mini two-dimensional gel electrophoresis approach. The minigel format, 8 x 10 cm, was found sufficiently resolving and reproducible to study complex biological samples while considerably increasing throughput and lowering costs compared to larger gel formats. Consequently, we used the minigel format to rapidly screen a large number of samples, of which only the most relevant were then analyzed by optimized, preparative two-dimensional gels. The accurate localization and relative quantification of tyrosine-phosphorylated proteins was performed using a nonradioactive triple labeling method. After transfer onto polyvinylidene difluoride (PVDF) membranes, proteins were stained with Sypro Ruby to verify the separation quality and to localize the general region of interest for immunostaining. The membranes were subsequently blocked with polyvinylpyrrolidone-40 and probed with the relevant antibodies for visualization of the phosphorylated proteins by chemiluminescence. Finally, membranes were stained with colloidal gold to obtain a pattern reminiscent of the silver staining of a polyacrylamide gel. We believe that the presented strategy can be generalized for any gel application in which a protein has to be detected and identified based on its immunoreactivity. PMID:10892730

  15. Two-dimensional polyacrylamide-gel electrophoresis of the proteins and glycoproteins of purified human platelet surface and intracellular membranes.

    PubMed

    Hack, N; Crawford, N

    1984-08-15

    By using highly purified surface and intracellular membrane fractions prepared from human platelets by free-flow electrophoresis, the polypeptide and glycopeptides of these membranes have been characterized by high-resolution gel electrophoresis under reducing and non-reducing conditions. Silver staining and a variety of glycoprotein-staining procedures have been applied to identify the major components. The principal finding was the clear disparity between the distribution patterns for these two membrane fractions. There are proportionately more low-Mr acidic components present in the intracellular membrane than in the surface-derived membrane. Of the major platelet surface glycoproteins GPIb, IIb, IIIa and IIIb (or IV) well expressed in the surface membrane only, GPIIb and IIIa appear as trace components in the intracellular membrane. The cytoskeleton proteins, actin, myosin, tropomyosin, actin-binding protein and alpha-actinin are prominent features of the surface membrane and essentially absent from the intracellular membrane. Neuraminidase treatment at the whole-cell level, before homogenization, which is an essential requirement for good resolution of the two membrane subfractions, modifies a number of the glycoprotein subunits with respect to their pI characteristics, suggesting much molecular micro-heterogeneity with respect to sialic acid content. A comparison of the staining characteristics of the major glycoproteins with periodic acid/Schiff's reagent and concanavalin A/peroxidase detection and a combined procedure revealed significant differences in associated carbohydrate structures, and the major concanavalin A-binding component was shown to be GPIIIa. These observations are discussed in the context of functional activities of both membrane systems in the physiological behaviour of the platelet.

  16. A visual detection of protein content based on titration of moving reaction boundary electrophoresis.

    PubMed

    Wang, Hou-Yu; Guo, Cheng-Ye; Guo, Chen-Gang; Fan, Liu-Yin; Zhang, Lei; Cao, Cheng-Xi

    2013-04-24

    A visual electrophoretic titration method was firstly developed from the concept of moving reaction boundary (MRB) for protein content analysis. In the developed method, when the voltage was applied, the hydroxide ions in the cathodic vessel moved towards the anode, and neutralized the carboxyl groups of protein immobilized via highly cross-linked polyacrylamide gel (PAG), generating a MRB between the alkali and the immobilized protein. The boundary moving velocity (V(MRB)) was as a function of protein content, and an acid-base indicator was used to denote the boundary displacement. As a proof of concept, standard model proteins and biological samples were chosen for the experiments to study the feasibility of the developed method. The experiments revealed that good linear calibration functions between V(MRB) and protein content (correlation coefficients R>0.98). The experiments further demonstrated the following merits of developed method: (1) weak influence of non-protein nitrogen additives (e.g., melamine) adulterated in protein samples, (2) good agreement with the classic Kjeldahl method (R=0.9945), (3) fast measuring speed in total protein analysis of large samples from the same source, and (4) low limit of detection (0.02-0.15 mg mL(-1) for protein content), good precision (R.S.D. of intra-day less than 1.7% and inter-day less than 2.7%), and high recoveries (105-107%).

  17. Capillary electrophoresis separation of neutral organic compounds, pharmaceutical drugs, proteins and peptides, enantiomers, and anions

    SciTech Connect

    Ding, W.L.

    1999-02-12

    Addition of a novel anionic surfactant, namely lauryl polyoxyethylene sulfate, to an aqueous-acetonitrile electrolyte makes it possible to separate nonionic organic compounds by capillary electrophoresis. Separation is based on differences in the association between analytes and the surfactant. Highly hydrophobic compounds such as polyaromatic hydrocarbons are well separated by this new surfactant. Migration times of analytes can be readily changed over an unusually large range by varying the additive concentration and the proportion of acetonitrile in the electrolyte. Several examples are given, including the separation of four methylbenz[a]anthracene isomers and the separation of normal and deuterated acetophenone. The effect of adding this new surfactant to the acidic electrolyte was also investigated. Incorporation of cetyltrimethylammonium bromide in the electrolyte is shown to dynamically coat the capillary and reverse electroosmotic flow. Chiral recognition mechanism is studied using novel synthetic surfactants as chiral selectors, which are made from amino acids reacting with alkyl chloroformates. A satisfactory separation of both inorganic and organic anions is obtained using electrolyte solutions as high as 5 M sodium chloride using direct photometric detection. The effect of various salts on electrophoretic and electroosmotic mobility is further discussed. Several examples are given under high-salt conditions.

  18. Phosphate-affinity electrophoresis on a microchip for determination of protein kinase activity.

    PubMed

    Han, Aishan; Hosokawa, Kazuo; Maeda, Mizuo

    2009-10-01

    We describe microchip-based phosphate-affinity electrophoresis (microPAE) for separation of peptides aimed at determination of kinase activity. The microPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form. We synthesized a Phos-tag-poly(dimethylacrylamide) conjugate. The conjugate and the sample solutions were autonomously injected into a PDMS-glass hybrid microchip. The two solutions were contacted together in the microchannel. When the peptides were electrophoresed into the Phos-tag-poly(dimethylacrylamide) region, the phosphorylated peptide was specifically trapped, and separated from the nonphosphorylated peptide in 10 s. The results were quantified by the areas of the fluorescence peaks. The calibration plot obtained with standard samples showed an excellent linearity and a LOD of 0.9% phosphorylated peptide among the total peptides. For c-Src-reacted samples, the results from the microPAE were in good agreement with those from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The microPAE was also successful in the presence of inhibitors for c-Src. The measured 50% inhibitory concentration values for staurosporine, PP2, and SU6656 were in good agreement with the literature values. PMID:19784951

  19. Evaluating two-dimensional electrophoresis profiles of the protein phaseolin as markers of genetic differentiation and seed protein quality in common bean (Phaseolus vulgaris L.).

    PubMed

    López-Pedrouso, María; Bernal, Javier; Franco, Daniel; Zapata, Carlos

    2014-07-23

    High-resolution two-dimensional electrophoresis (2-DE) profiles of the protein phaseolin, the major seed storage protein of common bean, display great number of spots with differentially glycosylated and phosphorylated α- and β-type polypeptides. This work aims to test whether these complex profiles can be useful markers of genetic differentiation and seed protein quality in bean populations. The 2-DE phaseolin profile and the amino acid composition were examined in bean seeds from 18 domesticated and wild accessions belonging to the Mesoamerican and Andean gene pools. We found that proteomic distances based on 2-DE profiles were successful in identifying the accessions belonging to each gene pool and outliers distantly related. In addition, accessions identified as outliers from proteomic distances showed the highest levels of methionine content, an essential amino acid deficient in bean seeds. These findings suggest that 2-DE phaseolin profiles provide valuable information with potential of being used in common bean genetic improvement.

  20. Evaluating two-dimensional electrophoresis profiles of the protein phaseolin as markers of genetic differentiation and seed protein quality in common bean (Phaseolus vulgaris L.).

    PubMed

    López-Pedrouso, María; Bernal, Javier; Franco, Daniel; Zapata, Carlos

    2014-07-23

    High-resolution two-dimensional electrophoresis (2-DE) profiles of the protein phaseolin, the major seed storage protein of common bean, display great number of spots with differentially glycosylated and phosphorylated α- and β-type polypeptides. This work aims to test whether these complex profiles can be useful markers of genetic differentiation and seed protein quality in bean populations. The 2-DE phaseolin profile and the amino acid composition were examined in bean seeds from 18 domesticated and wild accessions belonging to the Mesoamerican and Andean gene pools. We found that proteomic distances based on 2-DE profiles were successful in identifying the accessions belonging to each gene pool and outliers distantly related. In addition, accessions identified as outliers from proteomic distances showed the highest levels of methionine content, an essential amino acid deficient in bean seeds. These findings suggest that 2-DE phaseolin profiles provide valuable information with potential of being used in common bean genetic improvement. PMID:24983510

  1. Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities.

    PubMed

    Trotha, René; Reichl, Udo; Thies, Frank L; Sperling, Danuta; König, Wolfgang; König, Brigitte

    2002-04-01

    The analysis of microbial communities is of increasing importance in life sciences and bioengineering. Traditional techniques of investigations like culture or cloning methods suffer from many disadvantages. They are unable to give a complete qualitative and quantitative view of the total amount of microorganisms themselves, their interactions among each other and with their environment. Obviously, the determination of static or dynamic balances among microorganisms is of fast growing interest. The generation of species specific and fluorescently labeled 16S ribosomal DNA (rDNA) fragments by the terminal restriction fragment length polymorphism (T-RFLP) technique is a suitable tool to overcome the problems other methods have. For the separation of these fragments polyacrylamide gel sequencers are preferred as compared to capillary sequencers using linear polymers until now because of their higher electrophoretic resolution and therefore sizing accuracy. But modern capillary sequencers, especially multicapillary sequencers, offer an advanced grade of automation and an increased throughput necessary for the investigation of complex communities in long-time studies. Therefore, we adapted a T-RFLP technique to an automated high-throughput multicapillary electrophoresis device (ABI 3100 Genetic Analysis) with regard to a precise qualitative and quantitative characterization of microbial communities. PMID:11981854

  2. Electrophoresis and isoelectric focusing of whole cell and membrane proteins from the extremely halophilic archaebacteria

    NASA Technical Reports Server (NTRS)

    Stan-Lotter, Helga; Lang, Frank J., Jr.; Hochstein, Lawrence I.

    1989-01-01

    The subunits from two purified halobacterial membrane enzymes (ATPase and nitrate reductase) behaved differently with respect to isoelectric focusing, silver staining and interaction with ampholytes. Differential behavior was also observed in whole cell proteins from Halobacterium saccharovorum regarding resolution in two-dimensional gels and silver staining. It is proposed that these differences reflect the existence of two classes of halobacterial proteins.

  3. Quantitative estimation of urinary protein excretion by refractometry.

    PubMed

    Kumar, S; Visweswaran, K; Sobha, A; Sarasa, G; Nampoory, M R

    1992-09-01

    Quantitative estimation of proteinuria done by the refractometric method was compared with that done by the sulphosalycilic acid method and biuret method in 102 urine samples. The analysis of results by students' t test showed no statistically significant difference between the three methods. It is concluded that quantitative estimation of urinary protein excretion by refractometric method is a simple cheap and reliable method and can be performed easily in the outpatient clinic. The instrument is quite handy and can be carried in the pocket.

  4. Quantitation of carcinogen bound protein adducts by fluorescence measurements

    NASA Astrophysics Data System (ADS)

    Gan, Liang-Shang; Otteson, Michael S.; Doxtader, Mark M.; Skipper, Paul L.; Dasari, Ramachandra R.; Tannenbaum, Steven R.

    1989-01-01

    A highly significant correlation of aflatoxin B 1 serum albumin adduct level with daily aflatoxin B 1 intake was observed in a molecular epidemiological study of aflatoxin carcinogenesis which used conventional fluorescence spectroscopy methods for adduct quantitation. Synchronous fluorescence spectroscopy and laser induced fluorescence techniques have been employed to quantitate antibenzo[ a]pyrene diol epoxide derived globin peptide adducts. Fast and efficient methods to isolate the peptide adducts as well as eliminate protein fluorescence background are described. A detection limit of several femtomoles has been achieved. Experimental and technical considerations of low temperature synchronous fluorescence spectroscopy and fluorescence line narrowing to improve the detection sensitivities are also presented.

  5. Changes in muscle protein composition induced by disuse atrophy - Analysis by two-dimensional electrophoresis

    NASA Technical Reports Server (NTRS)

    Ellis, S.; Giometti, C. S.; Riley, D. A.

    1985-01-01

    Using 320 g rats, a two-dimensional electrophoretic analysis of muscle proteins in the soleus and EDL muscles from hindlimbs maintained load-free for 10 days is performed. Statistical analysis of the two-dimensional patterns of control and suspended groups reveals more protein alteration in the soleus muscle, with 25 protein differences, than the EDL muscle, with 9 protein differences, as a result of atrophy. Most of the soleus differences reside in minor components. It is suggested that the EDL may also show alteration in its two-dimensional protein map, even though no significant atrophy occurred in muscle wet weight. It is cautioned that strict interpretation of data must take into account possible endocrine perturbations.

  6. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

    PubMed Central

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide. PMID:26865351

  7. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry.

    PubMed

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-02-11

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide.

  8. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry.

    PubMed

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-01-01

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide. PMID:26865351

  9. Enantiomeric separation of free L- and D-amino acids in hydrolyzed protein fertilizers by capillary electrophoresis tandem mass spectrometry.

    PubMed

    Sánchez-Hernández, Laura; Serra, Nuria Sierras; Marina, María Luisa; Crego, Antonio L

    2013-05-29

    Two capillary electrophoresis-tandem mass spectrometry (CE-MS(2)) methods were optimized in this work using cyclodextrins (CDs) as chiral selectors in order to determine the degree of racemization of the free amino acids contained in different hydrolyzed protein fertilizers used as plant biostimulants. The methodologies developed were characterized by the specificity of MS(2) experiments enabling the identification of all protein amino acids, except for cysteine. The enantiomeric separation of up to 14 amino acids was achieved with resolutions above 1.0 and limits of detection between 0.02 and 0.8 μM. The methods were applied to the analysis of complex samples such as hydrolyzed protein fertilizers to evaluate the presence of d-amino acids after different kinds of hydrolysis treatments. The results corroborated the absence or almost negligible presence of enantiomeric conversions of the L-amino acids into D-amino acids in the case of fertilizers obtained by enzymatic hydrolysis, as well as the high racemization rate for those obtained through a chemical hydrolysis.

  10. Titanium Dioxide Photocatalytic Polymerization of Acrylamide for Gel Electrophoresis (TIPPAGE) of Proteins and Structural Identification by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhang, Wenyang; Yuan, Zhiwei; Huang, Lulu; Kang, Jie; Jiang, Ruowei; Zhong, Hongying

    2016-02-01

    Polyacrylamide gel electrophoresis (PAGE) coupled with mass spectrometry has been well established for separating, identifying and quantifying protein mixtures from cell lines, tissues or other biological samples. The copolymerization process of acrylamide and bis-acrylamide is the key to mastering this powerful technique. In general, this is a vinyl addition reaction initiated by free radical-generating reagents such as ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) under basic pH and degassing experimental condition. We report herein a photocatalytic polymerization approach that is based on photo-generated hydroxyl radicals with nanoparticles of titanium dioxide. It was shown that the polymerization process is greatly accelerated in acidic condition when ultraviolet light shots on the gel solution containing TiO2 nanoparticles without degassing. This feature makes it very useful in preparing Triton X-100 acid urea (TAU) gel that has been developed for separating basic proteins such as histones and variants in acidic experimental condition. Additionally, the presence of titanium dioxide in the gel not only improves mechanistic property of gels but also changes the migration pattern of different proteins that have different affinities to titanium dioxide.

  11. Global, quantitative and dynamic mapping of protein subcellular localization

    PubMed Central

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  12. The contribution of genetic and environmental factors to quantitative variability of erythrocyte membrane proteins in primary hypotension.

    PubMed

    Ivanov, V P; Polonikov, A V; Solodilova, M A

    2005-01-01

    Our previous studies have shown that, compared with healthy individuals, patients with primary arterial hypotension (PAH) have significant quantitative changes in erythrocyte membrane proteins. The purpose of the present study was to evaluate the contribution made by genetic and environmental factors to quantitative variation of erythrocyte membrane proteins in PAH. We studied 109 hypotensive patients, 124 normotensive subjects, 222 of their first-degree relatives and 24 twin pairs by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. The decomposition of total phenotypic variance of erythrocyte membrane proteins to genetic and environmental components was performed on the basis of correlations among first-degree relatives by the least squares method. The genetic dominance and shared environmental factors were found to influence the variability of cytoskeletal membrane proteins whose contents were changed in PAH. Furthermore, variations in alpha-spectrin, actin and anion exchanger in hypotensives were substantially influenced by major gene and maternal effects. Ankyrin 2.1 and actin content was under the control of common underlying genes. Variations in membrane-associated glutathione-S-transferase and tropomyosin were predominantly affected by polygenes. These findings suggest that the putative major genes with pleiotropic effects appear to be involved in the control of quantitative disorders of erythrocyte membrane proteins in primary hypotension. PMID:15638825

  13. Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

    PubMed

    Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

    2014-03-01

    Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

  14. Capillary gel electrophoresis for the quantification and purity determination of recombinant proteins in inclusion bodies.

    PubMed

    Espinosa-de la Garza, Carlos E; Perdomo-Abúndez, Francisco C; Campos-García, Víctor R; Pérez, Néstor O; Flores-Ortiz, Luis F; Medina-Rivero, Emilio

    2013-09-01

    In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-β 1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r(2) ) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-β 1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.

  15. Quantitating protein synthesis, degradation, and endogenous antigen processing.

    PubMed

    Princiotta, Michael F; Finzi, Diana; Qian, Shu-Bing; Gibbs, James; Schuchmann, Sebastian; Buttgereit, Frank; Bennink, Jack R; Yewdell, Jonathan W

    2003-03-01

    Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cell's 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cell's 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate. PMID:12648452

  16. Analysis of proteins in the extracellular matrix of the plant pathogenic fungus Bipolaris sorokiniana using 2-D gel electrophoresis and MS/MS.

    PubMed

    Apoga, D; Ek, B; Tunlid, A

    2001-04-13

    A method was developed for isolating and sequencing proteins present in the extracellular matrix (ECM) of germlings and hyphae of filamentous fungi. Surface proteins of the cereal pathogen Bipolaris sorokiniana were labelled with a membrane impermeable biotinylating agent and extracted using a glycine-HCl buffer. Extracted proteins were purified by affinity binding to streptavidin-conjugated magnetic beads or by two-dimensional gel electrophoresis. Four of the biotinylated proteins from the ECM of B. sorokiniana were isolated, in gel digested with trypsin and partly sequenced by tandem mass spectrometry. No significant sequence similarities to proteins in databases were obtained.

  17. Quantitative analysis of protein-ligand interactions by NMR.

    PubMed

    Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

    2016-08-01

    Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

  18. Genetic diversity in the Chinese pangolin (Manis pentadactyla) inferred from protein electrophoresis.

    PubMed

    Su, B; Liu, R Q; Wang, Y X; Shi, L M

    1994-10-01

    We examined protein polymorphism of Chinese pangolins (Manis pentadactyla) from Yunnan Province of China, including two forms of three brown and nine dusky Chinese pangolins. Sixty-two genetic loci were screened; 12 loci were found to be polymorphic. The percentage of polymorphic loci (P) is 0.194, the mean individual heterozygosity (H) is 0.078, and the mean number of alleles (A) is 1.258. Furthermore, we calculated the genetic distance (D) between the two forms and found a low level of genetic divergence (D = 0.0206) between them, which indicates an almost-indistinguishable divergence at the level of proteins.

  19. Nonurea sodium dodecyl sulfate-polyacrylamide gel electrophoresis with high-molarity buffers for the separation of proteins and peptides.

    PubMed

    Okajima, T; Tanabe, T; Yasuda, T

    1993-06-01

    A sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a discontinuous buffer system for separation of both peptides and proteins, which is a modification of the Laemmli system, is described. In the modified procedure, twofold higher concentrations of buffers in the separating gel and the running buffer solution allow superior resolution for peptides as small as M(r) 5000. The resolution of peptides was dependent on salt concentrations in the systems in which sodium chloride was partially substituted for Tris-HCl buffer and buffer concentrations were varied. In the stacking gel of the modified procedure, detection of peptides and SDS demonstrated a sharp stack of peptides at the trailing edge of the SDS stack. On the other hand, this SDS stack included peptides, forced them to diffuse, and produced a broad starting zone under Laemmli conditions. In addition, following expansion of the SDS stack impaired peptide resolution further in the separating gel. Accordingly, the different interaction with the SDS stack in the stacking process was found to produce different resolution of peptides in the electrophoretic procedures. The modified conditions have potential to provide a superior alternative to the Laemmli system for analysis of various proteins.

  20. Precolumn affinity capillary electrophoresis for the identification of clinically relevant proteins in human serum: application to human cardiac troponin I.

    PubMed

    Dalluge, J J; Sander, L C

    1998-12-15

    An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of capillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, at clinically relevant levels (2 nmol/L) in serum is demonstrated. The technique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (approximately 5 mm) packed bed of porous silica containing covalently immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex matrix. Following a rinsing step to eliminate nonspecifically bound serum proteins and other impurities from the column, desorption of the antigen into the separation region of the PA-CE capillary for subsequent measurement of femto-molar amounts of cTnI by CE is effected by the injection of an appropriate elution buffer. Advantages of this approach over previously reported affinity preconcentration techniques, related applications for PA-CE technology, and its potential for use in the development of a certified reference material for cTnI in serum are discussed. PMID:9868922

  1. Multi-dimension microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

    PubMed

    Wu, Ruige; Wang, Zhiping; Zhao, Wenfeng; Yeung, William Shu-Biu; Fung, Ying Sing

    2013-08-23

    To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, α-LA, β-LgA and β-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents.

  2. Imaging metals in proteins by combining electrophoresis with rapid x-ray fluorescence mapping.

    SciTech Connect

    Finney, L.; Chishti, Y.; Khare, T.; Giometti, C.; Levina, A.; Lay, P. A.; Vogt, S.; Univ. of Sydney; Northwestern Univ.

    2010-01-01

    Growing evidence points toward a very dynamic role for metals in biology. This suggests that physiological circumstance may mandate metal ion redistribution among ligands. This work addresses a critical need for technology that detects, identifies, and measures the metal-containing components of complex biological matrixes. We describe a direct, user-friendly approach for identifying and quantifying metal?protein adducts in complex samples using native- or SDS-PAGE, blotting, and rapid synchrotron X-ray fluorescence mapping with micro-XANES (X-ray absorption near-edge structure) of entire blots. The identification and quantification of each metal bound to a protein spot has been demonstrated, and the technique has been applied in two exemplary cases. In the first, the speciation of the in vitro binding of exogenous chromium to blood serum proteins was influenced markedly by both the oxidation state of chromium exposed to the serum proteins and the treatment conditions, which is of relevance to the biochemistry of Cr dietary supplements. In the second case, in vivo changes in endogenous metal speciation were examined to probe the influence of oxygen depletion on iron speciation in Shewanella oneidensis.

  3. Novel cationic coating agent for protein separation by capillary electrophoresis(†).

    PubMed

    Znaleziona, Joanna; Drahoňovský, Dušan; Drahoš, Bohuslav; Ševčík, Juraj; Maier, Vítězslav

    2016-06-01

    A novel positively charged surfactant N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride enabled the reversal of the electro-osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.

  4. Studies of proteinograms in dermatophytes by disc electrophoresis. 1. Protein bands in relation to growth phase

    NASA Technical Reports Server (NTRS)

    Danev, P.; Friedrich, E.; Balabanov, V.

    1983-01-01

    Homogenates were prepared from various growth phases of Microsporum gypseum grown on different amino acids as the nitrogen source. When analyzed on 7.5% polyacrylamide disc gels, the water-soluble proteins in these homogenates gave essentially identical banding patterns.

  5. Quantitative urinary protein excretion in chronic renal failure.

    PubMed

    McMorrow, R G; Galla, J H; Luke, R G

    1982-01-01

    The diagnostic value of the measurement of quantitative proteinuria in patients with a creatinine clearance of less than 10 ml/min was determined in patients seen in a single center over a 5-year period. All 126 patients in whom a definitive renal diagnosis was possible were included. Patients with glomerular disease excreted 6.1 +/- 0.6 g/day and patients with interstitial disease 1.5 +/- 0.3 g/day (p less than 0.001). In individual patients with end-stage renal disease, however, measurement of urinary protein excretion excluded (with 95% confidence levels) patients with interstitial diseases only when greater than 2.9 g/day. To examine the natural history of proteinuria in progressive renal disease, urinary protein, absolute and factored for glomerular filtration rate (GFR; creatinine clearance), was determined at 10 ml/min decrements in GFR for patients with membranoproliferative glomerulonephritis, idiopathic membranous glomerulonephritis and focal glomerulosclerosis. Quantitative urinary protein excretion was relatively constant as GFR fell but did fall significantly at less than 10 ml/min but only to 4.8-7.0 g/day at even that level. Urinary protein excretion/GFR increased as GFR fell, particularly at end stage where a highly significant four-fold rise was seen; an increase also occurred in patients with primary interstitial disease. Similar data were obtained for 34 randomly selected patients after at least 1 year of chronic hemodialysis. Although a significant decline in absolute urinary protein excretion occurred during the year of dialysis to levels not different between glomerular and interstitial disease, urinary protein excretion/unit GFR remained elevated. Increased urinary protein excretion/unit GFR may result from a functional adaptation of remaining nephrons in response to declining renal mass.

  6. Stress Responsive Proteins Are Actively Regulated during Rice (Oryza sativa) Embryogenesis as Indicated by Quantitative Proteomics Analysis

    PubMed Central

    Zi, Jin; Zhang, Jiyuan; Wang, Quanhui; Zhou, Baojin; Zhong, Junyan; Zhang, Chaoliang; Qiu, Xuemei; Wen, Bo; Zhang, Shenyan; Fu, Xiqin; Lin, Liang; Liu, Siqi

    2013-01-01

    Embryogenesis is the initial step in a plant’s life, and the molecular changes that occur during embryonic development are largely unknown. To explore the relevant molecular events, we used the isobaric tags for relative and absolute quantification (iTRAQ) coupled with the shotgun proteomics technique (iTRAQ/Shotgun) to study the proteomic changes of rice embryos during embryogenesis. For the first time, a total of 2 165 unique proteins were identified in rice embryos, and the abundances of 867 proteins were actively changed based on the statistical evaluation of the quantitative MS/MS signals. The quantitative data were then confirmed using multiple reactions monitoring (MRM) and were also supported by our previous study based on two-dimensional gel electrophoresis (2 DE). Using the proteome at 6 days after pollination (DAP) as a reference, cluster analysis of these differential proteins throughout rice embryogenesis revealed that 25% were up-regulated and 75% were down-regulated. Gene Ontology (GO) analysis implicated that most of the up-regulated proteins were functionally categorized as stress responsive, mainly including heat shock-, lipid transfer-, and reactive oxygen species-related proteins. The stress-responsive proteins were thus postulated to play an important role during seed maturation. PMID:24058531

  7. A strategy to quantitate global phosphorylation of bone matrix proteins.

    PubMed

    Sroga, Grażyna E; Vashishth, Deepak

    2016-04-15

    Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured "in bulk" are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.

  8. [Quantitative differences between genetic variants of milk proteins in Cholmogor cattle].

    PubMed

    Khaertdinov, R A

    1985-11-01

    The quantitative determination of genetical variants A and B of beta, kappa-caseins (beta-Cn, kappa-Cn), beta-lactoglobulin (beta-Lg) in Cholmogor cow's milk was carried out by means of polyacrylamide gel electrophoresis and direct densitometrication. In all milk proteins of heterozygous cows with beta-CnAB, kappa-CnAB, beta-LgAB the variant A content was higher than that of variant B (P less than 0.001). The variant A of beta-casein was 0.380 g/100 ml (54.6%), B-0.316 g/100 ml (45.4%); of kappa-casein A-0.208 g/100 ml (58.3%), B-0.149 g/100 ml (41.7%); of beta-lactoglobulin A-0.143 g/100 ml (54.8%), B-0.118 g/100 ml (45.2%). The alleles beta-CnA, kappa-CnA, beta-LgA ensure higher quantity of protein than beta-CnB, kappa-CnB, beta-LgB alleles.

  9. Fusion-Related Host Proteins Are Actively Regulated by NA during Influenza Infection as Revealed by Quantitative Proteomics Analysis

    PubMed Central

    Sui, Zhiwei; Wen, Bo; Gao, Zhimin; Chen, Quanjiao

    2014-01-01

    Three recombinant influenza A viruses with different neuraminidases (NAs) in the background of A/PR/8/34 (PR8), named rPR8-H5N1NA, rPR8-H9N2NA, and rPR8-H1N1NA, derived from H5N1, H9N2, H1N1 (swine) viruses, respectively, were constructed. We performed a quantitative proteomics analysis to investigate differential protein expression in Madin-Darby canine kidney (MDCK) cells infected with recombinant and wild-type influenza viruses to determine whether NA replacement would alter host cell gene expression. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-TOF MS) and two-dimensional gel electrophoresis (2-DE), we identified 12 up-regulated and 49 down-regulated protein spots, including cytoskeletal proteins, molecular biosynthesis proteins, ubiquitin-proteasome pathway proteins, and heat shock proteins. The most significant changes in infected cells were observed for molecular biosynthesis proteins. We found more differentially expressed protein spots in cells infected with rPR8-H5N1NA or rPR8-H9N2NA viruses than cells infected with wild-type virus. Many of those proteins are postulated to be involved in cell-cell fusion, but the full mechanism remains to be explored. Meanwhile, our data demonstrate that the wild-type virus has evolutionary advantages over recombinant viruses. PMID:25153908

  10. Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR ▿

    PubMed Central

    Martín, Rocío; Jiménez, Esther; Heilig, Hans; Fernández, Leonides; Marín, María L.; Zoetendal, Erwin G.; Rodríguez, Juan M.

    2009-01-01

    The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut. PMID:19088308

  11. Quantitative study of stereospecific binding of monoclonal antibody to anti-benzo(a)pyrene diol epoxide-N(2)-dG adducts by capillary electrophoresis immunoassay.

    PubMed

    Wang, Chao; Li, Tao; Wang, Zhixin; Feng, Feng; Wang, Hailin

    2010-04-01

    The stereospecific binding of monoclonal antibody (mAb) 8E11 to anti-benzo(a)pyrene diol epoxide (BPDE)-dG adducts in single nucleoside, long oligonucleotide, and genomic DNA were quantitatively evaluated using noncompetitive and competitive capillary electrophoresis (CE) immunoassays. Two single-stranded TMR-BPDE-90 mers containing a single anti-BPDE-dG adduct with defined stereochemistry and a fluorescent label at 5'-end were used as fluorescent probes for competitive CE immunoassay. To quantitatively evaluate the binding affinity through competitive CE immunoassays, a series of equations were derived according to the binding stoichiometry. The binding of mAb 8E11 to trans-(+)-anti-BPDE-dG displays strongest affinity (K(b): 3.57 x 10(8) M(-1)) among all four investigated anti-BPDE-dG mononucleoside adducts, and the cis-(-)-anti-BPDE-dG displays lowest affinity (K(b): 1.14 x10(7) M(-1)). The binding of monoclonal antibody (mAb) 8E11 to BPDE-dG adducts in long DNA (90 mer) preferentially forms the complex with a stoichiometry of 1:1, and that mAb 8E11 displays a slightly higher affinity with trans-(+)-anti-BPDE-90 mers (K(b): 6.36+/-0.54 x 10(8)M(-1)) than trans-(-)-anti-BPDE-90 mers (K(b): 4.52+/-0.52 x 10(8) M(-1)). The mAb 8E11 also displays high affinity with BPDE-dG adducts in genomic DNA (K(b): 3.74 x 10(8) M(-1)), indicating its promising applications for sensitive immuno-detection of BPDE-DNA adducts in genomic DNA. PMID:20223464

  12. [The novel copolymer coated capillary columns of electrophoresis and their applications to separation of proteins].

    PubMed

    Lu, G; Gao, D; Gu, J; Fu, R; Li, F; Zhang, H

    1999-01-01

    The copolymer of acrylonitrile, methyl acrylate, hydroxy ethyl acrylate (ZB-004), the copolymer of acrylonitrile, methyl acrylate, hydroxy ethyl acrylate, acrylamide (ZB-014) and the copolymer of acrylonitrile, hydroxy ethyl acrylate (ZB-016) were coated on the inner surface of fused-silica capillaries by just filling the capillary with solutions containing these copolymers followed by flushing the capillary with nitrogen. The physically adsorbed layer can reduce both protein adsorption and electroosmotic flow in the pH range of 3-5. Electroosmotic flow decreased by raising the concentrations of the copolymers. Separation performance of ZB-004 layer is better than those of other two layers due to its low hydrophilicity, but with higher pH values, appreciable peak deformation and increase in electroosmosis were observed. The intra day and inter day migration reproducibility were investigated in terms of relative standard deviation (RSD) with four basic proteins at pH 4.0. The RSDs of the intra day migration times were less than 2%. The RSDs of the inter day migration times were less than 4%. At pH 5.0, the RSDs of the migration times in two ZB-004-coated capillaries made on two different days were less than 1%. Separation efficiencies of four basic proteins in a ZB-004-coated capillary which stored in a buffer (pH 4.0) for fifteen days after being used for 14 days decreased 15%. These coatings were stable and exhibited reproducible separations from intra day, inter day and inter column under acidic conditions.

  13. Optimal protein extraction methods from diverse sample types for protein profiling by using Two-Dimensional Electrophoresis (2DE).

    PubMed

    Tan, A A; Azman, S N; Abdul Rani, N R; Kua, B C; Sasidharan, S; Kiew, L V; Othman, N; Noordin, R; Chen, Y

    2011-12-01

    There is a great diversity of protein samples types and origins, therefore the optimal procedure for each sample type must be determined empirically. In order to obtain a reproducible and complete sample presentation which view as many proteins as possible on the desired 2DE gel, it is critical to perform additional sample preparation steps to improve the quality of the final results, yet without selectively losing the proteins. To address this, we developed a general method that is suitable for diverse sample types based on phenolchloroform extraction method (represented by TRI reagent). This method was found to yield good results when used to analyze human breast cancer cell line (MCF-7), Vibrio cholerae, Cryptocaryon irritans cyst and liver abscess fat tissue. These types represent cell line, bacteria, parasite cyst and pus respectively. For each type of samples, several attempts were made to methodically compare protein isolation methods using TRI-reagent Kit, EasyBlue Kit, PRO-PREP™ Protein Extraction Solution and lysis buffer. The most useful protocol allows the extraction and separation of a wide diversity of protein samples that is reproducible among repeated experiments. Our results demonstrated that the modified TRI-reagent Kit had the highest protein yield as well as the greatest number of total proteins spots count for all type of samples. Distinctive differences in spot patterns were also observed in the 2DE gel of different extraction methods used for each type of sample. PMID:22433892

  14. Electrophoresis technology

    NASA Technical Reports Server (NTRS)

    Snyder, R. S.

    1985-01-01

    A new high resolution apparatus designed for space was built as a laboratory prototype. Using a moving wall with a low zeta potential coating, the major sources of flow distortion for an electrophoretic sample stream are removed. Highly resolved fractions, however, will only be produced in space because of the sensitivity of this chamber to buoyancy-induced convection in the laboratory. The second and third flights of the McDonnell Douglas Astronautics Corporation continuous flow electrophoresis system carried samples developed at MSFC intended to evaluate the broad capabilities of free flow electrophoresis in a reduced gravity environment. Biological model materials, hemoglobin and polystyrene latex microspheres, were selected because of their past use as electrophoresis standards and as visible markers for fluid flow due to electroosmosis, spacecraft acceleration or other factors. The dependence of the separation resolution on the properties of the sample and its suspension solution was assessed.

  15. Free-flow zone electrophoresis of peptides and proteins in PDMS microchip for narrow pI range sample prefractionation coupled with mass spectrometry.

    PubMed

    Song, Yong-Ak; Chan, Michael; Celio, Chris; Tannenbaum, Steven R; Wishnok, John S; Han, Jongyoon

    2010-03-15

    In this paper, we are evaluating the strategy of sorting peptides/proteins based on the charge to mass without resorting to ampholytes and/or isoelectric focusing, using a single- and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS.

  16. Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans.

    PubMed

    John, N E; Andersen, H U; Fey, S J; Larsen, P M; Roepstorff, P; Larsen, M R; Pociot, F; Karlsen, A E; Nerup, J; Green, I C; Mandrup-Poulsen, T

    2000-11-01

    Interleukin-1beta (IL-1beta) treatment of neonatal rat islets for 24 h induces changes in the expression of 105 of 2,200 proteins, as determined previously by two-dimensional (2D) gel electrophoresis. Nitric oxide (NO) has been implicated as one of the mediators of IL-1beta effects in insulin-containing cell lines and rat islets. The aims of this study were 1) to determine the involvement of NO in IL-1beta-induced alterations in protein expression and 2) to investigate the effects of chemically generated NO on protein expression by 2D gel electrophoresis of neonatal rat islet samples. IL-1beta-induced NO production was prevented by incubation of islets in arginine-free medium supplemented with the arginine analog NG-nitro-L-arginine. [35S]methionine-labeled islet proteins were separated using 2D gel electrophoresis and analyzed using the BioImage computer program. Analysis revealed that the expression levels of 23 protein spots of the 105 protein spots, altered by prior treatment with IL-1beta (60 U/ml) alone, were significantly affected (P < 0.01 [n = 4] and P < 0.05 [n = 19]) when NO production was prevented. The effects of chemically generated NO were investigated by exposing islets to the NO donor GSNO (100 micromol/l) for 24 h before labeling with [35S]methionine and 2D gel electrophoresis. Computer-based analysis identified alterations in the expression of 19 of a total of 1,600 detectable proteins in GSNO-treated islets (P < 0.01). We conclude 1) that the expression of up to 42 proteins is altered by cytokine-induced or chemically generated NO in the precise experimental conditions chosen and 2) that the majority of proteins altered by prior treatment with IL-1beta may be the result of NO-independent IL-1beta-mediated regulation of gene expression. This study demonstrates that the combination of 2D gel electrophoresis and mass spectrometry is a powerful tool in the identification of beta-cell proteins involved in the response to toxic mediators.

  17. Binary Oscillatory Crossflow Electrophoresis

    NASA Technical Reports Server (NTRS)

    Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.

    1997-01-01

    Electrophoresis has long been recognized as an effective analytic technique for the separation of proteins and other charged species, however attempts at scaling up to accommodate commercial volumes have met with limited success. In this report we describe a novel electrophoretic separation technique - Binary Oscillatory Crossflow Electrophoresis (BOCE). Numerical simulations indicate that the technique has the potential for preparative scale throughputs with high resolution, while simultaneously avoiding many problems common to conventional electrophoresis. The technique utilizes the interaction of an oscillatory electric field and a transverse oscillatory shear flow to create an active binary filter for the separation of charged protein species. An oscillatory electric field is applied across the narrow gap of a rectangular channel inducing a periodic motion of charged protein species. The amplitude of this motion depends on the dimensionless electrophoretic mobility, alpha = E(sub o)mu/(omega)d, where E(sub o) is the amplitude of the electric field oscillations, mu is the dimensional mobility, omega is the angular frequency of oscillation and d is the channel gap width. An oscillatory shear flow is induced along the length of the channel resulting in the separation of species with different mobilities. We present a model that predicts the oscillatory behavior of charged species and allows estimation of both the magnitude of the induced convective velocity and the effective diffusivity as a function of a in infinitely long channels. Numerical results indicate that in addition to the mobility dependence, the steady state behavior of solute species may be strongly affected by oscillating fluid into and out of the active electric field region at the ends of the cell. The effect is most pronounced using time dependent shear flows of the same frequency (cos((omega)t)) flow mode) as the electric field oscillations. Under such conditions, experiments indicate that

  18. [Identification of the apomixis in Poa pratensis L. using SDS-electrophoresis of endosperm reserve proteins].

    PubMed

    Agafonov, A V; Sukhareva, N B; Baturin, S O; Struzhkova, O A

    2004-01-01

    To determine the characteristics of variability of seed reproduction in the Kentucky bluegrass (Poa pratensis), individual seed variability with respect to the composition of endospermal reserve proteins was studied. Comparative analysis of caryopses obtained by self-fertilization and free fertilization of plants I1 (no. 2-4) and I2 (no. 2-4-7) of the wild-type specimen Murmanskii-95 was performed using SDS-PAGE. Using a cytoembryological express method, we demonstrated that facultative stimulation-autonomous apomeiotic apomixis, along with the formation of meiotic megasporocytes, is characteristic of the Kentucky bluegrass. This method made it possible to determine the consequences of meiotic processes in the maternal plant and to reveal the hybrid nature of seed endosperm.

  19. An Integrated Quantitative Proteomics and Systems Biology Approach to Explore Synaptic Protein Profile Changes During Morphine Exposure

    PubMed Central

    Stockton, Steven D; Devi, Lakshmi A

    2014-01-01

    Morphine is a classic analgesic for the treatment of chronic pain. However, its repeated use is known to produce tolerance, physical dependence, and addiction; these properties limit its long-term therapeutic use and this has led to a quest for therapeutics without these unwanted side effects. Understanding the molecular changes in response to long-term use of morphine is likely to aid in the development of novel therapeutics for the treatment of pain. Studies examining the effects of chronic morphine administration have reported alterations in gene expression, synapse morphology, and synaptic transmission implying changes in synaptic protein profile. To fully understand the changes in protein profiles, proteomic techniques have been used. Studies using two-dimensional gel electrophoresis of various brain regions combined with mass spectrometry have found alterations in the levels of a number of proteins. However, neither the changes in brain regions relevant to morphine effects nor changes in the abundance of synaptic proteins have been clearly delineated. Recent studies employing subcellular fractionation to isolate the striatal synapse, combined with quantitative proteomics and graph theory-inspired network analyses, have begun to quantify morphine-regulated changes in synaptic proteins and facilitate the generation of networks that could serve as targets for the development of novel therapeutics for the treatment of chronic pain. Thus, an integrated quantitative proteomics and systems biology approach can be useful to identify novel targets for the treatment of pain and other disorders of the brain. PMID:24045585

  20. Development of an SDS-gel electrophoresis method on SU-8 microchips for protein separation with LIF detection: Application to the analysis of whey proteins.

    PubMed

    Del Mar Barrios-Romero, Maria; Crevillén, Agustín G; Diez-Masa, José Carlos

    2013-08-01

    This work describes the development of an SDS-gel electrophoresis method for the analysis of major whey proteins (α-lactalbumin, β-lactoglobulin, and BSA) carried out in SU-8 microchips. The method uses a low-viscosity solution of dextran as a sieving polymer. A commercial coating agent (EOTrol LN) was added to the separation buffer to control the EOF of the chips. The potential of this coating agent to prevent protein adsorption on the walls of the SU-8 channels was also evaluated. Additionally, the fluorescence background of the SU-8 material was studied to improve the sensitivity of the method. By selecting an excitation wavelength of 532 nm at which the background fluorescence remains low and by replacing the mercury arc lamp by a laser in the detection system, an LOD in the nanomolar range was achieved for proteins derivatized with the fluorogenic reagent Chromeo P540. Finally, the method was applied to the analysis of milk samples, demonstrating the potential of SU-8 microchips for the analysis of proteins in complex food samples.

  1. Simulating Electrophoresis.

    ERIC Educational Resources Information Center

    Moertel, Cheryl; Frutiger, Bruce

    1996-01-01

    Describes a DNA fingerprinting simulation that uses vegetable food coloring and plastic food containers instead of DNA and expensive gel electrophoresis chambers. Allows students to decipher unknown combinations of dyes in a method similar to that used to decipher samples of DNA in DNA fingerprint techniques. (JRH)

  2. Heterogeneity mapping of protein expression in tumors using quantitative immunofluorescence.

    PubMed

    Faratian, Dana; Christiansen, Jason; Gustavson, Mark; Jones, Christine; Scott, Christopher; Um, InHwa; Harrison, David J

    2011-10-25

    Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor(1,2), and some of these sub-clones give rise to metastatic (and therefore lethal) disease. Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension(3), or on macrodissection(4). A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue(5), providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative

  3. Polymerization of SDS-PAGE gel by gamma irradiation and its use for characterization by electrophoresis of a protein [rapid communication

    NASA Astrophysics Data System (ADS)

    Kim, Dong-Ho; Kim, Jin-Hee; Seo, Ji-Hyun; Lee, Ju-Woon; Lim, Sang-Yong; Lee, Ho-Joon; Byun, Myung-Woo

    2005-12-01

    An SDS-PAGE gel strip was polymerized using a gamma irradiation process and used for electrophoresis. The relative mobility (Rf) and resolution of marker proteins were determined. Polymerization was induced by gamma irradiation in an acrylamide and N'-methylene bisacrylamide mixture with and without the polymerization initiators, ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED). The calibration curves of log 10 molecular weight of the protein versus the distance of the migration showed higher correlations in the gamma irradiated gel than in that of the APS-TEMED polymerized control. The Rf value of the protein was increased in the gel polymerized by gamma-irradiation.

  4. Quantitative thermophoretic study of disease-related protein aggregates

    PubMed Central

    Wolff , Manuel; Mittag, Judith J.; Herling, Therese W.; Genst, Erwin De; Dobson, Christopher M.; Knowles, Tuomas P. J.; Braun, Dieter; Buell, Alexander K.

    2016-01-01

    Amyloid fibrils are a hallmark of a range of neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases. A detailed understanding of the physico-chemical properties of the different aggregated forms of proteins, and of their interactions with other compounds of diagnostic or therapeutic interest, is crucial for devising effective strategies against such diseases. Protein aggregates are situated at the boundary between soluble and insoluble structures, and are challenging to study because classical biophysical techniques, such as scattering, spectroscopic and calorimetric methods, are not well adapted for their study. Here we present a detailed characterization of the thermophoretic behavior of different forms of the protein α-synuclein, whose aggregation is associated with Parkinson’s disease. Thermophoresis is the directed net diffusional flux of molecules and colloidal particles in a temperature gradient. Because of their low volume requirements and rapidity, analytical methods based on this effect have considerable potential for high throughput screening for drug discovery. In this paper we rationalize and describe in quantitative terms the thermophoretic behavior of monomeric, oligomeric and fibrillar forms of α-synuclein. Furthermore, we demonstrate that microscale thermophoresis (MST) is a valuable method for screening for ligands and binding partners of even such highly challenging samples as supramolecular protein aggregates. PMID:26984748

  5. Capillary electrophoresis with laser-induced fluorescence detection of proteins from two types of complex sample matrices: food and biological fluids.

    PubMed

    Garrido-Medina, Raul; Puerta, Angel; Pelaez-Lorenzo, Cristina; Rivera-Monroy, Zuly; Guttman, Andras; Diez-Masa, Jose Carlos; de Frutos, Mercedes

    2013-01-01

    Sample preparation and laser-induced fluorescence detection are two key steps of the analytical methodology to determine by capillary electrophoresis low concentrations of proteins in complex sample matrices. In this chapter the options of performing both steps in different ways are shown by detailing the analysis of the allergen β-lactoglobulin in food products for infants and the analysis of the isoforms of alpha 1-acid glycoprotein, a potential biomarker, in serum and secretome.

  6. Electrophoresis in space.

    PubMed

    Bauer, J; Hymer, W C; Morrison, D R; Kobayashi, H; Seaman, G V; Weber, G

    1999-01-01

    Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed. Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground. Some studies have shown that resolution is also increased. However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation. Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity. If this result is confirmed, a further cellular mechanism of "sensing" the low gravity environment will have been found. Several free fluid electrophoresis devices are now available. Most have been tried at least once in microgravity. Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing. The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity. Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit. Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware. A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities--equipment like bioreactors and protein crystal growth chambers. The authors feel that such capabilities will lead to

  7. On-line coupling of continuous-flow gel electrophoresis with inductively coupled plasma-mass spectrometry to quantitatively evaluate intracellular metal binding properties of metallochaperones HpHypA and HpHspA in E. coli cells.

    PubMed

    Wang, Yuchuan; Hu, Ligang; Yang, Xinming; Chang, Yuen-Yan; Hu, Xuqiao; Li, Hongyan; Sun, Hongzhe

    2015-10-01

    On-line coupling of gel electrophoresis with inductively coupled plasma-mass spectrometry (GE-ICP-MS) offers a strategy to monitor intracellular metals and their associated proteins simultaneously. Herein, we examine the feasibility of the GE-ICP-MS system in the quantitative analysis of intracellular metal binding properties using two Helicobacter pylori metallochaperones HypA and HspA overexpressed in E. coli cells as showcases. We show that parallel detection of metal and sulfur signals allows accurate quantification of intracellular metal-protein stoichiometries, even for metalloproteins that bind metal ions with micromolar affinities. Using this approach, we demonstrate that only a trace amount of Ni(2+) is associated with HpHypA in cells, distinct from the in vitro observation of stoichiometric binding, while HpHypA exhibits high fidelity towards its structural metal Zn(2+) with stoichiometric Zn(2+) binding. In contrast, HpHspA associates with Zn(2+), Ni(2+), Cu(2+) and Co(2+) from an essential metal pool with ca. 0.5 molar equivalents of total metals bound per HpHspA monomer. The metal binding properties of both HpHypA and HpHspA were altered by Bi(3+). The binding of both Zn(2+) and Ni(2+) to HpHypA was suppressed under the stress of Bi(3+) in cells, different from in vitro studies that showed that Bi(3+) interfered with Zn(2+) but not Ni(2+) binding. This study provides an analytical approach to investigate the intracellular metal selectivity of overexpressed metalloproteins.

  8. Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing.

    PubMed

    Nehmé, Hala; Nehmé, Reine; Lafite, Pierre; Routier, Sylvain; Morin, Philippe

    2013-11-01

    A capillary electrophoresis (CE)-based enzyme assay method has been developed to screen protein kinase inhibitors. Four human kinases GSK3β, DYRK1A, CDK5/p25 and CDK1/cyclin B were chosen to test this novel method. These enzymes have been identified as very promising targets to develop treatments against cancer and neurodegenerative diseases. The efficiency of drugs against these relevant biological targets has never been carried out by CE. For this proposal, the capillary was used as a nanoreactor in which four reactants (the enzyme, its two substrates and its potential inhibitor) were successively injected, mixed by using transverse diffusion of laminar flow profiles and incubated. The adenosine 5'-diphosphate (ADP) formed during the enzymatic reaction was detected by UV and quantified. The efficiency of the developed CE method was validated by determining the IC50 values of a wide variety of inhibitors covering a large domain of affinity toward kinases and containing representative and chemically divergent skeletons. Excellent agreement was found between the results obtained by CE and those reported in the literature when using conventional radiometric enzyme assays. Moreover, CE was successfully used to determine the inhibitory effect of several potential inhibitors that was not yet assessed by conventional methods and is crucial for structure activity relation studies. This novel CE method is simple, rapid, very economic (few tens of nanoliters per IC50) and eco-friendly since no radioactivity was required. It could be extended to high-throughput screening of kinase inhibitors, which is of great interest for biomedical and pharmaceutical research fields. PMID:24075461

  9. Quantitative densitometry of proteins stained with coomassie blue using a Hewlett Packard scanjet scanner and Scanplot software.

    PubMed

    Vincent, S G; Cunningham, P R; Stephens, N L; Halayko, A J; Fisher, J T

    1997-01-01

    In the present study we evaluated the performance of a software/scanner system that employed the Hewlett Packard (HP) ScanJet Plus and Scanplot Software for densitometric quantification of protein loads stained with Coomassie Brilliant Blue following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels with bovine serum albumin (BSA) standards, ranging from 0.125 to 10 micrograms, were scanned using reflectance densitometry with 127 microns step size in both the x and y directions and a resolution of 200 dots per inch. Densitometric volume was calculated for each protein band from scanner output in the tagged image file format (TIFF) by a customized software package, Scanplot V. 4.05 (Cunningham Engineering). Protein loads between 0.125 and 10.0 micrograms vs. volume were fit by a second-order regression: Volume = -0.58 x protein load2 + 16.82 x protein load + 7.87 (r = 0.991, p < 0.01). The same gels were scanned and quantified using a transmittance laser densitometer; densitometric volumes measured by both systems were highly correlated (r2 = 0.981, p < 0.01). Additional gels of BSA, smooth muscle myosin heavy chain (myosin), and actin displayed linear relationships between protein loads up to 4.0 micrograms and densitometric volume reflecting unique dye binding properties. We conclude that accurate and reproducible quantitative densitometry of SDS-PAGE can be performed using the HP ScanJet Plus scanner and Scanplot software.

  10. Quantitative analysis of protein-protein interactions by split firefly luciferase complementation in plant protoplasts.

    PubMed

    Li, Jian-Feng; Zhang, Dandan

    2014-07-01

    This unit describes the split firefly luciferase complementation (SFLC) assay, a high-throughput quantitative method that can be used to investigate protein-protein interactions (PPIs) in plant mesophyll protoplasts. In SFLC, the two proteins to be tested for interaction are expressed as chimeric proteins, each fused to a different half of firefly luciferase. If the proteins interact, a functional luciferase can be transitorily reconstituted, and is detected using the cell-permeable substrate D-luciferin. An advantage of the SFLC assay is that dynamic changes in PPIs in a cell can be detected in a near real-time manner. Another advantage is the unusually high DNA co-transfection and protein expression efficiencies that can be achieved in plant protoplasts, thereby enhancing the throughput of the method.

  11. Quantitative Measurement of Protein Relocalization in Live Cells

    PubMed Central

    Bush, Alan; Colman-Lerner, Alejandro

    2013-01-01

    Microscope cytometry provides a powerful means to study signaling in live cells. Here we present a quantitative method to measure protein relocalization over time, which reports the absolute fraction of a tagged protein in each compartment. Using this method, we studied an essential step in the early propagation of the pheromone signal in Saccharomyces cerevisiae: recruitment to the membrane of the scaffold Ste5 by activated Gβγ dimers. We found that the dose response of Ste5 recruitment is graded (EC50 = 0.44 ± 0.08 nM, Hill coefficient = 0.8 ± 0.1). Then, we determined the effective dissociation constant (Kde) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. Kde changed during the first minutes from a high affinity of <0.65 nM to a steady-state value of 17 ± 9 nM. During the same period, the total number of binding sites decreased slightly, from 1940 ± 150 to 1400 ± 200. This work shows how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system. PMID:23442923

  12. High pressure electrophoresis in narrow bore glass tubes: One- and two-dimensional separations of protein subunits

    NASA Astrophysics Data System (ADS)

    Erijman, Leonardo; Clegg, Robert M.

    1996-03-01

    A mini-gel tube electrophoresis apparatus that is easily constructed and simple to operate has been developed. The system can be accommodated in standard commercially available high pressure tubing, and has been tested at up to 200 MPa. The narrow diameter of the glass tubes allows rapid and efficient dissipation of heat. Adequate buffer capacity is maintained in the low volume anode reservoir by increasing the concentration of the buffer. Analytical separations can be achieved in short times with high resolution. After the electrophoresis has been carried out at elevated pressure, the gel can easily be extruded from the tube and loaded onto a standard slab gel for a second-dimensional run at atmospheric pressure. We illustrate the application of this apparatus with the high pressure gel electrophoresis separation and subsequent identification of the constituent subunits of E. coli RNA polymerase.

  13. Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients.

    PubMed

    Rabilloud, T

    1994-02-01

    A modified procedure for the two-dimensional electrophoretic analysis of basic polypeptides is described. This method uses isoelectric focusing with carrier ampholytes in the first dimension, and sodium dodecyl sulfate-electrophoresis in the second dimension. Counteraction of the cathodic drift is achieved by glass tube treatment (silanization), electrolyte modification (use of weak bases and acids), protection of the catholyte from carbon dioxide, and the addition of glycerol to the gel mix. Better resolution and reproducibility are obtained than with nonequilibrium pH gradient electrophoresis, since quasi equilibrium focusing can be obtained. PMID:8026444

  14. Electrophoresis experiments in microgravity

    NASA Technical Reports Server (NTRS)

    Snyder, Robert S.; Rhodes, Percy H.

    1991-01-01

    The use of the microgravity environment to separate and purify biological cells and proteins has been a major activity since the beginning of the NASA Microgravity Science and Applications program. Purified populations of cells are needed for research, transplantation and analysis of specific cell constituents. Protein purification is a necessary step in research areas such as genetic engineering where the new protein has to be separated from the variety of other proteins synthesized from the microorganism. Sufficient data are available from the results of past electrophoresis experiments in space to show that these experiments were designed with incomplete knowledge of the fluid dynamics of the process including electrohydrodynamics. However, electrophoresis is still an important separation tool in the laboratory and thermal convection does limit its performance. Thus, there is a justification for electrophoresis but the emphasis of future space experiments must be directed toward basic research with model experiments to understand the microgravity environment and fluid analysis to test the basic principles of the process.

  15. Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.

    PubMed

    Ku, Hyung-Keun; Lim, Hyuk-Min; Oh, Kyong-Hwa; Yang, Hyo-Jin; Jeong, Ji-Seon; Kim, Sook-Kyung

    2013-03-01

    The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards.

  16. [Urine protein analysis with the sodium-dodecyl-sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) in healthy cats and cats with kidney diseases].

    PubMed

    Meyer-Lindenberg, A; Wohlsein, P; Trautwein, G; Nolte, I

    1997-03-01

    In this investigation, the value of urine protein analysis by means of molecular-weight related sodium dodecyl-polyacryl gradient gel electrophoresis (SDS-PAGE) was examined with regard to its applicability and diagnostic significance in nephropathy in the cat. A total of 87 cats was included in the study, 30 of them that were clinically healthy served as the control group. The urine protein pattern of this group had, besides the band representing the market albumin, and additional broad band within the size of the marker transferrin. In some cases, weak bands were present within the range of the Tamm-Horsfall-protein and immunoglobulin G. Micromolecular protein bands were not demonstrable. The remaining 57 animals had a histologically proven nephropathy. Thirty-eight cats had elevated urea and/or creatinine values in the plasma (group 1), and 19 animals had values within the reference range (group 2). The urine protein pattern as evidenced by SDS-urine electrophoresis was altered in all cats with histologically proven nephropathy, and it is thus concluded that with this technique a nephropathy can be diagnosed very early and prior to changes of plasma urea and creatinine (group 2). Moreover, in most of the cases, the nephrological changes can be classified as glomerular or tubulo-interstitial (group 1 and group 2). However, it is not possible to draw exact conclusions concerning the underlying morphological changes, nor can the severity of the disease be correctly assessed. PMID:9123982

  17. Quantitative analysis of flagellar proteins in Drosophila sperm tails.

    PubMed

    Mendes Maia, Teresa; Paul-Gilloteaux, Perrine; Basto, Renata

    2015-01-01

    The cilium has a well-defined structure, which can still accommodate some morphological and molecular composition diversity to suit the functional requirements of different cell types. The sperm flagellum of the fruit fly Drosophila melanogaster appears as a good model to study the genetic regulation of axoneme assembly and motility, due to the wealth of genetic tools publically available for this organism. In addition, the fruit fly's sperm flagellum displays quite a long axoneme (∼1.8mm), which may facilitate both histological and biochemical analyses. Here, we present a protocol for imaging and quantitatively analyze proteins, which associate with the fly differentiating, and mature sperm flagella. We will use as an example the quantification of tubulin polyglycylation in wild-type testes and in Bug22 mutant testes, which present defects in the deposition of this posttranslational modification. During sperm biogenesis, flagella appear tightly bundled, which makes it more challenging to get accurate measurements of protein levels from immunostained specimens. The method we present is based on the use of a novel semiautomated, macro installed in the image processing software ImageJ. It allows to measure fluorescence levels in closely associated sperm tails, through an exact distinction between positive and background signals, and provides background-corrected pixel intensity values that can directly be used for data analysis. PMID:25837396

  18. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions

    DOE PAGES

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary; Geller, Jil; Fisher, Susan; Hall, Steven; Hazen, Terry C.; et al

    2016-04-20

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification ofmore » endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.« less

  19. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions.

    PubMed

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary E; Geller, Jil T; Fisher, Susan J; Hall, Steven C; Hazen, Terry C; Brenner, Steven E; Butland, Gareth; Jin, Jian; Witkowska, H Ewa; Chandonia, John-Marc; Biggin, Mark D

    2016-06-01

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR. PMID:27099342

  20. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions

    SciTech Connect

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary; Geller, Jil; Fisher, Susan; Hall, Steven; Hazen, Terry C; Brenner, Steven; Butland, Gareth; Jin, Jian; Witkowska, H. Ewa; Chandonia, John-Marc; Biggin, Mark D.

    2016-01-01

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

  1. Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions*

    PubMed Central

    Shatsky, Maxim; Dong, Ming; Liu, Haichuan; Yang, Lee Lisheng; Choi, Megan; Singer, Mary E.; Geller, Jil T.; Fisher, Susan J.; Hall, Steven C.; Hazen, Terry C.; Brenner, Steven E.; Butland, Gareth; Jin, Jian; Witkowska, H. Ewa; Chandonia, John-Marc; Biggin, Mark D.

    2016-01-01

    Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris. These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR. PMID:27099342

  2. Biomarker discovery and identification in laser microdissected head and neck squamous cell carcinoma with ProteinChip technology, two-dimensional gel electrophoresis, tandem mass spectrometry, and immunohistochemistry.

    PubMed

    Melle, Christian; Ernst, Gunther; Schimmel, Bettina; Bleul, Annett; Koscielny, Sven; Wiesner, Andreas; Bogumil, Ralf; Moller, Ursula; Osterloh, Dirk; Halbhuber, Karl-Jurgen; von Eggeling, Ferdinand

    2003-07-01

    Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip technology. For this, cryostat sections from head and neck tumors (n = 57) and adjacent mucosa (n = 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p = 0.000029) was isolated by two-dimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexin-positive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal pharyngeal epithelium and tumor tissue can be detected, and it will become possible to educe a tumor-associated protein pattern that might be used as a marker for tumorigenesis and progression. PMID:12824440

  3. Beyond hairballs: the use of quantitative mass spectrometry data to understand protein-protein interactions

    PubMed Central

    Gingras, Anne-Claude; Raught, Brian

    2012-01-01

    The past 10 years have witnessed a dramatic proliferation in the availability of protein interaction data. However, for interaction mapping based on affinity purification coupled with mass spectrometry (AP-MS), there is a wealth of information present in the datasets that often goes unrecorded in public repositories, and as such remains largely unexplored. Further, how this type of data is represented and used by bioinformaticians has not been well established. Here, we point out some common mistakes in how AP-MS data are handled, and describe how protein complex organization and interaction dynamics can be inferred using quantitative AP-MS approaches. PMID:22710165

  4. Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae

    PubMed Central

    2013-01-01

    Background Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae. Results Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI > 7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to

  5. Proteomic analysis of halotolerant proteins under high and low salt stress in Dunaliella salina using two-dimensional differential in-gel electrophoresis

    PubMed Central

    Jia, Yan-Long; Chen, Hui; Zhang, Chong; Gao, Li-Jie; Wang, Xi-Cheng; Qiu, Le-Le; Wu, Jun-Fang

    2016-01-01

    Abstract Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE) was used to investigate the expression of halotolerant proteins under high (3 M NaCl) and low (0.75 M NaCl) salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress. PMID:27192131

  6. Proteomic analysis of halotolerant proteins under high and low salt stress in Dunaliella salina using two-dimensional differential in-gel electrophoresis.

    PubMed

    Jia, Yan-Long; Chen, Hui; Zhang, Chong; Gao, Li-Jie; Wang, Xi-Cheng; Qiu, Le-Le; Wu, Jun-Fang

    2016-05-13

    Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE) was used to investigate the expression of halotolerant proteins under high (3 M NaCl) and low (0.75 M NaCl) salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress. PMID:27192131

  7. Proteomic analysis of halotolerant proteins under high and low salt stress in Dunaliella salina using two-dimensional differential in-gel electrophoresis.

    PubMed

    Jia, Yan-Long; Chen, Hui; Zhang, Chong; Gao, Li-Jie; Wang, Xi-Cheng; Qiu, Le-Le; Wu, Jun-Fang

    2016-05-13

    Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE) was used to investigate the expression of halotolerant proteins under high (3 M NaCl) and low (0.75 M NaCl) salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress.

  8. Quantitative analysis of pheromone-binding protein specificity

    PubMed Central

    Katti, S.; Lokhande, N.; González, D.; Cassill, A.; Renthal, R.

    2012-01-01

    Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone-binding proteins (OBPs), using β-cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila OBP that binds the pheromone 11-cis vaccenyl acetate (cVA). Refolding of LUSH expressed in E. coli was assessed by measuring N-phenyl-1-naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β-cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β-cyclodextrin-cVA dissociation constant, gives the LUSH-cVA dissociation constant: ~100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH-ligand dissociation constants were determined to be ~200 nM for the silk moth pheromone bombykol and ~90 nM for methyl oleate. The results indicate that the ligand-binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the pheromone receptor model proposed by Laughlin et al. (Cell 133: 1255–65, 2008) are discussed. PMID:23121132

  9. A subtle calculation method for nanoparticle’s molar extinction coefficient: The gift from discrete protein-nanoparticle system on agarose gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Zhong, Ruibo; Yuan, Ming; Gao, Haiyang; Bai, Zhijun; Guo, Jun; Zhao, Xinmin; Zhang, Feng

    2016-03-01

    Discrete biomolecule-nanoparticle (NP) conjugates play paramount roles in nanofabrication, in which the key is to get the precise molar extinction coefficient of NPs. By making best use of the gift from a specific separation phenomenon of agarose gel electrophoresis (GE), amphiphilic polymer coated NP with exact number of bovine serum albumin (BSA) proteins can be extracted and further experimentally employed to precisely calculate the molar extinction coefficient of the NPs. This method could further benefit the evaluation and extraction of any other dual-component NP-containing bio-conjugates.

  10. Graft copolymer composed of cationic backbone and bottle brush-like side chains as a physically adsorbed coating for protein separation by capillary electrophoresis.

    PubMed

    Zhou, Dan; Xiang, Lina; Zeng, Rongju; Cao, Fuhu; Zhu, Xiaoxi; Wang, Yanmei

    2011-12-01

    To stabilize electroosmotic flow (EOF) and suppress protein adsorption onto the silica capillary inner wall, a cationic hydroxyethylcellulose-graft-poly (poly(ethylene glycol) methyl ether methacrylate) (cat-HEC-g-PPEGMA) graft copolymer composed of cationic backbone and bottle brush-like side chains was synthesized for the first time and used as a novel physically adsorbed coating for protein separation by capillary electrophoresis. Reversed (anodal) and very stable EOF was obtained in cat-HEC-g-PPEGMA-coated capillary at pH 2.2-7.8. The effects of degree of cationization, PEGMA grafting ratio, PEGMA molecular mass, and buffer pH on the separation of basic proteins were investigated. A systematic comparative study of protein separation in bare and HEC-coated capillaries and in cat-HEC-g-PPEGMA-coated capillary was also performed. The basic proteins can be well separated in cat-HEC-g-PPEGMA-coated capillary over the pH range of 2.8-6.8 with good repeatability and high separation efficiency, because the coating combines good protein-resistant property of bottle brush-like PPEGMA side chains with excellent coating ability of cat-HEC backbone. Besides its success in separation of basic proteins, the cat-HEC-g-PPEGMA coating was also superior in the fast separation of other protein samples, such as protein mixture, egg white, and saliva, which indicates that it is a promising coating for further proteomics analysis. PMID:22038787

  11. A two-dimensional electrophoresis proteomic reference map and systematic identification of 1367 proteins from a cell suspension culture of the model legume Medicago truncatula.

    PubMed

    Lei, Zhentian; Elmer, Aaron M; Watson, Bonnie S; Dixon, Richard A; Mendes, Pedro J; Sumner, Lloyd W

    2005-11-01

    The proteome of a Medicago truncatula cell suspension culture was analyzed using two-dimensional electrophoresis and nanoscale HPLC coupled to a tandem Q-TOF mass spectrometer (QSTAR Pulsar i) to yield an extensive protein reference map. Coomassie Brilliant Blue R-250 was used to visualize more than 1661 proteins, which were excised, subjected to in-gel trypsin digestion, and analyzed using nanoscale HPLC/MS/MS. The resulting spectral data were queried against a custom legume protein database using the MASCOT search engine. A total of 1367 of the 1661 proteins were identified with high rigor, yielding an identification success rate of 83% and 907 unique protein accession numbers. Functional annotation of the M. truncatula suspension cell proteins revealed a complete tricarboxylic acid cycle, a nearly complete glycolytic pathway, a significant portion of the ubiquitin pathway with the associated proteolytic and regulatory complexes, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, chalcone, and lignin biosynthesis. Proteins were also identified from most other functional classes including primary metabolism, energy production, disease/defense, protein destination/storage, protein synthesis, transcription, cell growth/division, and signal transduction. This work represents the most extensive proteomic description of M. truncatula suspension cells to date and provides a reference map for future comparative proteomic and functional genomic studies of the response of these cells to biotic and abiotic stress.

  12. The Isotope-Coded Affinity Tag Method for Quantitative Protein Profile Comparison and Relative Quantitation of Cysteine Redox Modifications.

    PubMed

    Chan, James Chun Yip; Zhou, Lei; Chan, Eric Chun Yong

    2015-11-02

    The isotope-coded affinity tag (ICAT) technique has been applied to measure pairwise changes in protein expression through differential stable isotopic labeling of proteins or peptides followed by identification and quantification using a mass spectrometer. Changes in protein expression are observed when the identical peptide from each of two biological conditions is identified and a difference is detected in the measurements comparing the peptide labeled with the heavy isotope to the one with a normal isotopic distribution. This approach allows the simultaneous comparison of the expression of many proteins between two different biological states (e.g., yeast grown on galactose versus glucose, or normal versus cancer cells). Due to the cysteine-specificity of the ICAT reagents, the ICAT technique has also been applied to perform relative quantitation of cysteine redox modifications such as oxidation and nitrosylation. This unit describes both protein quantitation and profiling of cysteine redox modifications using the ICAT technique.

  13. Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

    PubMed

    Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

    2016-02-01

    Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances.

  14. Evaluation of two-dimensional electrophoresis and liquid chromatography – tandem mass spectrometry for tissue-specific protein profiling of laser-microdissected plant samples

    SciTech Connect

    Schad, Martina; Lipton, Mary S.; Giavalisco, Patrick; Smith, Richard D.; Kehr, Julia

    2005-07-14

    Laser microdissection (LM) allows the collection of homogeneous tissue- and cell specific plant samples. The employment of this technique with subsequent protein analysis has thus far not been reported for plant tissues, probably due to the difficulties associated with defining a reasonable cellular morphology and, in parallel, allowing efficient protein extraction from tissue samples. The relatively large sample amount needed for successful proteome analysis is an additional issue that complicates protein profiling on a tissue- or even cell-specific level. In contrast to transcript profiling that can be performed from very small sample amounts due to efficient amplification strategies, there is as yet no amplification procedure for proteins available. In the current study, we compared different tissue preparation techniques prior to LM/laser pressure catapulting (LMPC) with respect to their suitability for protein retrieval. Cryosectioning was identified as the best compromise between tissue morphology and effective protein extraction. After collection of vascular bundles from Arabidopsis thaliana stem tissue by LMPC, proteins were extracted and subjected to protein analysis, either by classical two-dimensional gel electrophoresis (2-DE), or by high-efficiency liquid chromatography (LC) in conjunction with tandem mass spectrometry (MS/MS). Our results demonstrate that both methods can be used with LMPC collected plant material. But because of the significantly lower sample amount required for LC-MS/MS than for 2-DE, the combination of LMPC and LC-MS/MS has a higher potential to promote comprehensive proteome analysis of specific plant tissues.

  15. Comparison of liver mitochondrial proteins derived from newborn cloned calves and from cloned adult cattle by two-dimensional differential gel electrophoresis.

    PubMed

    Takeda, Kumiko; Tasai, Mariko; Akagi, Satoshi; Watanabe, Shinya; Oe, Mika; Chikuni, Koichi; Ohnishi-Kameyama, Mayumi; Hanada, Hirofumi; Nakamura, Yoshiaki; Tagami, Takahiro; Nirasawa, Keijiro

    2011-04-01

    Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. The inability to establish functional interactions between donor nucleus and recipient mitochondria is also likely responsible for such a developmental deficiency. However, detailed knowledge of protein expression during somatic cell nuclear transfer (SCNT) in cattle is lacking. In the present study, variations in mitochondrial protein levels between SCNT-derived and control cattle, and from calves derived by artificial insemination were investigated. Mitochondrial fractions were prepared from frozen liver samples and subjected to two-dimensional (2-D) fluorescence differential gel electrophoresis (DIGE) using CyDye™ dyes. Protein expression changes were confirmed with a volume ratio greater than 2.0 (P < 0.05). 2D-DIGE analysis revealed differential expression of three proteins for SCNT cattle (n = 4) and seven proteins for SCNT calves (n = 6) compared to controls (P < 0.05). Different protein patterning was observed among SCNT animals even if animals were generated from the same donor cell source. No differences were detected in two of the SCNT cattle. Moreover, there was no novel protein identified in any of the SCNT cattle or calves. In conclusion, variation in mitochondrial protein expression concentrations was observed in non-viable, neonatal SCNT calves and among SCNT individuals. This result implicates mitochondrial-related gene expression in early developmental loss of SCNT embryos. Comparative proteomic analysis represents an important tool for further studies on SCNT animals.

  16. Effectiveness of charged noncovalent polymer coatings against protein adsorption to silica surfaces studied by evanescent-wave cavity ring-down spectroscopy and capillary electrophoresis.

    PubMed

    Haselberg, Rob; van der Sneppen, Lineke; Ariese, Freek; Ubachs, Wim; Gooijer, Cees; de Jong, Gerhardus J; Somsen, Govert W

    2009-12-15

    Protein adsorption to silica surfaces is a notorious problem in analytical separations. Evanescent-wave cavity ring-down spectroscopy (EW-CRDS) and capillary electrophoresis (CE) were employed to investigate the capability of positively charged polymer coatings to minimize the adsorption of basic proteins. Adsorption of cytochrome c (cyt c) to silica coated with a single layer of polybrene (PB), or a triple layer of PB, dextran sulfate (DS), and PB, was studied and compared to bare silica. Direct analysis of silica surfaces by EW-CRDS revealed that both coatings effectively reduce irreversible protein adsorption. Significant adsorption was observed only for protein concentrations above 400 microM, whereas the PB-DS-PB coating was shown to be most effective and stable. CE analyses of cyt c were performed with and without the respective coatings applied to the fused-silica capillary wall. Monitoring of the electroosmotic flow and protein peak areas indicated a strong reduction of irreversible protein adsorption by the positively charged coatings. Determination of the electrophoretic mobility and peak width of cyt c revealed reversible protein adsorption to the PB coating. It is concluded that the combination of results from EW-CRDS and CE provides highly useful information on the adsorptive characteristics of bare and coated silica surfaces toward basic proteins.

  17. Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry.

    PubMed

    Bak-Jensen, Kristian Sass; Laugesen, Sabrina; Roepstorff, Peter; Svensson, Birte

    2004-03-01

    A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in-gel digestion by trypsin. In addition, the N-terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2-D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting. PMID:14997495

  18. Quantitative double-label radiography of two-dimensional protein gels using color negative film and computer analysis.

    PubMed

    Goldman, R C; Trus, B L; Leive, L

    1983-04-01

    We have devised a method of data collection and computer analysis which allows utilization of the resolving power of two-dimensional gel electrophoresis of proteins, in conjunction with the versatility of using two different radionuclides simultaneously. Cultures of Escherichia coli growing with exponential growth rate constants (mu) of 0.32 and 1.43 were labeled with [3H]leucine and [14C]leucine, respectively; these samples were mixed, and cell protein was separated on a two-dimensional gel. Spacial and quantitative data for both radionuclides were recorded on color negative film by radiographic exposure. Data for 14C alone were then collected photographically from the red-light-sensitive layer of the film using a red filter, while data for 3H and spillover of 14C were collected photographically from the blue-light-sensitive layer using a blue filter. These two data sets were analyzed by CINT, a computer program for analysis of two-dimensional gels, and quantitative data for 3H were calculated after determination of spillover of 14C in a manner analogous to quantification of 3H and 14C by liquid scintillation counting. Quantitative data from over 1000 protein spots representing from 0.002% to 10% of the total 3H or 14C, respectively, are available in a matter of hours. We have used this method to analyze the effect of growth rate and medium composition on the relative levels of individual proteins in a pathogenic strain of E. coli which contains group 111 O-antigen. As expected, the relative levels of aminoacyl-tRNA synthetases, protein chain elongation factors, ribosomal proteins, and the alpha-subunit of RNA polymerase are all increased with increased growth rate; the magnitude of these changes agreed with previous data derived using other strains of E. coli. Alterations in the levels of other proteins identified on the two-dimensional gels could be interpreted in terms of changes in medium composition. When compared to manual data collection by excising

  19. Quantitative Characterization of Heparin Binding to Tau Protein

    PubMed Central

    Zhu, Hai-Li; Fernández, Cristina; Fan, Jun-Bao; Shewmaker, Frank; Chen, Jie; Minton, Allen P.; Liang, Yi

    2010-01-01

    Neurofibrillary tangles, principally composed of bundles of filaments formed by the microtubule-associated protein Tau, are a hallmark of a group of neurodegenerative diseases such as Alzheimer disease. Polyanionic cofactors such as heparin can induce Tau filament formation in vitro. Here we quantitatively characterize the interaction between recombinant human Tau fragment Tau244–372 and heparin (average molecular mass = 7 kDa) as well as heparin-induced fibril formation by using static light scattering, isothermal titration calorimetry, turbidity assays, and transmission electron microscopy. Our data clearly show that at physiological pH, heparin 7K, and human Tau244–372 form a tight 1:1 complex with an equilibrium association constant exceeding 106 m−1 under reducing conditions, triggering Tau fibrillization. In the absence of dithiothreitol, heparin shows a moderate binding affinity (105 m−1) to Tau244–372, similarly triggering Tau fibrillization. Further fibrillization kinetics analyses show that the lag time appears to be approximately invariant up to a molar ratio of 2:1 and then increases at larger ratios of heparin/Tau. The maximum slope representing the apparent rate constant for fibril growth increases sharply with substoichiometric ratios of heparin/Tau and then decreases to some extent with ratios of >1:1. The retarding effect of heparin in excess is attributed to the large increase in ionic strength of the medium arising from free heparin. Together, these results suggest that the formation of the 1:1 complex of Tau monomer and heparin plays an important role in the inducer-mediated Tau filament formation, providing clues to understanding the pathogenesis of neurodegenerative diseases. PMID:19959468

  20. An algorithmic approach using kappa/lambda ratios to improve the diagnostic accuracy of urine protein electrophoresis and to reduce the volume required for immunoelectrophoresis.

    PubMed

    Levinson, S S

    1997-06-27

    The most sensitive routine method for identifying urinary monoclonal immunoglobulin kappa and lambda light chains, called Bence Jones proteins (BJPs), in clinical laboratories is immunofixation electrophoresis (IFE), but this procedure is time-consuming and expensive. As a result, many laboratories screen for paraproteins with urine protein electrophoresis (UPE), which is insensitive when low concentrations of BJP are present and is difficult to interpret with severe proteinuria. The purpose of this study was to determine whether kappa/lambda ratios can be used in conjunction with UPE to improve diagnostic reliability in identifying paraproteins, and decrease the need for IFE on all samples. Urine specimens from 243 patients were examined by UPE and kappa/lambda ratios, and compared with IFE. Due to poor analytical sensitivity, the urinary kappa or lambda concentrations could not be determined in many cases. As a result, many specimens showed kappa/lambda ratios that were indeterminate. Nevertheless, when both urinary kappa and lambda concentrations were undetectable, a BJP could be ruled out. A urinary kappa/lambda ratio between 0.75-3 also ruled out a BJP. The use of kappa/lambda ratios, in conjunction with UPE, resulted in a 52% decrease in the volume of IFE during the course of this study, with 100% sensitivity for detecting BJP.

  1. Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis

    SciTech Connect

    Giometti, C.S.; Willard, K.E.; Anderson, N.L.

    1982-04-01

    Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with /sup 125/I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.

  2. Identification of differentially expressed proteins in blood plasma of control and cigarette smoke-exposed mice by 2D differential in-gel electrophoresis/MS

    PubMed Central

    Tewari, Arun K.; Popova-Butler, Alexandra; El-Mahdy, Mohamed A.; Zweier, Jay L.

    2014-01-01

    Cigarette smoke exposure is known to induce obstructive lung disease and several cardiovascular disease states in humans and also in animal models. Smoking leads to oxidative stress and inflammation that are important in triggering pulmonary and cardiovascular disease. The objective of the current study was to quantify differences in expression levels of plasma proteins of cigarette smoke exposed and control mice, at the time of disease onset, and identify these proteins for use as potential biomarkers of the onset of smoking-induced disease. We utilized two dimensional difference in-gel electrophoresis/ mass spectroscopy (2D-DIGE/MS) to characterize these proteomic changes. 2D-DIGE of plasma samples identified 11 differentially expressed proteins in cigarette smoke exposed mice. From these 11 proteins, 9 were down-regulated and two were up-regulated. The proteins identified are involved in vascular function, coagulation, metabolism, and immune function. Among these, the alterations in fibrinogen (2.2 fold decrease), alpha-1-antitrypsin (1.8 fold increase) and arginase (4.5 fold decrease) are of particular interest since these have been directly linked to cardiovascular and lung pathology. Differences in expression levels of these proteins were also confirmed by immunoblotting. Thus, we observe that chronic cigarette smoke exposure in mice leads to prominent changes in the protein expression profile of blood plasma and these changes in turn can potentially serve as markers predictive of the onset and progression of cardiovascular and pulmonary disease. PMID:21500341

  3. Interactions by 2D Gel Electrophoresis Overlap (iGEO): a novel high fidelity approach to identify constituents of protein complexes

    PubMed Central

    2013-01-01

    Background Here we describe a novel approach used to identify the constituents of protein complexes with high fidelity, using the integrin-associated scaffolding protein PINCH as a test case. PINCH is comprised of five LIM domains, zinc-finger protein interaction modules. In Drosophila melanogaster, PINCH has two known high-affinity binding partners—Integrin-linked kinase (ILK) that binds to LIM1 and Ras Suppressor 1 (RSU1) that binds to LIM5—but has been postulated to bind additional proteins as well. Results To purify PINCH complexes, in parallel we fused different affinity tags (Protein A and Flag) to different locations within the PINCH sequence (N- and C-terminus). We expressed these tagged versions of PINCH both in cell culture (overexpressed in Drosophila S2 cell culture in the presence of endogenous PINCH) and in vivo (at native levels in Drosophila lacking endogenous PINCH). After affinity purification, we analyzed PINCH complexes by a novel 2D-gel electrophoresis analysis, iGEO (interactions by 2D Gel Electrophoresis Overlap), with mass spectrometric identification of individual spots of interest. iGEO allowed the identification of protein partners that associate with PINCH under two independent purification strategies, providing confidence in the significance of the interaction. Proteins identified by iGEO were validated against a highly inclusive list of candidate PINCH interacting proteins identified in previous analyses by MuDPIT mass spectrometry. Conclusions The iGEO strategy confirmed a core complex comprised of PINCH, RSU1, ILK, and ILK binding partner Parvin. Our iGEO method also identified five novel protein partners that specifically interacted with PINCH in Drosophila S2 cell culture. Because of the improved reproducibility of 2D-GE methodology and the increasing affordability of the required labeling reagents, iGEO is a method that is accessible to most moderately well-equipped biological laboratories. The biochemical co

  4. Quantitative phosphoproteomics of protein kinase SnRK1 regulated protein phosphorylation in Arabidopsis under submergence

    PubMed Central

    Cho, Hsing-Yi; Wen, Tuan-Nan; Wang, Ying-Tsui; Shih, Ming-Che

    2016-01-01

    SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 K48M, mpk6, and PTP1 S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence. PMID:27029354

  5. Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping.

    PubMed

    Liang, S; Li, X; Cao, M; Xie, J; Chen, P; Huang, R

    2000-04-01

    Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3-10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight >10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots. PMID:10981815

  6. Exposures of Sus scrofa to a TASER(®) conducted electrical weapon: no effects on 2-dimensional gel electrophoresis patterns of plasma proteins.

    PubMed

    Jauchem, James R; Cerna, Cesario Z; Lim, Tiffany Y; Seaman, Ronald L

    2014-12-01

    In an earlier study, we found significant changes in red-blood-cell, leukocyte, and platelet counts, and in red-blood-cell membrane proteins, following exposures of anesthetized pigs to a conducted electrical weapon. In the current study, we examined potential changes in plasma proteins [analyzed via two-dimensional gel electrophoresis (2-DGE)] following two 30 s exposures of anesthetized pigs (Sus scrofa) to a TASER (®) C2 conducted electrical weapon. Patterns of proteins, separated by 2-DGE, were consistent and reproducible between animals and between times of sampling. We determined that the blood plasma collection, handling, storage, and processing techniques we used are suitable for swine blood. There were no statistically significant changes in plasma proteins following the conducted-electrical-weapon exposures. Overall gel patterns of fibrinogen were similar to results of other studies of both pigs and humans (in control settings, not exposed to conducted electrical weapons). The lack of significant changes in plasma proteins may be added to the body of evidence regarding relative safety of TASER C2 device exposures.

  7. Electrophoresis. [in microgravity environment

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1977-01-01

    Ground-based techniques for electrophoresis take account of the need either to circumvent the effects of gravity to prevent convection, or to use gravity for fluid stabilization through artificial density gradients. The microgravity environments of orbiting spacecraft provides a new alternative for electrophoresis by avoiding the need for either of these two approaches. The paper presents some theoretical considerations concerning electrophoresis, examines certain experimental techniques (zone and high density gel electrophoresis, isoelectric focusing and isotachophoresis), and examines the electrophoresis of living cells.

  8. Using capillary electrophoresis to characterize polymeric particles.

    PubMed

    Riley, Kathryn R; Liu, Sophia; Yu, Guo; Libby, Kara; Cubicciotti, Roger; Colyer, Christa L

    2016-09-01

    Capillary electrophoresis (CE) was used for the characterization of a variety of polymeric micron and sub-micron particles based on size, surface functionality, and binding properties. First, a robust capillary zone electrophoresis (CZE) method was developed for the baseline separation and quantitation of commercially available polystyrene particles with various surface modifications (including amino, carboxylate, and sulfate functional groups) and various sizes (0.2, 0.5, 1.0, and 3.0μm). The separation of DNA-templated polyacrylamide particles from untemplated particles (as used for the Ion Torrent Personal Genome Machine) was demonstrated. Finally, using the 29-base thrombin aptamer and thrombin protein as a model system, a study was undertaken to determine dissociation constants for the aptamer and protein in free solution and when the aptamer was conjugated to a particle, with the goal of better understanding how the use of solid substrates, like particles, affects selection and binding processes. Dissociation constants were determined and were found to be approximately 5-fold higher for the aptamer conjugated to a particle relative to that in free solution. PMID:27543386

  9. Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry.

    PubMed Central

    Sonnenberg, M G; Belisle, J T

    1997-01-01

    A number of the culture filtrate proteins secreted by Mycobacterium tuberculosis are known to contribute to the immunology of tuberculosis and to possess enzymatic activities associated with pathogenicity. However, a complete analysis of the protein composition of this fraction has been lacking. By using two-dimensional polyacrylamide gel electrophoresis, detailed maps of the culture filtrate proteins of M. tuberculosis H37Rv were generated. In total, 205 protein spots were observed. The coupling of this electrophoretic technique with Western blot analysis allowed the identification and mapping of 32 proteins. Further molecular characterization of abundant proteins within this fraction was achieved by N-terminal amino acid sequencing and liquid chromatography-mass spectrometry. Eighteen proteins were subjected to N-group analysis; of these, only 10 could be sequenced by Edman degradation. Among the most interesting were a novel 52-kDa protein demonstrating significant homology to an alpha-hydroxysteroid dehydrogenase of Eubacterium sp. strain VPI 12708, a 25-kDa protein corresponding to open reading frame 28 of the M. tuberculosis cosmid MTCY1A11, and a 31-kDa protein exhibiting an amino acid sequence identical to that of antigen 85A and 85B. This latter product migrated with an isoelectric point between those of antigen 85A and 85C but did not react with the antibody specific for this complex, suggesting that there is a fourth member of the antigen 85 complex. Novel N-terminal amino acid sequences were obtained for three additional culture filtrate proteins; however, these did not yield significant homology to known protein sequences. A protein cluster of 85 to 88 kDa, recognized by the monoclonal antibodies IT-57 and IT-42 and known to react with sera from a large proportion of tuberculosis patients, was refractory to N-group analysis. Nevertheless, mass spectrometry of peptides obtained from one member of this complex identified it as the M. tuberculosis Kat

  10. Analysis of the mouse proteome. (I) Brain proteins: separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation.

    PubMed

    Gauss, C; Kalkum, M; Löwe, M; Lehrach, H; Klose, J

    1999-03-01

    The total protein of the mouse brain was fractionated into three fractions, supernatant, pellet extract and rest pellet suspension, by a procedure that avoids any loss of groups or classes of proteins. The supernatant proteins were resolved to a maximum by large-gel two-dimensional electrophoresis. Two-dimensional patterns from ten individual mice of the commonly used inbred strain C57BL/6 (species: Mus musculus) were prepared. The master pattern was subjected to densitometry, computer-assisted image analysis and treatment with our spot detection program. The resulting two-dimensional pattern, a standard pattern for mouse brain supernatant proteins, was divided into 40 squares, calibrated, and specified by providing each spot with a number. The complete pattern and each of the 40 squares are shown in our homepage (http://www.charite.de/ humangenetik). The standard pattern comprises 8767 protein spots. To identify the proteins known so far in the brain fraction investigated, a first set of 200 spots was analyzed by matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) after in-gel digestion. By screening protein databases 115 spots were identified; by extending the analysis to selected, genetically variant protein spots, 166 spots (including some spot series) were identified in total. This number was increased to 331 by adding protein spots identified indirectly by a genetic approach. By comparing the two-dimensional patterns from C57BL/6 mice with those of another mouse species (Mus spretus), more than 1000 genetically variant spots were detected. The genetic analysis allowed us to recognize spot families, i.e., protein spots that represent the same protein but that are post-translationally modified. If some members of the family were identified, the whole family was considered as being identified. Spot families were investigated in more detail, and interpreted as the result of protein modification or degradation. Genetic analysis led to the

  11. Analysis of automatically generated peptide mass fingerprints of cellular proteins and antigens from Helicobacter pylori 26695 separated by two-dimensional electrophoresis.

    PubMed

    Krah, Alexander; Schmidt, Frank; Becher, Dörte; Schmid, Monika; Albrecht, Dirk; Rack, Axel; Büttner, Knut; Jungblut, Peter R

    2003-12-01

    Helicobacter pylori is a causative agent of severe diseases of the gastric tract ranging from chronic gastritis to gastric cancer. Cellular proteins of H. pylori were separated by high resolution two-dimensional gel electrophoresis. A dataset of 384 spots was automatically picked, digested, spotted, and analyzed by matrix-assisted laser desorption ionization mass spectrometry peptide mass fingerprint in triple replicates. This procedure resulted in 960 evaluable mass spectra. Using a new version of our data analysis software MS-Screener we improved identification and tested reliability of automatically generated data by comparing with manually produced data. Antigenic proteins from H. pylori are candidates for vaccines and diagnostic tests. Previous immunoproteomics studies of our group revealed antigen candidates, and 24 of them were now closely analyzed using the MS-Screener software. Only in three spots minor components were found that may have influenced their antigenicities. These findings affirm the value of immunoproteomics as a hypothesis-free approach. Additionally, the protein species distribution of the known antigen GroEL was investigated, dimers of the protein alkyl hydroperoxide reductase were found, and the fragmentation of gamma-glutamyltranspeptidase was demonstrated.

  12. 2-D difference gel electrophoresis approach to assess protein expression profiles in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vents.

    PubMed

    Company, Rui; Antúnez, Oreto; Bebianno, Maria João; Cajaraville, Miren P; Torreblanca, Amparo

    2011-11-18

    Hydrothermal vent mussels Bathymodiolus azoricus are naturally exposed to toxic chemical species originated directly from vent chimneys. The amount of toxic elements varies significantly among vent sites along the Mid-Atlantic Ridge and B. azoricus must be able to adapt to changes in hydrothermal fluid composition, temperature and pressure. The aim of this work was to study changes in the proteome in the "gill-bacteria complex" of mussels B. azoricus from three hydrothermal vent sites with distinct environmental characteristics using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Results showed that 31 proteins had different expression profiles among vent sites and both cluster and principal component analysis confirm a clear separation of mussels between sites. This suggests the existence of specific parameters grouping individuals from the same hydrothermal site. Protein spots of the more abundant differentially expressed proteins were excised, digested with trypsin and identified by mass spectrometry. All identified proteins (actin, ubiquinone, S-adenosylhomocysteine hydrolase, cysteine peptidases, chaperonin and catalase) have been related previously with oxidative stress conditions and are known to be affected by ROS inducing stressors, including metals. Results point out to specific adaptations at the proteome level of B. azoricus depending on the level of toxicants present in their environment.

  13. Neurodegenerative diseases: quantitative predictions of protein-RNA interactions.

    PubMed

    Cirillo, Davide; Agostini, Federico; Klus, Petr; Marchese, Domenica; Rodriguez, Silvia; Bolognesi, Benedetta; Tartaglia, Gian Gaetano

    2013-02-01

    Increasing evidence indicates that RNA plays an active role in a number of neurodegenerative diseases. We recently introduced a theoretical framework, catRAPID, to predict the binding ability of protein and RNA molecules. Here, we use catRAPID to investigate ribonucleoprotein interactions linked to inherited intellectual disability, amyotrophic lateral sclerosis, Creutzfeuld-Jakob, Alzheimer's, and Parkinson's diseases. We specifically focus on (1) RNA interactions with fragile X mental retardation protein FMRP; (2) protein sequestration caused by CGG repeats; (3) noncoding transcripts regulated by TAR DNA-binding protein 43 TDP-43; (4) autogenous regulation of TDP-43 and FMRP; (5) iron-mediated expression of amyloid precursor protein APP and α-synuclein; (6) interactions between prions and RNA aptamers. Our results are in striking agreement with experimental evidence and provide new insights in processes associated with neuronal function and misfunction.

  14. Capillary Electrophoresis in Metabolomics.

    PubMed

    Maier, Tanja Verena; Schmitt-Kopplin, Philippe

    2016-01-01

    Metabolomics is an analytical toolbox to describe (all) low-molecular-weight compounds in a biological system, as cells, tissues, urine, and feces, as well as in serum and plasma. To analyze such complex biological samples, high requirements on the analytical technique are needed due to the high variation in compound physico-chemistry (cholesterol derivatives, amino acids, fatty acids as SCFA, MCFA, or LCFA, or pathway-related metabolites belonging to each individual organism) and concentration dynamic range. All main separation techniques (LC-MS, GC-MS) are applied in routine to metabolomics hyphenated or not to mass spectrometry, and capillary electrophoresis is a powerful high-resolving technique but still underused in this field of complex samples. Metabolomics can be performed in the non-targeted way to gain an overview on metabolite profiles in biological samples. Targeted metabolomics is applied to analyze quantitatively pre-selected metabolites. This chapter reviews the use of capillary electrophoresis in the field of metabolomics and exemplifies solutions in metabolite profiling and analysis in urine and plasma. PMID:27645748

  15. Apparatus for electrophoresis separation

    DOEpatents

    Anderson, Norman L.

    1978-01-01

    An apparatus is disclosed for simultaneously performing electrophoresis separations on a plurality of slab gels containing samples of protein, protein subunits or nucleic acids. A reservoir of buffer solution is divided into three compartments by two parallel partitions having vertical slots spaced along their length. A sheet of flexible, electrically insulative material is attached to each partition and is provided with vertical slits aligned with the slots. Slab-gel holders are received within the slots with the flexible material folded outwardly as flaps from the slits to overlay portions of the holder surfaces and thereby act as electrical and liquid seals. An elongated, spaghetti-like gel containing a sample of specimen that was previously separated by isoelectric focusing techniques is vertically positioned along a marginal edge portion of the slab gel. On application of an electrical potential between the two outer chambers of buffer solution, a second dimensional electrophoresis separation in accordance with molecular weight occurs as the specimen molecules migrate across the slab gel.

  16. Quantitative characterization of protein-protein complexes involved in base excision DNA repair.

    PubMed

    Moor, Nina A; Vasil'eva, Inna A; Anarbaev, Rashid O; Antson, Alfred A; Lavrik, Olga I

    2015-07-13

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein-protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

  17. Getting the Most out of Electrophoresis Units

    ERIC Educational Resources Information Center

    Mulvihill, Charlotte

    2007-01-01

    At Oklahoma City Community College, they have developed gel electrophoresis activities that support active learning of many scientific concepts, including: pH, electrolysis, oxidation reduction, electrical currents, potentials, conductivity, molarity, gel electrophoresis, DNA and protein separation, and DNA fingerprinting. This article presents…

  18. Analytical biotechnology: Capillary electrophoresis and chromatography

    SciTech Connect

    Horvath, C.; Nikelly, J.G.

    1990-01-01

    The papers describe the separation, characterization, and equipment required for the electrophoresis or chromatography of cyclic nucleotides, pharmaceuticals, therapeutic proteins, recombinant DNA products, pheromones, peptides, and other biological materials. One paper, On-column radioisotope detection for capillary electrophoresis, has been indexed separately for inclusion on the data base.

  19. Discrimination of Glycoproteins from Unglycosylated Proteins in Capillary Electrophoresis: Two-Color LIF Detection Coupled with Post-column Derivatization.

    PubMed

    Kaneta, Takashi

    2016-01-01

    Glycosylation is one of the most important posttranslational modifications (PTMs) which lead to the functionalization of proteins. Here, we describe one method for discriminating glycosylated proteins from unglycosylated ones in their mixture sample by capillary electrophoretic separation and two-color laser-induced fluorescence detection coupled with post-column derivatization. Two lasers emitting at 450 and 532 nm permit the detection of amino groups of proteins derivatized by naphthalene-2,3-dicarboxaldehyde and a fluorescently labeled lectin, tetramethylrhodamine-labeled concanavalin A (Rh-Con A), respectively. When a protein mixture react with Rh-Con A, the glycoproteins bound with Rh-Con A exhibit signals at the same migration time in two electropherograms obtained by 450- and 532-nm lasers whereas unbound proteins show a signal only in the electropherogram of the 450-nm laser. So, when one protein is glycosylated it is detected at the same migration time in the electropherograms obtained by two lasers. PMID:27473478

  20. Gold nanoparticle extraction followed by capillary electrophoresis to determine the total, free, and protein-bound aminothiols in plasma.

    PubMed

    Chang, Chung-Wei; Tseng, Wei-Lung

    2010-04-01

    This study describes the use of Tween 20-capped gold nanoparticles (Tween 20-AuNPs) for the selective extraction and enrichment of five aminothiols, including glutathione, gamma-glutamylcysteine, cysteine, homocysteine, and cysteineglycine, prior to analysis by capillary electrophoresis with UV detection. Tween 20-AuNPs are capable of extracting aminothiols from a complicated matrix because a Tween 20 capping layer can inhibit effectively the nonspecific adsorption. Moreover, Tween 20-AuNPs had better aminothiol loading compared to Zonyl FSN-100- and Triton X-100-capped AuNPs. The extraction efficiency of aminothiols was highly dependent on the number of Tween 20-AuNPs, the concentration of dithiothreitol, and the type of surfactant (i.e., capping agent). Under optimal extraction conditions, the limits of detection at a signal-to-noise ratio of 3 for five aminothiols were down to 10-65 nM. Total and free aminothiols in plasma were determined by varying the order of disulfide reduction with tris(2-carboxyethyl)phosphine. Efficient separation of total and free aminothiols with baseline resolution was successfully achieved by the addition of cationic polyelectrolyte, poly(diallyldimethylammonium chloride), to the background electrolyte. Because this extraction method provided no matrix effect, the concentrations of total and free aminothiols in plasma can be quantified with an external calibration method. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, this proposed method may have great potential for disease diagnosis.

  1. Quantitative proteomic analysis of cold-responsive proteins in rice.

    PubMed

    Neilson, Karlie A; Mariani, Michael; Haynes, Paul A

    2011-05-01

    Rice is susceptible to cold stress and with a future of climatic instability we will be unable to produce enough rice to satisfy increasing demand. A thorough understanding of the molecular responses to thermal stress is imperative for engineering cultivars, which have greater resistance to low temperature stress. In this study we investigated the proteomic response of rice seedlings to 48, 72 and 96 h of cold stress at 12-14°C. The use of both label-free and iTRAQ approaches in the analysis of global protein expression enabled us to assess the complementarity of the two techniques for use in plant proteomics. The approaches yielded a similar biological response to cold stress despite a disparity in proteins identified. The label-free approach identified 236 cold-responsive proteins compared to 85 in iTRAQ results, with only 24 proteins in common. Functional analysis revealed differential expression of proteins involved in transport, photosynthesis, generation of precursor metabolites and energy; and, more specifically, histones and vitamin B biosynthetic proteins were observed to be affected by cold stress. PMID:21433000

  2. Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.

    PubMed

    Ream, Jennifer A; Lewis, L Kevin; Lewis, Karen A

    2016-10-15

    Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5-10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments. PMID:27495142

  3. Polymorphic AP-1 binding site in bovine CSN1S1 shows quantitative differences in protein binding associated with milk protein expression.

    PubMed

    Kuss, A W; Gogol, J; Bartenschlager, H; Geldermann, H

    2005-06-01

    Polymorphisms in 5'-flanking regions of milk protein encoding genes can influence the binding activity of the affected response elements and thus have an impact on the expression of the gene products. However, precise quantitative data concerning the binding properties of such variable response elements have so far not been described. In this study we present the results of a quantitative fluorescent electromobility shift assay comparing the allelic variants of a polymorphic activator protein-1 binding site in the promoter region of the bovine alphas1-casein encoding gene (CSN1S1), which is affected by an A-->G exchange at -175 bp (CSN1S1(-175bp)). A supershift assay using a commercial c-jun antibody was carried out to verify the specificity of protein binding. The gel shift analysis revealed specific and significantly reduced protein binding of oligonucleotides containing the G variant of the CSN1S1(-175bp) binding site. Further investigations comprised genotyping of the variable CSN1S1(-175bp) activator protein-1 element by an NmuCl restriction fragment length polymorphism in 62 cows of the breed Simmental and 80 cows of the breed German Holstein. Single milk proteins from at least 4 milk samples per cow were quantified by alkaline urea polyacrylamide gel electrophoresis. Homozygotes for CSN1S1(-175bp)*G were not observed, and the allele frequencies were 0.19 in Simmental and 0.05 in German Holstein. Carriers of CSN1S1(-175bp)*G showed higher content (%) as well as quantity (g/d) of alphas1-casein than CSN1S1(-175bp)*A homozygotes, independent of breed. We assume that the positive association of the CSN1S1(-175bp)*G variant with CSN1S1 expression is likely to be caused by a reduced affinity of the affected response element to a c-jun-containing CSN1S1 dimer with repressor properties. PMID:15905454

  4. Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size

    SciTech Connect

    Zhang, Haizhen; Burnum, Kristin E.; Luna, Maria L.; Petritis, Brianne O.; Kim, Jong Seo; Qian, Weijun; Moore, Ronald J.; Heredia-Langner, Alejandro; Webb-Robertson, Bobbie-Jo M.; Thrall, Brian D.; Camp, David G.; Smith, Richard D.; Pounds, Joel G.; Liu, Tao

    2011-12-01

    In biofluids (e.g., blood plasma) nanoparticles are readily embedded in layers of proteins that can affect their biological activity and biocompatibility. Herein, we report a study on the interactions between human plasma proteins and nanoparticles with a controlled systematic variation of properties using stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) based quantitative proteomics. Novel protocol has been developed to simplify the isolation of nanoparticle bound proteins and improve the reproducibility. Plasma proteins associated with polystyrene nanoparticles with three different surface chemistries and two sizes as well as for four different exposure times (for a total of 24 different samples) were identified and quantified by LC-MS analysis. Quantitative comparison of relative protein abundances were achieved by spiking an 18 O-labeled 'universal reference' into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantitation across the sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive pattern that classifies the nanoparticles based on their surface properties and size. In addition, data on the temporal study indicated that the stable protein 'corona' that was isolated for the quantitative analysis appeared to be formed in less than 5 minutes. The comprehensive results obtained herein using quantitative proteomics have potential implications towards predicting nanoparticle biocompatibility.

  5. High Blood Pressure Effects on the Blood to Cerebrospinal Fluid Barrier and Cerebrospinal Fluid Protein Composition: A Two-Dimensional Electrophoresis Study in Spontaneously Hypertensive Rats

    PubMed Central

    González-Marrero, Ibrahim; Castañeyra-Ruiz, Leandro; González-Toledo, Juan M.; Castañeyra-Ruiz, Agustín; de Paz-Carmona, Hector; Castro, Rafael; Hernandez-Fernaud, Juan R.; Castañeyra-Perdomo, Agustín; Carmona-Calero, Emilia M.

    2013-01-01

    The aim of the present work is to analyze the cerebrospinal fluid proteomic profile, trying to find possible biomarkers of the effects of hypertension of the blood to CSF barrier disruption in the brain and their participation in the cholesterol and β-amyloid metabolism and inflammatory processes. Cerebrospinal fluid (CSF) is a system linked to the brain and its composition can be altered not only by encephalic disorder, but also by systemic diseases such as arterial hypertension, which produces alterations in the choroid plexus and cerebrospinal fluid protein composition. 2D gel electrophoresis in cerebrospinal fluid extracted from the cistern magna before sacrifice of hypertensive and control rats was performed. The results showed different proteomic profiles between SHR and WKY, that α-1-antitrypsin, apolipoprotein A1, albumin, immunoglobulin G, vitamin D binding protein, haptoglobin and α-1-macroglobulin were found to be up-regulated in SHR, and apolipoprotein E, transthyretin, α-2-HS-glycoprotein, transferrin, α-1β-glycoprotein, kininogen and carbonic anhidrase II were down-regulated in SHR. The conclusion made here is that hypertension in SHR produces important variations in cerebrospinal fluid proteins that could be due to a choroid plexus dysfunction and this fact supports the close connection between hypertension and blood to cerebrospinal fluid barrier disruption. PMID:23401751

  6. Stage-specific analysis of plasma protein profiles in ovarian cancer: Difference in-gel electrophoresis analysis of pooled clinical samples

    PubMed Central

    Bailey, Mark J.; Shield-Artin, Kristy L.; Oliva, Karen; Ayhan, Mustafa; Reisman, Simone; Rice, Gregory E.

    2013-01-01

    Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE) protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4). The expression of TPM4 increased by 2-fold in Stage 2 before returning to “normal” levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage. PMID:23858298

  7. Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

    PubMed

    Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

    2004-09-01

    We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

  8. How Many proteins are Missed in Quantitative proteomics Based on Ms/Ms sequencing Methods?

    PubMed Central

    Mulvey, Claire; Thur, Bettina; Crawford, Mark; Godovac-Zimmermann, Jasminka

    2014-01-01

    Current bottom-up quantitative proteomics methods based on MS/MS sequencing of peptides are shown to be strongly dependent on sample preparation. Using cytosolic proteins from MCF-7 breast cancer cells, it is shown that protein pre-fractionation based on pI and MW is more effective than pre-fractionation using only MW in increasing the number of observed proteins (947 vs. 704 proteins) and the number of spectral counts per protein. Combination of MS data from the different pre-fractionation methods results in further improvements (1238 proteins). We discuss that at present the main limitation on quantitation by MS/MS sequencing is not MS sensitivity and protein abundance, but rather extensive peptide overlap and limited MS/MS sequencing throughput, and that this favors internally calibrated methods such as SILAC, ICAT or ITRAQ over spectral counting methods in attempts to drastically improve proteome coverage of biological samples. PMID:25729266

  9. Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling

    PubMed Central

    Tannu, Nilesh S; Hemby, Scott E

    2007-01-01

    Quantitative proteomics is the workhorse of the modern proteomics initiative. The gel-based and MuDPIT approaches have facilitated vital advances in the measurement of protein expression alterations in normal and disease phenotypic states. The methodological advance in two-dimensional gel electrophoresis (2DGE) has been the multiplexing fluorescent two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). 2D-DIGE is based on direct labeling of lysine groups on proteins with cyanine CyDye DIGE Fluor minimal dyes before isoelectric focusing, enabling the labeling of 2–3 samples with different dyes and electrophoresis of all the samples on the same 2D gel. This capability minimizes spot pattern variability and the number of gels in an experiment while providing simple, accurate and reproducible spot matching. This protocol can be completed in 3–5 weeks depending on the sample size of the experiment and the level of expertise of the investigator. PMID:17487156

  10. Evaluation and Comparison of Four Protein Extraction Protocols for Mono- and Two-Dimensional Electrophoresis in Mytilus Galloprovincialis

    PubMed Central

    Chirollo, Claudia; Boccia, Federica; Smaldone, Giorgio; Marrone, Raffaele; Pepe, Tiziana

    2015-01-01

    In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i) TRIzol reagent; ii) Lysis buffer; iii) phenylmethanesulfonyl fluoride; iv) trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis. PMID:27800405

  11. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1980-01-01

    The following aspects of kidney cell electrophoresis are discussed: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characterization of kidney cells.

  12. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1979-01-01

    A kidney cell electrophoresis technique is described in four parts: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characteristics of kidney cells.

  13. An Economical Electrophoresis Apparatus

    ERIC Educational Resources Information Center

    Andrews, I. M.

    1975-01-01

    Describes the production of an electrophoresis apparatus from commonly discarded articles. Outlines paper and gel electrophoresis and its application to the separation of amino acids and intestinal enzymes. (GS)

  14. One step physically adsorbed coating of silica capillary with excellent stability for the separation of basic proteins by capillary zone electrophoresis.

    PubMed

    Guo, Xiao-Feng; Guo, Xiao-Mei; Wang, Hong; Zhang, Hua-Shan

    2015-11-01

    The coating of capillary inner surface is considered to be an effective approach to suppress the adsorption of proteins on capillary inner surface in CE. However, most of coating materials reported are water-soluble, which may dissolve in BGE during the procedure of electrophoresis. In this study, a novel strategy for selection of physically coating materials has been illustrated to get coating layer with excellent stability using materials having poor solubility in commonly used solvents. Taking natural chitin as example (not hydrolyzed water soluble chitosan), a simple one step coating method using chitin solution in hexafluoroisopropanol was adopted within only 21 min with good coating reproducibility (RSDs of EOF for within-batch coated capillaries of 1.55% and between-batch coated capillaries of 2.31%), and a separation of four basic proteins on a chitin coated capillary was performed to evaluate the coating efficacy. Using chitin coating, the adsorption of proteins on capillary inner surface was successfully suppressed with reversed and stable EOF, and four basic proteins including lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A were baseline separated within 16 min with satisfied separation efficiency using 20 mM pH 2.0 H3PO4-Na2HPO4 as back ground electrolyte and 20 kV as separation voltage. What is more important, the chitin coating layer could be stable for more than two months during this study, which demonstrates that chitin is an ideal material for preparing semi-permanent coating on bare fused silica capillary inner wall and has hopeful potential in routine separation of proteins with CE.

  15. One step physically adsorbed coating of silica capillary with excellent stability for the separation of basic proteins by capillary zone electrophoresis.

    PubMed

    Guo, Xiao-Feng; Guo, Xiao-Mei; Wang, Hong; Zhang, Hua-Shan

    2015-11-01

    The coating of capillary inner surface is considered to be an effective approach to suppress the adsorption of proteins on capillary inner surface in CE. However, most of coating materials reported are water-soluble, which may dissolve in BGE during the procedure of electrophoresis. In this study, a novel strategy for selection of physically coating materials has been illustrated to get coating layer with excellent stability using materials having poor solubility in commonly used solvents. Taking natural chitin as example (not hydrolyzed water soluble chitosan), a simple one step coating method using chitin solution in hexafluoroisopropanol was adopted within only 21 min with good coating reproducibility (RSDs of EOF for within-batch coated capillaries of 1.55% and between-batch coated capillaries of 2.31%), and a separation of four basic proteins on a chitin coated capillary was performed to evaluate the coating efficacy. Using chitin coating, the adsorption of proteins on capillary inner surface was successfully suppressed with reversed and stable EOF, and four basic proteins including lysozyme, cytochrome c, ribonuclease A and α-chymotrypsinogen A were baseline separated within 16 min with satisfied separation efficiency using 20 mM pH 2.0 H3PO4-Na2HPO4 as back ground electrolyte and 20 kV as separation voltage. What is more important, the chitin coating layer could be stable for more than two months during this study, which demonstrates that chitin is an ideal material for preparing semi-permanent coating on bare fused silica capillary inner wall and has hopeful potential in routine separation of proteins with CE. PMID:26452799

  16. Kidney cell electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P. W.

    1985-01-01

    Tasks were undertaken in support of two objectives. They are: (1) to carry out electrophoresis experiments on cells in microgravity; and (2) assess the feasibility of using purified kidney cells from embryonic kidney cultures as a source of important cell products. Investigations were carried out in the following areas: (1) ground based electrophoresis technology; (2) cell culture technology; (3) electrophoresis of cells; (4) urokinase assay research; (5) zero-g electrophoresis; and (6) flow cytometry.

  17. Influence of the ionic strength of acidic background electrolytes on the separation of proteins by capillary electrophoresis.

    PubMed

    Bekri, Samya; Leclercq, Laurent; Cottet, Hervé

    2016-02-01

    The ionic strength is one of the key parameters for optimizing CE separations. However, only a few data are available in the literature about the ionic strength effect on the separation of proteins. The effect of ionic strength on separation performances is rather complex since many different parameters are involved: such as the protein effective mobility, the electroosmotic mobility, the separation efficiency via the electromigration dispersion, as well as the viscosity and temperature of the background electrolyte. In the present work, the influence of ionic strength on the electrophoretic separation of five model proteins has been investigated in acidic conditions, on successive multi-ionic layers coated capillary, in counter-electroosmotic mode with anodic electroosmotic flow. The decrease in effective and electroosmotic mobilities with increasing ionic strength were compared using the slope-plot approach, which is very helpful for understanding the observed changes in apparent selectivity and resolution. The relative decrease of the protein effective mobility was about 30-40% of the mobility determined at 5mM ionic strength per ionic strength decade. It was found that relatively low ionic strength (∼5-10mM) was preferable to optimize the overall separation of the five model proteins. PMID:26780847

  18. Secondary Reactions and Strategies to Improve Quantitative Protein Footprinting

    SciTech Connect

    Xu,G.; Kiselar, J.; He, Q.; Chance, M.

    2005-01-01

    Hydroxyl radical-mediated footprinting permits detailed examination of structure and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides are governed by changes in the solvent accessibility of the side-chain probe residues. The precise and accurate determination of peptide reaction rates is essential to successfully probing protein structure using footprinting. In this study, we specifically examine the magnitude and mechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroxide and other oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but not aromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed by adding trace amounts of catalase or millimolar Met-NH{sub 2} (or Met-OH) buffer immediately after irradiation; this leads to greatly improved adherence to first-order kinetics and more precise observed oxidation rates. The strategy is shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disulfide as the principal product. The disulfides can be reduced before mass spectrometric analysis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.

  19. Quantitative protein composition and baking quality of winter wheat as affected by late sulfur fertilization.

    PubMed

    Zörb, Christian; Steinfurth, Dorothee; Seling, Simone; Langenkämper, Georg; Koehler, Peter; Wieser, Herbert; Lindhauer, Meinolf G; Mühling, Karl H

    2009-05-13

    Increasing prices for wheat products and fertilizers, as well as reduced sulfur (S) contributions from the atmosphere, call for an improvement of product quality and agricultural management. To detect the impact of a time-dependent S fertilization, the quantitative protein composition and the baking quality of two different wheat cultivars, Batis and Turkis, were evaluated. The glutathione concentration in grains serves as a reliable marker of the need for added S fertilizer. The quantitation of gliadins and glutenin subunits by reversed-phase high-performance liquid chromatography confirmed that S-rich proteins significantly increased with S fertilization, whereas the S-poor proteins significantly decreased. Proteome analysis by means of high-resolution protein profiles detected 55 and 37 proteins from Batis and Turkis changed by late S fertilization. A microscale baking test using wholemeal flour was implemented for the evaluation of baking quality, and late S fertilization was found to improve the composition of gluten proteins and baking quality.

  20. Simultaneous separation of acidic and basic proteins using gemini pyrrolidinium surfactants and hexafluoroisopropanol as dynamic coating additives in capillary electrophoresis.

    PubMed

    Tian, Yu; Li, Yunfang; Mei, Jie; Cai, Bo; Dong, Jinfeng; Shi, Zhiguo; Xiao, Yuxiu

    2015-09-18

    The separation of acidic and basic proteins using CE has been limited in part due to the adsorption of proteins onto the capillary wall. In this work, the efficient control of EOF and the simultaneous separation of acidic and basic proteins are achieved by use of C18-4-C18PB as a dynamic coating additive, which is a representative surfactant for 1,1'-(butane-1,s-alkyl)bis(1-alkylpyrrolidinium) bromide (Cn-4-CnPB, n=10, 12, 14, 16 and 18). C18-4-C18PB exhibits a powerful capability in the reversal of EOF, and a low concentration even less than 0.001 mM is sufficient to reverse EOF at the tested pH values (3.0-9.0). Baseline separation of eight proteins with sharp peaks and high efficiencies (54,000-297,000 plates/m) is obtained with 30 mM NaH2PO4 buffer (pH 5.0) containing 4 mM C18-4-C18PB. At the same buffer condition, the Cn-4-CnPB with shorter alkyl chain (n=10, 12, 14, 16) cannot achieve the same effective protein separation as C18-4-C18PB. However, the combined use of small amounts (≤0.5%, v/v) of hexafluoroisopropanol (HFIP) and Cn-4-CnPB (n=10, 12, 14, 16) as additives can completely separate all eight proteins with high efficiencies of 81,000-318,000 plates/m. The RSDs of migration time are less than 0.80% and 5.84% for run-to-run and day-to-day assays (n=5), respectively, and the protein recoveries are larger than 90.15%. To the best of our knowledge, this is the first report on the simultaneous separation of acidic and basic proteins using Cn-4-CnPB surfactants or Cn-4-CnPB surfactants combined with HFIP as dynamic coating additives.

  1. Self-assembled covalent capillary coating of diazoresin/carboxyl fullerene for analysis of proteins by capillary electrophoresis and a comparison with diazoresin/graphene oxide coating.

    PubMed

    Yu, Bing; Shu, Xi; Cong, Hailin; Chen, Xin; Liu, Huwei; Yuan, Hua; Chi, Ming

    2016-03-11

    Self-assembled and covalently linked capillary coatings of carboxyl fullerenes (C60-COOH) were prepared using photosensitive diazoresin (DR) as a coupling agent. Layer by layer (LBL) self-assembled DR/C60-COOH coatings based on ionic bonding was fabricated first on the inner surface of silica capillary, and subsequently converted into covalent bonding after treatment with UV light through a unique photochemistry reaction of DR. The covalently bonded coatings had the ability of suppressing protein adsorption on the inner surface of silica capillary, and thus the baseline separation of lysozyme (Lys), cytochrome c (Cyt-c), bovine serum albumin (BSA) and myoglobin (Mb) was achieved within 13min by using capillary electrophoresis (CE). The covalently linked DR/C60-COOH capillary coatings presented good chemical stability and repeatability. The reproducibility of the separation of proteins was less than 1%, 2.5%, and 3.5%, respectively, for run-to-run, day-to-day, capillary-to-capillary, respectively; and the RSD of migration time for the proteins are all less than 2.5% after a continuous 100 times running in a coating column. Compared with DR/graphene oxide (GO) coatings prepared by the same method, the DR/C60-COOH capillary coatings showed excellent protein separation performance due to a self-lubrication based anti-fouling mechanism. Because of the replacement of highly toxic and moisture sensitive silane coupling agent by DR in the covalent coating preparation, this method may provide an environmentally friendly and simple way to prepare the covalently coated capillaries for CE.

  2. ProCoCoA: A quantitative approach for analyzing protein core composition.

    PubMed

    Bottini, Silvia; Bernini, Andrea; De Chiara, Matteo; Garlaschelli, Diego; Spiga, Ottavia; Dioguardi, Marco; Vannuccini, Elisa; Tramontano, Anna; Niccolai, Neri

    2013-04-01

    Defining the amino acid composition of protein cores is fundamental for understanding protein folding, as different architectures might achieve structural stability only in the presence of specific amino acid networks. Quantitative characterization of protein cores in relation to the corresponding structures and dynamics is needed to increase the reliability of protein engineering procedures. Unambiguous criteria based on atom depth considerations were established to assign amino acid residues to protein cores and, hence, for classifying inner and outer molecular moieties. These criteria were summarized in a new tool named ProCoCoA, Protein Core Composition Analyzer. An user-friendly web interface was developed, available at the URL: http://www.sbl.unisi.it/prococoa. An accurate estimate of protein core composition for six protein architectures selected from the CATH database of solved structures has been carried out, and the obtained results indicate the presence of specific patterns of amino acid core composition in different protein folds.

  3. The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis.

    PubMed

    Plowman, Jeffrey E; Flanagan, Leanne M; Paton, Louise N; Fitzgerald, Anna C; Joyce, Nigel I; Bryson, Warren G

    2003-06-01

    Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new

  4. Supported Molecular Matrix Electrophoresis.

    PubMed

    Matsuno, Yu-Ki; Kameyama, Akihiko

    2015-01-01

    Mucins are difficult to separate using conventional gel electrophoresis methods such as SDS-PAGE and agarose gel electrophoresis, owing to their large size and heterogeneity. On the other hand, cellulose acetate membrane electrophoresis can separate these molecules, but is not compatible with glycan analysis. Here, we describe a novel membrane electrophoresis technique, termed "supported molecular matrix electrophoresis" (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used to achieve separation. This description includes the separation, visualization, and glycan analysis of mucins with the SMME technique. PMID:26139278

  5. Quantitative methods for the analysis of protein phosphorylation in drug development.

    PubMed

    Olive, D Michael

    2004-10-01

    Most signal transduction and cell signaling pathways are mediated by protein kinases. Protein kinases have emerged as important cellular regulatory proteins in many aspects of neoplasia. Protein kinase inhibitors offer the opportunity to target diseases such as cancer with chemotherapeutic agents specific for the causative molecular defect. In order to identify possible targets and assess kinase inhibitors, quantitative methods for analyzing protein phosphorylation have been developed. This review examines some of the current formats used for quantifying kinase function for drug development. PMID:15966829

  6. Quantitative proteomic analysis of mice corneal tissues reveals angiogenesis-related proteins involved in corneal neovascularization.

    PubMed

    Shen, Minqian; Tao, Yimin; Feng, Yifan; Liu, Xing; Yuan, Fei; Zhou, Hu

    2016-07-01

    Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n=5) and five corresponding neovascularized corneal tissues (model group mice n=5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis.

  7. Current two-dimensional electrophoresis technology for proteomics.

    PubMed

    Görg, Angelika; Weiss, Walter; Dunn, Michael J

    2004-12-01

    Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where Mr and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037-1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5-12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the

  8. Coparative assessment of irradiated proteins in potato tuber with untreated control by High Performance Liquid Chromatography (HPLC) and gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Ghojaie, M.; Sayhoon, M.

    1995-02-01

    About 2% of the weight of potato tuber is composed of proteins. In spite of their low quantity the proteins play a key role in the physiological activities leading to the break of the dormancy period and start of the cell division. This causes sprouting and also greening due to chlorophyll formation. This in turn is always accompanied by the production of the glycoalkaloid solanine in the flesh of tuber. For evaluation of radiation effect (dose range 50-250 Gy) and probable structural changes (amino acid release), analysis of selected proteins (molecular range 5 × 10^4 - 2 × 10^5 Dalton) of potato tuber in both irradiated and control type by HPLC showed no considerable changes in retention times, but qualitative assessment of amino acids by Pico-Tag^TM Pre-derivatizing method had some changes in quantity of amino acids like lysine which was increased 1 month after irradiation while Glutamic acid had considerable decreasment after the same time of irradiation.

  9. Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase

    PubMed Central

    Burchett, Gina G.; Folsom, Charles G.; Lane, Kimberly T.

    2015-01-01

    β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins. PMID:26121040

  10. Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

    NASA Astrophysics Data System (ADS)

    Wang, Evelyn H.; Combe, Peter C.; Schug, Kevin A.

    2016-05-01

    Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

  11. Capillary electrophoresis with capacitively coupled contactless conductivity detection applied to the quantitation and to the determination of physical-chemical properties of peroxycarboxylates in aqueous medium.

    PubMed

    Vidal, Denis T R; do Lago, Claudimir L

    2013-07-01

    CE with C⁴D (CE-C⁴D) was successfully applied to the investigation of performate, peracetate, and perpropionate in aqueous medium. Ionic mobilities, diffusion coefficients, and hydrodynamic radii were obtained for the first time for these species. CE-C⁴D was also used to estimate the pKa values of the peroxycarboxylic acids. Because the peroxycarboxylates (POCs) undergoes hydrolysis while migrating, a simple calibration curve cannot be used for quantitation. Thus, an indirect calibration approach was used. The new method was used to monitor the formation of peroxycarboxylic acids from hydrogen peroxide and the carboxylic acid as well as to the quantitation of peracetic acid in a commercial sample. The CE-C⁴D method compares favorably with the conventional titration method because of the possibility of speciation of the POC, the low sample consumption, and the low LOD (14, 8, and 24 μmol/L for performate, peracetate, and perpropionate, respectively). Although POCs are structural isomers of monoalkyl carbonates, they have greater hydrodynamic radii, which suggests that the positions of the oxygen atoms in the molecules have a direct impact in the charge density and consequently on the hydration atmosphere. PMID:23595363

  12. A Novel Systems-Biology Algorithm for the Analysis of Coordinated Protein Responses Using Quantitative Proteomics.

    PubMed

    García-Marqués, Fernando; Trevisan-Herraz, Marco; Martínez-Martínez, Sara; Camafeita, Emilio; Jorge, Inmaculada; Lopez, Juan Antonio; Méndez-Barbero, Nerea; Méndez-Ferrer, Simón; Del Pozo, Miguel Angel; Ibáñez, Borja; Andrés, Vicente; Sánchez-Madrid, Francisco; Redondo, Juan Miguel; Bonzon-Kulichenko, Elena; Vázquez, Jesús

    2016-05-01

    The coordinated behavior of proteins is central to systems biology. However, the underlying mechanisms are poorly known and methods to analyze coordination by conventional quantitative proteomics are still lacking. We present the Systems Biology Triangle (SBT), a new algorithm that allows the study of protein coordination by pairwise quantitative proteomics. The Systems Biology Triangle detected statistically significant coordination in diverse biological models of very different nature and subjected to different kinds of perturbations. The Systems Biology Triangle also revealed with unprecedented molecular detail an array of coordinated, early protein responses in vascular smooth muscle cells treated at different times with angiotensin-II. These responses included activation of protein synthesis, folding, turnover, and muscle contraction - consistent with a differentiated phenotype-as well as the induction of migration and the repression of cell proliferation and secretion. Remarkably, the majority of the altered functional categories were protein complexes, interaction networks, or metabolic pathways. These changes could not be detected by other algorithms widely used by the proteomics community, and the vast majority of proteins involved have not been described before to be regulated by AngII. The unique capabilities of The Systems Biology Triangle to detect functional protein alterations produced by the coordinated action of proteins in pairwise quantitative proteomics experiments make this algorithm an attractive choice for the biological interpretation of results on a routine basis.

  13. Quantification of PEGylated proteases with varying degree of conjugation in mixtures: An analytical protocol combining protein precipitation and capillary gel electrophoresis.

    PubMed

    Morgenstern, Josefine; Busch, Markus; Baumann, Pascal; Hubbuch, Jürgen

    2016-09-01

    PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times. In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase(®) which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis. PMID:27521256

  14. Mid-scale free-flow electrophoresis with gravity-induced uniform flow of background buffer in chamber for the separation of cells and proteins.

    PubMed

    Dong, Yu-Chao; Shao, Jing; Yin, Xiao-Yang; Fan, Liu-yin; Cao, Cheng-Xi

    2011-07-01

    A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.

  15. Quantification of PEGylated proteases with varying degree of conjugation in mixtures: An analytical protocol combining protein precipitation and capillary gel electrophoresis.

    PubMed

    Morgenstern, Josefine; Busch, Markus; Baumann, Pascal; Hubbuch, Jürgen

    2016-09-01

    PEGylation, i.e. the covalent attachment of chemically activated polyethylene glycol (PEG) to proteins, is a technique commonly used in biopharmaceutical industry to improve protein stability, pharmacokinetics and resistance to proteolytic degradation. Therefore, PEGylation represents a valuable strategy to reduce autocatalysis of biopharmaceutical relevant proteases during production, purification and storage. In case of non-specific random conjugation the existence of more than one accessible binding site results in conjugates which vary in position and number of attached PEG molecules. These conjugates may differ considerably in their physicochemical properties. Optimizing the reaction conditions with respect to the degree of PEGylation (number of linked PEG molecules) using high-throughput screening (HTS) technologies requires a fast and reliable analytical method which allows stopping the reaction at defined times. In this study an analytical protocol for PEGylated proteases is proposed combining preservation of sample composition by trichloroacetic acid (TCA) precipitation with high-throughput capillary gel electrophoresis (HT-CGE). The well-studied protein hen egg-white lysozyme served as a model system for validating the newly developed analytical protocol for 10kDa mPEG-aldehyde conjugates. PEGamer species were purified by chromatographic separation for calibrating the HT-CGE system. In a case study, the serine protease Savinase(®) which is highly sensitive to autocatalysis was randomly modified with 5kDa and 10kDa mPEG-aldehyde and analyzed. Using the presented TCA protocol baseline separation between PEGamer species was achieved allowing for the analysis of heterogeneous PEGamer mixtures while preventing protease autocatalysis.

  16. Proteomic and mass spectroscopic quantitation of protein S-nitrosation differentiates NO-donors

    PubMed Central

    Sinha, Vaishali; Wijewickrama, Gihani T.; Chandrasena, R. Esala P.; Xu, Hua; Edirisinghe, Praneeth D.; Schiefer, Isaac T.; Thatcher, Gregory R. J.

    2010-01-01

    Protein S-nitrosation has been argued to be the most important signaling pathway mediating the bioactivity of NO. This post-translational modification of protein thiols is the result of chemical nitrosation of cysteine residues. The term NO-donors covers very different chemical classes: from clinical therapeutics to probes of routine use in chemical biology; their different chemistry is predicted to result in distinctive biology regulated by protein S-nitrosation. To measure the extent of protein S-nitrosation by NO-donors, a proteomic mass spectrometry method was developed, which quantitates free thiol versus nitrosothiol for each modified cysteine residue, coined d-Switch. This method is adapted from the biotin switch (BST) method, used extensively to identify S-nitrosated proteins in complex biological mixtures, however, BST does not quantitate free thiol. Since glutathione-S-transferase P1-1 (GST-P1) has been proposed to be a biological “NO carrier”, GST-P1 was used as a reporter protein. The 5 different chemical classes of NO-donors compared by d-Switch demonstrated very different profiles of protein S-nitrosation and response to O2 and cysteine, although, all NO-donors were oxidants towards GST-P1. The low limits of detection and the ability to use established MS database searching allowed facile generalization of the d-Switch method, therefore after incubation of neuronal cell cultures with nitrosothiol, it was possible not only to quantitate S-nitrosation of GST-P1, but also many other proteins, including novel targets such as ubiquitin carboxyl-terminal esterase L1 (UCHL1), moreover d-Switch also allowed identification of non-nitrosated proteins and quantitation of degree of nitrosation for individual protein thiols. PMID:20524644

  17. Measurement of neonatal equine immunoglobulins for assessment of colostral immunoglobulin transfer: comparison of single radial immunodiffusion with the zinc sulfate turbidity test, serum electrophoresis, refractometry for total serum protein, and the sodium sulfite precipitation test.

    PubMed

    Rumbaugh, G E; Ardans, A A; Ginno, D; Trommershausen-Smith, A

    1978-02-01

    Four procedures for assessment of adequacy of colostral immunoglobulin (Ig) transfer in foals were evaluated. Results of zinc sulfate turbidity test, serum electrophoresis, total serum protein refractometry, and sodium sulfite precipitation test were compared with immunoglobulin G content determined by single radial immunodiffusion. The zinc sulfate turbidity test gave acceptable results for IgG, except that hemolyzed serum samples gave higher than expected values. A correction factor for hemolyzed serum was found to be useful. Serum electrophoresis was a satisfactory method of estimating IgG content. Total serum protein values may not be a valid basis for estimating IgG content, inasmuch as postsuckling total protein values were found to decrease in some foals in which passive transfer of IgG had been adequate. Sodium sulfite precipitation reactions were too unpredictable to be of value for determination of neonatal IgG concentration.

  18. Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

    PubMed

    Xu, Ming; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra

    2014-10-01

    Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism. PMID:25059147

  19. Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting

    PubMed Central

    Jacob, Thomas; Agarwal, Anupriya; Ramunno-Johnson, Damien; O’Hare, Thomas; Gönen, Mehmet; Tyner, Jeffrey W.; Druker, Brian J.; Vu, Tania Q.

    2016-01-01

    Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells. PMID:27320899

  20. Laboratory and field validation of a Cry1Ab protein quantitation method for water.

    PubMed

    Strain, Katherine E; Whiting, Sara A; Lydy, Michael J

    2014-10-01

    The widespread planting of crops expressing insecticidal proteins derived from the soil bacterium Bacillus thuringiensis (Bt) has given rise to concerns regarding potential exposure to non-target species. These proteins are released from the plant throughout the growing season into soil and surface runoff and may enter adjacent waterways as runoff, erosion, aerial deposition of particulates, or plant debris. It is crucial to be able to accurately quantify Bt protein concentrations in the environment to aid in risk analyses and decision making. Enzyme-linked immunosorbent assay (ELISA) is commonly used for quantitation of Bt proteins in the environment; however, there are no published methods detailing and validating the extraction and quantitation of Bt proteins in water. The objective of the current study was to optimize the extraction of a Bt protein, Cry1Ab, from three water matrices and validate the ELISA method for specificity, precision, accuracy, stability, and sensitivity. Recovery of the Cry1Ab protein was matrix-dependent and ranged from 40 to 88% in the validated matrices, with an overall method detection limit of 2.1 ng/L. Precision among two plates and within a single plate was confirmed with a coefficient of variation less than 20%. The ELISA method was verified in field and laboratory samples, demonstrating the utility of the validated method. The implementation of a validated extraction and quantitation protocol adds consistency and reliability to field-collected data regarding transgenic products. PMID:25059137

  1. Absolute quantitation of isoforms of post-translationally modified proteins in transgenic organism.

    PubMed

    Li, Yaojun; Shu, Yiwei; Peng, Changchao; Zhu, Lin; Guo, Guangyu; Li, Ning

    2012-08-01

    Post-translational modification isoforms of a protein are known to play versatile biological functions in diverse cellular processes. To measure the molar amount of each post-translational modification isoform (P(isf)) of a target protein present in the total protein extract using mass spectrometry, a quantitative proteomic protocol, absolute quantitation of isoforms of post-translationally modified proteins (AQUIP), was developed. A recombinant ERF110 gene overexpression transgenic Arabidopsis plant was used as the model organism for demonstration of the proof of concept. Both Ser-62-independent (14)N-coded synthetic peptide standards and (15)N-coded ERF110 protein standard isolated from the heavy nitrogen-labeled transgenic plants were employed simultaneously to determine the concentration of all isoforms (T(isf)) of ERF110 in the whole plant cell lysate, whereas a pair of Ser-62-dependent synthetic peptide standards were used to quantitate the Ser-62 phosphosite occupancy (R(aqu)). The P(isf) was finally determined by integrating the two empirically measured variables using the following equation: P(isf) = T(isf) · R(aqu). The absolute amount of Ser-62-phosphorylated isoform of ERF110 determined using AQUIP was substantiated with a stable isotope labeling in Arabidopsis-based relative and accurate quantitative proteomic approach. The biological role of the Ser-62-phosphorylated isoform was demonstrated in transgenic plants.

  2. Deep proteomics of mouse skeletal muscle enables quantitation of protein isoforms, metabolic pathways, and transcription factors.

    PubMed

    Deshmukh, Atul S; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T; Cox, Jürgen; Mann, Matthias

    2015-04-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms.

  3. Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors*

    PubMed Central

    Deshmukh, Atul S.; Murgia, Marta; Nagaraj, Nagarjuna; Treebak, Jonas T.; Cox, Jürgen; Mann, Matthias

    2015-01-01

    Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms. PMID:25616865

  4. Molecular beacon aptamers for direct and universal quantitation of recombinant proteins from cell lysates.

    PubMed

    Tan, Xiaohong; Chen, Weijun; Lu, Shun; Zhu, Zhi; Chen, Tao; Zhu, Guizhi; You, Mingxu; Tan, Weihong

    2012-10-01

    Western blot, enzyme linked immunosorbent assay (ELISA), and fluorescent fusion proteins are currently the most common methods for detecting recombinant proteins. However, the former two are cumbersome and time-consuming, and the latter method may interfere with the trafficking and function of the fused recombinant proteins. We report here a rapid, inexpensive, and simple approach to detect and quantify recombinant proteins using an anti-His-tag molecular beacon aptamer (HMBA). We demonstrated the technique by detection and quantitation of expressed recombinant proteins directly from E. coli cell lysate. The amount of expressed P78-His was determined to be 1.49 μg from the 20 μg cell lysate proteins. To the best of our knowledge, this is the first example directly measuring the concentration and expression yield of recombinant proteins from cell lysate, and the entire procedure required only 5 min.

  5. A statistical framework for protein quantitation in bottom-up MS-based proteomics

    SciTech Connect

    Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas; Huang, Jianhua; Adkins, Joshua N.; Ansong, Charles; Heffron, Fred; Metz, Thomas O.; Qian, Weijun; Yoon, Hyunjin; Smith, Richard D.; Dabney, Alan R.

    2009-08-15

    ABSTRACT Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and confidence measures. Challenges include the presence of low-quality or incorrectly identified peptides and widespread, informative, missing data. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model for protein abundance in terms of peptide peak intensities, applicable to both label-based and label-free quantitation experiments. The model allows for both random and censoring missingness mechanisms and provides naturally for protein-level estimates and confidence measures. The model is also used to derive automated filtering and imputation routines. Three LC-MS datasets are used to illustrate the methods. Availability: The software has been made available in the open-source proteomics platform DAnTE (Polpitiya et al. (2008)) (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu

  6. Quantitative Analysis of Spatial Protein-protein Proximity in Fluorescence Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Yong; Liu, Yi-Kuang; Eghbali, Mansoureh; Stefani, Enrico

    2009-02-01

    To quantify spatial protein-protein proximity (colocalization) in fluorescence microscopic images, cross-correlation and autocorrelation functions were decomposed into fast and slowly decaying components. The fast component results from clusters of proteins specifically labeled and the slow one from background/image heterogeneity. We show that the calculation of the protein-protein proximity index and the correlation coefficient are more reliably determined by extracting the fast-decaying component.

  7. A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC).

    PubMed

    Orton, Dennis J; Doucette, Alan A

    2013-03-15

    As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications.

  8. A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC).

    PubMed

    Orton, Dennis J; Doucette, Alan A

    2013-03-15

    As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications. PMID:23435344

  9. Kidney Cell Electrophoresis

    NASA Technical Reports Server (NTRS)

    Todd, P.

    1985-01-01

    Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated, ground support in the form of analytical cell electrophoresis and flow cytometry was provided and cells returned from space flight were analyzed. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. The protocol established and utilized is given.

  10. Quantitative analysis of protein-RNA interactions by gel mobility shift

    PubMed Central

    Ryder, Sean P.; Recht, Michael I.; Williamson, James R.

    2010-01-01

    The gel mobility shift assay is routinely used to visualize protein-RNA interactions. Its power resides in the ability to resolve free from bound RNA with high resolution in a gel matrix. In this chapter, we review the quantitative application of this approach to elucidate thermodynamic properties of protein-RNA complexes. Assay designs for titration, competition, and stoichiometry experiments are presented for two unrelated model complexes. PMID:18982286

  11. Quantitative imaging of protein targets in the human brain with PET

    NASA Astrophysics Data System (ADS)

    Gunn, Roger N.; Slifstein, Mark; Searle, Graham E.; Price, Julie C.

    2015-11-01

    PET imaging of proteins in the human brain with high affinity radiolabelled molecules has a history stretching back over 30 years. During this period the portfolio of protein targets that can be imaged has increased significantly through successes in radioligand discovery and development. This portfolio now spans six major categories of proteins; G-protein coupled receptors, membrane transporters, ligand gated ion channels, enzymes, misfolded proteins and tryptophan-rich sensory proteins. In parallel to these achievements in radiochemical sciences there have also been significant advances in the quantitative analysis and interpretation of the imaging data including the development of methods for image registration, image segmentation, tracer compartmental modeling, reference tissue kinetic analysis and partial volume correction. In this review, we analyze the activity of the field around each of the protein targets in order to give a perspective on the historical focus and the possible future trajectory of the field. The important neurobiology and pharmacology is introduced for each of the six protein classes and we present established radioligands for each that have successfully transitioned to quantitative imaging in humans. We present a standard quantitative analysis workflow for these radioligands which takes the dynamic PET data, associated blood and anatomical MRI data as the inputs to a series of image processing and bio-mathematical modeling steps before outputting the outcome measure of interest on either a regional or parametric image basis. The quantitative outcome measures are then used in a range of different imaging studies including tracer discovery and development studies, cross sectional studies, classification studies, intervention studies and longitudinal studies. Finally we consider some of the confounds, challenges and subtleties that arise in practice when trying to quantify and interpret PET neuroimaging data including motion artifacts

  12. Quantitative imaging of protein targets in the human brain with PET.

    PubMed

    Gunn, Roger N; Slifstein, Mark; Searle, Graham E; Price, Julie C

    2015-11-21

    PET imaging of proteins in the human brain with high affinity radiolabelled molecules has a history stretching back over 30 years. During this period the portfolio of protein targets that can be imaged has increased significantly through successes in radioligand discovery and development. This portfolio now spans six major categories of proteins; G-protein coupled receptors, membrane transporters, ligand gated ion channels, enzymes, misfolded proteins and tryptophan-rich sensory proteins. In parallel to these achievements in radiochemical sciences there have also been significant advances in the quantitative analysis and interpretation of the imaging data including the development of methods for image registration, image segmentation, tracer compartmental modeling, reference tissue kinetic analysis and partial volume correction. In this review, we analyze the activity of the field around each of the protein targets in order to give a perspective on the historical focus and the possible future trajectory of the field. The important neurobiology and pharmacology is introduced for each of the six protein classes and we present established radioligands for each that have successfully transitioned to quantitative imaging in humans. We present a standard quantitative analysis workflow for these radioligands which takes the dynamic PET data, associated blood and anatomical MRI data as the inputs to a series of image processing and bio-mathematical modeling steps before outputting the outcome measure of interest on either a regional or parametric image basis. The quantitative outcome measures are then used in a range of different imaging studies including tracer discovery and development studies, cross sectional studies, classification studies, intervention studies and longitudinal studies. Finally we consider some of the confounds, challenges and subtleties that arise in practice when trying to quantify and interpret PET neuroimaging data including motion artifacts

  13. Quantitative measurement of intracellular protein dynamics using photobleaching or photoactivation of fluorescent proteins.

    PubMed

    Matsuda, Tomoki; Nagai, Takeharu

    2014-12-01

    Unlike in vitro protein dynamics, intracellular protein dynamics are intricately regulated by protein-protein interactions or interactions between proteins and other cellular components, including nucleic acids, the plasma membrane and the cytoskeleton. Alteration of these dynamics plays a crucial role in physiological phenomena such as gene expression and cell division. Live-cell imaging via microscopy with the inherent properties of fluorescent proteins, i.e. photobleaching and photoconversion, or fluorescence correlation spectroscopy, provides insight into the movement of proteins and their interactions with cellular components. This article reviews techniques based on photo-induced changes in the physicochemical properties of fluorescent proteins to measure protein dynamics inside living cells, and it also discusses the strengths and weaknesses of these techniques.

  14. ReAsH as a Quantitative Probe of In-Cell Protein Dynamics.

    PubMed

    Gelman, Hannah; Wirth, Anna Jean; Gruebele, Martin

    2016-04-01

    The tetracysteine (tc) tag/biarsenical dye system (FlAsH or ReAsH) promises to combine the flexibility of fluorescent protein tags with the small size of dye labels, allowing in-cell study of target proteins that are perturbed by large protein tags. Quantitative thermodynamic and kinetic studies in-cell using FlAsH and ReAsH have been hampered by methodological complexities presented by the fluorescence properties of the tag-dye complex probed by either Förster resonance energy transfer (FRET) or direct excitation. We label the model protein phosphoglycerate kinase (PGK) with AcGFP1 and ReAsH for direct comparison with AcGFP1/mCherry-labeled PGK. We find that fast relaxation imaging (FReI), combining millisecond temperature jump kinetics with fluorescence microscopy detection, circumvents many of the difficulties encountered working with the ReAsH system, allowing us to obtain quantitative FRET measurements of protein stability and kinetics both in vitro and in cells. We also demonstrate the to us surprising result that fluorescence from directly excited, unburied ReAsH at the C-terminus of the model protein also reports on folding in vitro and in cells. Comparing the ReAsH-labeled protein to a construct labeled with two fluorescent protein tags allows us to evaluate how a bulkier protein tag affects protein dynamics in cells and in vitro. We find that the average folding rate in the cell is closer to the in vitro rate with the smaller tag, highlighting the effect of tags on quantitative in-cell measurements. PMID:26959408

  15. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    PubMed

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  16. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  17. Quantitative analysis of peptides and proteins in biomedicine by targeted mass spectrometry

    PubMed Central

    Gillette, Michael A; Carr, Steven A

    2014-01-01

    Targeted mass spectrometry (MS) is becoming widely used in academia and in pharmaceutical and biotechnology industries for sensitive and quantitative detection of proteins, peptides and post-translational modifications. Here we describe the increasing importance of targeted MS technologies in clinical proteomics and the potential key roles these techniques will have in bridging biomedical discovery and clinical implementation. PMID:23269374

  18. Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae

    PubMed Central

    Yin, Qing Yuan; de Groot, Piet W J; de Jong, Luitzen; Klis, Frans M; De Koster, Chris G

    2007-01-01

    The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 × 103, 44 × 103, 38 × 103, 11 × 103 and 6.5 × 103 of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 × 103 copies μm−2. For relative quantitation, we compared wild-type cells to gas1Δ cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi. PMID:17617218

  19. A quantitative liposome microarray to systematically characterize protein-lipid interactions.

    PubMed

    Saliba, Antoine-Emmanuel; Vonkova, Ivana; Ceschia, Stefano; Findlay, Greg M; Maeda, Kenji; Tischer, Christian; Deghou, Samy; van Noort, Vera; Bork, Peer; Pawson, Tony; Ellenberg, Jan; Gavin, Anne-Claude

    2014-01-01

    Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds and signaling molecules, but they are still largely under-represented in known biological networks. Here we describe a liposome microarray-based assay (LiMA), a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed and high-throughput manner.

  20. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells

    PubMed Central

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  1. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells.

    PubMed

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  2. Qualitative and Quantitative Protein Complex Prediction Through Proteome-Wide Simulations.

    PubMed

    Rizzetto, Simone; Priami, Corrado; Csikász-Nagy, Attila

    2015-10-01

    Despite recent progress in proteomics most protein complexes are still unknown. Identification of these complexes will help us understand cellular regulatory mechanisms and support development of new drugs. Therefore it is really important to establish detailed information about the composition and the abundance of protein complexes but existing algorithms can only give qualitative predictions. Herein, we propose a new approach based on stochastic simulations of protein complex formation that integrates multi-source data--such as protein abundances, domain-domain interactions and functional annotations--to predict alternative forms of protein complexes together with their abundances. This method, called SiComPre (Simulation based Complex Prediction), achieves better qualitative prediction of yeast and human protein complexes than existing methods and is the first to predict protein complex abundances. Furthermore, we show that SiComPre can be used to predict complexome changes upon drug treatment with the example of bortezomib. SiComPre is the first method to produce quantitative predictions on the abundance of molecular complexes while performing the best qualitative predictions. With new data on tissue specific protein complexes becoming available SiComPre will be able to predict qualitative and quantitative differences in the complexome in various tissue types and under various conditions.

  3. Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins.

    PubMed

    Capriotti, Anna Laura; Caracciolo, Giulio; Caruso, Giuseppe; Cavaliere, Chiara; Pozzi, Daniela; Samperi, Roberto; Laganà, Aldo

    2013-01-01

    A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA-cationic lipid complexes (lipoplexes), and lipid-polycation-DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.

  4. Reverse Phase Protein Arrays—Quantitative Assessment of Multiple Biomarkers in Biopsies for Clinical Use

    PubMed Central

    Boellner, Stefanie; Becker, Karl-Friedrich

    2015-01-01

    Reverse Phase Protein Arrays (RPPA) represent a very promising sensitive and precise high-throughput technology for the quantitative measurement of hundreds of signaling proteins in biological and clinical samples. This array format allows quantification of one protein or phosphoprotein in multiple samples under the same experimental conditions at the same time. Moreover, it is suited for signal transduction profiling of small numbers of cultured cells or cells isolated from human biopsies, including formalin fixed and paraffin embedded (FFPE) tissues. Owing to the much easier sample preparation, as compared to mass spectrometry based technologies, and the extraordinary sensitivity for the detection of low-abundance signaling proteins over a large linear range, RPPA have the potential for characterization of deregulated interconnecting protein pathways and networks in limited amounts of sample material in clinical routine settings. Current aspects of RPPA technology, including dilution curves, spotting, controls, signal detection, antibody validation, and calculation of protein levels are addressed. PMID:27600215

  5. Reverse Phase Protein Arrays—Quantitative Assessment of Multiple Biomarkers in Biopsies for Clinical Use

    PubMed Central

    Boellner, Stefanie; Becker, Karl-Friedrich

    2015-01-01

    Reverse Phase Protein Arrays (RPPA) represent a very promising sensitive and precise high-throughput technology for the quantitative measurement of hundreds of signaling proteins in biological and clinical samples. This array format allows quantification of one protein or phosphoprotein in multiple samples under the same experimental conditions at the same time. Moreover, it is suited for signal transduction profiling of small numbers of cultured cells or cells isolated from human biopsies, including formalin fixed and paraffin embedded (FFPE) tissues. Owing to the much easier sample preparation, as compared to mass spectrometry based technologies, and the extraordinary sensitivity for the detection of low-abundance signaling proteins over a large linear range, RPPA have the potential for characterization of deregulated interconnecting protein pathways and networks in limited amounts of sample material in clinical routine settings. Current aspects of RPPA technology, including dilution curves, spotting, controls, signal detection, antibody validation, and calculation of protein levels are addressed.

  6. Quantitation of protein post-translational modifications using isobaric tandem mass tags.

    PubMed

    Liang, Hui-Chung; Lahert, Emma; Pike, Ian; Ward, Malcolm

    2015-01-01

    Post-translational modifications (PTMs) of proteins are known to modulate many cellular processes and their qualitative and quantitative evaluation is fundamental for understanding the mechanisms of biological events. Over the past decade, improvements in sample preparation techniques and enrichment strategies, the development of quantitative labeling strategies, the launch of a new generation of mass spectrometers and the creation of bioinformatics tools for the interrogation of ever larger datasets has established MS-based quantitative proteomics as a powerful workflow for global proteomics, PTM analysis and the elucidation of key biological mechanisms. With the advantage of their multiplexing capacity and the flexibility of an ever-growing family of different peptide-reactive groups, isobaric tandem mass tags facilitate quantitative proteomics and PTM experiments and enable higher sample throughput. In this review, we focus on the technical concept and utility of the isobaric tandem mass tag labeling approach to PTM analysis, including phosphorylation, glycosylation and S-nitrosylation. PMID:25697195

  7. Two-dimensional peptide separation improving sensitivity of selected reaction monitoring-based quantitative proteomics in mouse liver tissue: comparing off-gel electrophoresis and strong cation exchange chromatography.

    PubMed

    Schäfer, Alexander; von Toerne, Christine; Becker, Silke; Sarioglu, Hakan; Neschen, Susanne; Kahle, Melanie; Hauck, Stefanie M; Ueffing, Marius

    2012-10-16

    Protein expression analysis is one of the most powerful tools to further the understanding of biological systems. Progress in the field of mass spectrometry has shifted focus from gel-based approaches to the upcoming LC-selected reaction monitoring (SRM) technique which combines high technical accuracy with absolute quantification of proteins and the capability for high-throughput analyses. Due to these properties, LC-SRM has the potential to become the foundation for biomarker analysis, targeted hypothesis driven proteomic studies and contribute to the field of systems biology. While the performance of LC-SRM applied to samples from various bodily fluids, particularly plasma, and microorganisms has been extensively investigated, there is only little experience with its application to animal tissue samples. Here, we show that a conventional one-dimensional LC-SRM workflow applied to mouse liver tissue suffers from a shortcoming in terms of sensitivity for lower abundance proteins. This problem could be solved through the extension of the standard workflow by an additional dimension of separation at the peptide level prior to online LC-SRM. For this purpose, we used off-gel electrophoresis (OGE) which is also shown to outperform strong cation exchange (SCX) in terms of resolution, gain of signal intensity, and predictability of separation. The extension of the SRM workflow by a high resolving peptide separation technique is an ideal combination as it allows the addition of stable isotope standards directly after trytic digestion and will increase the dynamic range of protein abundances amenable by SRM in animal tissue.

  8. Quantitative assessment of the p53-Mdm2 feedback loop using protein lysate microarrays.

    PubMed

    Ramalingam, Sundhar; Honkanen, Peter; Young, Lynn; Shimura, Tsutomu; Austin, John; Steeg, Patricia S; Nishizuka, Satoshi

    2007-07-01

    Mathematical simulations of the p53-Mdm2 feedback loop suggest that both proteins will exhibit impulsive expression characteristics in response to high cellular stress levels. However, little quantitative experimental evaluation has been done, particularly of the phosphorylated forms. To evaluate the mathematical models experimentally, we used lysate microarrays from an isogenic pair of gamma-ray-irradiated cell lysates from HCT116 (p53(+/+) and p53(-/-)). Both p53 and Mdm2 proteins showed expected pulses in the wild type, whereas no pulses were seen in the knockout. Based on experimental observations, we determined model parameters and generated an in silico "knockout," reflecting the experimental data, including phosphorylated proteins.

  9. Quantitative and Functional Characterization of the Hyper-Conserved Protein of Prochlorococcus and Marine Synechococcus

    PubMed Central

    Zorz, Jackie K.; Joy, Andrew P.; Barnett, David A.; Johnson, Milo S.; Zhaxybayeva, Olga; Cockshutt, Amanda M.

    2014-01-01

    A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly. PMID:25360678

  10. Quantitative Glycomics Strategies*

    PubMed Central

    Mechref, Yehia; Hu, Yunli; Desantos-Garcia, Janie L.; Hussein, Ahmed; Tang, Haixu

    2013-01-01

    The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed. PMID:23325767

  11. Quantitative Determination of Spatial Protein-Protein Correlations in Fluorescence Confocal Microscopy

    PubMed Central

    Wu, Yong; Eghbali, Mansoureh; Ou, Jimmy; Lu, Rong; Toro, Ligia; Stefani, Enrico

    2010-01-01

    Abstract To quantify spatial protein-protein proximity (colocalization) in paired microscopic images of two sets of proteins labeled by distinct fluorophores, we showed that the cross-correlation and the autocorrelation functions of image intensity consisted of fast and slowly decaying components. The fast component resulted from clusters of proteins specifically labeled, and the slow component resulted from image heterogeneity and a broadly-distributed background. To better evaluate spatial proximity between the two specifically labeled proteins, we extracted the fast-decaying component by fitting the sharp peak in correlation functions to a Gaussian function, which was then used to obtain protein-protein proximity index and the Pearson's correlation coefficient. We also employed the median-filter method as a universal approach for background reduction to minimize nonspecific fluorescence. We illustrated our method by analyzing computer-simulated images and biological images. PMID:20141764

  12. Automatic multiple applicator electrophoresis

    NASA Technical Reports Server (NTRS)

    Grunbaum, B. W.

    1977-01-01

    Easy-to-use, economical device permits electrophoresis on all known supporting media. System includes automatic multiple-sample applicator, sample holder, and electrophoresis apparatus. System has potential applicability to fields of taxonomy, immunology, and genetics. Apparatus is also used for electrofocusing.

  13. Electrophoresis of biological materials

    NASA Technical Reports Server (NTRS)

    1975-01-01

    The selection of biological products was studied for electrophoresis in space. Free flow electrophoresis, isoelectric focusing, and isotachophoresis are described. The candidates discussed include: immunoglobulins and gamma globulins; isolated islet of langerhans from pancreas; bone marrow; tumor cells; kidney cells, cryoprecipitate; and column separated cultures.

  14. Improved Electrophoresis Cell

    NASA Technical Reports Server (NTRS)

    Rhodes, P. H.; Snyder, R. S.

    1982-01-01

    Several proposed modifications are expected to improve performance of a continous-flow electrophoresis cell. Changes would allow better control of buffer flow and would increase resolution by suppressing thermal gradients. Improved electrophoresis device would have high resolution and be easy to operate. Improvements would allow better flow control and heat dissipation.

  15. Electrophoresis for biological production

    NASA Technical Reports Server (NTRS)

    Mccreight, L. R.

    1977-01-01

    Preparative electrophoresis may provide a unique method for meeting ever more stringent purity requirements. Prolonged near zero gravity in space may permit the operation of preparative electrophoresis equipment with 100 times greater throughput than is currently available. Some experiments with influenza Virus Antigen, Erythropoietin and Antihemophaliac Factor, along with process and economic projections, are briefly reviewed.

  16. Capillary electrophoresis coupled with end-column electrochemiluminescence for the determination of ephedrine in human urine, and a study of its interactions with three proteins.

    PubMed

    Yang, Ran; Zeng, Hua-Jin; Li, Jian-Jun; Zhang, Ying; Li, Shi-Jun; Qu, Ling-Bo

    2011-01-01

    A tris(2,2-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)-based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10⁻⁸-6.0 × 10⁻⁶ g/mL. The detection limit was 4.5 × 10⁻⁹ g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt-C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt-C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴  mol/L, 6.58 × 10³  mol/L and 1.59 × 10⁴  mol/L, respectively.

  17. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    PubMed

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-01

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress. PMID:26549328

  18. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    PubMed

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-01

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

  19. Quantitative Analysis of Age Specific Variation in the Abundance of Human Female Parotid Salivary Proteins

    PubMed Central

    Ambatipudi, Kiran S.; Lu, Bingwen; Hagen, Fred K; Melvin, James E.; Yates, John R.

    2010-01-01

    Summary Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20–30 and 55–65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high from low abundant proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g. lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women. PMID:19764810

  20. Quantitative Fluorescent Labeling of Aldehyde-Tagged Proteins for Single-Molecule Imaging

    PubMed Central

    Shi, Xinghua; Jung, Yonil; Lin, Li-Jung; Liu, Cheng; Wu, Cong; Cann, Isaac K. O.; Ha, Taekjip

    2012-01-01

    A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescent labeling with high efficiency, specificity, and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ~100% efficiency while maintaining the biological function. We demonstrate that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We further show the unique power of our method in a series of single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor. PMID:22466795

  1. Quantitative Proteomics of Sleep-Deprived Mouse Brains Reveals Global Changes in Mitochondrial Proteins

    PubMed Central

    Li, Tie-Mei; Zhang, Ju-en; Lin, Rui; Chen, She; Luo, Minmin; Dong, Meng-Qiu

    2016-01-01

    Sleep is a ubiquitous, tightly regulated, and evolutionarily conserved behavior observed in almost all animals. Prolonged sleep deprivation can be fatal, indicating that sleep is a physiological necessity. However, little is known about its core function. To gain insight into this mystery, we used advanced quantitative proteomics technology to survey the global changes in brain protein abundance. Aiming to gain a comprehensive profile, our proteomics workflow included filter-aided sample preparation (FASP), which increased the coverage of membrane proteins; tandem mass tag (TMT) labeling, for relative quantitation; and high resolution, high mass accuracy, high throughput mass spectrometry (MS). In total, we obtained the relative abundance ratios of 9888 proteins encoded by 6070 genes. Interestingly, we observed significant enrichment for mitochondrial proteins among the differentially expressed proteins. This finding suggests that sleep deprivation strongly affects signaling pathways that govern either energy metabolism or responses to mitochondrial stress. Additionally, the differentially-expressed proteins are enriched in pathways implicated in age-dependent neurodegenerative diseases, including Parkinson’s, Huntington’s, and Alzheimer’s, hinting at possible connections between sleep loss, mitochondrial stress, and neurodegeneration. PMID:27684481

  2. Quantitative Analysis of Protein-DNA Interaction by qDPI-ELISA.

    PubMed

    Fischer, Stefan M; Böser, Alexander; Hirsch, Jan P; Wanke, Dierk

    2016-01-01

    The specific binding of DNA-binding proteins to their cognate DNA motifs is a crucial step for gene expression control and chromatin organization in vivo. The development of methods for the identification of in vivo binding regions by, e.g. chromatin immunoprecipitation (ChIP) or DNA adenine methyltransferase identification (Dam-ID) added an additional level of qualitative information for data mining in systems biology or applications in synthetic biology. In this respect, the in vivo techniques outpaced methods for thorough characterization of protein-DNA interaction and, especially, of the binding motifs at single base-pair resolution. The elucidation of DNA-binding capacities of proteins is frequently done with methods such as yeast one-hybrid, electrophoretic mobility shift assay (EMSA) or systematic evolution of ligands by exponential enrichment (SELEX) that provide only qualitative binding information and are not suited for automation or high-throughput screening of several DNA motifs. Here, we describe the quantitative DNA-protein-Interaction-ELISA (qDPI-ELISA) protocol, which makes use of fluorescent fusion proteins and, hence, is faster and easier to handle than the classical DPI-ELISA. Although every DPI-ELISA experiment delivers quantitative information, the qDPI-ELISA has an increased consistency, as it does not depend on immunological detection. We demonstrate the high comparability between probes and different protein extracts in qDPI-ELISA experiments. PMID:27557760

  3. Quantitative Proteomic Analysis of Ovarian Cancer Cells Identified Mitochondrial Proteins Associated with Paclitaxel Resistance

    PubMed Central

    Tian, Yuan; Tan, Aik-Choon; Sun, Xiaer; Olson, Matthew T; Xie, Zhi; Jinawath, Natini; Chan, Daniel W.; Shih, Ie-Ming; Zhang, Zhen; Zhang, Hui

    2010-01-01

    Paclitaxel has been widely used as an anti-mitotic agent in chemotherapy for a variety of cancers and adds substantial efficacy as the first-line chemotherapeutic regimen for ovarian cancers. However, the frequent occurrence of paclitaxel resistance limits its function in long-term management. Despite abundant clinical and cellular demonstration of paclitaxel resistant tumors, the molecular mechanisms leading to paclitaxel resistance are poorly understood. Using genomic approaches, we have previously identified an association between a BTB/POZ gene, Nac1, and paclitaxel resistance in ovarian cancer. The experiments presented here have applied multiple quantitative proteomic methods to identify protein changes associated with paclitaxel resistance and Nac1 function. The SKOV-3 ovarian serous carcinoma cell line, which has inducible expression of dominant negative Nac1, was used to determine the paclitaxel treatment associated changes in the presence and absence of functional Nac1. Quantitative proteomic analyses were performed using iTRAQ labeling and mass spectrometry. Two label-free quantitative proteomic methods: LC-MS and spectral count were used to increase confidence of proteomic quantification. A total of 1371 proteins were quantified by at least one of the quantitative proteomic methods. Candidate proteins related to paclitaxel and NAC1 function were identified in this study. Go analysis of the protein changes identified upon paclitaxel resistance revealed that cell component enrichment related to mitochondria. Moreover, tubulin and mitochondrial proteins were the major cellular components with changes associated with paclitaxel treatment. This suggests that mitochondria may play a role in paclitaxel resistance. PMID:21113235

  4. Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria). Ib. Gel antibodies against proteins (hemoglobins).

    PubMed

    Takátsy, Anikó; Végvári, Akos; Hjertén, Stellan; Kilár, Ferenc

    2007-07-01

    Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation.

  5. Universal method for synthesis of artificial gel antibodies by the imprinting approach combined with a unique electrophoresis technique for detection of minute structural differences of proteins, viruses and cells (bacteria). Ib. Gel antibodies against proteins (hemoglobins).

    PubMed

    Takátsy, Anikó; Végvári, Akos; Hjertén, Stellan; Kilár, Ferenc

    2007-07-01

    Using the molecular imprinting approach, we have shown that polyacrylamide-based artificial antibodies against human and bovine hemoglobin have a very high selectivity, as revealed by the free-zone electrophoresis in a revolving capillary. By the same technique we have previously synthesized gel antibodies not only against proteins but also against viruses and bacteria. The synthesis is thus universal, i.e., it has the great advantage of not requiring a modification - or only a slight one - for each particular antigen. The combination synthesis of artificial gel antibodies and electrophoretic analysis reveals small discrepancies in shape and chemical composition not only of proteins, as shown here and in paper Ia, but also of viruses and bacteria, to be illustrated in papers II and III in this series. Upon rehydration, the freeze-dried gel antibodies, selective for human hemoglobin, regain their selectivity. The gel antibodies can repeatedly be used following the removal of the antigen (protein in this study) from the complex gel antibody/antigen by an SDS washing or an enzymatic degradation. PMID:17476715

  6. A Miniaturized Technique for Assessing Protein Thermodynamics and Function Using Fast Determination of Quantitative Cysteine Reactivity

    PubMed Central

    Isom, Daniel G.; Marguet, Philippe R.; Oas, Terrence G.; Hellinga, Homme W.

    2010-01-01

    Protein thermodynamic stability is a fundamental physical characteristic that determines biological function. Furthermore, alteration of thermodynamic stability by macromolecular interactions or biochemical modifications is a powerful tool for assessing the relationship between protein structure, stability, and biological function. High-throughput approaches for quantifying protein stability are beginning to emerge that enable thermodynamic measurements on small amounts of material, in short periods of time, and using readily accessible instrumentation. Here we present such a method, fast quantitative cysteine reactivity (fQCR), which exploits the linkage between protein stability, sidechain protection by protein structure, and structural dynamics to characterize the thermodynamic and kinetic properties of proteins. In this approach, the reaction of a protected cysteine and thiol-reactive fluorogenic indicator is monitored over a gradient of temperatures after a short incubation time. These labeling data can be used to determine the midpoint of thermal unfolding, measure the temperature dependence of protein stability, quantify ligand-binding affinity, and, under certain conditions, estimate folding rate constants. Here, we demonstrate the fQCR method by characterizing these thermodynamic and kinetic properties for variants of Staphylococcal nuclease and E. coli ribose-binding protein engineered to contain single, protected cysteines. These straightforward, information-rich experiments are likely to find applications in protein engineering and functional genomics. PMID:21387407

  7. Preparative electrophoresis experiment design

    NASA Technical Reports Server (NTRS)

    Thiehler, A.

    1972-01-01

    A multifaceted study supporting the NASA programs to develop a space electrophoresis capability has been conducted. The study involved principally the technique of continuous free electrophoresis. It comprised a critical review of the art, study of new techniques for enhancing resolution and stability, and construction and initial testing of a high resolution cell. The effort resulted in a significant advance in free electrophoresis technique. It has provided also a much improved base for developments exploiting the added advantages of a zero-gravity environment.

  8. Microfluidic free-flow electrophoresis for the discovery and characterisation of calmodulin binding partners

    NASA Astrophysics Data System (ADS)

    Herling, Therese; Linse, Sara; Knowles, Tuomas

    2015-03-01

    Non-covalent and transient protein-ligand interactions are integral to cellular function and malfunction. Key steps in signalling and regulatory pathways rely on reversible non-covalent protein-protein binding or ion chelation. Here we present a microfluidic free-flow electrophoresis method for detecting and characterising protein-ligand interactions in solution. We apply this method to probe the binding equilibria of calmodulin, a central protein to calcium signalling pathways. In this study we characterise the specific binding of calmodulin to phosphorylase kinase, a known target, and creatine kinase, which we identify as a putative binding partner through a protein array screen and surface plasmon resonance experiments. We verify the interaction between calmodulin and creatine kinase in solution using free-flow electrophoresis and investigate the effect of calcium and sodium chloride on the calmodulin-ligand binding affinity in free solution without the presence of a potentially interfering surface. Our results demonstrate the general applicability of quantitative microfluidic electrophoresis to characterise binding equilibria between biomolecules in solution.

  9. Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics.

    PubMed

    Li, Zhao Bo; Flint, Paul W; Boluyt, Marvin O

    2005-09-01

    Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.

  10. Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials.

    PubMed

    Farris, M Heath; Ford, Kara A; Doyle, Richard C

    2016-01-01

    Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

  11. Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

    PubMed Central

    Farris, M. Heath; Ford, Kara A.; Doyle, Richard C.

    2016-01-01

    Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest. PMID:27166738

  12. Quantitative variability of 342 plasma proteins in a human twin population

    PubMed Central

    Liu, Yansheng; Buil, Alfonso; Collins, Ben C; Gillet, Ludovic CJ; Blum, Lorenz C; Cheng, Lin-Yang; Vitek, Olga; Mouritsen, Jeppe; Lachance, Genevieve; Spector, Tim D; Dermitzakis, Emmanouil T; Aebersold, Ruedi

    2015-01-01

    The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis-SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood-based biomarker studies. PMID:25652787

  13. Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

    PubMed

    Barbeau, D; Marcellus, R C; Bacchetti, S; Bayley, S T; Branton, P E

    1992-01-01

    Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably. PMID:1297336

  14. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  15. A Turn-On Resonance Raman Scattering (BCS/Cu+) Sensor for Quantitative Determination of Proteins.

    PubMed

    Chen, Lei; Xue, Xiangxin; Jiang, Dayu; Yang, Jin; Zhao, Bing; Han, Xiao Xia; Mee Jung, Young

    2016-02-01

    In this study, a new method for the quantitative evaluation of proteins is developed using competitive resonance Raman spectroscopy. A chelation reaction between bathocuproine disulfonate (BCS) and Cu(+) which is reduced by proteins in an alkaline environment, is utilized to create a BCS-Cu(+) complex that has strong resonance Raman activity. As a result, the method presented here enables protein detection over a much wider concentration range than conventional methods. Furthermore, the resonance Raman-based method can provide an improved lower limit of detection (500 pg/mL) compared to that obtained by colorimetric and ultraviolet- (UV-) based methods. Additionally, protein-to-protein variation can be determined using a standard curve created from known concentrations of bovine serum albumin (BSA), and excellent protein recovery is observed. More importantly, the proposed method was employing to the real sample (fetal bovine serum [FBS]). Based on the calibration curve from this method, it is extremely accurate for the determination of total protein concentrations in FBS. Results of this study demonstrate that the proposed method provides a novel and highly sensitive protocol for reagent-based protein assays.

  16. Quantitative Proteomic Analysis of Differentially Expressed Protein Profiles Involved in Pancreatic Ductal Adenocarcinoma

    PubMed Central

    Kuo, Kung-Kai; Kuo, Chao-Jen; Chiu, Chiang-Yen; Liang, Shih-Shin; Huang, Chun-Hao; Chi, Shu-Wen; Tsai, Kun-Bow; Chen, Chiao-Yun; Hsi, Edward; Cheng, Kuang-Hung; Chiou, Shyh-Horng

    2016-01-01

    Objectives The aim of this study was to identify differentially expressed proteins among various stages of pancreatic ductal adenocarcinoma (PDAC) by shotgun proteomics using nano-liquid chromatography coupled tandem mass spectrometry and stable isotope dimethyl labeling. Methods Differentially expressed proteins were identified and compared based on the mass spectral differences of their isotope-labeled peptide fragments generated from protease digestion. Results Our quantitative proteomic analysis of the differentially expressed proteins with stable isotope (deuterium/hydrogen ratio, ≥2) identified a total of 353 proteins, with at least 5 protein biomarker proteins that were significantly differentially expressed between cancer and normal mice by at least a 2-fold alteration. These 5 protein biomarker candidates include α-enolase, α-catenin, 14-3-3 β, VDAC1, and calmodulin with high confidence levels. The expression levels were also found to be in agreement with those examined by Western blot and histochemical staining. Conclusions The systematic decrease or increase of these identified marker proteins may potentially reflect the morphological aberrations and diseased stages of pancreas carcinoma throughout progressive developments leading to PDAC. The results would form a firm foundation for future work concerning validation and clinical translation of some identified biomarkers into targeted diagnosis and therapy for various stages of PDAC. PMID:26262590

  17. Quantitative Proteomics Reveals the Roles of Peroxisome-associated Proteins in Antiviral Innate Immune Responses.

    PubMed

    Zhou, Mao-Tian; Qin, Yue; Li, Mi; Chen, Chen; Chen, Xi; Shu, Hong-Bing; Guo, Lin

    2015-09-01

    Compared with whole-cell proteomic analysis, subcellular proteomic analysis is advantageous not only for the increased coverage of low abundance proteins but also for generating organelle-specific data containing information regarding dynamic protein movement. In the present study, peroxisome-enriched fractions from Sendai virus (SeV)-infected or uninfected HepG2 cells were obtained and subjected to quantitative proteomics analysis. We identified 311 proteins that were significantly changed by SeV infection. Among these altered proteins, 25 are immune response-related proteins. Further bioinformatic analysis indicated that SeV infection inhibits cell cycle-related proteins and membrane attack complex-related proteins, all of which are beneficial for the survival and replication of SeV within host cells. Using Luciferase reporter assays on several innate immune-related reporters, we performed functional analysis on 11 candidate proteins. We identified LGALS3BP and CALU as potential negative regulators of the virus-induced activation of the type I interferons.

  18. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions

    PubMed Central

    Vickers, Timothy A.; Crooke, Stanley T.

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  19. Serum globulin electrophoresis

    MedlinePlus

    ... may indicate: Acute infection Bone marrow cancer called multiple myeloma Chronic inflammatory disease (for example, rheumatoid arthritis and ... test Hemoglobin Hyperimmunization Immunoelectrophoresis - ... electrophoresis - serum Rheumatoid arthritis Systemic lupus erythematosus ...

  20. Electrophoresis operations in space

    NASA Technical Reports Server (NTRS)

    Richman, D. W.

    1982-01-01

    Application of electrophoresis in space processing is described. Spaceborne experiments in areas such as biological products and FDA approved drugs are discussed. These experiments will be carried on shuttle payloads.

  1. A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization.

    PubMed

    Dreger, Mathias; Leung, Bo Wah; Brownlee, George G; Deng, Tao

    2009-07-01

    We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N-hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions-at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein-protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs. PMID:19517532

  2. Improved Protein Arrays for Quantitative Systems Analysis of the Dynamics of Signaling Pathway Interactions

    SciTech Connect

    YANG, CHIN-RANG

    2013-12-11

    Astronauts and workers in nuclear plants who repeatedly exposed to low doses of ionizing radiation (IR, <10 cGy) are likely to incur specific changes in signal transduction and gene expression in various tissues of their body. Remarkable advances in high throughput genomics and proteomics technologies enable researchers to broaden their focus from examining single gene/protein kinetics to better understanding global gene/protein expression profiling and biological pathway analyses, namely Systems Biology. An ultimate goal of systems biology is to develop dynamic mathematical models of interacting biological systems capable of simulating living systems in a computer. This Glue Grant is to complement Dr. Boothman’s existing DOE grant (No. DE-FG02-06ER64186) entitled “The IGF1/IGF-1R-MAPK-Secretory Clusterin (sCLU) Pathway: Mediator of a Low Dose IR-Inducible Bystander Effect” to develop sensitive and quantitative proteomic technology that suitable for low dose radiobiology researches. An improved version of quantitative protein array platform utilizing linear Quantum dot signaling for systematically measuring protein levels and phosphorylation states for systems biology modeling is presented. The signals are amplified by a confocal laser Quantum dot scanner resulting in ~1000-fold more sensitivity than traditional Western blots and show the good linearity that is impossible for the signals of HRP-amplification. Therefore this improved protein array technology is suitable to detect weak responses of low dose radiation. Software is developed to facilitate the quantitative readout of signaling network activities. Kinetics of EGFRvIII mutant signaling was analyzed to quantify cross-talks between EGFR and other signaling pathways.

  3. Quantitative Fluorescence Studies in Living Cells: Extending Fluorescence Fluctuation Spectroscopy to Peripheral Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Smith, Elizabeth Myhra

    The interactions of peripheral membrane proteins with both membrane lipids and proteins are vital for many cellular processes including membrane trafficking, cellular signaling, and cell growth/regulation. Building accurate biophysical models of these processes requires quantitative characterization of the behavior of peripheral membrane proteins, yet methods to quantify their interactions inside living cells are very limited. Because peripheral membrane proteins usually exist both in membrane-bound and cytoplasmic forms, the separation of these two populations is a key challenge. This thesis aims at addressing this challenge by extending fluorescence fluctuation spectroscopy (FFS) to simultaneously measure the oligomeric state of peripheral membrane proteins in the cytoplasm and at the plasma membrane. We developed a new method based on z-scan FFS that accounts for the fluorescence contributions from cytoplasmic and membrane layers by incorporating a fluorescence intensity z-scan through the cell. H-Ras-EGFP served as a model system to demonstrate the feasibility of the technique. The resolvability and stability of z-scanning was determined as well as the oligomeric state of H-Ras-EGFP at the plasma membrane and in the cytoplasm. Further, we successfully characterized the binding affinity of a variety of proteins to the plasma membrane by quantitative analysis of the z-scan fluorescence intensity profile. This analysis method, which we refer to as z-scan fluorescence profile deconvoution, was further used in combination with dual-color competition studies to determine the lipid specificity of protein binding. Finally, we applied z-scan FFS to provide insight into the early assembly steps of the HTLV-1 retrovirus.

  4. Recent advances in preparative electrophoresis

    NASA Technical Reports Server (NTRS)

    Mosher, Richard A.; Thormann, Wolfgang; Egen, Ned B.; Couasnon, Pascal; Sammons, David W.

    1987-01-01

    Various approaches for preparative electrophoresis, and three new instruments for preparative electrophoresis are discussed. Consideration is given to isoelectric focusing, isotachophoresis, and zone electrophoresis, three gel-based electrophoresis methods. The design, functions, and performance of the Elphor VaP 21 device of Hannig (1982), the shear-stabilized BIOSTREAM separator of Thompson (1983), and the recycling isoelectric focusing device are described.

  5. Micromorphological characterization and label-free quantitation of small rubber particle protein in natural rubber latex.

    PubMed

    Wang, Sai; Liu, Jiahui; Wu, Yanxia; You, Yawen; He, Jingyi; Zhang, Jichuan; Zhang, Liqun; Dong, Yiyang

    2016-04-15

    Commercial natural rubber is traditionally supplied by Hevea brasiliensis, but now there is a big energy problem because of the limited resource and increasing demand. Intensive study of key rubber-related substances is urgently needed for further research of in vitro biosynthesis of natural rubber. Natural rubber is biosynthesized on the surface of rubber particles. A membrane protein called small rubber particle protein (SRPP) is a key protein associated closely with rubber biosynthesis; however, SRPP in different plants has been only qualitatively studied, and there are no quantitative reports so far. In this work, H. brasiliensis was chosen as a model plant. The microscopic distribution of SRPP on the rubber particles during the washing process was investigated by transmission electron microscopy-immunogold labeling. A label-free surface plasmon resonance (SPR) immunosensor was developed to quantify SRPP in H. brasiliensis for the first time. The immunosensor was then used to rapidly detect and analyze SRPP in dandelions and prickly lettuce latex samples. The label-free SPR immunosensor can be a desirable tool for rapid quantitation of the membrane protein SRPP, with excellent assay efficiency, high sensitivity, and high specificity. The method lays the foundation for further study of the functional relationship between SRPP and natural rubber content. PMID:26844871

  6. High Throughput Quantitative Analysis of Serum Proteins using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

    SciTech Connect

    Zhang, Hui; Yi, Eugene C.; Li, Xiao-jun; Mallick, Parag; Kelly-Spratt, Karen S.; Masselon, Christophe D.; Camp, David G.; Smith, Richard D.; Kemp, Christopher; Aebersold, Ruedi

    2005-02-01

    It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease, and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible and robust to detect potential biomarkers below the level of highly expressed proteins, to generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. In this paper, we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these, no de-glycosylated peptides by LC-ESI-MS, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared to their control littermates.

  7. High Throughput Quantitative Analysis of Serum Proteins Using Glycopeptide Capture and Liquid Chromatography Mass Spectrometry

    SciTech Connect

    Zhang, Hui; Yi, Eugene C.; Li, Xiao-jun; Mallick, Parag; Kelly-Spratt, Karen S.; Masselon, Christophe D.; Camp, David G.; Smith, Richard D.; Kemp, Christopher J.; Aebersold, Reudi

    2005-02-01

    It is expected that the composition of the serum proteome can provide valuable information about the state of the human body in health and disease and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible, and robust to detect potential biomarkers below the level of highly expressed proteins, generate data sets that are comparable between experiments and laboratories, and have high throughput to support statistical studies. Here we report a method for high throughput quantitative analysis of serum proteins. It consists of the selective isolation of peptides that are N-linked glycosylated in the intact protein, the analysis of these now deglycosylated peptides by liquid chromatography electrospray ionization mass spectrometry, and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced and the sensitivity and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We provide data that document the performance of the method and show that sera from untreated normal mice and genetically identical mice with carcinogen-induced skin cancer can be unambiguously discriminated using unsupervised clustering of the resulting peptide patterns. We further identify, by tandem mass spectrometry, some of the peptides that were consistently elevated in cancer mice compared with their control littermates.

  8. A Statistical Framework for Protein Quantitation in Bottom-Up MS-Based Proteomics

    SciTech Connect

    Karpievitch, Yuliya; Stanley, Jeffrey R.; Taverner, Thomas; Huang, Jianhua; Adkins, Joshua N.; Ansong, Charles; Heffron, Fred; Metz, Thomas O.; Qian, Weijun; Yoon, Hyunjin; Smith, Richard D.; Dabney, Alan R.

    2009-08-15

    Motivation: Quantitative mass spectrometry-based proteomics requires protein-level estimates and associated confidence measures. Challenges include the presence of low quality or incorrectly identified peptides and informative missingness. Furthermore, models are required for rolling peptide-level information up to the protein level. Results: We present a statistical model that carefully accounts for informative missingness in peak intensities and allows unbiased, model-based, protein-level estimation and inference. The model is applicable to both label-based and label-free quantitation experiments. We also provide automated, model-based, algorithms for filtering of proteins and peptides as well as imputation of missing values. Two LC/MS datasets are used to illustrate the methods. In simulation studies, our methods are shown to achieve substantially more discoveries than standard alternatives. Availability: The software has been made available in the opensource proteomics platform DAnTE (http://omics.pnl.gov/software/). Contact: adabney@stat.tamu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

  9. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  10. Simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and charge-coupled device, or photomultiplier for determining proteins in capillary electrophoresis: a critical comparison.

    PubMed

    Casado-Terrones, Silvia; Fernández-Sánchez, Jorge F; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

    2007-06-01

    The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices use commercially available, reasonably priced optical components that can be used by nonexperts. After fine optimization of several optical and separation parameters in both devices, a zone capillary electrophoresis methodology was achieved with 50mM borate buffer (pH 8.4) and 10mM phytic acid for the determination of two phycobiliproteins. Detection limits of 0.50 and 0.64microg/ml for R-phycoerythrin and B-phycoerythrin, respectively, were achieved by using the LED-induced fluorescence capillary electrophoresis (LED-IF-CE) system, and corresponding detection limits of 2.73 and 2.16microg/ml were achieved by using the Xe lamp-IF-CE system. Analytical performance and other parameters, such as cost and potential to miniaturization, are compared for both devices.

  11. Quantitative Analysis of Multivalent Ligand Presentation on Gold Glyconanoparticles and Their Effects on Protein Binding

    NASA Astrophysics Data System (ADS)

    Wang, Xin; Ramström, Olof; Yan, Mingdi

    2010-03-01

    Bio-functionalized nanomaterials, which combine functions of biological ligands and unique properties of nano-sized building blocks, have exhibited increased potential applications in biosensing, therapeutics, and diagnostics. Glyconanoparitcles carrying a monolayer of carbohydrate ligands on nanoparticles provide an excellent platform for sensitive protein recognitions. Using Au nanoparticles as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, quantitative analysis especially the binding affinity of the resulting glyconanoparticles is challenging to determine. Here we present a new characterization technique, based on fluorescent competition binding assays, for measuring dissociation constants for glyconanoparticles-protein interactions. Au nanoparticles coupled with a series of un-derivatized carbohydrates were prepared by a photocoupling chemistry. Dramatic binding affinity enhancement was observed due to the high ligand density on nanoparticles, which was highly relevant to ligand display, controlled by the linker type, chain length, ligand size and density.

  12. A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization

    PubMed Central

    Dreger, Mathias; Leung, Bo Wah; Brownlee, George G; Deng, Tao

    2009-01-01

    We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N-hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described. Five regions in the PB1 monomer showed altered reactivity to NHS-biotin when compared with the [PB1-PA] heterodimer. Mutational analysis of residues in two such regions—at K265 and K481 of PB1, which were about three- and twofold, respectively, less accessible to biotinylation in the PB1-PA heterodimer compared with the PB1 monomer, demonstrated that both K265 and K481 were crucial for polymerase function. This novel assay of quantitative profiling of biotinylation patterns (Q-POP assay) highlights likely conformational changes at important functional sites, as observed here for PB1, and may provide information on protein–protein interaction interfaces. The Q-POP assay should be a generally applicable approach and may detect novel functional sites suitable for targeting by drugs. PMID:19517532

  13. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    PubMed Central

    Zhang, Pengfei; Bao, Yan; Draz, Mohamed Shehata; Lu, Huiqi; Liu, Chang; Han, Huanxing

    2015-01-01

    Convenient and rapid immunofiltration assays (IFAs) enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs)-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP). CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG) and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test. PMID:26491289

  14. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay.

    PubMed

    Zhang, Pengfei; Bao, Yan; Draz, Mohamed Shehata; Lu, Huiqi; Liu, Chang; Han, Huanxing

    2015-01-01

    Convenient and rapid immunofiltration assays (IFAs) enable on-site "yes" or "no" determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs)-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP). CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG) and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.

  15. Quantitative proteomics reveals novel insights into isoniazid susceptibility in mycobacteria mediated by a universal stress protein.

    PubMed

    Hu, Xinling; Li, Xiaojing; Huang, Lige; Chan, John; Chen, Yuling; Deng, Haiteng; Mi, Kaixia

    2015-03-01

    Tuberculosis (TB) is caused by the ancient pathogen, Mycobacterium tuberculosis, and is one of the most serious infectious diseases in the world. Isoniazid (INH) is an important first-line drug for the treatment of active and latent TB. INH resistance is an increasing problem in the treatment of TB. Phenotypic resistance to INH, however, is poorly understood. In this study, we constructed a strain of Mycobacterium bovis BCG that overexpresses the latency-related universal stress protein (USP), BCG_2013, and designated this strain BCG-2013. BCG_2013 overexpression increased susceptibility to INH compared with that of the wild-type strain, BCG-pMV261. Quantitative proteomic analysis revealed that BCG_2013 overexpression resulted in the upregulation of 50 proteins and the downregulation of 26 proteins among the 1500 proteins identified. Upregulation of catalase-peroxidase KatG expression in BCG-2013 was observed and confirmed by qPCR, whereas expression of other INH resistance-related proteins did not change. In addition, differential expression of the mycobacterial persistence regulator MprA and its regulatory proteins was observed. BCG_2013 and katG mRNA levels increased in a Wayne dormancy model, whereas MprA mRNA levels decreased. Taken together, our results suggest that the increase in KatG levels induced by increased BCG_2013 levels underlies the phenotypic susceptibility of mycobacteria to INH.

  16. Complex mixture analysis using protein expression as a qualitative and quantitative tool

    SciTech Connect

    Bradley, B.P.; Gonzalez, C.M.; Bond, J.A. . Dept. of Biological Sciences); Tepper, B.E. . Paper Products Division)

    1994-07-01

    Some proteins in organisms exposed to chemicals in stressful amounts or toxic concentrations show increased expression; others show decreased expression. These inducible and repressible proteins together potentially provide qualitative and quantitative diagnoses of components in complex mixtures of chemicals. The authors examined sets of proteins synthesized by Daphnia magna after exposure to mixtures of a cationic polyamide epichlorhydrin adduct (Kymene) and a combined assortment of water-extractable substances from chemi-thermal-mechanical pulp (CTMP) in lab water. Proteins were identified, after extracting from Daphnia magna, by gel filtration and silver staining, or by radiolabeling and then gel separation. Patterns of proteins induced by Kymene[reg sign] and by CTMP extracts were distinguishable in lab water, but there was interaction between them. The method of identifying and quantifying Kymene, however, was successful using lab simulations of mixtures. The method was tested using wastewater samples from a paper manufacturing plant. Kymene could be detected against variable levels and types of additional substances. But, again, there was interference, perhaps due to Kymene binding to other anionic polymers sometimes present in the samples. Interpretation from analyses of protein expression were consistent with results from sublethal Ceriodaphnia dubia assays.

  17. Quantitative changes in sets of proteins as markers of biological response

    SciTech Connect

    Giometti, C.S.; Taylor, J.; Gemmell, M.A.; Tollaksen, S.L. ); Lalwani, N.D.; Reddy, J.K. )

    1990-01-01

    Exposure to either physical or chemical insults triggers a cascade of bio-chemical events within the target cell. This response requires adjustment within the protein population of the cell, some proteins becoming more abundant (those involved in the cellular response), others less abundant (those not required or counterproductive to the response). Thus, quantitative changes in the global protein population of an exposed biological system may well serve as an indicator of exposure, provided the alterations observed are selective and dose-dependent. In this paper we present results from a study in which liver protein changes induced by exposure of mice to chemicals known to cause peroxisome proliferation and subsequent hepatocellular carcinoma where monitored. Clofibrate, and its chemical analog ciprofibrate, are hypolipidemic drugs. Di-(ethylhexyl)phthalate (DEHP) is a plasticizer used widely in disposable containers for blood products. WY-14643 is a chemical shown to cause hypolipidemic and peroxisome proliferation, similar to clofibrate, ciprofibrate and DEHP, but structurally different from these three chemicals. Thus, two of the four chemicals are structurally similar while the remaining two are very distinct, although all four chemicals cause the same gross biological response. Our results show that although common protein effects are observed in mice exposed to these chemicals, each chemical also causes specific alterations in selective subsets of proteins that could serve as markers of a particular exposure. 13 refs., 4 figs., 1 tab.

  18. Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions.

    PubMed

    Yamniuk, Aaron P; Newitt, John A; Doyle, Michael L; Arisaka, Fumio; Giannetti, Anthony M; Hensley, Preston; Myszka, David G; Schwarz, Fred P; Thomson, James A; Eisenstein, Edward

    2015-12-01

    A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions. PMID:26543437

  19. Development of a Model Protein Interaction Pair as a Benchmarking Tool for the Quantitative Analysis of 2-Site Protein-Protein Interactions

    PubMed Central

    Newitt, John A.; Doyle, Michael L.; Arisaka, Fumio; Giannetti, Anthony M.; Hensley, Preston; Myszka, David G.; Schwarz, Fred P.; Thomson, James A.; Eisenstein, Edward

    2015-01-01

    A significant challenge in the molecular interaction field is to accurately determine the stoichiometry and stepwise binding affinity constants for macromolecules having >1 binding site. The mission of the Molecular Interactions Research Group (MIRG) of the Association of Biomolecular Resource Facilities (ABRF) is to show how biophysical technologies are used to quantitatively characterize molecular interactions, and to educate the ABRF members and scientific community on the utility and limitations of core technologies [such as biosensor, microcalorimetry, or analytic ultracentrifugation (AUC)]. In the present work, the MIRG has developed a robust model protein interaction pair consisting of a bivalent variant of the Bacillus amyloliquefaciens extracellular RNase barnase and a variant of its natural monovalent intracellular inhibitor protein barstar. It is demonstrated that this system can serve as a benchmarking tool for the quantitative analysis of 2-site protein-protein interactions. The protein interaction pair enables determination of precise binding constants for the barstar protein binding to 2 distinct sites on the bivalent barnase binding partner (termed binase), where the 2 binding sites were engineered to possess affinities that differed by 2 orders of magnitude. Multiple MIRG laboratories characterized the interaction using isothermal titration calorimetry (ITC), AUC, and surface plasmon resonance (SPR) methods to evaluate the feasibility of the system as a benchmarking model. Although general agreement was seen for the binding constants measured using solution-based ITC and AUC approaches, weaker affinity was seen for surface-based method SPR, with protein immobilization likely affecting affinity. An analysis of the results from multiple MIRG laboratories suggests that the bivalent barnase-barstar system is a suitable model for benchmarking new approaches for the quantitative characterization of complex biomolecular interactions. PMID:26543437

  20. Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Li, Wenjing; Yu, Jianshi; Kane, Maureen A.

    2016-10-01

    Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein-ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range (~1-10 μM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system.

  1. Protein adducts of malondialdehyde and 4-hydroxynonenal in livers of iron loaded rats: quantitation and localization.

    PubMed

    Khan, M Firoze; Wu, Xiaohong; Tipnis, Ulka R; Ansari, G A S; Boor, Paul J

    2002-05-01

    Pathophysiological mechanisms for hepatocellular injury, fibrosis and/or cirrhosis in hepatic iron overload are poorly understood. An increase in intracellular transit pool of iron can catalyze peroxidation of lipids to produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Covalent binding of such lipid aldehydes with proteins may cause impairment in cellular function and integrity. This investigation was focused on quantitative determination of MDA and HNE-protein adducts, and to establish a correlation between iron deposition and formation and localization of MDA and HNE-protein adducts, using immunohistochemistry. To achieve iron overload, male SD rats were fed a 2.5% carbonyl iron-supplemented diet for six weeks, while control animals received standard diet. Total iron as well as low molecular weight chelatable iron (LMWC-Fe) in the hepatic tissue of rats fed the iron supplemented diet increased significantly ( approximately 14- and approximately 15-fold, respectively). Quantitative ELISA for MDA-and HNE-protein adducts showed remarkable increases of 186 and 149%, respectively, in the liver homogenates of rats fed the iron-supplemented diet. Sections of liver stained for iron showed striking iron deposits in periportal (zone 1) hepatocytes, which was less dramatic in midzonal (zone 2) cells. Livers from iron-loaded rats showed strong, diffuse staining for both MDA and HNE adducts, which was highly pronounced in centrilobular (zone 3) hepatocytes, but was also evident in midzonal cells (zone 2). The demonstration of greater formation of both MDA and HNE-protein adducts provides evidence of iron-catalyzed lipid peroxidation in vivo. Although in this model of iron overload there was no evidence of tissue injury, our results provide an account of some of the initiating factors or early molecular events in hepatocellular damage that may lead to the pathological manifestations seen in chronic iron overload.

  2. Quantitative mass spectrometry measurements reveal stoichiometry of principal postsynaptic density proteins.

    PubMed

    Lowenthal, Mark S; Markey, Sanford P; Dosemeci, Ayse

    2015-06-01

    Quantitative studies are presented of postsynaptic density (PSD) fractions from rat cerebral cortex with the ultimate goal of defining the average copy numbers of proteins in the PSD complex. Highly specific and selective isotope dilution mass spectrometry assays were developed using isotopically labeled polypeptide concatemer internal standards. Interpretation of PSD protein stoichiometry was achieved as a molar ratio with respect to PSD-95 (SAP-90, DLG4), and subsequently, copy numbers were estimated using a consensus literature value for PSD-95. Average copy numbers for several proteins at the PSD were estimated for the first time, including those for AIDA-1, BRAGs, and densin. Major findings include evidence for the high copy number of AIDA-1 in the PSD (144 ± 30)-equivalent to that of the total GKAP family of proteins (150 ± 27)-suggesting that AIDA-1 is an element of the PSD scaffold. The average copy numbers for NMDA receptor sub-units were estimated to be 66 ± 18, 27 ± 9, and 45 ± 15, respectively, for GluN1, GluN2A, and GluN2B, yielding a total of 34 ± 10 NMDA channels. Estimated average copy numbers for AMPA channels and their auxiliary sub-units TARPs were 68 ± 36 and 144 ± 38, respectively, with a stoichiometry of ∼1:2, supporting the assertion that most AMPA receptors anchor to the PSD via TARP sub-units. This robust, quantitative analysis of PSD proteins improves upon and extends the list of major PSD components with assigned average copy numbers in the ongoing effort to unravel the complex molecular architecture of the PSD.

  3. Clinical laboratory standard capillary protein electrophoresis alerted of a low C3 state and lead to the identification of a Factor I deficiency due to a novel homozygous mutation.

    PubMed

    Franco-Jarava, Clara; Colobran, Roger; Mestre-Torres, Jaume; Vargas, Victor; Pujol-Borrell, Ricardo; Hernández-González, Manuel

    2016-06-01

    Complement factor I (CFI) deficiency is typically associated to recurrent infections with encapsulated microorganisms and, less commonly, to autoimmunity. We report a 53-years old male who, in a routine control for non-alcoholic fatty liver disease, presented a flat beta-2 fraction at the capillary protein electropherogram. Patient's clinical records included multiple oropharyngeal infections since infancy and an episode of invasive meningococcal infection. Complement studies revealed reduced C3, low classical pathway activation and undetectable Factor I. CFI gene sequencing showed a novel inherited homozygous deletion of 5 nucleotides in exon 12, causing a frameshift leading to a truncated protein. This study points out that capillary protein electrophoresis can alert of possible states of low C3, which, once confirmed and common causes ruled out, can lead to CFI and other complement deficiency diagnosis. This is important since they constitute a still underestimated risk of invasive meningococcemia that can be greatly reduced by vaccination. PMID:27091480

  4. Toxicity challenges in environmental chemicals: Prediction of human plasma protein binding through quantitative structure-activity relationship (QSAR) models

    EPA Science Inventory

    The present study explores the merit of utilizing available pharmaceutical data to construct a quantitative structure-activity relationship (QSAR) for prediction of the fraction of a chemical unbound to plasma protein (Fub) in environmentally relevant compounds. Independent model...

  5. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

    PubMed Central

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-01-01

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

  6. Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks.

    PubMed

    Jenkins, Janelle E; Creager, Melinda S; Lewis, Randolph V; Holland, Gregory P; Yarger, Jeffery L

    2010-01-11

    Synthetic spider silk holds great potential for use in various applications spanning medical uses to ultra lightweight armor; however, producing synthetic fibers with mechanical properties comparable to natural spider silk has eluded the scientific community. Natural dragline spider silks are commonly made from proteins that contain highly repetitive amino acid motifs, adopting an array of secondary structures. Before further advances can be made in the production of synthetic fibers based on spider silk proteins, it is imperative to know the percentage of each amino acid in the protein that forms a specific secondary structure. Linking these percentages to the primary amino acid sequence of the protein will establish a structural foundation for synthetic silk. In this study, nuclear magnetic resonance (NMR) techniques are used to quantify the percentage of Ala, Gly, and Ser that form both beta-sheet and helical secondary structures. The fraction of these three amino acids and their secondary structure are quantitatively correlated to the primary amino acid sequence for the proteins that comprise major and minor ampullate silk from the Nephila clavipes spider providing a blueprint for synthetic spider silks. PMID:20000730

  7. Identification of cypermethrin induced protein changes in green algae by iTRAQ quantitative proteomics.

    PubMed

    Gao, Yan; Lim, Teck Kwang; Lin, Qingsong; Li, Sam Fong Yau

    2016-04-29

    Cypermethrin (CYP) is one of the most widely used pesticides in large scale for agricultural and domestic purpose and the residue often seriously affects aquatic system. Environmental pollutant-induced protein changes in organisms could be detected by proteomics, leading to discovery of potential biomarkers and understanding of mode of action. While proteomics investigations of CYP stress in some animal models have been well studied, few reports about the effects of exposure to CYP on algae proteome were published. To determine CYP effect in algae, the impact of various dosages (0.001μg/L, 0.01μg/L and 1μg/L) of CYP on green algae Chlorella vulgaris for 24h and 96h was investigated by using iTRAQ quantitative proteomics technique. A total of 162 and 198 proteins were significantly altered after CYP exposure for 24h and 96h, respectively. Overview of iTRAQ results indicated that the influence of CYP on algae protein might be dosage-dependent. Functional analysis of differentially expressed proteins showed that CYP could induce protein alterations related to photosynthesis, stress responses and carbohydrate metabolism. This study provides a comprehensive view of complex mode of action of algae under CYP stress and highlights several potential biomarkers for further investigation of pesticide-exposed plant and algae. PMID:26961939

  8. Identification of cypermethrin induced protein changes in green algae by iTRAQ quantitative proteomics.

    PubMed

    Gao, Yan; Lim, Teck Kwang; Lin, Qingsong; Li, Sam Fong Yau

    2016-04-29

    Cypermethrin (CYP) is one of the most widely used pesticides in large scale for agricultural and domestic purpose and the residue often seriously affects aquatic system. Environmental pollutant-induced protein changes in organisms could be detected by proteomics, leading to discovery of potential biomarkers and understanding of mode of action. While proteomics investigations of CYP stress in some animal models have been well studied, few reports about the effects of exposure to CYP on algae proteome were published. To determine CYP effect in algae, the impact of various dosages (0.001μg/L, 0.01μg/L and 1μg/L) of CYP on green algae Chlorella vulgaris for 24h and 96h was investigated by using iTRAQ quantitative proteomics technique. A total of 162 and 198 proteins were significantly altered after CYP exposure for 24h and 96h, respectively. Overview of iTRAQ results indicated that the influence of CYP on algae protein might be dosage-dependent. Functional analysis of differentially expressed proteins showed that CYP could induce protein alterations related to photosynthesis, stress responses and carbohydrate metabolism. This study provides a comprehensive view of complex mode of action of algae under CYP stress and highlights several potential biomarkers for further investigation of pesticide-exposed plant and algae.

  9. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response.

    PubMed

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R; Fox, Paul L; Gerson, Stanton L; Hoppel, Charles L; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. PMID:26595448

  10. Gel Electrophoresis on a Budget to Dye for

    ERIC Educational Resources Information Center

    Yu, Julie H.

    2010-01-01

    Gel electrophoresis is one of the most important tools used in molecular biology and has facilitated the entire field of genetic engineering by enabling the separation of nucleic acids and proteins. However, commercial electrophoresis kits can cost up to $800 for each setup, which is cost prohibitive for most classroom budgets. This article…

  11. Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

    PubMed

    Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

    2015-12-10

    D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression.

  12. Quantitative, fluorescence-based in-situ assessment of protein expression.

    PubMed

    Moeder, Christopher B; Giltnane, Jennifer M; Moulis, Sharon Pozner; Rimm, David L

    2009-01-01

    As companion diagnostics grow in prevalence and importance, the need for accurate assessment of in situ protein concentrations has increased. Traditional immunohistochemistry (IHC), while valuable for assessment of context of expression, is less valuable for quantification. The lack of rigorous quantitative potential of traditional IHC led to our development of an immunofluorescence-based method now commercialized as the AQUA technology. Immunostaining of tissue samples, image acquisition, and use of AQUA software allow investigators to quickly, efficiently, and accurately measure levels of expression within user-defined subcellular or architectural compartments. IHC analyzed by AQUA shows high reproducibility and demonstrates protein measurement accuracy similar to ELISA assays. The process is largely automated, eliminating potential error, and the resultant scores are exported on a continuous scale. There are now numerous published examples where observations made with this technology are not seen by traditional methods.

  13. A quantitative autoradiographic method for the measurement of local rates of brain protein synthesis

    SciTech Connect

    Dwyer, B.E.; Donatoni, P.; Wasterlain, C.G.

    1982-05-01

    We have developed a new method for measuring local rates of brain protein synthesis in vivo. It combines the intraperitoneal injection of a large dose of low specific activity amino acid with quantitative autoradiography. This method has several advantages: 1) It is ideally suited for young or small animals or where immobilizing an animal is undesirable. 2 The amino acid injection ''floods'' amino acid pools so that errors in estimating precursor specific activity, which is especially important in pathological conditions, are minimized. 3) The method provides for the use of a radioautographic internal standard in which valine incorporation is measured directly. Internal standards from experimental animals correct for tissue protein content and self-absorption of radiation in tissue sections which could vary under experimental conditions.

  14. Quantitative analysis of gene expression in preimplantation mouse embryos using green fluorescent protein reporter.

    PubMed

    Medvedev, Serguei Yuri; Tokunaga, Tomoyuki; Schultz, Richard M; Furukawa, Tsutomu; Nagai, Takashi; Yamaguchi, Manabu; Hosoe, Misa; Yakovlev, Alexander F; Takahashi, Seiya; Izaike, Yoshiaki

    2002-07-01

    We have developed a method to monitor noninvasively, quantitatively, and in real-time transcription in living preimplantation mouse embryos by measuring expression of a short half-life form of enhanced green fluorescent protein (EGFP) following microinjection of a plasmid-borne EGFP reporter gene. A standard curve was established by injecting known amounts of recombinant green fluorescent protein, and transcriptional activity was then determined by interpolating the amount of fluorescence in the DNA-injected embryos. This approach permitted multiple measurements in single embryos with no significant detrimental effect on embryonic development as long as light exposure was brief (<30 sec) and no more than two measurements were made each day. This method should facilitate analysis of the regulation of gene expression in preimplantation embryos; in particular, during the maternal-to-zygotic transition, and in other species in which limited numbers of embryos are available. PMID:12080029

  15. Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins*

    PubMed Central

    Mehus, Aaron A.; Muhonen, Wallace W.; Garrett, Scott H.; Somji, Seema; Sens, Donald A.; Shabb, John B.

    2014-01-01

    Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14N- or 15N-iodoacetamide. Absolute quantitation was achieved using 15N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These

  16. DNA ELECTROPHORESIS AT SURFACES

    SciTech Connect

    RAFAILOVICH, MIRIAM; SOKOLOV, JONATHAN; GERSAPPE, DILIP

    2003-09-01

    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  17. Application of Microchip Electrophoresis for Clinical Tests

    NASA Astrophysics Data System (ADS)

    Yatsushiro, Shouki; Kataoka, Masatoshi

    Microchip electrophoresis has recently attracted much attention in the field of nuclear acid analysis due to its high efficiency, ease of operation, low consumption of samples and reagents, and relatively low costs. In addition, the analysis has expanded to an analytical field like not only the analysis of DNA but also the analysis of RNA, the protein, the sugar chain, and the cellular function, etc. In this report, we showed that high-performance monitoring systems for human blood glucose levels and α-amylase activity in human plasma using microchip electrophoresis.

  18. Identification and comparative proteomic study of quail and duck egg white protein using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis.

    PubMed

    Hu, S; Qiu, N; Liu, Y; Zhao, H; Gao, D; Song, R; Ma, M

    2016-05-01

    A proteomic study of egg white proteins from 2 major poultry species, namely quail (Coturnix coturnix) and duck (Anas platyrhynchos), was performed with comparison to those of chicken (Gallus gallus) through 2-dimensional polyacrylamide gel electrophoresis (2-DE) analysis. By using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), 29 protein spots representing 10 different kinds of proteins as well as 17 protein spots designating 9 proteins were successfully identified in quail and duck egg white, respectively. This report suggested a closer relationship between quail and chicken egg white proteome patterns, whereas the duck egg white protein distribution on the 2-DE map was more distinct. In duck egg white, some well-known major proteins, such as ovomucoid, clusterin, extracellular fatty acid-binding protein precursor (ex-FABP), and prostaglandin D2 synthase (PG D2 synthase), were not detected, while two major protein spots identified as "deleted in malignant brain tumors 1" protein (DMBT1) and vitellogenin-2 were found specific to duck in the corresponding range on the 2-DE gel map. These interspecies diversities may be associated with the egg white protein functions in cell defense or regulating/supporting the embryonic development to adapt to the inhabiting environment or reproduction demand during long-term evolution. The findings of this work will give insight into the advantages involved in the application on egg white proteins from various egg sources, which may present novel beneficial properties in the food industry or related to human health.

  19. Identification and comparative proteomic study of quail and duck egg white protein using 2-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry analysis

    PubMed Central

    Hu, Shan; Qiu, Ning; Liu, Yaping; Zhao, Hongyan; Gao, Dan; Song, Rui; Ma, Meihu

    2016-01-01

    A proteomic study of egg white proteins from 2 major poultry species, namely quail (Coturnix coturnix) and duck (Anas platyrhynchos), was performed with comparison to those of chicken (Gallus gallus) through 2-dimensional polyacrylamide gel electrophoresis (2-DE) analysis. By using matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS), 29 protein spots representing 10 different kinds of proteins as well as 17 protein spots designating 9 proteins were successfully identified in quail and duck egg white, respectively. This report suggested a closer relationship between quail and chicken egg white proteome patterns, whereas the duck egg white protein distribution on the 2-DE map was more distinct. In duck egg white, some well-known major proteins, such as ovomucoid, clusterin, extracellular fatty acid-binding protein precursor (ex-FABP), and prostaglandin D2 synthase (PG D2 synthase), were not detected, while two major protein spots identified as “deleted in malignant brain tumors 1” protein (DMBT1) and vitellogenin-2 were found specific to duck in the corresponding range on the 2-DE gel map. These interspecies diversities may be associated with the egg white protein functions in cell defense or regulating/supporting the embryonic development to adapt to the inhabiting environment or reproduction demand during long-term evolution. The findings of this work will give insight into the advantages involved in the application on egg white proteins from various egg sources, which may present novel beneficial properties in the food industry or related to human health. PMID:26957635

  20. Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma

    PubMed Central

    Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M.; Davies, Anthony J.; Weetman, Malcolm; Garden, Oliver A.; Masters, John R.; Thrasivoulou, Christopher; Ahmed, Aamir

    2016-01-01

    Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

  1. Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

    PubMed

    Giuliano, Antonio; Swift, Rebecca; Arthurs, Callum; Marote, Georgina; Abramo, Francesca; McKay, Jenny; Thomson, Calum; Beltran, Mariana; Millar, Michael; Priestnall, Simon; Dobson, Jane; Costantino-Casas, Fernando; Petrou, Terry; McGonnell, Imelda M; Davies, Anthony J; Weetman, Malcolm; Garden, Oliver A; Masters, John R; Thrasivoulou, Christopher; Ahmed, Aamir

    2016-01-01

    Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease. PMID:27559731

  2. Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

  3. Genetic mapping and confirmation of quantitative trait loci for seed protein and oil contents and seed weight in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Demand for soybean [Glycine max (L.) Merr.] meal has increased worldwide and soybean importers often offer premiums for soybean containing higher contents of protein and oil. Objectives were to detect quantitative trait loci (QTL) associated with soybean seed protein, oil, and seed weight in a soyb...

  4. Quantitative proteomic analysis of wheat grain proteins reveals differential effects of silencing of omega-5 gliadin genes in transgenic lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Novel wheat lines with altered flour compositions can be used to decipher the roles of specific gluten proteins in flour quality. Grain proteins from transgenic wheat lines in which genes encoding the omega-5 gliadins were silenced by RNA interference (RNAi) were analyzed in detail by quantitative 2...

  5. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

    PubMed Central

    Zhuang, Min; Wiita, Arun P.; O’Donoghue, Anthony J.; Knudsen, Giselle M.; Craik, Charles S.; Wells, James A.

    2016-01-01

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events. PMID:27006500

  6. Quantitation of phosphorothioate oligonucleotides in human plasma.

    PubMed

    Leeds, J M; Graham, M J; Truong, L; Cummins, L L

    1996-03-01

    Methods are presented for the extraction of phosphorothioate oligonucleotides from human plasma to permit quantitation by capillary gel electrophoresis. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reverse-phase column to remove salts. A second desalting step, achieved by dialysis utilizing a membrane with a molecular weight cutoff of 2500 Da floating on distilled water, was required to remove residual ionic material from the extracted sample. This method should be generally applicable to the analysis and quantitation of phosphorothioate oligonucleotides. PMID:8850544

  7. Protein self-association induced by macromolecular crowding: a quantitative analysis by magnetic relaxation dispersion.

    PubMed

    Snoussi, Karim; Halle, Bertil

    2005-04-01

    In the presence of high concentrations of inert macromolecules, the self-association of proteins is strongly enhanced through an entropic, excluded-volume effect variously called macromolecular crowding or depletion attraction. Despite the predicted large magnitude of this universal effect and its far-reaching biological implications, few experimental studies of macromolecular crowding have been reported. Here, we introduce a powerful new technique, fast field-cycling magnetic relaxation dispersion, for investigating crowding effects on protein self-association equilibria. By recording the solvent proton spin relaxation rate over a wide range of magnetic field strengths, we determine the populations of coexisting monomers and decamers of bovine pancreatic trypsin inhibitor in the presence of dextran up to a macromolecular volume fraction of 27%. Already at a dextran volume fraction of 14%, we find a 30-fold increase of the decamer population and 510(5)-fold increase of the association constant. The analysis of these results, in terms of a statistical-mechanical model that incorporates polymer flexibility as well as the excluded volume of the protein, shows that the dramatic enhancement of bovine pancreatic trypsin inhibitor self-association can be quantitatively rationalized in terms of hard repulsive interactions. PMID:15665132

  8. Functional region identification in proteins by accumulative-quantitative peptide mapping using RP-HPLC-MS.

    PubMed

    Kuipers, Bas J H; Bakx, E J; Gruppen, Harry

    2007-11-14

    A new method was developed to identify regions in proteins from which peptides are derived with specific functional properties. This method is applicable for systems in which peptides of a hydrolyzed protein possess specific functional properties, but are too large to be sequenced directly and/or the peptide mixture is too complex to purify and characterize each peptide individually. In the present work, aggregating peptides obtained by proteolytic hydrolysis of soy glycinin were used as a case study. The aggregating peptides are isolated and subsequently further degraded with trypsin to result in peptides with a mass <5000 Da to enable sequence identification using RP-HPLC-MS in combination with MS/MS. Prior to RP-HPLC the peptides are fractionated using anion and cation exchange chromatography. The fractions obtained are analyzed with RP-HPLC-MS. The peptides, with identified sequences, were quantified using the peak areas of the RP-HPLC chromatograms measured at 214 nm. Next, the peak areas were corrected for the molar extinction coefficient of the individual peptides, followed by accumulative-quantitative peptide mapping. The results show that in complex systems, based on the method described, the regions in the parental protein from which the functional peptides originate can be properly identified.

  9. Quantitative Phosphoproteomics Reveals the Role of Protein Arginine Phosphorylation in the Bacterial Stress Response*

    PubMed Central

    Schmidt, Andreas; Trentini, Débora Broch; Spiess, Silvia; Fuhrmann, Jakob; Ammerer, Gustav; Mechtler, Karl; Clausen, Tim

    2014-01-01

    Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response. PMID:24263382

  10. A new quantitative method to measure activity of ice structuring proteins using differential scanning calorimetry.

    PubMed

    Hassa-Roudsari, Majid; Goff, H Douglas

    2012-01-01

    There are very few quantitative assays to measure the activity of antifreeze proteins (AFPs, or Ice Structuring Proteins, ISPs) and these can be prone to various inaccuracies and inconsistencies. Some methods rely only on unassisted visual assessment. When microscopy is used to measure ice crystal size, it is critical that standardized procedures be adopted, especially when image analysis software is used to quantify sizes. Differential Scanning Calorimetry (DSC) has been used to measure the thermal hysteresis activity (TH) of AFPs. In this study, DSC was used isothermally to measure enthalpic changes associated with structural rearrangements as a function of time. Differences in slopes of isothermal heat flow vs. time between winter wheat ISP or AFP type I containing samples, and those without ISP or AFP type I were demonstrated. ISP or AFP type I containing samples had significantly higher slopes compared to those without ISP or AFP type I. Samples with higher concentration of ISP or AFP type I showed higher slope values during the first hour and took up to 3 hr to attain equilibrium. Differences were attributed to activity of the proteins at the ice interface. Proteinaceous activity of ISPs or AFP type I was confirmed by loss of activity after treatment with protease.

  11. Analysis of electrophoresis performance

    NASA Technical Reports Server (NTRS)

    Roberts, G. O.

    1984-01-01

    The SAMPLE computer code models electrophoresis separation in a wide range of conditions. Results are included for steady three dimensional continuous flow electrophoresis (CFE), time dependent gel and acetate film experiments in one or two dimensions and isoelectric focusing in one dimension. The code evolves N two dimensional radical concentration distributions in time, or distance down a CFE chamber. For each time or distance increment, there are six stages, successively obtaining the pH distribution, the corresponding degrees of ionization for each radical, the conductivity, the electric field and current distribution, and the flux components in each direction for each separate radical. The final stage is to update the radical concentrations. The model formulation for ion motion in an electric field ignores activity effects, and is valid only for low concentrations; for larger concentrations the conductivity is, therefore, also invalid.

  12. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    PubMed Central

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Wei-Jun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F; Fredrickson, James K.; Paša-Tolić, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-01-01

    We report a mass spectrometry-based strategy for quantitative analysis of cell surface membrane proteome changes. The strategy includes enrichment of surface membrane proteins using a membrane-impermeable chemical probe followed by stable isotope 18O labeling and LC-MS analysis. We applied this strategy for enriching membrane proteins expressed by Shewanella oneidensis MR-1, a gram-negative bacterium with known metal-reduction capability via extracellular electron transfer between outer membrane proteins and extracellular electron receptors. LC/MS/MS analysis resulted in the identification of about 400 proteins with 79% of them being predicted to be membrane localized. Quantitative aspects of the membrane enrichment were shown by peptide level 16O and 18O labeling of proteins from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) prior to LC-MS analysis. Using a chemical probe labeled pure protein as an internal standard for normalization, the quantitative data revealed reduced abundances in ΔgspD mutant cells of many outer membrane proteins including the outer membrane c-cype cytochromes OmcA and MtrC, in agreement with previously investigation demonstrating that these proteins are substrates of the type II secretion system. PMID:20380418

  13. Happy bicentennial, electrophoresis!

    PubMed

    Righetti, Pier Giorgio

    2009-12-01

    A short survey of electrophoresis and a celebration of its bicentennial, with some remarkable mementos and a list of books that shaped the field. Where one also learns of a secret production plant with a huge-scale electrophoretic apparatus for skimming of latex from Hevea brasiliensis and keeping the wheels of the Ally Army running during World War II. And of cyber (mammoth) 2D gels of 1.5 x 1 m in size accommodating >12,000 spots. PMID:19938305

  14. Capillary zone electrophoresis

    SciTech Connect

    Jorgenson, J.W.; Lukacs, K.D.

    1983-10-21

    Zone electrophoresis in capillaries is a technique complementary to electrophoresis in supporting media, and each approach has its own particular advantages. Efficient heat transfer from small-diameter capillaries permits use of unusually high voltages, resulting in both high resolution and rapid analysis. Capillaries also seem well suited for automation. Our present electromigration injection technique is relatively straightforward and should be simple to automate. Capillaries are reusable, which is an advantage over gels. On-line electronic detection permits good quantification, further enhancing possibilities for fully automatic operation. The greatest obstacle to further development and utilization of capillaries is the requirement of extremely sensitive detectors, and more types of detectors with higher sensitivity are greatly needed. A better understanding of capillary surface modification will also be important, both for improved capillary surface deactivation and for better control over electroosmotic flow. Capillaries should provide an ideal system in which to explore nonaqueous separation media. The prospects for nonaqueous media in electrophoresis are similar to those in electrochemistry, and capillaries should prove an excellent system in which to begin their study. 18 refs., 8 figs.

  15. Preparative electrophoresis for space

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H.; Snyder, Robert S.

    1987-01-01

    A premise of continuous flow electrophoresis is that removal of buoyancy-induced thermal convection caused by axial and lateral temperature gradients results in ideal performance of these instruments in space. Although these gravity dependent phenomena disturb the rectilinear flow in the separation chamber when high voltage gradients or thick chambers are used, distortion of the injected sample stream due to electrohydrodynamic effects cause major broadening of the separated bands. The electrophoresis separation process is simple, however flow local to the sample filament produced by the applied electric field have not been considered. These electrohydrodynamic flows distort the sample stream and limit the separation. Also, electroosmosis and viscous flow combine to further distort the process. A moving wall concept is being proposed for space which will eliminate and control the disturbances. The moving wall entrains the fluid to move as a rigid body and produces a constant residence time for all samples distributed across the chamber thickness. The moving wall electrophoresis chamber can only be operated in space because there is no viscous flow in the chamber to stabilize against thermal convection.

  16. Preparative electrophoresis for space

    NASA Technical Reports Server (NTRS)

    Rhodes, Percy H.; Snyder, Robert S.

    1988-01-01

    A premise of continuous flow electrophoresis is that removal of buoyance-induced thermal convection caused by axial and lateral temperature gradients results in ideal performance of these instruments in space. Although these gravity dependent phenomena disturb the rectilinear flow in the separation chamber when high voltage gradients or thick chamber are used, distortion of the injected sample stream due to electrodynamic effects cause major broadening of the separated bands. The electrophoresis separation process is simple, however flow local to the sample filament produced by the applied electric field were not considered. These electrohydrodynamic flows distort the sample stream and limit the separation. Also, electroosmosis and viscous flow combine to further distort the process. A moving wall concept is being proposed for space which will eliminate and control the disturbances. The moving wall entrains the fluid to move as a rigid body and produces a constant residence time for all samples distributed across the chamber thickness. The moving wall electrophoresis chamber can only be operated in space because there is no viscous flow in the chamber to stabilize against thermal convection.

  17. Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips

    PubMed Central

    Cheng, Xianglin; Pu, Xu; Jun, Pen; Zhu, XiaoBo; Zhu, Di; Chen, Ming

    2014-01-01

    Background Rapid immunochromatographic tests can detect disease markers in 10–15 minutes, which facilitates clinical diagnosis and treatment programs. However, most immunochromatographic tests employ gold nanoparticles as reporters, and these have only moderate sensitivity and act as qualitative methods for analyzing high biomarker concentrations. Methods In this study, we introduce quantum dots (QDs) as fluorescent probes and immunochromatographic strips to develop quantitative fluorescence point-of-care tests (QF-POCT) to analyze C-reactive protein (CRP) levels. Goat anti-rabbit IgG and rabbit IgG were used as control antibodies, and mouse monoclonal CRP antibody pairs were used for disease marker detection. One monoclonal CRP antibody was conjugated with QDs and served as a signal antibody, and the other monoclonal CRP antibody was dispensed onto the nitrocellulose membrane and served as a capturing antibody. In the presence of CRP, the fluorescence intensity of the monoclonal antibody-CRP-monoclonal antibody sandwich complex captured on the nitrocellulose membrane was determined using the fluorescence strip reader. Results QF-POCT assays could quantitatively analyze the concentration of CRP in 15 minutes had a detection limit of 0.25 mg/L, and had a wide detection linearity range (0.5–300 mg/L). The intra-assay and interassay coefficients of variation were 8.95% and 9.86% at 0.5 mg/L, 6.47% and 8.66% at 10 mg/L, and 6.81% and 9.10% at 60 mg/L, respectively. In a comparison between clinical samples, the results of this QD-based assay of CRP levels were significantly correlated with those of an Immulite 2000 assay (R=0.993, P<0.001). Conclusion Our results demonstrated that the QD-based immunochromatographic test is a rapid, sensitive, accurate, and quantitative method for the detection of disease biomarkers. PMID:25506215

  18. Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

    SciTech Connect

    Li, Zhou; Czarnecki, Olaf; Chourey, Karuna; Yang, Jun; Tuskan, Gerald A; Hurst, Gregory {Greg} B; Pan, Chongle; Chen, Jay

    2014-01-01

    Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

  19. Quantitative Investigation of Protein-Nucleic Acid Interactions by Biosensor Surface Plasmon Resonance

    PubMed Central

    Wang, Shuo; Poon, Gregory M. K.; Wilson, W. David

    2015-01-01

    Biosensor-surface plasmon resonance (SPR) technology has emerged as a powerful label-free approach for the study of nucleic acid interactions in real time. The method provides simultaneous equilibrium and kinetic characterization for biomolecular interactions with minimal materials and without an external probe. A detailed and practical guide for protein-DNA interaction analyses using biosensor-SPR methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips and samples, as well as extensive information on experimental design, quantitative and qualitative data analyses and presentation. A specific example of the interaction of a transcription factor with DNA is shown with results evaluated by both kinetic and steady-state SPR methods. PMID:26404159

  20. Methods for quantitative evaluation of dynamics of repair proteins within irradiated cells

    NASA Astrophysics Data System (ADS)

    Hable, V.; Dollinger, G.; Greubel, C.; Hauptner, A.; Krücken, R.; Dietzel, S.; Cremer, T.; Drexler, G. A.; Friedl, A. A.; Löwe, R.

    2006-04-01

    Living HeLa cells are irradiated well directed with single 100 MeV oxygen ions by the superconducting ion microprobe SNAKE, the Superconducting Nanoscope for Applied Nuclear (=Kern-) Physics Experiments, at the Munich 14 MV tandem accelerator. Various proteins, which are involved directly or indirectly in repair processes, accumulate as clusters (so called foci) at DNA-double strand breaks (DSBs) induced by the ions. The spatiotemporal dynamics of these foci built by the phosphorylated histone γ-H2AX are studied. For this purpose cells are irradiated in line patterns. The γ-H2AX is made visible under the fluorescence microscope using immunofluorescence techniques. Quantitative analysis methods are developed to evaluate the data of the microscopic images in order to analyze movement of the foci and their changing size.

  1. Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the integrated stress response

    PubMed Central

    Gao, Xing-Huang; Krokowski, Dawid; Guan, Bo-Jhih; Bederman, Ilya; Majumder, Mithu; Parisien, Marc; Diatchenko, Luda; Kabil, Omer; Willard, Belinda; Banerjee, Ruma; Wang, Benlian; Bebek, Gurkan; Evans, Charles R.; Fox, Paul L.; Gerson, Stanton L.; Hoppel, Charles L.; Liu, Ming; Arvan, Peter; Hatzoglou, Maria

    2015-01-01

    The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the integrated stress response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism. DOI: http://dx.doi.org/10.7554/eLife.10067.001 PMID:26595448

  2. Multiplexed Western Blotting Using Microchip Electrophoresis.

    PubMed

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-01

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps. PMID:27270033

  3. Characterization of Membrane Protein Interactions in Plasma Membrane Derived Vesicles with Quantitative Imaging FRET

    PubMed Central

    Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

    2016-01-01

    CONSPECTUS Here we describe an experimental tool, termed Quantitative Imaging Förster Resonance Energy Transfer (QI-FRET), which enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles which bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), an RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor

  4. Two-Dimensional Gel Electrophoresis Analyses of pH-Dependent Protein Expression in Facultatively Alkaliphilic Bacillus pseudofirmus OF4 Lead to Characterization of an S-Layer Protein with a Role in Alkaliphily

    PubMed Central

    Gilmour, Raymond; Messner, Paul; Guffanti, Arthur A.; Kent, Rebecca; Scheberl, Andrea; Kendrick, Nancy; Krulwich, Terry Ann

    2000-01-01

    The large majority of proteins of alkaliphilic Bacillus pseudofirmus OF4 grown at pH 7.5 and 10.5, as studied by two-dimensional gel electrophoresis analyses, did not exhibit significant pH-dependent variation. A new surface layer protein (SlpA) was identified in these studies. Although the prominence of some apparent breakdown products of SlpA in gels from pH 10.5-grown cells led to discovery of the alkaliphile S-layer, the largest and major SlpA forms were present in large amounts in gels from pH 7.5-grown cells as well. slpA RNA abundance was, moreover, unchanged by growth pH. SlpA was similar in size to homologues from nonalkaliphiles but contained fewer Arg and Lys residues. An slpA mutant strain (RG21) lacked an exterior S-layer that was identified in the wild type by electron microscopy. Electrophoretic analysis of whole-cell extracts further indicated the absence of a 90-kDa band in the mutant. This band was prominent in wild-type extracts from both pH 7.5- and 10.5-grown cells. The wild type grew with a shorter lag phase than RG21 at either pH 10.5 or 11 and under either Na+-replete or suboptimal Na+ concentrations. The extent of the adaptation deficit increased with pH elevation and suboptimal Na+. By contrast, the mutant grew with a shorter lag and faster growth rate than the wild type at pH 7.5 under Na+-replete and suboptimal Na+ conditions, respectively. Logarithmically growing cells of the two strains exhibited no significant differences in growth rate, cytoplasmic pH regulation, starch utilization, motility, Na+-dependent transport of α-aminoisobutyric acid, or H+-dependent synthesis of ATP. However, the capacity for Na+-dependent pH homeostasis was diminished in RG21 upon a sudden upward shift of external pH from 8.5 to 10.5. The energy cost of retaining the SlpA layer at near-neutral pH is apparently adverse, but the constitutive presence of SlpA enhances the capacity of the extremophile to adjust to high pH. PMID:11029415

  5. Quantitative single-molecule detection of protein based on DNA tetrahedron fluorescent nanolabels.

    PubMed

    Ding, Yongshun; Liu, Xingti; Zhu, Jing; Wang, Lei; Jiang, Wei

    2014-07-01

    A highly sensitive method for single-molecule quantitative detection of human IgG is presented by the employment of a new fluorescent nanolabel. In this method, fluorescent nanolabels were assembled by inserting SYBR Green I into DNA tetrahedron nanostructure. The bio-nanolabels were attached to the streptavidin-antihuman antibody by a specific reaction between biotin and streptavidin. The antibody was combined with the target antigen, human IgG, which was immobilized on the silanized glass subtrate surface. Finally, epi-fluorescence microscopy (EFM) coupled with an electron multiplying charge-coupled device was employed for fluorescence imaging. The fluorescent spots corresponding to single protein molecule on images were counted and further used for the quantitative detection. It was found that the new nanolabel shows good photostability, biocompatiblity and exhibits no blinking compared to traditional labels like fluorescence dyes and quantum dot (QDs). In addition, the number of fluorescence spots on the images has a linear relationship with the concentration of human IgG in the range of 3.0×10(-14) to 1.0×10(-12)mol L(-1). What is more, this method showed an excellent specificity and a low matrix effect.

  6. Comparison of protein patterns of xrs-5, a radiosensitive Chinese hamster ovary cell line, and CHO-K1, its radioresistant parent, using two-dimensional gel-electrophoresis

    SciTech Connect

    Kramer, J.M. . Dept. of Zoology)

    1991-01-01

    X-ray sensitive strains of Chinese hamster ovary cell lines have been used to analyze radiation repair mechanisms. One cell line, xrs-5, has been shown to be very sensitive to ionizing radiation and radical forming chemical mutagens. This sensitivity is thought to be a result a mutation in the DNA double strand break (DSB) repair mechanism, and its characterization has been a goal of several repair mechanism studies. Using two-dimensional gel electrophoresis, we have detected a protein (MW approximately 55KD) in the DNA/Nuclear Matrix (nucleoid) cell fraction of CHO-Kl cells that is absent in the nucleoid fraction of xrs-5. This protein is present, however, in both CHO-Kl and xrs-5 whole cell protein maps. To determine whether the 55KD protein is responsible for the radiosensitive and defective DSB repair phenotype of xrs-5 cells, studies are now underway to analyze revertants of xrs-5 that are proficient in DSB repair. Furthermore, an effort to sequence the protein in question is planned. 23 refs., 2 figs.

  7. Unbiased Quantitative Models of Protein Translation Derived from Ribosome Profiling Data

    PubMed Central

    Gritsenko, Alexey A.; Hulsman, Marc; Reinders, Marcel J. T.; de Ridder, Dick

    2015-01-01

    Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of protein synthesis. Although a number of computational models of the process have been proposed, their application is limited by the assumptions they make. Ribosome profiling (RP), a relatively new sequencing-based technique capable of recording snapshots of the locations of actively translating ribosomes, is a promising source of information for deriving unbiased data-driven translation models. However, quantitative analysis of RP data is challenging due to high measurement variance and the inability to discriminate between the number of ribosomes measured on a gene and their speed of translation. We propose a solution in the form of a novel multi-scale interpretation of RP data that allows for deriving models with translation dynamics extracted from the snapshots. We demonstrate the usefulness of this approach by simultaneously determining for the first time per-codon translation elongation and per-gene translation initiation rates of Saccharomyces cerevisiae from RP data for two versions of the Totally Asymmetric Exclusion Process (TASEP) model of translation. We do this in an unbiased fashion, by fitting the models using only RP data with a novel optimization scheme based on Monte Carlo simulation to keep the problem tractable. The fitted models match the data significantly better than existing models and their predictions show better agreement with several independent protein abundance datasets than existing models. Results additionally indicate that the tRNA pool adaptation hypothesis is incomplete, with evidence suggesting that tRNA post-transcriptional modifications and codon context may play a role in determining codon elongation rates. PMID:26275099

  8. Guided protein extraction from formalin-fixed tissues for quantitative multiplex analysis avoids detrimental effects of histological stains.

    PubMed

    Becker, Karl-Friedrich; Schott, Christina; Becker, Ingrid; Höfler, Heinz

    2008-05-01

    Formalin fixed and paraffin embedded (FFPE) tissues are the basis for histopathological diagnosis of many diseases around the world. For translational research and routine diagnostics, protein analysis from FFPE tissues is very important. We evaluated the potential influence of six histological stains, including hematoxylin (Mayer and Gill), fast red, light green, methyl blue and toluidine blue, for yield, electrophoretic mobility in 1-D gels, and immunoreactivity of proteins isolated from formalin-fixed breast cancer tissues. Proteins extracted from stained FFPE tissues using a recently established technique were compared with proteins obtained from the same tissues but without prior histological staining. Western blot and quantitative protein lysate microarray analysis demonstrated that histological staining can result in decreased protein yield but may not have much influence on immunoreactivity and electrophoretic mobility. Interestingly, not all staining protocols tested are compatible with subsequent protein analysis. The commonly used hematoxylin staining was found to be suitable for multiplexed quantitative protein measurement technologies although protein extraction was less efficient. For best results we suggest a guided protein extraction method, in which an adjacent hematoxylin/eosin-stained tissue section is used to control dissection of an unstained specimen for subsequent protein extraction and quantification for research and diagnosis.

  9. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    EPA Science Inventory

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  10. Quantitative Assessment of Effect of Preanalytic Cold Ischemic Time on Protein Expression in Breast Cancer Tissues

    PubMed Central

    2012-01-01

    Background Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. Methods A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided. Results We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series. Conclusions Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive

  11. A tunable isoelectric focusing via moving reaction boundary for two-dimensional gel electrophoresis and proteomics.

    PubMed

    Guo, Chen-Gang; Shang, Zhi; Yan, Jian; Li, Si; Li, Guo-Qing; Liu, Rong-Zhong; Qing, Ying; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-05-01

    Routine native immobilized pH gradient isoelectric focusing (IPG-IEF) and two-dimensional gel electrophoresis (2DE) are still suffering from unfortunate reproducibility, poor resolution (caused by protein precipitation) and instability in characterization of intact protein isoforms and posttranslational modifications. Based on the concept of moving reaction boundary (MRB), we firstly proposed a tunable non-IPG-IEF system to address these issues. By choosing proper pairs of catholyte and anolyte, we could achieve desired cathodic and anodic migrating pH gradients in non-IPG-IEF system, effectively eliminating protein precipitation and uncertainty of quantitation existing in routine IEF and 2DE, and enhancing the resolution and sensitivity of IEF. Then, an adjustable 2DE system was developed by combining non-IPG-IEF with polyacrylamide gel electrophoresis (PAGE). The improved 2DE was evaluated by testing model proteins and colon cancer cell lysates. The experiments revealed that (i) a tunable pH gradient could be designed via MRB; (ii) up to 1.65 fold improvement of resolution was achieved via non-IPG-IEF; (iii) the sensitivity of developed techniques was increased up to 2.7 folds; and (iv) up to about 16.4% more protein spots could be observed via the adjustable 2DE as compared with routine one. The developed techniques might contribute to complex proteome research, especially for screening of biological marker and analysis of extreme acidic/alkaline proteins.

  12. xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry.

    PubMed

    Walzthoeni, Thomas; Joachimiak, Lukasz A; Rosenberger, George; Röst, Hannes L; Malmström, Lars; Leitner, Alexander; Frydman, Judith; Aebersold, Ruedi

    2015-12-01

    Chemical cross-linking in combination with mass spectrometry generates distance restraints of amino acid pairs in close proximity on the surface of native proteins and protein complexes. In this study we used quantitative mass spectrometry and chemical cross-linking to quantify differences in cross-linked peptides obtained from complexes in spatially discrete states. We describe a generic computational pipeline for quantitative cross-linking mass spectrometry consisting of modules for quantitative data extraction and statistical assessment of the obtained results. We used the method to detect conformational changes in two model systems: firefly luciferase and the bovine TRiC complex. Our method discovers and explains the structural heterogeneity of protein complexes using only sparse structural information. PMID:26501516

  13. xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry

    PubMed Central

    Walzthoeni, Thomas; Joachimiak, Lukasz A; Rosenberger, George; Röst, Hannes L; Malmström, Lars; Leitner, Alexander; Frydman, Judith; Aebersold, Ruedi

    2016-01-01

    Chemical cross-linking in combination with mass spectrometry generates distance restraints of amino acid pairs in close proximity on the surface of native proteins and protein complexes. In this study we used quantitative mass spectrometry and chemical cross-linking to quantify differences in cross-linked peptides obtained from complexes in spatially discrete states. We describe a generic computational pipeline for quantitative cross-linking mass spectrometry consisting of modules for quantitative data extraction and statistical assessment of the obtained results. We used the method to detect conformational changes in two model systems: firefly luciferase and the bovine TRiC complex. Our method discovers and explains the structural heterogeneity of protein complexes using only sparse structural information. PMID:26501516

  14. Dissociation coefficients of protein adsorption to nanoparticles as quantitative metrics for description of the protein corona: A comparison of experimental techniques and methodological relevance.

    PubMed

    Hühn, Jonas; Fedeli, Chiara; Zhang, Qian; Masood, Atif; Del Pino, Pablo; Khashab, Niveen M; Papini, Emanuele; Parak, Wolfgang J

    2016-06-01

    Protein adsorption to nanoparticles is described as a chemical reaction in which proteins attach to binding sites on the nanoparticle surface. This process is defined by a dissociation coefficient, which tells how many proteins are adsorbed per nanoparticle in dependence of the protein concentration. Different techniques to experimentally determine dissociation coefficients of protein adsorption to nanoparticles are reviewed. Results of more than 130 experiments in which dissociation coefficients have been determined are compared. Data show that different methods, nanoparticle systems, and proteins can lead to significantly different dissociation coefficients. However, we observed a clear tendency of smaller dissociation coefficients upon less negative towards more positive zeta potentials of the nanoparticles. The zeta potential thus is a key parameter influencing protein adsorption to the surface of nanoparticles. Our analysis highlights the importance of the characterization of the parameters governing protein-nanoparticle interaction for quantitative evaluation and objective literature comparison. PMID:26748245

  15. Quantitative Description of a Protein Fitness Landscape Based on Molecular Features.

    PubMed

    Meini, María-Rocío; Tomatis, Pablo E; Weinreich, Daniel M; Vila, Alejandro J

    2015-07-01

    Understanding the driving forces behind protein evolution requires the ability to correlate the molecular impact of mutations with organismal fitness. To address this issue, we employ here metallo-β-lactamases as a model system, which are Zn(II) dependent enzymes that mediate antibiotic resistance. We present a study of all the possible evolutionary pathways leading to a metallo-β-lactamase variant optimized by directed evolution. By studying the activity, stability and Zn(II) binding capabilities of all mutants in the preferred evolutionary pathways, we show that this local fitness landscape is strongly conditioned by epistatic interactions arising from the pleiotropic effect of mutations in the different molecular features of the enzyme. Activity and stability assays in purified enzymes do not provide explanatory power. Instead, measurement of these molecular features in an environment resembling the native one provides an accurate description of the observed antibiotic resistance profile. We report that optimization of Zn(II) binding abilities of metallo-β-lactamases during evolution is more critical than stabilization of the protein to enhance fitness. A global analysis of these parameters allows us to connect genotype with fitness based on quantitative biochemical and biophysical parameters.

  16. Quantitative Description of a Protein Fitness Landscape Based on Molecular Features

    PubMed Central

    Meini, María-Rocío; Tomatis, Pablo E.; Weinreich, Daniel M.; Vila, Alejandro J.

    2015-01-01

    Understanding the driving forces behind protein evolution requires the ability to correlate the molecular impact of mutations with organismal fitness. To address this issue, we employ here metallo-β-lactamases as a model system, which are Zn(II) dependent enzymes that mediate antibiotic resistance. We present a study of all the possible evolutionary pathways leading to a metallo-β-lactamase variant optimized by directed evolution. By studying the activity, stability and Zn(II) binding capabilities of all mutants in the preferred evolutionary pathways, we show that this local fitness landscape is strongly conditioned by epistatic interactions arising from the pleiotropic effect of mutations in the different molecular features of the enzyme. Activity and stability assays in purified enzymes do not provide explanatory power. Instead, measurement of these molecular features in an environment resembling the native one provides an accurate description of the observed antibiotic resistance profile. We report that optimization of Zn(II) binding abilities of metallo-β-lactamases during evolution is more critical than stabilization of the protein to enhance fitness. A global analysis of these parameters allows us to connect genotype with fitness based on quantitative biochemical and biophysical parameters. PMID:25767204

  17. Quantitative Description of a Protein Fitness Landscape Based on Molecular Features.

    PubMed

    Meini, María-Rocío; Tomatis, Pablo E; Weinreich, Daniel M; Vila, Alejandro J

    2015-07-01

    Understanding the driving forces behind protein evolution requires the ability to correlate the molecular impact of mutations with organismal fitness. To address this issue, we employ here metallo-β-lactamases as a model system, which are Zn(II) dependent enzymes that mediate antibiotic resistance. We present a study of all the possible evolutionary pathways leading to a metallo-β-lactamase variant optimized by directed evolution. By studying the activity, stability and Zn(II) binding capabilities of all mutants in the preferred evolutionary pathways, we show that this local fitness landscape is strongly conditioned by epistatic interactions arising from the pleiotropic effect of mutations in the different molecular features of the enzyme. Activity and stability assays in purified enzymes do not provide explanatory power. Instead, measurement of these molecular features in an environment resembling the native one provides an accurate description of the observed antibiotic resistance profile. We report that optimization of Zn(II) binding abilities of metallo-β-lactamases during evolution is more critical than stabilization of the protein to enhance fitness. A global analysis of these parameters allows us to connect genotype with fitness based on quantitative biochemical and biophysical parameters. PMID:25767204

  18. Nearly 1000 Protein Identifications from 50 ng of Xenopus laevis Zygote Homogenate Using Online Sample Preparation on a Strong Cation Exchange Monolith Based Microreactor Coupled with Capillary Zone Electrophoresis.

    PubMed

    Zhang, Zhenbin; Sun, Liangliang; Zhu, Guijie; Cox, Olivia F; Huber, Paul W; Dovichi, Norman J

    2016-01-01

    A sulfonate-silica hybrid strong cation exchange monolith microreactor was synthesized and coupled to a linear polyacrylamide coated capillary for online sample preparation and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) bottom-up proteomic analysis. The protein sample was loaded onto the microreactor in an acidic buffer. After online reduction, alkylation, and digestion with trypsin, the digests were eluted with 200 mM ammonium bicarbonate at pH 8.2 for CZE-MS/MS analysis using 1 M acetic acid as the background electrolyte. This combination of basic elution and acidic background electrolytes results in both sample stacking and formation of a dynamic pH junction. 369 protein groups and 1274 peptides were identified from 50 ng of Xenopus laevis zygote homogenate, which is comparable with an offline sample preparation method, but the time required for sample preparation was decreased from over 24 h to less than 40 min. Dramatically improved performance was produced by coupling the reactor to a longer separation capillary (∼100 cm) and a Q Exactive HF mass spectrometer. 975 protein groups and 3749 peptides were identified from 50 ng of Xenopus protein using the online sample preparation method.

  19. Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics.

    PubMed

    Nanjo, Yohei; Nakamura, Takuji; Komatsu, Setsuko

    2013-11-01

    Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean.

  20. Quantitative characterization of protein–protein complexes involved in base excision DNA repair

    PubMed Central

    Moor, Nina A.; Vasil'eva, Inna A.; Anarbaev, Rashid O.; Antson, Alfred A.; Lavrik, Olga I.

    2015-01-01

    Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process. PMID:26013813

  1. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

    PubMed Central

    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (P<0.05). Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the

  2. Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

    PubMed Central

    Xie, Xitao; Yi, Zhengping; Bowen, Benjamin; Wolf, Cassandra; Flynn, Charles R.; Sinha, Sandeep; Mandarino, Lawrence J.; Meyer, Christian

    2010-01-01

    Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to 1) comprehensively characterize the human adipocyte proteome for the first time, and 2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and High Performance Liquid Chromatography -Electron Spray Ionization - tandem Mass Spectrometry (HPLC-ESI-MS/MS). In adipocytes isolated from ~0.5 g subcutaneous abdominal adipose tissue of three healthy, lean subjects we identified a total of 1493 proteins. Triplicate analysis indicated a 22.5% coefficient of variation of protein abundances. Proteins ranged from 5.8 to 629 kDa and included a large number of proteins involved in lipid metabolism, such as fatty acid transport, fatty acid oxidation, lipid storage, lipolysis and lipid droplet maintenance. Furthermore, we found most glycolysis enzymes and numerous proteins associated with oxidative stress, protein synthesis and degradation as well as some adipokines. 22% of all proteins were of mitochondrial origin. These results provide the first detailed characterization of the human adipocyte proteome, suggest an important role of adipocyte mitochondria, and demonstrate feasibility of this approach to examine alterations of adipocyte protein abundances in human diseases. PMID:20812759

  3. Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera.

    PubMed

    Sanchez-Campillo, M; Bini, L; Comanducci, M; Raggiaschi, R; Marzocchi, B; Pallini, V; Ratti, G

    1999-08-01

    Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL-like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface-exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK-like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N-terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha-subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF-Tu, seven (41%) a putative stress-induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl-like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates. PMID:10493131

  4. A New Strategy of Using O18-Labeled Iodoacetic Acid for Mass Spectrometry-Based Protein Quantitation

    NASA Astrophysics Data System (ADS)

    Wang, Shunhai; Kaltashov, Igor A.

    2012-07-01

    A new O18 labeling protocol is designed to assist quantitation of cysteine-containing proteins using LC/MS. Unlike other O18 labeling strategies, the labeling is carried out at the intact protein level (prior to its digestion) during reduction/alkylation of cysteine side chains using O18-labeled iodoacetic acid (IAA). The latter can be easily prepared by exchanging carboxylic oxygen atoms of commercially available IAA in O18-enriched water at low pH. Since incorporation of the O18 label in the protein occurs at the whole protein, rather than peptide level, the quantitation results are not peptide-dependent. The excellent stability of the label in mild pH conditions provides flexibility and robustness needed of sample processing steps following the labeling. In contrast to generally costly isotope labeling reagents, this approach uses only two relatively inexpensive commercially available reagents (IAA and H2O18). The feasibility of the new method is demonstrated using an 80 kDa human serum transferrin (hTf) as a model, where linear quantitation is achieved across a dynamic range spanning three orders of magnitude. The new approach can be used in quantitative proteomics applications and is particularly suitable for a variety of tasks in the biopharmaceutical sector, ranging from pharmacokinetic studies to quality control of protein therapeutics.

  5. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Li, Q.; Lu, X.

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis