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Sample records for quantitative protein electrophoresis

  1. Fish Muscle Proteins: Extraction, Quantitation, and Electrophoresis

    NASA Astrophysics Data System (ADS)

    Smith, Denise

    Electrophoresis can be used to separate and visualize proteins. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins are separated based on size. When protein samples are applied to such gels, it is usually necessary to know the protein content of the sample. This makes it possible to apply a volume of sample to the gel such that samples have a comparable amount of total protein. While it is possible to use an official method of protein analysis (e.g., Kjeldahl, N combustion) for such an application, it often is convenient to use a rapid spectroscopic protein analysis that requires only a small amount of sample. The bicinchoninic acid (BCA) assay method will be used for this purpose.

  2. 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.

  3. 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. PMID:26796977

  4. Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

    PubMed

    Kralj, Jason G; Munson, Matthew S; Ross, David

    2014-07-01

    We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods. PMID:24648165

  5. 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...

  6. Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection

    SciTech Connect

    Lee, T.T.; Yeung, E.S. Iowa State Univ., Ames )

    1992-12-01

    Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A[sub 0], and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A[sub 0] reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A[sub 0] for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage. 35 refs., 10 figs., 1 tab.

  7. Protein electrophoresis - serum

    MedlinePlus

    ... digestive tract to absorb proteins ( protein-losing enteropathy ) Malnutrition Kidney disorder called nephrotic syndrome Scarring of the ... may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone marrow ...

  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. Protein electrophoresis - urine

    MedlinePlus

    ... nephropathy Kidney failure Multiple myeloma Nephrotic syndrome Acute urinary tract infection Risks There are no risks associated with this ... Primary amyloidosis Protein in diet Protein urine test Urinary tract infection - adults Update Date 5/29/2014 Updated by: ...

  10. Protein electrophoresis - serum

    MedlinePlus

    Normal value ranges are: Total protein: 6.4 to 8.3 g/dL (grams per deciliter) Albumin: 3.5 to 5.0 g/dL Alpha-1 ... Decreased total protein may indicate: Abnormal loss of protein from the digestive tract or the inability of the digestive tract ...

  11. Non-denaturating isoelectric focusing gel electrophoresis for uranium-protein complexes quantitative analysis with LA-ICP MS.

    PubMed

    Xu, Ming; Frelon, Sandrine; Simon, Olivier; Lobinski, Ryszard; Mounicou, Sandra

    2014-02-01

    A non-denaturating isoelectric focusing (ND-IEF) gel electrophoresis protocol has been developed to study and identify uranium (U)-protein complexes with laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS) and electrospray ionization mass spectrometry (ESI-MS). The ND-IEF-LA-ICP MS methodology set-up was initiated using in vitro U-protein complex standards (i.e., U-bovine serum albumin and U-transferrin) allowing the assessment of U recovery to 64.4 ± 0.4 %. This methodology enabled the quantification of U-protein complexes at 9.03 ± 0.23, 15.27 ± 0.36, and 177.31 ± 25.51 nmol U L(-1) in digestive gland cytosols of the crayfish, Procambarus clarkii, exposed respectively to 0, 0.12, and 2.5 μmol of waterborne depleted U L(-1) during 10 days. ND-IEF-LA-ICP MS limit of detection was 19.3 pmol U L(-1). Elemental ICP MS signals obtained both in ND-IEF electropherograms and in size exclusion chromatograms of in vivo U-protein complexes revealed interactions between U- and Fe- and Cu-proteins. Moreover, three proteins (hemocyanin, pseudohemocyanin-2, and arginine kinase) out of 42 were identified as potential uranium targets in waterborne-exposed crayfish cytosols by microbore reversed phase chromatography coupled to molecular mass spectrometry (µRPC-ESI-MS/MS) after ND-IEF separation. PMID:23665639

  12. Electronic imaging systems for quantitative electrophoresis of DNA

    SciTech Connect

    Sutherland, J.C.

    1989-01-01

    Gel electrophoresis is one of the most powerful and widely used methods for the separation of DNA. During the last decade, instruments have been developed that accurately quantitate in digital form the distribution of materials in a gel or on a blot prepared from a gel. In this paper, I review the various physical properties that can be used to quantitate the distribution of DNA on gels or blots and the instrumentation that has been developed to perform these tasks. The emphasis here is on DNA, but much of what is said also applies to RNA, proteins and other molecules. 36 refs.

  13. 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.

  14. 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

  15. Serum protein electrophoresis in spontaneous canine hyperadrenocorticalism.

    PubMed

    van den Broek, A H; Lida, J

    1989-01-01

    The serum protein concentrations of dogs with confirmed spontaneous hyperadrenocorticalism were determined by agarose gel electrophoresis before and during treatment with mitotane. In untreated animals a significant increase was detected in the mean concentration of total protein and the mean concentration and percentage of alpha-2 globulin. The mean concentration and percentage of albumin and gamma-globulin were significantly decreased. In animals on treatment the mean concentration of total proteins and the mean concentration and percentage of beta-2 globulin were significantly reduced. PMID:2466309

  16. 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)

  17. 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

  18. Quantitative analysis of plasma membrane proteome using two-dimensional difference gel electrophoresis.

    PubMed

    Tang, Wenqiang

    2012-01-01

    The plasma membrane (PM) controls cell's exchange of both material and information with the outside environment, and PM-associated proteins play key roles in cellular regulation. Numerous cell surface receptors allow cells to perceive and respond to various signals from neighbor cells, pathogens, or the environment; large numbers of transporter and channel proteins control material uptake or release. Quantitative proteomic analysis of PM-associated proteins can identify key proteins involved in signal transduction and cellular regulation. Here, we describe a protocol for quantitative proteomic analysis of PM proteins using two-dimensional difference gel electrophoresis. The protocol has been successfully employed to identify new components of the brassinosteroid signaling pathway, and should also be applicable to the studies of other plant signal transduction pathways and regulatory mechanisms. PMID:22576086

  19. Accessing Protein Methyltransferase and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis

    PubMed Central

    Wigle, Tim J.; Provencher, Laurel M.; Norris, Jacqueline L.; Jin, Jian; Brown, Peter J.; Frye, Stephen V.; Janzen, William P.

    2010-01-01

    Summary The discovery of small molecules targeting the > 80 enzymes that add (methyltransferases) or remove (demethylases) methyl marks from lysine and arginine residues, most notably present in histone tails, may yield unprecedented chemotherapeutic agents and facilitate regenerative medicine. To better enable chemical exploration of these proteins, we have developed a novel and highly quantitative microfluidic capillary electrophoresis assay to enable full mechanistic studies of these enzymes and the kinetics of their inhibition. This technology separates small biomolecules, i.e., peptides, based on their charge-to-mass ratio. Methylation, however, does not alter the charge of peptide substrates. To overcome this limitation, we have employed a methylation-sensitive endoproteinase strategy to separate methylated from unmethylated peptides. The assay was validated on a lysine methyltransferase (G9a) and a lysine demethylase (LSD1) and was employed to investigate the inhibition of G9a by small molecules. PMID:20659682

  20. Rapid inorganic ion analysis using quantitative microchip capillary electrophoresis.

    PubMed

    Vrouwe, Elwin X; Luttge, Regina; Olthuis, Wouter; van den Berg, Albert

    2006-01-13

    Rapid quantitative microchip capillary electrophoresis (CE) for online monitoring of drinking water enabling inorganic ion separation in less than 15 s is presented. Comparing cationic and anionic standards at different concentrations the analysis of cationic species resulted in non-linear calibration curves. We interpret this effect as a variation in the volume of the injected sample plug caused by changes of the electroosmotic flow (EOF) due to the strong interaction of bivalent cations with the glass surface. This explanation is supported by the observation of severe peak tailing. Conducting microchip CE analysis in a glass microchannel, optimized conditions are received for the cationic species K+, Na+, Ca2+, Mg2+ using a background electrolyte consisting of 30 mmol/L histidine and 2-(N-morpholino)ethanesulfonic acid, containing 0.5 mmol/L potassium chloride to reduce surface interaction and 4 mmol/L tartaric acid as a complexing agent resulting in a pH-value of 5.8. Applying reversed EOF co-migration for the anionic species Cl-, SO42- and HCO3- optimized separation occurs in a background electrolyte consisting of 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mmol/L HEPES sodium salt, containing 0.05 mmol/L CTAB (cetyltrimethylammonium bromide) resulting in a pH-value of 7.5. The detection limits are 20 micromol/L for the monovalent cationic and anionic species and 10 micromol/L for the divalent species. These values make the method very suitable for many applications including the analysis of abundant ions in tap water as demonstrated in this paper. PMID:16310794

  1. 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

  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. Protein Separation by Capillary Gel Electrophoresis: A Review

    PubMed Central

    Zhu, Zaifang; Lu, Joann J.; Liu, Shaorong

    2011-01-01

    Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples. PMID:22122927

  4. Size separation of proteins by capillary zone electrophoresis with cationic hitchhiking (CZECH)

    PubMed Central

    Dolnik, Vladislav; Gurske, William A.

    2012-01-01

    The paper describes a method of size separation of proteins by capillary sieving electrophoresis with cationic surfactant. Proteins are separated within 12 minutes with repeatability of migration times better than 0.2%. Some proteins achieve the separation efficiency of 200,000 theoretical plates. The method can be used for determination of protein relative molecular masses. The accuracy of the determined relative molecular masses and the limitation of the method were investigated by the analysis of more than 60 proteins. The method also allows separation of protein oligomers. Proteins can be quantitated after the electrokinetic injection in the concentration range 0.07–0.43 g/L. The average detection limit is about 2 mg/L. PMID:21948216

  5. 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.

  6. Phylogenetic reconstruction of South American felids defined by protein electrophoresis.

    PubMed

    Slattery, J P; Johnson, W E; Goldman, D; O'Brien, S J

    1994-09-01

    Phylogenetic associations among six closely related South American felid species were defined by changes in protein-encoding gene loci. We analyzed proteins isolated from skin fibroblasts using two-dimensional electrophoresis and allozymes extracted from blood cells. Genotypes were determined for multiple individuals of ocelot, margay, tigrina, Geoffroy's cat, kodkod, and pampas cat at 548 loci resolved by two-dimensional electrophoresis and 44 allozyme loci. Phenograms were constructed using the methods of Fitch-Margoliash and neighbor-joining on a matrix of Nei's unbiased genetic distances for all pairs of species. Results of a relative-rate test indicate changes in two-dimensional electrophoresis data are constant among all South American felids with respect to a hyena outgroup. Allelic frequencies were transformed to discrete character states for maximum parsimony analysis. Phylogenetic reconstruction indicates a major split occurred approximately 5-6 million years ago, leading to three groups within the ocelot lineage. The earliest divergence led to Leopardus tigrina, followed by a split between an ancestor of an unresolved trichotomy of three species (Oncifelis guigna, O. geoffroyi, and Lynchailuris colocolo) and a recent common ancestor of Leopardus pardalis and L. wiedii. The results suggest that modern South American felids are monophyletic and evolved rapidly after the formation of the Panama land bridge between North and South America. PMID:7932791

  7. [Does bilirubin interfere with capillary electrophoresis of serum proteins?].

    PubMed

    Hellara, Ilhem; Fekih, Ons; Triki, Sonia; Elmay, Ahlem; Neffati, Fadoua; Najjar, Mohamed Fadhel

    2014-01-01

    Capillary electrophoresis of serum proteins is a fast, reliable and simple technique, but many interference exist. The objective of our work is to study the interference of bilirubin on this technique; 70 icteric sera were analysed on Capillarys ™ (Sebia). A second electrophoresis was performed on 40 samples after bilirubin photodegradation. The bilirubin and serum proteins were determinated respectively by Jendrassik and Grof and biuret methods on Konélab 20i ™ (Thermo Electron Corporation). We found abnormal spreading of the albumin fraction of the anode side wich constitute sometimes an isolated fraction in the traditional area of pre-albumin migration. This fraction varies from 2.0 ± 2.0% (0.0 to 7.3%) or 0.98 ± 1.53 g/L (0 to 5.3 g/L) and it seems to be related to the direct bilirubin since, following overloading sera with a solution of bilirubin, no further fraction was recovered. An average decrease of bilirubin after photodegradation of 58 ± 17% (26-89%) is followed by a decrease in the same order 64 ± 38% (10-100%) of the additional fraction. Acetate cellulose electrophoresis of the same samples showed no variation. The high bilirubin levels seem modify slightly the electrophoretic profile. However the impact of the interference on the interpretation of electrophoretic trace is negligible. PMID:24492101

  8. 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. PMID:10344261

  9. Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis.

    PubMed

    Barron, Tamara L; Turco, Salvatore J

    2006-04-01

    The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms. PMID:16310310

  10. Protein separation by continuous-flow electrophoresis in microgravity.

    PubMed

    Clifton, M J; Roux-de Balmann, H; Sanchez, V

    1996-07-01

    During the IML-2 space shuttle mission, the RAMSES instrument was operated in the Spacelab module. This continuous-flow electrophoresis device performs separation and purification of protein solutions on a preparative scale. Samples containing artificial mixtures of pure proteins were used to test the capabilities of the device, and useful separations were obtained for proteins having a mobility difference of only 3 x 10(-9) m2 V-1 s-1. Operating conditions that cannot be applied on earth were explored for two different sample concentrations, one of which is too high to allow treatment on earth. It agrees well with a previously published numerical model in that the main cause of loss in resolution in this process is the electrohydrodynamic spreading of the protein filaments. PMID:11539848

  11. 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

  12. 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.

  13. 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

  14. Moving towards harmonized reporting of serum and urine protein electrophoresis.

    PubMed

    Moss, Michael A

    2016-06-01

    During the last decade, surveys by questionnaire in Canada, Australia and New Zealand revealed wide variation in reporting practices by laboratories and individual practitioners in the interpretation of serum and urine protein electrophoresis (PE). Such variation has potential to adversely impact patient outcomes if report structure is inconsistent or if the messaging is incorrectly perceived by the receiving physician. Concerted efforts have been initiated to promote harmonization in the use of interpretative comments. The primary goal is to add value through clear communication with requesting physicians in the interest of quality patient care. Resistance to a harmonized approach largely reflects longstanding personal reporting habits and preferences but change can be more readily embraced if the new system is intuitive, easy to use and saves time in reporting. PMID:26824981

  15. Protein and cholesterol electrophoresis of plasma samples from captive cownose ray (Rhinoptera bonasus).

    PubMed

    Cray, Carolyn; Rodriguez, Marilyn; Field, Cara; McDermott, Alexa; Leppert, Lynda; Clauss, Tonya; Bossart, Gregory D

    2015-11-01

    Our study was undertaken to assess the application of semiautomated methods available at the reference laboratory level for the evaluation of plasma protein and cholesterol via electrophoresis in samples from cownose rays (Rhinoptera bonasus). Three groups of animals were assessed: clinically normal, clinically abnormal, and parasitized with leeches. As reported previously, the albumin band was negligible; the protein electrophoretograms were dominated by a large beta-globulin fraction. While the group of samples from the leech-parasitized rays did not show any large differences, the abnormal group exhibited significantly elevated total solids and cholesterol levels. The latter was related to a significant increase in very low density lipoprotein levels. The results demonstrate the potential application of these laboratory methods in quantitation of plasma proteins and cholesterol fractions in subclass Elasmobranchii. PMID:26450839

  16. Rapid separation and quantification of major caseins and whey proteins of bovine milk by capillary electrophoresis.

    PubMed

    Vallejo-Cordoba, B

    1997-01-01

    A rapid capillary zone electrophoresis (CZE) method was established for separating and quantifying major casein and whey proteins in milk. Optimum sample preparation and electrophoretic conditions in a coated capillary maintained at 40 degrees C allowed accurate and reproducible quantification of milk proteins in a single analysis. Sample and run buffer allowed caseins to be maintained in solution by using a combination of urea and a nonionic detergent in phosphate buffer at pH 2.5. Quantitative CZE protein data were derived by calculating percentages and concentrations (mg/mL) of alpha-casein, beta-casein, alpha-lactalbumin, and beta-lactoglobulin. Calibration curves followed linear relationships with highly significant (p < 0.1) correlation coefficients. Relative standard deviations of less than 0.82 (%) for migration times and 2.18 (%) for percent protein indicated that the technique was reproducible. Electrophoretic protein profiles of fresh bovine milk and rehydrated dry milk showed marked quantitative differences in whey protein concentrations. Whey protein represented 12.37 +/- 0.07% beta-lactoglobulin and 3.05 +/- 0.08% alpha-lactalbumin of total protein in typical fresh milk, while only 1.90 +/- 0.16% beta-lactoglobulin and 0.86 +/- 0.04% alpha-lactalbumin of total protein were detected in a commercial rehydrated milk powder. By quantifying these differences, the established technique may allow the detection of substitution of fresh milk with rehydrated milk powder. The accuracy and reproducibility of the technique permitted the quantitation of individual protein concentrations in milk samples, which agreed with ranges reported in the literature. CZE may be well suited for routine use by dairies and regulatory agencies, since it allows the determination of milk proteins in less than 60 min. PMID:9725120

  17. 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. PMID:26414025

  18. 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. PMID:26174138

  19. Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels

    SciTech Connect

    Sutherland, B.M.; Shih, A.G.

    1983-02-15

    We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs. DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels. From their migration relative to known molecular weight standards, their median molecular weight and thus the number of dimers per DNA molecule in each sample can be calculated. Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing. In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.

  20. Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis.

    PubMed

    Leach, T J; Glaser, R L

    1998-10-15

    Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect. PMID:9753752

  1. Electrophoresis of proteins and protein-protein complexes in native polyacrylamide gels using a horizontal gel apparatus.

    PubMed

    Su, C; Wang, F; Ciolek, D; Pan, Y C

    1994-11-15

    Electrophoresis of proteins and protein-protein complexes in polyacrylamide gels under native conditions using a horizontal gel apparatus is described. The advantage of this system is that it permits the detection of both negatively and positively charged proteins as well as protein-protein complexes in the same gel. During electrophoresis, a continuous gel sandwiched between two glass plates is placed horizontally on the platform and submerged in a reservoir buffer. The sample wells are made along the center of the gel, allowing positively and negatively charged proteins to migrate toward the cathode and anode, respectively. Several proteins with varying molecular weights and isoelectric point (pI) values and pairs of proteins capable of forming protein-protein complexes were chosen as model systems to illustrate the methodology. The effects of several parameters on the performance of the gel system including protein molecular weight, pI, and gel concentration were also examined and the results obtained by this method are comparable to those obtained by the vertical system. Following electrophoresis, both negatively and positively charged proteins as well as protein-protein complexes can be transferred by electroblotting onto polyvinylidene difluoride membranes for further analyses. PMID:7695108

  2. 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

  3. Capillary electrophoresis methods for the determination of covalent polyphenol-protein complexes.

    PubMed

    Trombley, John D; Loegel, Thomas N; Danielson, Neil D; Hagerman, Ann E

    2011-09-01

    The bioactivities and bioavailability of plant polyphenols including proanthocyanidins and other catechin derivatives may be affected by covalent reaction between polyphenol and proteins. Both processing conditions and gastrointestinal conditions may promote formation of covalent complexes for polyphenol-rich foods and beverages such as wine. Little is known about covalent reactions between proteins and tannin, because suitable methods for quantitating covalent complexes have not been developed. We established capillary electrophoresis methods that can be used to distinguish free protein from covalently bound protein-polyphenol complexes and to monitor polyphenol oxidation products. The methods are developed using the model protein bovine serum albumin and the representative polyphenol (-)epigallocatechin gallate. By pairing capillaries with different diameters with appropriate alkaline borate buffers, we are able to optimize resolution of either the protein-polyphenol complexes or the polyphenol oxidation products. This analytical method, coupled with purification of the covalent complexes by diethylaminoethyl cellulose chromatography, should facilitate characterization of covalent complexes in polyphenol-rich foods and beverages such as wine. PMID:21400190

  4. 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.

  5. Protein-protein interaction studies based on molecular aptamers by affinity capillary electrophoresis.

    PubMed

    Huang, Chih-Ching; Cao, Zehui; Chang, Huan-Tsung; Tan, Weihong

    2004-12-01

    Protein-DNA/protein-protein interactions play critical roles in many biological processes. We report here the investigation of protein-protein interactions using molecular aptamers with affinity capillary electrophoresis (ACE). A human alpha-thrombin binding aptamer was labeled with 6-carboxyfluorescein and exploited as a selective fluorescent probe for studying thrombin-protein interactions using capillary electrophoresis with laser-induced fluorescence. A 15-mer binding DNA aptamer can be separated into two peaks in CE that correspond to the linear aptamer (L-Apt) and the thrombin-binding G-quadruplex structure in the presence of K(+) or Ba(2+). In a bare capillary, the peak area of G-quadruplex aptamer (G-Apt) was found to decrease with the addition of thrombin while that of L-Apt remained unchanged. Even though the peak of the G-Apt/thrombin binding complex is broad due to a weaker binding affinity between aptamer and thrombin, we were still able to quantify the thrombin and anti-thrombin proteins (human anti-thrombin III, AT III) based on the peak areas of free G-Apt. The detection limits of thrombin and AT III were 9.8 and 2.1 nM, respectively. The aptamer-based competitive ACE assay has also been applied to quantify thrombin-anti-thrombin III interaction and to monitor this reaction in real time. The addition of poly(ethylene glycol) to the sample matrix stabilized the complex of the G-Aptthrombin. This assay can be used to study the interactions between thrombin and proteins that do not disrupt G-Apt binding property at Exosit I site of the thrombin. Our aptamer-based ACE assay can be an effective approach for studying protein-protein interactions and for analyzing binding site and binding constant information in protein-protein and protein-DNA interaction studies. PMID:15571349

  6. 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.

  7. On-column enzyme-catalyzed microreactions using capillary electrophoresis: quantitative studies.

    PubMed

    Zhang, Ying; Kaddis, John; Silverio, Catherine; Zurita, Cecilia; Gomez, Frank A

    2002-01-01

    Capillary electrophoresis and on-column enzyme-catalyzed microreactor techniques were used to quantitate the reaction projects resulting from three model systems: i) the conversion of nicotinamide adenine dinucleotide (NAD) to nicotinamide adenine dinucleotide, reduced form (NADH) in the oxidation of glucose-6-phosphate (glc-6-p) to 6-phosphogluconate by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49); ii) the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) by hexokinase (HK, EC 2.7.1.1) and apyrase (APY, EC 3.6.1.5), respectively, in the conversion of glucose to glucose-6-phosphate and inorganic phosphate, respectively, and; iii) the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13). Single and double microreactor techniques employing direct or indirect detection were used to follow the conversion of substrate to product(s). In addition, electrophoresis conditions including voltage, enzyme concentration, and mixing time of the reaction, were correlated to product distribution profiles. PMID:11936761

  8. Two-dimensional electrophoresis of plant proteins with phastsystem using nonequilibrium pH gradient separation.

    PubMed

    Ferullo, J M; Nespoulous, L

    1991-10-01

    We have adapted a two-dimensional electrophoretic technique described by P. Z. O'Farrell et al. (Cell 12, 1133-1142, 1977) to Phastsystem, resolving both acidic and basic proteins by using nonequilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. Protein separation was optimized for the analysis of plant proteins. The use of the Phastsystem apparatus reduced times of preparation and separation, allowing the rapid screening of plant proteins on a large scale of isoelectric points. This technique was used for the immunodetection and characterization of two stress-induced proteins in irradiated tomato leaves. PMID:1789413

  9. 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

  10. 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

  11. Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides.

    PubMed

    Grossman, P D; Colburn, J C; Lauer, H H; Nielsen, R G; Riggin, R M; Sittampalam, G S; Rickard, E C

    1989-06-01

    The application of free solution capillary electrophoresis (FSCE) to the separation of protein and peptide mixtures is presented. Both qualitative and quantitative aspects of FSCE separations are considered. In addition, a brief introduction describing the separation principle behind FSCE separations and a discussion of electrophoretic mobility are included. The applications were chosen in order to highlight the selectivity of FSCE separations and to demonstrate applications of potential practical interest to the bioanalytical chemist. Comparison of FSCE relative to traditional analytical separation alternatives is stressed throughout. The examples are presented in three broad categories: protein separations, peptide separations, and the application of both to the analysis of recombinant protein products. In the first section, FSCE separations of peptide mixtures are presented which demonstrate the suitability of FSCE for the analysis of the purity of peptide samples, the homogeneity of peptide samples prior to sequencing, the identity of peptides by using electrophoretic mobility values, and the reduction of an intrachain disulfide bridge. In the second section, protein separations are presented that show the resolution of glycoproteins having the same primary structure and the separation of immune complexes from free unreacted antibody and antigen. In the final section, highly purified and well-characterized samples of biosynthetic human insulin (BHI), biosynthetic human growth hormone (hGH), and their derivatives were used to evaluate FSCE as a complement and/or alternative to conventional analytical separation techniques for the determination of purity and identity of biosynthetic human proteins. In addition, the quantitative aspects of FSCE analysis such as linearity of response, precision, and limit of detection were examined. PMID:2757205

  12. 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...

  13. [Protein analysis of 6 crude drugs and their processed products by polyacrylamide gel electrophoresis technique].

    PubMed

    Shi, J; Sun, L; Jing, X

    1995-09-01

    In this paper, the proteins in 6 crude drugs (Prunus persica; P. armeniaca; Dolichos lablab; Strychnos nux-vomica; Mylabris phalerata; Whitmania pigra) and their processed products were analysed by polyacrylamide gel electrophoresis technique, and the effect of different processing methods on the quantity and kind of protein was explored. Protein electrophorograms of 20 samples are drawn. PMID:8679088

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

    PubMed

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

    2013-03-01

    Sieving of proteins in silica colloidal crystals of millimeter 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

  15. 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

  16. The determination of molecular weights of biologically active proteins by cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis.

    PubMed

    Akin, D T; Shapira, R; Kinkade, J M

    1985-02-15

    A novel cetyltrimethylammonium bromide-polyacrylamide gel electrophoresis system which is useful for the separation of native forms of proteins consistent with their molecular weights is reported here. Many proteins examined in this system demonstrated the same association patterns which have been shown by other techniques to exist under nondenaturing conditions. In addition, biological activity could be assayed directly in the gel after electrophoresis. Based on the peculiar characteristics of cetyltrimethylammonium bromide, a possible explanation which may account for the behavior of proteins in this system is presented. PMID:4003759

  17. 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 ...

  18. Intact Protein Quantitation Using Pseudoisobaric Dimethyl Labeling.

    PubMed

    Fang, Houqin; Xiao, Kaijie; Li, Yunhui; Yu, Fan; Liu, Yan; Xue, Bingbing; Tian, Zhixin

    2016-07-19

    Protein structural and functional studies rely on complete qualitative and quantitative information on protein species (proteoforms); thus, it is important to quantify differentially expressed proteins at their molecular level. Here we report our development of universal pseudoisobaric dimethyl labeling (pIDL) of amino groups at both the N-terminal and lysine residues for relative quantitation of intact proteins. Initial proof-of-principle study was conducted on standard protein myoglobin and hepatocellular proteomes (HepG2 vs LO2). The amino groups from both the N-terminal and lysine were dimethylated with HXHO (X = (13)C or C) and NaBY3CN (Y = H or D). At the standard protein level, labeling efficiency, effect of product ion size, and mass resolution on quantitation accuracy were explored; and a good linear quantitation dynamic range up to 50-fold was obtained. For the hepatocellular proteome samples, 33 proteins were quantified with RSD ≤ 10% from one-dimensional reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis of the 1:1 mixed samples. The method in this study can be extended to quantitation of other intact proteome systems. The universal "one-pot" dimethyl labeling of all the amino groups in a protein without the need of preblocking of those on the lysine residues is made possible by protein identification and quantitation analysis using ProteinGoggle 2.0 with customized databases of both precursor and product ions containing heavy isotopes. PMID:27359340

  19. Quantification by PIXE of metallic sites in proteins separated by electrophoresis

    NASA Astrophysics Data System (ADS)

    Strivay, D.; Schoefs, B.; Weber, G.

    1998-03-01

    Electrophoresis on polyacrylamide gel (PAGE) is widely used in life sciences to determine the molecular weight of proteins in solution by separating them into different bands. By coupling electrophoresis and Particle Induced X-ray Emission (PIXE), the nature and the quantity of metals contained in proteins can be investigated. After the electrophoresis, the gel is dried and each track is scanned with a 2.5 MeV proton beam which induces X-ray emission. Analysis of these spectra allows the determination of the metals contained in an electrophoretic band. The metal content in each band is obtained by comparing the characteristic X-ray peak area with those obtained with polyacrylamide gels doped with the same metal. Finally, the relative concentration of each protein is determined by densitometry in order to compute the protein/metal ratio. An example of metallic site determination is presented. This procedure seems to be a very useful multielementary method for the determination of the metal amounts inside proteins after their separation by electrophoresis. Furthermore it allows to check if metals remain bound to proteins.

  20. 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

  1. 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. PMID:12580515

  2. Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

    PubMed Central

    Velapatiño, Billie; Zlosnik, James E. A.; Hird, Trevor J.; Speert, David P.

    2013-01-01

    The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining. PMID:24192802

  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. 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

  5. Targeted quantitation of proteins by mass spectrometry.

    PubMed

    Liebler, Daniel C; Zimmerman, Lisa J

    2013-06-01

    Quantitative measurement of proteins is one of the most fundamental analytical tasks in a biochemistry laboratory, but widely used immunochemical methods often have limited specificity and high measurement variation. In this review, we discuss applications of multiple-reaction monitoring (MRM) mass spectrometry, which allows sensitive, precise quantitative analyses of peptides and the proteins from which they are derived. Systematic development of MRM assays is permitted by databases of peptide mass spectra and sequences, software tools for analysis design and data analysis, and rapid evolution of tandem mass spectrometer technology. Key advantages of MRM assays are the ability to target specific peptide sequences, including variants and modified forms, and the capacity for multiplexing that allows analysis of dozens to hundreds of peptides. Different quantitative standardization methods provide options that balance precision, sensitivity, and assay cost. Targeted protein quantitation by MRM and related mass spectrometry methods can advance biochemistry by transforming approaches to protein measurement. PMID:23517332

  6. Bargain Electrophoresis.

    ERIC Educational Resources Information Center

    Maderia, Vitor M. C.; Pires, Euclides M. V.

    1986-01-01

    Discusses the value of electrophoresis in the fields of protein chemistry and biochemistry. Describes how to build an inexpensive electrophoresis setup for use in either research or teaching activities. Details the construction of both the separating device and the power supply. (TW)

  7. Quantitation of specific proteins in polyacrylamide gels by the elution of Fast Green FCF.

    PubMed

    Gilmore, L B; Hook, G E

    1981-07-01

    The quantitation of proteins in polyacrylamide gels stained with Fast green FCF has been investigated using a modification of the elution technique originally described by Fenner et al. (Fenner, C., Traut, R.R., Mason, D.T. and Wikman-Coffelt, J. (1975) Anal. Biochem. 63, 595--602) for Coomassie Blue and adapted by Medugorac (Medugorac, I. (1979) Basic Res. Cardiol. 74, 406--416) for use with proteins stained with Fast Green FCF. The elution of dye from stained protein was accomplished using 1.0 M NaOH instead of aqueous pyridine as required by the original method. The primary advantages of our modification are that the time required for protein quantitation has been considerably reduced and the use of toxic organic solvents has been eliminated. We have investigated the applicability of the method of several different proteins and our results indicate: (a) The quantity of Fast Green FCF eluted from specific proteins is proportional to the quantity of protein applied to the gel, but varies for each individual protein. (b) The method allows quantitation over a very wide range of protein (1--800 micrograms). (c) Quantitation of protein is independent of the width of the stained bands as well as acrylamide concentration. (d) The method is applicable to gels of many types including disc, slab and continuous gradient gels. (e) Protein can be estimated from the patterns obtained by two-dimensional polyacrylamide gel electrophoresis. (f) The presence of Triton X-100 in gel and protein sample does not affect quantitation; the method is applicable to gels containing SDS provided that SDS is removed prior to staining. (g) Precipitation of protein with 12.5% TCA following electrophoresis does not interfere with quantitation. (h) The reproducibility of the technique is excellent, with standard deviations being less than 10% of the mean in all cases. This method appears highly versatile but requires appropriate standards for the quantitation of individual proteins. PMID:7276424

  8. Calf-ovary protein kinases dependent on adenosine 3':5' -monophosphate. Analysis by electrophoresis and electro-focusing on polyacrylamide get.

    PubMed

    Salokangas, A; Talmadge, K; Bechtel, E; Eppenberger, U; Chrambach, A

    1977-03-01

    High resolving power and quantitative application polyacrylamide-gel electrophopresis at various pore sizes and electrofocusing provide resolution of a calf-ovarian protein-kinase system at an increased level of magnification, as well as optimal preparative routes. Three protein kinases dependent on adenosine 3':5' -monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5--10.2, appear to be aggregates of a commonsubmit or monomer. The three kinases are, by the criteria of polyacylamide gel electrophoresis, distinct from three adenosine-3':5' -monophosphate-binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins. PMID:191253

  9. Acute phase protein and protein electrophoresis values for captive Grant's zebra (Equus burchelli).

    PubMed

    Cray, Carolyn; Hammond, Elizabeth; Haefele, Holly

    2013-12-01

    Grant's zebra (Equus burchelli) are commonly kept in zoos and are subject to routine health monitoring and research studies. Recently, assays for acute phase proteins (APP) have been described in many wildlife species, and specific assays for serum amyloid A (SAA) have been well validated and studied in horses (Equus ferus caballus), in which it serves as a major APP. In the present study, serum samples from 26 Grant's zebra were subject to analysis by using assays for SAA, haptoglobin (HP), and protein electrophoresis. Reference intervals were calculated by using the robust method: SAA 1.8-31.4 mg/L and HP 0.37-1.58 mg/ml. Significant differences in SAA and HP were observed in clinically abnormal zebra; in some cases, these differences were marked and were noted in the absence of abnormal values for protein electrophoretic fractions. These data indicate that APP may be a valuable and sensitive tool in monitoring inflammation in this species. PMID:24450080

  10. 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.

  11. Identification of methanococcus jannaschii proteins in 2-D gel electrophoresis patterns by mass spectrometry.

    SciTech Connect

    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.

  12. Polyacrylamide Slab Gel Electrophoresis of Soluble Proteins for Studies of Bacterial Floras

    PubMed Central

    Moore, W. E. C.; Hash, D. E.; Holdeman, Lillian V.; Cato, Elizabeth P.

    1980-01-01

    A polyacrylamide slab gel electrophoresis procedure was used to compare cellular proteins from bacterial isolates of gingival crevice floras. Isolates with identical protein patterns consistently were shown to be members of the same species. When used to screen isolates, the procedure reduced total analytical time and expense without sacrificing accuracy, and it provided additional verification of the identity of strains characterized by conventional phenotypic tests. Images PMID:16345555

  13. Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis.

    PubMed

    An, Philip; Winters, Dwight; Walker, Kenneth W

    2016-04-01

    Modern molecular genetics technology has made it possible to swiftly sequence, clone and mass-produce recombinant DNA for the purpose of expressing heterologous genes of interest; however, recombinant protein production systems have struggled to keep pace. Mammalian expression systems are typically favored for their ability to produce and secrete proteins in their native state, but bacterial systems benefit from rapid cell line development and robust growth. The primary drawback to prokaryotic expression systems are that recombinant proteins are generally not secreted at high levels or correctly folded, and are often insoluble, necessitating post-expression protein folding to obtain the active product. In order to harness the advantages of prokaryotic expression, high-throughput methods for executing protein folding screens and the subsequent analytics to identify lead conditions are required. Both of these tasks can be accomplished using a Biomek 3000 liquid handling robot to prepare the folding screen and to subsequently prepare the reactions for assessment using Caliper microfluidic capillary electrophoresis. By augmenting a protein folding screen with automation, the primary disadvantage of Escherichia coli expression has been mitigated, namely the labor intensive identification of the required protein folding conditions. Furthermore, a rigorous, quantitative method for identifying optimal protein folding buffer aids in the rapid development of an optimal production process. PMID:26678961

  14. Use of capillary electrophoresis and indirect detection to quantitate in-capillary enzyme-catalyzed microreactions.

    PubMed

    Zhang, Y; el-Maghrabi, M R; Gomez, F A

    2000-04-01

    The use of capillary electrophoresis and indirect detection to quantify reaction products of in-capillary enzyme-catalyzed microreactions is described. Migrating in a capillary under conditions of electrophoresis, plugs of enzyme and substrate are injected and allowed to react. Capillary electrophoresis is subsequently used to measure the extent of reaction. This technique is demonstrated using two model systems: the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate by fructose-biphosphate aldolase (ALD, EC 4.1.2.13), and the conversion of fructose-1,6-bisphosphate to fructose-6-phosphate by fructose-1,6-bisphospatase (FBPase, EC 3.1.3.11). These procedures expand the use of the capillary as a microreactor and offer a new approach to analyzing enzyme-mediated reactions. PMID:10892022

  15. 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

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

    PubMed Central

    Singh, Pramod Kumar; Shrivastava, Nidhi; Chaturvedi, Krishna

    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

  17. Modified capillary electrophoresis system for peptide, protein and double-stranded DNA analysis.

    PubMed

    Belenkii, B G; Kassalainen, G E; Nasledov, D G

    2000-05-26

    The results of high-performance capillary electrophoresis (HPCE) studies of peptide, protein and double-stranded DNA separations on a laboratory-made HPCE system are presented. Parameters of the HPCE system are given. The new method of capillary surface modification by grafting poly(glycidyl methacrylate) is described. The problems of HPCE biopolymer analysis connected with the sample-wall interactions are discussed. PMID:10893035

  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. Role of charge suppression and ionic strength in free zone electrophoresis of proteins.

    PubMed

    Compton, B J; O'Grady, E A

    1991-11-15

    The free zone electrophoretic mobility of proteins can be predicted from the protein's amino acid content by applying a model based on the Debye-Hückle-Henry theory and Henderson-Hasselbalch equation. Calculated mobilities are always greater than actual mobility but a pH-independent proportionality (described by the constant FZ) is found between the two. Thus, determination of a protein's mobility at one pH allows, with the use of the model and FZ, calculation of its mobility at other pH conditions. This leads directly to optimum conditions for the electrophoretic resolution of proteins in capillary zone electrophoresis. The fundamental nature of FZ is examined and found to be a function of a proteins molecular weight, charge, and solution ionic strength. This work aids in explaining the form of previously proposed empirically based equations for peptide and protein mobility. PMID:1776698

  2. Proteomic Profiling Of Two-Dimensional Gel Electrophoresis Protein Expression Data

    NASA Astrophysics Data System (ADS)

    Ahmad, Norhaiza; Zhang, J.; Brown, P. J.; James, D. C.; Birch, J. R.; Racher, A. J.; Smales, C. M.

    2008-01-01

    We have undertaken two-dimensional gel electrophoresis (2-DE) proteomic profiling on a series of cell lines with different recombinant antibody production rates. Due to the nature of 2-DE proteomic investigations there will always be `process variability' factors in any data set collected in this way. Some of this variation will arise during sample preparation, gel running and staining, while further variation will arise from the gel analysis procedure. Therefore, in order to identify all significant changes in protein expression between biological samples when analysed by 2-DE, the system precision or `error', and how this correlates to protein abundance, must be known. Only then can the system be considered robust and investigators accurately and confidently report all observable statistically significant changes in protein expression. We introduce an expression variability test to identify protein spots whose expression correlates with increased antibody production. The results have highlighted a small number of candidate proteins for further investigation.

  3. 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

  4. 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. PMID:25959826

  5. 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

  6. Capillary electrophoresis of proteins in buffers containing high concentrations of zwitterionic salts.

    PubMed

    Bushey, M M; Jorgenson, J W

    1989-10-20

    A method for improving protein separations in capillary zone electrophoresis utilizing high concentrations of zwitterionic buffer additives was examined. Lysozyme and alpha-chymotrypsinogen A were used as test proteins in untreated fused-silica capillaries in buffers of pH ca. 7.0 and 9.0 The zwitterion-containing buffers were compared with buffers containing high ionic salt concentrations and a buffer containing a combination of high ionic salt and high zwitterion concentrations. Over 100,000 theoretical plates were obtained in less than 30 min. for both test proteins in a pH 7 buffer containing both trimethylglycine and potassium sulfate. The advantages and disadvantages of this technique compared with those of other methods used to prevent protein adsorption are discussed. PMID:2592485

  7. 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.

  8. High resolution protein electrophoresis of 100 paired canine cerebrospinal fluid and serum.

    PubMed

    Behr, Sébastien; Trumel, Cathy; Cauzinille, Laurent; Palenché, Florence; Braun, Jean-Pierre

    2006-01-01

    This study was performed to investigate the diagnostic relevance of cerebrospinal fluid (CSF) high resolution electrophoresis. The laboratory technique was applied to 100 paired samples of canine CSF and serum, with paired samples tested during the same analytical run, as recommended in human medicine. Ninety four of the dogs had a neurological disease and 6 healthy dogs served as a control group. A strong linear correlation between CSF total protein concentration and the albumin quota (AQ) was found in the control group and in the inflammatory (infectious or noninfectious), neoplastic, and miscellaneous groups: AQ = 0.015 CSF total protein--0.102, r = 0.990. This correlation suggests that an increased CSF total protein concentration can be an indicator of blood brain barrier dysfunction. The highest median AQ value was found in the aseptic suppurative meningitis group, but no statistical differences were found between this and the other groups. The AQ, calculated with this technique, did not provide any additional information. Moreover, although unexpected, the electrophoretic profiles were not characteristic of any particular disease. In conclusion, this study did not confirm high resolution electrophoresis of paired CSF and serum samples to be a valuable ancillary diagnostic tool for canine neurological diseases. PMID:16734104

  9. Silica colloidal crystals as emerging materials for high-throughput protein electrophoresis.

    PubMed

    Njoya, Nadine K; Birdsall, Robert E; Wirth, Mary J

    2013-10-01

    Silica colloidal crystals are a new type of media for protein electrophoresis, and they are assessed for their promise in rapidly measuring aggregation of monoclonal antibodies. The nature of silica colloidal crystals is described in the context of the need for a high-throughput separation tool for optimizing the formulations of protein drugs for minimal aggregation. The fundamental relations between molecular weight and mobility in electrophoresis are used to make a theoretical comparison of selectivity between gels and colloidal crystals. The results show that the selectivity is similar for these media, but slightly higher, 10%, for gels, and the velocity is inherently lower than that for gels due to the smaller free volume fraction. These factors are more than compensated for by lower broadening in colloidal crystals. These new media give plate heights of only 0.15 μm for the antibody monomer and 0.42 μm for the antibody dimer. The monoclonal antibody is separated from its dimer in 72 s over a distance of only 6.5 mm. This is five times faster than size-exclusion chromatography, with more than tenfold miniaturization, and amenable to parallel separations, all of which are promising for the design of high-throughput devices for optimizing protein drug formulations. PMID:23800834

  10. 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

  11. Should routine laboratories stop doing screening serum protein electrophoresis and replace it with screening immune-fixation electrophoresis? No quick fixes: Counterpoint.

    PubMed

    Smith, Joel D; Raines, Geoffrey; Schneider, Hans G

    2016-06-01

    Monoclonal gammopathies are characterised by the production of a monoclonal immunoglobulin or free light chains by an abnormal plasma cell or B-cell clone and may indicate malignancy or a precursor (MGUS). There is currently no consensus on the initial test or combination of tests to be performed in suspected monoclonal gammopathies but serum protein electrophoresis and urine protein electrophoresis are commonly requested as initial investigations. If abnormal, immunofixation electrophoresis is then performed to confirm the presence of paraprotein and to determine its heavy and light chain type. Recently, some groups have developed simplified "screening" IFE methods for use in parallel to SPEP for the detection monoclonal gammopathies. We argue here that screening IFE may be of benefit in clinical laboratories using SPEP with poor resolution in the β-region, assisting in the detection of mainly IgA paraprotein, but may be of less benefit in laboratories utilising higher resolution gels. Further it may increase the detection of trace bands of questionable clinical significance, representing transient phenomena in infectious and auto-immune conditions or very low risk MGUS. The increased detection of these bands using screening IFE would require further patient follow up, possibly causing unnecessary patient anxiety and additional follow up healthcare costs. PMID:26677889

  12. Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection.

    PubMed

    Kaneta, Takashi; Yamamoto, Daisuke; Imasaka, Totaro

    2009-11-01

    The separation methods for proteins with high resolution and sensitivity are absolutely important in the field of biological sciences. Capillary sieving electrophoresis (CSE) is an excellent separation technique for DNA and proteins with high resolution, while LIF permits the most sensitive detection in CSE. Therefore, proteins have to be labeled with fluorescent or fluorogenic reagent to produce fluorescent derivatives. Both precolumn and oncolumn derivatization have been employed for the labeling of proteins in CSE. However, there is no report on the postcolumn derivatization due to the limitation in the use of a standard migration buffer, despite it being a promising method for sensitive detection of proteins. Here, we show a novel postcolumn derivatization method for protein separation by CSE, using a tertiary amine as a buffer component in the running buffer. Tris, which is commonly used as a base in CSE separation buffers, was substituted by tertiary amines, 2-(diethylamino)ethanol and triethanolamine. A buffer solution containing 2-(diethylamino)ethanol or triethanolamine can be used for the CSE separation followed by the postcolumn derivatization of proteins, since both reagents are unreactive toward a fluorogenic labeling reagent, naphthalene-2,3-dicarbaldehyde. Thus, LIF detection using the postcolumn derivatization permits significant reduction in the LOD (by a factor of 2.4-28) of proteins, compared with conventional absorbance detection. PMID:19862753

  13. Analysis of Blastocladiella emersonii ribosomal proteins in four two-dimensional gel electrophoresis systems.

    PubMed

    Bonato, M C; Maia, J C; Juliani, M H

    1985-01-01

    Ribosomal proteins of the aquatic fungus Blastocladiella emersonii were isolated and characterized on four different two-dimensional polyacrylamide gel electrophoresis systems. 40S and 60S ribosomal subunit proteins from zoospores were identified. The position of every protein was determined in each electrophoretic system using the "four-corners" method (Madjar et al., Molecular and General Genetics, 171: 121-134, 1979). Thirty-two and 39 proteins were identified in the 40S and 60S ribosomal subunits, respectively. The molecular weights of individual proteins in the 40S subunit ranged from 10 000 to 37 000, with a number-average molecular weight of 20 000. The molecular weight range for the 60S subunit was 13 000-51 000 with a number-average molecular weight of 21 000. Proteins from ribosomes of different cell types were compared and found to be qualitatively indistinguishable. The only consistent difference in the patterns of proteins was in the S6 protein of the 40S subunit, which is the major phosphoprotein of Blastocladiella ribosomes. PMID:3830281

  14. Automated SDS Depletion for Mass Spectrometry of Intact Membrane Proteins though Transmembrane Electrophoresis.

    PubMed

    Kachuk, Carolyn; Faulkner, Melissa; Liu, Fang; Doucette, Alan A

    2016-08-01

    Membrane proteins are underrepresented in proteome analysis platforms because of their hydrophobic character, contributing to decreased solubility. Sodium dodecyl sulfate is a favored denaturant in proteomic workflows, facilitating cell lysis and protein dissolution; however, SDS impedes MS detection and therefore must be removed prior to analysis. Although strategies exist for SDS removal, they provide low recovery, purity, or reproducibility. Here we present a simple automated device, termed transmembrane electrophoresis (TME), incorporating the principles of membrane filtration, but with an applied electric current to ensure near-complete (99.9%) removal of the surfactant, including protein-bound SDS. Intact proteins are recovered in solution phase in high yield (90-100%) within 1 h of operation. The strategy is applied to protein standards and proteome mixtures, including an enriched membrane fraction from E. coli, resulting in quality MS spectra free of SDS adducts. The TME platform is applicable to both bottom-up MS/MS as well as LC-ESI-MS analysis of intact proteins. SDS-depleted fractions reveal a similar number of protein identifications (285) compared wit a non-SDS control (280), being highly correlated in terms of protein spectral counts. This fully automated approach to SDS removal presents a viable tool for proteome sample processing ahead of MS analysis. Data are available via ProteomeXchange, identifier PXD003941. PMID:27376408

  15. Quantitation of active ingredients and excipients in nasal sprays by high-performance liquid chromatography, capillary electrophoresis and UV spectroscopy.

    PubMed

    Bernal, J L; del Nozal, M J; Martín, M T; Diez-Masa, J C; Cifuentes, A

    1998-10-01

    A study on the use of different analytical methodologies to determine active ingredients and excipients found in commercial nasal sprays is presented. Two of the developed methodologies consisted of separation techniques, i.e. high-performance liquid chromatography and capillary electrophoresis, and the third one involved a UV-spectroscopic multicomponent procedure. The samples studied are characterized by a high viscosity and the existence of a large number of particles in suspension; therefore, special emphasis is paid on the sample preparation required by each methodology. Advantages and drawbacks of each analytical technique are also discussed in terms of speed of analysis, sensitivity and reproducibility. From this work it is observed that although the UV method needs the most laborious sample preparation, the total time required per analysis is the shortest one. The best reproducibility in terms of analysis time and quantitation of the analyzed compounds is obtained using HPLC. CE allows the determination of more components in the same sample. PMID:9818419

  16. 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. PMID:25314816

  17. 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

  18. Two-dimensional polyacylamide gel electrophoresis of envelope proteins of Escherichia coli.

    PubMed

    Johnson, W C; Silhavy, T J; Boos, W

    1975-03-01

    A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight. PMID:803821

  19. Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: part 3: anions.

    PubMed

    Xu, Yuanhong; Redweik, Sabine; El-Hady, Deia Abd; Albishri, Hassan M; Preu, Lutz; Wätzig, Hermann

    2014-08-01

    The binding of physiologically anionic species or negatively charged drug molecules to proteins is of great importance in biochemistry and medicine. Since affinity capillary electrophoresis (ACE) has already proven to be a suitable analytical tool to study the influence of ions on proteins, this technique was applied here for comprehensively studying the influence of various anions on proteins of BSA, β-lactoglobulin, ovalbumin, myoglobin, and lysozyme. The analysis was performed using different selected anions of succinate, glutamate, phosphate, acetate, nitrate, iodide, thiocyanate, and pharmaceuticals (salicylic acid, aspirin, and ibuprofen) that exist in the anionic form at physiological pH 7.4. Due to the excellent repeatability and precision of the ACE measurements, not necessarily strong but significant influences of the anions on the proteins were found in many cases. Different influences in the observed bindings indicated change of charge, mass, or conformational changes of the proteins due to the binding with the studied anions. Combining the mobility-shift and pre-equilibrium ACE modes, rapidity and reversibility of the protein-anion bindings were discussed. Further, circular dichroism has been used as an orthogonal approach to characterize the interactions between the studied proteins and anions to confirm the ACE results. Since phosphate and various anions from amino acids and small organic acids such as succinate or acetate are present in very high concentrations in the cellular environment, even weak influences are certainly relevant as well. PMID:24436007

  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. Immunofixation electrophoresis: a technique for the study of protein polymorphism. Vox Sang 1969:17:445-52.

    PubMed

    Alper, C A; Johnson, A M

    1993-01-01

    A technique is described which allows direct visualization of individual proteins in mixtures by specific antiserum after electrophoresis. By minimizing diffusion it permits rapid, direct, and clear detection of genetic polymorphism and 'conversion' of proteins in the complement and coagulation systems. PMID:8362522

  3. Quantitative Proteomics of Caveolin-1-regulated Proteins

    PubMed Central

    Dávalos, Alberto; Fernández-Hernando, Carlos; Sowa, Grzegorz; Derakhshan, Behrad; Lin, Michelle I.; Lee, Ji Y.; Zhao, Hongyu; Luo, Ruiyan; Colangelo, Christopher; Sessa, William C.

    2010-01-01

    Caveolae are organelles abundant in the plasma membrane of many specialized cells including endothelial cells (ECs), epithelial cells, and adipocytes, and in these cells, caveolin-1 (Cav-1) is the major coat protein essential for the formation of caveolae. To identify proteins that require Cav-1 for stable incorporation into membrane raft domains, a quantitative proteomics analysis using isobaric tagging for relative and absolute quantification was performed on rafts isolated from wild-type and Cav-1-deficient mice. In three independent experiments, 117 proteins were consistently identified in membrane rafts with the largest differences in the levels of Cav-2 and in the caveola regulatory proteins Cavin-1 and Cavin-2. Because the lung is highly enriched in ECs, we validated and characterized the role of the newly described protein Cavin-1 in several cardiovascular tissues and in ECs. Cavin-1 was highly expressed in ECs lining blood vessels and in cultured ECs. Knockdown of Cavin-1 reduced the levels of Cav-1 and -2 and weakly influenced the formation of high molecular weight oligomers containing Cav-1 and -2. Cavin-1 silencing enhanced basal nitric oxide release from ECs but blocked proangiogenic phenotypes such as EC proliferation, migration, and morphogenesis in vitro. Thus, these data support an important role of Cavin-1 as a regulator of caveola function in ECs. PMID:20585024

  4. 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. PMID:24607654

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

    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.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

  6. Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry.

    PubMed

    Mamone, Gianfranco; Addeo, Francesco; Chianese, Lina; Di Luccia, Aldo; De Martino, Alessandra; Nappo, Annunziata; Formisano, Annarita; De Vivo, Pasqualina; Ferranti, Pasquale

    2005-07-01

    A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties. PMID:15952231

  7. Difference gel electrophoresis identifies differentially expressed proteins in endoscopically collected pancreatic fluid.

    PubMed

    Paulo, Joao A; Lee, Linda S; Banks, Peter A; Steen, Hanno; Conwell, Darwin L

    2011-08-01

    Alterations in the pancreatic fluid proteome of individuals with chronic pancreatitis (CP) may offer insights into the development and progression of the disease. The endoscopic pancreatic function test (ePFT) can safely collect large volumes of pancreatic fluid that are potentially amenable to proteomic analyses using difference gel electrophoresis (DIGE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pancreatic fluid was collected endoscopically using the ePFT method following secretin stimulation from three individuals with severe CP and three chronic abdominal pain (CAP) controls. The fluid was processed to minimize protein degradation and the protein profiles of each cohort, as determined by DIGE and LC-MS/MS, were compared. This DIGE-LC-MS/MS analysis reveals proteins that are differentially expressed in CP compared with CAP controls. Proteins with higher abundance in pancreatic fluid from CP individuals include: actin, desmoplankin, α-1-antitrypsin, SNC73, and serotransferrin. Those of relatively lower abundance include carboxypeptidase B, lipase, α-1-antichymotrypsin, α-2-macroglobulin, actin-related protein (Arp2/3) subunit 4, glyceraldehyde-3-phosphate dehydrogenase, and protein disulfide isomerase. Endoscopic collection (ePFT) in tandem with DIGE-LC-MS/MS is a suitable approach for pancreatic fluid proteome analysis; however, further optimization of our protocol, as outlined herein, may improve proteome coverage in future analyses. PMID:21792986

  8. Serum protein electrophoresis under effective control of HIV-1 disease progression

    PubMed Central

    Adedeji, Adebayo Lawrence; Adenikinju, Rufus Omotayo; Ajele, Joshua Olufemi; Olawoye, Theophilus Ladapo

    2014-01-01

    In this report, we compared the serum protein electrophoresis (SPE) patterns in a subset of HIV-1-infected subjects who did not progress to AIDS without antiretroviral treatment with those in whose control of disease progression was achieved by highly active antiretroviral therapy (HAART). SPE and immunofixation electrophoresis were performed on Helena Electrophoresis System according to manufacturer’s instructions. The percentage of SPE abnormalities, resembling chronic inflammation, was significantly higher in HIV-1-infected subject without HAART compared with those under HAART (p = 0.001). The majority of individuals under HAART showed evidence of oligoclonal bands on the γ-band against a polyclonal background compared with those without HAART but ß-γ-band bridging was more evident. Immunofixation pattern was consistent with oligoclonal hypergammaglobulinaemia of IgG kappa type, which was found to be more intense in group without HAART. HIV clinical status did not show appreciable effect on the SPE pattern in subjects without HAART. However, under effective HAART, subjects with better CD4 T-cell count were associated with higher γ-globulin band. In group without HAART, acute infection was found to be associated the higher γ-globulin fraction compared with chronic infection. The opposite was the case under effective HAART. HIV infected subjects that did not progress to AIDS were associated with markedly abnormal SPE pattern. Overall results reflect the host ability compensate defective cellular immunity in HIV-1 infection with humoral immune responses. These findings underscore the usefulness of SPE monitoring HIV disease management and identifying individuals that may not progress to full-blown AIDS in the absence of treatment. PMID:26417299

  9. 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. PMID:21755171

  10. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary protein in acute kidney injury.

    PubMed

    Suhail, Sufi M; Woo, K T; Tan, H K; Wong, K S

    2011-07-01

    Recent experimental and clinical studies have shown the importance of urinary proteomics in acute kidney injury (AKI). We analyzed the protein in urine of patients with clinical AKI using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for its diagnostic value, and followed them up for 40 months to evaluate prognosis. Urine from 31 consecutive cases of AKI was analyzed with SDS-PAGE to determine the low, middle and high molecular weight proteins. Fractional excretion of sodium (FENa) was estimated from serum and urine creatinine and sodium (Na). The cases were followed-up for 40 months from the end of the recruitment of study cases. Glomerular protein was higher in the hematuria group when compared with the non-hematuria group (P <0.04) and in the AKI group than in the acute on chronic renal failure (AKI-on-CRF) group (P <0.002). Tubular protein was higher in the AKI-on-CRF group (P <0.003) than in the AKI group. Tubular protein correlated with FENa in groups with diabetes mellitus (DM), AKI-on-CRF, and without hematuria (P <0.03, P <0.02 and P <0.004, respectively). Pattern of protein did not differ between groups with and without DM and clinical acute tubular necrosis (ATN). At the end of 40 months follow-up, category with predominantly glomerular protein progressed to chronic renal failure (CRF) or end-stage renal failure in higher proportion (P <0.05). In clinical AKI, we observed that glomerular protein dominated in cases with glomerular insult, as indicated by hematuria. Tubular protein was common in the study cases with CRF, DM and cases without hematuria. This indicates tubulo-interstitial injury for AKI in these cases. Patients with predominantly glomerular protein had an adverse outcome. PMID:21743220

  11. 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

  12. Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence.

    PubMed

    Wang, Siyang; Circu, Magdalena L; Zhou, Hu; Figeys, Daniel; Aw, Tak Y; Feng, June

    2011-09-23

    S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions. PMID:21820121

  13. Evaluation of capillary zone electrophoresis for the determination of protein composition in therapeutic immunoglobulins and human albumins.

    PubMed

    Christians, Stefan; van Treel, Nadine Denise; Bieniara, Gabriele; Eulig-Wien, Annika; Hanschmann, Kay-Martin; Giess, Siegfried

    2016-07-01

    Capillary zone electrophoresis (CZE) provides an alternative means of separating native proteins on the basis of their inherent electrophoretic mobilities. The major advantage of CZE is the quantification by UV detection, circumventing the drawbacks of staining and densitometry in the case of gel electrophoresis methods. The data of this validation study showed that CZE is a reliable assay for the determination of protein composition in therapeutic preparations of human albumin and human polyclonal immunoglobulins. Data obtained by CZE are in line with "historical" data obtained by the compendial method, provided that peak integration is performed without time correction. The focus here was to establish a rapid and reliable test to substitute the current gel based zone electrophoresis techniques for the control of protein composition of human immunoglobulins or albumins in the European Pharmacopoeia. We believe that the more advanced and modern CZE method described here is a very good alternative to the procedures currently described in the relevant monographs. PMID:27156142

  14. 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. PMID:23409432

  15. Identification of Drosophila indirect flight muscle myofibrillar proteins by means of two-dimensional electrophoresis.

    PubMed

    Mogami, K; Fujita, S C; Hotta, Y

    1982-02-01

    When proteins of whole Drosophila thorax were analyzed by two-dimensional gel electrophoresis, 186 spots were detected by protein staining with Coomassie brilliant blue R-250. Two methods were developed to identify proteins which exist in indirect flight muscle (IFM) and its myofibrils. 1) A whole fly was freeze-dried in a dry ice-acetone mixture, and indirect flight muscle fibers were cleanly dissected out from the thorax. The muscle cells and the rest of the thorax were analyzed separately. The muscle contained 146 polypeptides, of which 12 were not detected elsewhere. 2) Flies were frozen in liquid nitrogen and shaken vigorously so that their thoraces broke off from heads and abdomens. The thoraces were separated from the rest by sieving and centrifugation. After homogenization of the thorax, myofibrils were prepared by centrifugation in a discontinuous sucrose density gradient. The myofibril fraction contained at least 20 proteins. There were two types of actin (II and III), myosin heavy chain, tropomyosin and paramyosin. Nine of the other myofibrillar proteins were specific to this muscle. PMID:6802813

  16. 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.

  17. In-gel staining of proteins in native polyacrylamide gel electrophoresis using meso-tetrakis(4-sulfonatophenyl) porphyrin.

    PubMed

    Divakar, K; Devi, G Nandhini; Gautam, Pennathur

    2012-01-01

    Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining, and destaining of the gel, which are time-consuming and cumbersome. A new method for direct visualization of protein bands in PAGE has been developed using meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a dye without the need for any post-electrophoretic steps; thus, separation and recovery of enzymes become much easier for further analysis. Activity staining was carried out to show that the biochemical activity of the enzymes was preserved after electrophoresis. PMID:22585523

  18. In-gel staining of proteins in native poly acryl amide gel electrophoresis using tetrakis(4-sulfonato phenyl)porphyrin.

    PubMed

    Divakar, Kalivarathan; Sujatha, Vijayan; Barath, Sridhar; Srinath, Krishnamurthy; Gautam, Pennathur

    2011-01-01

    Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining and destaining of the gel, which are time-consuming and cumbersome. We have developed a method for direct visualization of protein bands in PAGE using tetrakis(4-sulfonato phenyl)porphyrin (TPPS) as a dye without the need for any post electrophoretic steps, where separation and recovery of enzymes become much easier for further analysis. Activity staining was done to prove that the biochemical activity of the enzymes was preserved after electrophoresis. PMID:21233569

  19. Definition of the quantitative contents of gossypol in selection samples of cotton by capillary electrophoresis method

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oil-seed meal from cotton seed contains high-quality protein and can be used in the animal industry. However, its application is limited by the presence of a poisonous substance called gossypol. There is a need to analyze the amount of gossypol in cottonseed as part of the current breeding program...

  20. Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two-dimensional gel electrophoresis.

    PubMed

    Parkhey, Suruchi; Chandrakar, Vibhuti; Naithani, S C; Keshavkant, S

    2015-10-01

    Protein extraction for two-dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two-dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one-dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77-95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants. PMID:26257211

  1. 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

  2. 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. PMID:25863608

  3. 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.

  4. Non-denaturing gel electrophoresis system for the purification of membrane bound proteins

    SciTech Connect

    Cavinato, A.G.; Macleod, R.M.; Ahmed, M.S.

    1988-01-01

    A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an /sup 3/H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.

  5. Previsible silver staining of protein in electrophoresis gels with mass spectrometry compatibility.

    PubMed

    Jin, Li-Tai; Li, Xiao-Kun; Cong, Wei-Tao; Hwang, Sun-Young; Choi, Jung-Kap

    2008-12-15

    A convenient silver staining method for protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels is described. The method is previsible, sensitive, and mass spectrometry (MS) compatible. Two visible counter ion dyes, ethyl violet (EV) and zincon (ZC), were used in the first staining solution with a detection limit of 2 to 8 ng/band in approximately 1h. The dye-stained gel can be further stained by silver staining, which is based on acidic silver staining employing ZC with sodium thiosulfate as silver ion sensitizers. Especially, ZC has silver ion reducing power by cleavage of the diazo bond of the dye during silver reduction. The second silver staining can be completed in approximately 1h with a detection limit of 0.2 ng/band. PMID:18804088

  6. Electrophoresis of DNA-protein complexes in polymer solutions: from free-flow to gels

    NASA Astrophysics Data System (ADS)

    Slater, Gary W.; Desruisseaux, Claude; Drouin, Guy

    2000-03-01

    We previously showed that labeling one of the ends of single-stranded DNA molecules with a neutral label like the protein streptavidin increases the interband separation of these hybrid molecules when they are electrophoresed in gels because of strong steric trapping effects. In 1999, we also demonstrated that these labeled DNA molecules can be sequenced in free-solution, a novel separation process that we called ELFSE. Here, we examine the fascinating intermediate regime where the streptavidin-DNA molecules are electrophoresed in polymer solutions of increasing concentrations, from ultra-dilute to fully entangled conditions. Our capillary electrophoresis results clarify the respective roles of friction, polymer capture,reptation and steric trapping. In some cases, two separation regimes coexist and the mobility becomes a non-monotonic function of the DNA size. A universal relationship is found to relate the mobility of labeled and unlabeled DNA molecules for all systems.

  7. Performing Isoelectric Focusing and Simultaneous Fractionation of Proteins on A Rotary Valve Followed by Sodium Dodecyl – Polyacrylamide Gel Electrophoresis

    PubMed Central

    Wang, Wei; Lu, Joann J.; Gu, Congying; Zhou, Lei; Liu, Shaorong

    2013-01-01

    In this technical note, we design and fabricate a novel rotary valve and demonstrate its feasibility for performing isoelectric focusing and simultaneous fractionation of proteins, followed by sodium dodecyl – polyacrylamide gel electrophoresis. The valve has two positions. In one position, the valve routes a series of capillary loops together into a single capillary tube where capillary isoelectric focusing (CIEF) is performed. By switching the valve to another position, the CIEF-resolved proteins in all capillary loops are isolated simultaneously, and samples in the loops are removed and collected in vials. After the collected samples are briefly processed, they are separated via sodium dodecyl – polyacrylamide gel electrophoresis (SDS-PAGE, the 2nd-D separation) on either a capillary gel electrophoresis instrument or a slab-gel system. The detailed valve configuration is illustrated, and the experimental conditions and operation protocols are discussed. PMID:23819755

  8. 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

  9. 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

  10. 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.

  11. Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide.

    PubMed

    Wu, D; Regnier, F E

    1992-09-11

    Proteins with relative molecular masses of 14,000 to 205,000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved. PMID:1430034

  12. Surface-modified poly(methyl methacrylate) capillary electrophoresis microchips for protein and peptide analysis.

    PubMed

    Liu, Jikun; Pan, Tao; Woolley, Adam T; Lee, Milton L

    2004-12-01

    Polymeric materials have emerged as appealing alternatives to conventional inorganic substrates for the fabrication of microscale analytical systems; however, native polymeric surfaces typically require covalent modification to ensure optimum biocompatibility. 2-Bromoisobutyryl bromide was immobilized on poly(methyl methacrylate) (PMMA) substrates activated using an oxygen plasma. Atom-transfer radical polymerization was then performed to graft poly(ethylene glycol) (PEG) on the PMMA surface. PMMA microcapillary electrophoresis (muCE) devices made with the covalently modified surfaces exhibited substantially reduced electroosmotic flow and nonspecific adsorption of proteins on microchannel surfaces. Experiments using fluorescein isothiocyanate-conjugated bovine serum albumin indicated that both column efficiency and migration time reproducibility were 1 order of magnitude better with derivatized compared to untreated PMMA muCE chips. Fast, reproducible, and efficient separations of proteins and peptides were demonstrated using the PEG-grafted PMMA muCE chips. All analyses were completed in less than 60 s, and separation efficiencies as high as 5.2 x10(4) plates for a 3.5-cm-long separation channel were obtained. These results demonstrate the general applicability of surface-grafted PMMA microdevices for a broad range of protein analyses. PMID:15571346

  13. 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

  14. Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis.

    PubMed

    Jaskowski, Troy D; Litwin, Christine M; Hill, Harry R

    2006-02-01

    Recently, turbidimetric immunoassays for detecting and quantifying kappa and lambda free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound kappa and lambda light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the kappa-FLC and lambda-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal kappa/lambda FLC ratios using the turbidimetric immunoassays. In conclusion, the kappa and lambda FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the kappa/lambda ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal kappa/lambda FLC ratios. PMID:16467338

  15. 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.

  16. 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. PMID:25638307

  17. 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.

  18. 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.

  19. A Novel Gaussian Extrapolation Approach for 2-D Gel Electrophoresis Saturated Protein Spots.

    PubMed

    Natale, Massimo; Caiazzo, Alfonso; Ficarra, Elisa

    2016-01-01

    Analysis of images obtained from two-dimensional gel electrophoresis (2-D GE) is a topic of utmost importance in bioinformatics research, since commercial and academic software currently available have proven to be neither completely effective nor fully automatic, often requiring manual revision and refinement of computer generated matches. In this chapter, we present an effective technique for the detection and the reconstruction of over-saturated protein spots. Firstly, the algorithm reveals overexposed areas, where spots may be truncated, and plateau regions caused by smeared and overlapping spots. Next, it reconstructs the correct distribution of pixel values in these overexposed areas and plateau regions, using a two-dimensional least-squares fitting based on a generalized Gaussian distribution. Pixel correction in saturated and smeared spots allows more accurate proteins quantification, providing more reliable image analysis results. The method is validated for processing highly exposed 2-D GE images, comparing reconstructed spots with the corresponding non-saturated image. The results demonstrate that the algorithm enables correct spot quantification. PMID:26611417

  20. 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.

  1. 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

  2. Hybrid Phospholipid Bilayer Coatings for Separations of Cationic Proteins in Capillary Zone Electrophoresis

    PubMed Central

    Gallagher, Elyssia S.; Adem, Seid M.; Bright, Leonard K.; Calderon, Isen A. C.; Mansfield, Elisabeth; Aspinwall, Craig A.

    2014-01-01

    Protein separations in capillary zone electrophoresis (CZE) suffer from non-specific adsorption of analytes to the capillary surface. Semi-permanent phospholipid bilayers (PLBs) have been used to minimize adsorption, but must be regenerated regularly to ensure reproducibility. We investigated the formation, characterization, and use of hybrid phospholipid bilayers (HPBs) as more stable biosurfactant capillary coatings for CZE protein separations. HPBs are formed by covalently modifying a support with a hydrophobic monolayer onto which a self-assembled lipid monolayer is deposited. Monolayers prepared in capillaries using 3-cyanopropyldimethylchlorosilane (CPDCS) or n-octyldimethylchlorosilane (ODCS) yielded hydrophobic surfaces with lowered surface free energies of 6.0 ± 0.3 or 0.2 ± 0.1 mJ m−2, respectively, compared to 17 ± 1 mJ m−2 for bare silica capillaries. HPBs were formed by subsequently fusing vesicles comprised of 1,2-dilauroyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine to CPDCS- or ODCS-modified capillaries. The resultant HPB coatings shielded the capillary surface and yielded reduced electroosmotic mobility (1.3 – 1.9 × 10−4 cm2 V−1s−1) compared to CPDCS- and ODCS-modified or bare capillaries (3.6 ± 0.2 × 10−4 cm2 V−1s−1, 4.8 ± 0.4 × 10−4 cm2 V−1s−1, and 6.0 ± 0.2 × 10−4 cm2 V−1s−1, respectively), with increased stability compared to PLB coatings. HPB-coated capillaries yielded reproducible protein migration times (RSD ≤ 3.6 %, n ≥ 6) with separation efficiencies as high as 200,000 plates m−1. PMID:24459085

  3. 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

  4. 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.

  5. [Genetic diversity and phylogenetic relationships among Chinese Macacas based on protein electrophoresis].

    PubMed

    Su, B; Wang, W; Lan, H; Zhang, Y

    1997-04-01

    In this paper, using protein electrophoresis method, we studied proteinpolymorphism and genetic divergence of 5 species in Genus Macaca: M. mulatta, M. arctoides, M. assamensis, M. thibetana, M. fascicularis. A total of 30 genetic loci were analyzed for 29 individuals, including 4 Nycticebus pygmaeus as outgroup. For the 19 M. mulatta, 9 loci were found to be polymorphic. Accordingly, the percentage of polymorphic loci, P = 0.3; the mean number of alleles, A = 1.4, and the mean heterozygosity, H = 0.1045, indicating a rather high level of genetic diversity in this species. Furthermore, 10 loci showed polymorphic among the 5 species, which can be used as information loci for phylogenetic reconstruction. Three programs (conml, neighbor, fitch) in PHYLIP 3.5 c were chosen to construct phylogenetic trees. All of the three trees show support a close relationship between M. mulatta and M. fascicularis. However, two trees have the same topology, suggesting that M. arctoides belongs to an independent species group, while M. assamensis and M. thibetana are closely related and belong to another species group, and the other tree gives a different topology which implies that M. arctoides, M. assamensis and M. thibetana belong to one species group. PMID:9254965

  6. Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis.

    PubMed

    Antonioli, Paolo; Mendieta, Martha E; Sebastiano, Roberto; Citterio, Attilio; Peltre, Gabriel; Busnel, Jean-Marc; Descroix, Stephanie; Candiano, Giovanni; Righetti, Pier Giorgio

    2006-03-20

    Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall. PMID:16289957

  7. Whole cell, label free protein quantitation with data independent acquisition: quantitation at the MS2 level.

    PubMed

    McQueen, Peter; Spicer, Vic; Schellenberg, John; Krokhin, Oleg; Sparling, Richard; Levin, David; Wilkins, John A

    2015-01-01

    Label free quantitation by measurement of peptide fragment signal intensity (MS2 quantitation) is a technique that has seen limited use due to the stochastic nature of data dependent acquisition (DDA). However, data independent acquisition has the potential to make large scale MS2 quantitation a more viable technique. In this study we used an implementation of data independent acquisition--SWATH--to perform label free protein quantitation in a model bacterium Clostridium stercorarium. Four tryptic digests analyzed by SWATH were probed by an ion library containing information on peptide mass and retention time obtained from DDA experiments. Application of this ion library to SWATH data quantified 1030 proteins with at least two peptides quantified (∼ 40% of predicted proteins in the C. stercorarium genome) in each replicate. Quantitative results obtained were very consistent between biological replicates (R(2) ∼ 0.960). Protein quantitation by summation of peptide fragment signal intensities was also highly consistent between biological replicates (R(2) ∼ 0.930), indicating that this approach may have increased viability compared to recent applications in label free protein quantitation. SWATH based quantitation was able to consistently detect differences in relative protein quantity and it provided coverage for a number of proteins that were missed in some samples by DDA analysis. PMID:25348682

  8. Streamlined sign-out of capillary protein electrophoresis using middleware and an open-source macro application

    PubMed Central

    Mathur, Gagan; Haugen, Thomas H.; Davis, Scott L.; Krasowski, Matthew D.

    2014-01-01

    Background: Interfacing of clinical laboratory instruments with the laboratory information system (LIS) via “middleware” software is increasingly common. Our clinical laboratory implemented capillary electrophoresis using a Sebia® Capillarys-2™ (Norcross, GA, USA) instrument for serum and urine protein electrophoresis. Using Data Innovations Instrument Manager, an interface was established with the LIS (Cerner) that allowed for bi-directional transmission of numeric data. However, the text of the interpretive pathology report was not properly transferred. To reduce manual effort and possibility for error in text data transfer, we developed scripts in AutoHotkey, a free, open-source macro-creation and automation software utility. Materials and Methods: Scripts were written to create macros that automated mouse and key strokes. The scripts retrieve the specimen accession number, capture user input text, and insert the text interpretation in the correct patient record in the desired format. Results: The scripts accurately and precisely transfer narrative interpretation into the LIS. Combined with bar-code reading by the electrophoresis instrument, the scripts transfer data efficiently to the correct patient record. In addition, the AutoHotKey script automated repetitive key strokes required for manual entry into the LIS, making protein electrophoresis sign-out easier to learn and faster to use by the pathology residents. Scripts allow for either preliminary verification by residents or final sign-out by the attending pathologist. Conclusions: Using the open-source AutoHotKey software, we successfully improved the transfer of text data between capillary electrophoresis software and the LIS. The use of open-source software tools should not be overlooked as tools to improve interfacing of laboratory instruments. PMID:25337433

  9. Population proteomics: quantitative variation within and among populations in cardiac protein expression.

    PubMed

    Rees, Bernard B; Andacht, Tracy; Skripnikova, Elena; Crawford, Douglas L

    2011-03-01

    Population analysis of gene expression is typically achieved by quantifying levels of mRNA; however, gene expression is also a function of protein translation and turnover. Therefore, a complete understanding of population variation in gene expression requires quantitative knowledge of protein expression within and among natural populations. We used two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) to quantitatively compare expression of heart ventricle proteins among 18 individuals in three populations of the teleost fish Fundulus. Among populations, expressions between orthologous proteins and mRNAs were generally positively correlated. Additionally, similar to the pattern of cardiac mRNA expression for the same populations, we found considerable variation in protein expression both within and among populations: Of 408 protein features in 2D gels, 34% are significantly different (P < 0.01) among individuals within a population, 9% differ between populations, and 12% have a pattern of expression that suggests they have evolved by natural selection. Although similar to mRNA expression, the frequency of significant differences among populations is larger for proteins. Similar to mRNA expressions, expressions of most proteins are correlated to the expressions of many other proteins. However, the correlations among proteins are more extensive than the correlation for similar RNAs. These correlations suggest a greater coordinate regulation of protein than mRNA expression. The larger frequency of significant differences among populations and the greater frequency of correlated expression among proteins versus among RNAs suggest that the molecular mechanisms affecting protein expression enhance the differences among populations, and these regulatory steps could be a source of variation for adaptation. PMID:21109588

  10. 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) ...

  11. 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

  12. The use of seed protein electrophoresis in the study of phylogenetic relationships in Chili pepper (Capsicum L.).

    PubMed

    Panda, R C; Aniel Kumar, O; Raja Rao, K G

    1986-08-01

    The seed protein profile of eight taxa of Chili peppers obtained by disc electrophoresis was found to be a diagnostic character in the study of phylogenetic relationships. The distinctness of each species and the wild and cultivated nature of concerned taxa has been confirmed. While the clustering of wildC. annuum var. 'glabriusculum' withC. baccatum types indicated that the former is the progenitor of the latter group, the marked differences discernible in the seed protein profile of all other taxa suggest a polyphyletic origin for the genusCapsicum. PMID:24248078

  13. Serum protein electrophoresis: an interesting diagnosis tool to distinguish viral from bacterial community-acquired pneumonia.

    PubMed

    Davido, B; Badr, C; Lagrange, A; Makhloufi, S; De Truchis, P; Perronne, C; Salomon, J; Dinh, A

    2016-06-01

    29-69 % of pneumonias are microbiologically documented because it can be considered as an invasive procedure with variable test sensitivity. However, it drastically impacts therapeutic strategy in particular the use of antibiotics. Serum protein electrophoresis (SPEP) is a routine and non-invasive test commonly used to identify serum protein disorders. As virus and bacteria may induce different globulins production, we hypothesize that SPEP can be used as an etiological diagnosis test. Retrospective study conducted from 1/1/13 until 5/1/15 among patient hospitalized for an acute community-acquired pneumonia based on fever, crackles and radiological abnormalities. α/β, α/γ, β/γ globulins and albumin/globulin (A/G) ratio were calculated from SPEP. Data were analyzed in 3 groups: documented viral (DVP) or bacterial pneumonia (DBP) and supposedly bacterial pneumonia (SBP). We used ANOVA statistic test with multiple comparisons using CI95 and ROC curve to compare them. 109 patients included divided into DBP (n = 16), DVP (n = 26) and SBP (n = 67). Mean age was 62 ± 18 year-old with a sex ratio M/F of 1.3. Underlying conditions (e.g. COPD, diabetes) were comparable between groups in multivariate analysis. Means of A/G ratio were 0.80 [0.76-0.84], 0.96 [0.91-1.01], 1.08 [0.99-1.16] respectively for DBP, SBP and DVP (p = 0.0002). A/G ratio cut-off value of 0.845 has a sensitivity of 87.5 % and a specificity of 73.1 %. A/G ratio seems to be an easy diagnostic tool to differentiate bacterial from viral pneumonia. A/G ratio cut-off value below 0.845 seems to be predictable of a bacterial origin and support the use of antibiotics. PMID:26936614

  14. Binding isotherms of sodium dodecyl sulfate to protein polypeptides with special reference to SDS-polyacylamide gel electrophoresis.

    PubMed

    Takagi, T; Tsujii, K; Shirahama, K

    1975-05-01

    To clarify the mode of interaction between sodium dodecyl sulfate (SDS) and protein polypeptides with special reference to SDS-polyacrylamide gel electrophoresis, the binding of SDS to several protein polypeptides was investigated by the equilibrium dialysis technique. Each of the binding isotherms was characterized by the presence of two phases: an initial gradual increase in the amount of binding to 0.3-0.6 g/g (first phase) and a subsequent steep increase to 1.2-1.5 g/g (second phase). The binding was completed at a concentration of SDS below the critical micelle concentration. Throughout the first and second phases, the isotherms obtained were different for each kind of protein. On the basis of experiments with bovine serum albumin and ribonuclease (EC 3.1.4.22], the isotherms were profoundly affected by the method used for modification of the sulfhydryl groups. The claim of Reynolds and Tanford (Proc. Natl, Acad. Sci. U.S., 66, 1002 (1970)) that the isotherms are virtually identical for many kinds of proteins was not supported by the present data. Changes in the gross and local conformations were examined with reference to the isotherms by measurements of CD spectrum, free boundary electrophoresis, and gel filtration. The results obtained were collectively interpreted based on the model of SDS-protein polypeptide complexes proposed by the present authors (J. Biochem., 75, 309 (1974)). PMID:1158859

  15. Quantitation of protein 3 content of circulating erythrocytes at the single-cell level

    SciTech Connect

    Jennings, L.K.; Brown, L.K.; Dockter, M.E.

    1985-05-01

    The density and size of human erythrocytes has been roughly correlated with cell age, with the denser and smaller cells being older. Observations of this type have led to a hypothesis that the membranes of circulating erythrocytes are dynamic with respect to composition and that material is lost from the membrane during cell maturation and circulation. In this study, flow cytofluorimetry was used to investigate the distribution of the human erythrocyte anion transport protein (protein 3) in heterogeneous samples of circulating red cells. We verified that protein 3 can be specifically and quantitatively labeled in intact human erythrocytes with eosin-5-maleimide, a luminescent probe. Individual cells were accordingly analyzed for size by forward light scattering and for protein 3 content by quantitation of eosin fluorescence. Initial results indicated that the smallest erythrocytes had a protein 3 content equal to that of the largest circulating erythrocytes. This result was independently verified by light scatter-activated cell sorting; direct measurement of cell diameters by microscopy verified that the cell sizes of erythrocytes showing the 10% greatest and 10% smallest light-scattering signal were indeed distinct. Independent analysis of the size-sorted erythrocytes for protein 3 content was accomplished by gel electrophoresis of stroma from 150,000 large and small erythrocytes. Quantitative scanning densitometry of silver-stained gels of prepared stroma showed that protein 3 content of each set of fractionated cells was equal and did not vary as a function of cell size. Taken in combination with the reported correlation between increasing red blood cell age and decreasing cell size, these results indicate that any loss of membranous material during the cell aging process is not random.

  16. 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

  17. Sex-specific and blood meal-induced proteins of Anopheles gambiae midguts: analysis by two-dimensional gel electrophoresis

    PubMed Central

    Prévot, GI; Laurent-Winter, C; Rodhain, F; Bourgouin, C

    2003-01-01

    Background Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite. Methods We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood. Results Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females. Conclusion Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity. PMID:12605724

  18. Novel application of Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE).

    PubMed

    Wang, Yanan; Zhang, Jing; Huang, Lingyun; He, Dacheng; Ma, Lin; Ouyang, Jin; Jiang, Fubin

    2012-01-27

    We have developed a novel application for DNA oligonucleotide-stabilized Ag nanoclusters in fluorescent imaging of human serum proteins after native polyacrylamide gel electrophoresis (PAGE). Oligonucleotide-stabilized Ag nanoclusters were used as fluorescent probes for direct detection of proteins after native PAGE. Some relatively low-abundance proteins, such as α-1-antichymotrypsin (ACT) and α-2-glycoprotein 1, zinc (ZAG) were easily detected by oligonucleotide-stabilized Ag nanocluster-based fluorescent imaging and identified by MS and MS/MS techniques, without the need of expensive antibodies or tedious immunoassay procedures. The pH condition for the oligonucleotide-stabilized Ag nanocluster solution was optimized and the possible mechanism of interaction between proteins and DNA oligonucleotide-stabilized Ag nanoclusters was analyzed. As a novel fluorescent detection method it is simple, fast, nontoxic and sensitive, and it shows great analytical potential in proteome research and in biochemistry. PMID:22249908

  19. Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis.

    PubMed

    Sano, Osamu; Kunikata, Toshio; Kohno, Keizo; Iwaki, Kanso; Ikeda, Masao; Kurimoto, Masashi

    2004-01-14

    In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species. PMID:14709007

  20. Two-Dimensional Electrophoresis for Analysis of Mycobacterium tuberculosis Culture Filtrate and Purification and Characterization of Six Novel Proteins

    PubMed Central

    Weldingh, Karin; Rosenkrands, Ida; Jacobsen, Susanne; Rasmussen, Peter Birk; Elhay, Martin J.; Andersen, Peter

    1998-01-01

    Culture filtrate from Mycobacterium tuberculosis contains molecules which promote high levels of protective immunity in animal models of subunit vaccination against tuberculosis. We have used two-dimensional electrophoresis for analysis and purification of six novel M. tuberculosis culture filtrate proteins (CFPs): CFP17, CFP20, CFP21, CFP22, CFP25, and CFP28. The proteins were tested for recognition by M. tuberculosis-reactive memory cells from different strains of inbred mice and for their capacity to induce a skin test response in M. tuberculosis-infected guinea pigs. CFP17, CFP20, CFP21 and CFP25 induced both a high gamma interferon release and a strong delayed-type hypersensitivity response, and CFP21 was broadly recognized by different strains of inbred mice. N-terminal sequences were obtained for the six proteins, and the corresponding genes were identified in the Sanger M. tuberculosis genome database. In parallel we established a two-dimensional electrophoresis reference map of short-term culture filtrate components and mapped novel proteins as well as already-known CFP. PMID:9673225

  1. 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

  2. [Analysis of proteins synthesized in aggregates of dissociated blastula and gastrula cells of Pleurodeles waltlii (Amphibia, urodela) by electrophoresis in polyacrylamide gradients].

    PubMed

    Boucaut, J C; Desvaux, F X

    1976-09-20

    The protein synthesis manifested by aggregates of blastula and gastrula dissociated cells were studied by means of polyacylamide gradient gel electrophoresis. This method permits a comparative analysis of protein electrophoretic mobilities in aggregates and controls. In all cases, the data obtained establish the identity of protein banding and confirm an egg laying polymorphism. PMID:825304

  3. 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

  4. Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast.

    PubMed

    Joubert, R; Strub, J M; Zugmeyer, S; Kobi, D; Carte, N; Van Dorsselaer, A; Boucherie, H; Jaquet-Guffreund, L

    2001-08-01

    As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins. PMID:11565791

  5. Quantitative determination of the β-methyl carbapenem doripenem in powder for injection by a stability-indicating capillary zone electrophoresis method.

    PubMed

    Paliosa, P K; Garcia, C V; Schapoval, E E S; Mendez, A S L; Steppe, M

    2015-09-01

    A capillary zone electrophoresis method for quantitative determination of doripenem in synthetic matrix was developed. The stability-indicating capability was performed applying stress testing protocols. The selected analytical conditions include 100 mM sodium borate buffer (pH 8.0) as run electrolyte, voltage of +15 kV, hydrodynamic injection of 5s (50 mBar), detection at 298 nm and temperature of analysis of 25 degrees C. The electrophoretic separation was carried out in a fused silica capillary (effective length 40 cm, 50 μm i.d.), using procainamide hydrochloride as internal standard. The proposed method showed quickness and reproducibility, with an analytical run in a total time of 5 min. The percentage of drug amount estimated was 101.33% (RSD = 0.80), with satisfactory intra-day and inter-day precision. In the recovery test, the method was found to be reliable and accurate in the drug quantitation (mean recovery = 101.86%). The robustness was performed applying the Plackett-Burman experimental design which confirmed the assay reliability. Based on results from forced degradation study, the stability-indicating capability was established, being observed a major degradation in alkaline, photolytic and thermal conditions. In comparison to HPLC method previously developed, the proposed capillary electrophoresis assay is statistically equivalent. PMID:26492640

  6. Capillary electrophoresis

    SciTech Connect

    Warner, M.

    1988-10-15

    Rapid instrumental methods for performing electrophoretic separations in capillary tubes have recently been developed, making capillary electrophoresis one of the most exciting new techniques available to analytical chemists. This article discusses detection methods, applications, and the future of capillary electrophoresis.

  7. 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. PMID:22949109

  8. 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

  9. Quantitative Packaging of Active Enzymes into a Protein Cage.

    PubMed

    Azuma, Yusuke; Zschoche, Reinhard; Tinzl, Matthias; Hilvert, Donald

    2016-01-22

    Genetic fusion of cargo proteins to a positively supercharged variant of green fluorescent protein enables their quantitative encapsulation by engineered lumazine synthase capsids possessing a negatively charged lumenal surface. This simple tagging system provides a robust and versatile means of creating hierarchically ordered protein assemblies for use as nanoreactors. The generality of the encapsulation strategy and its effect on enzyme function were investigated with eight structurally and mechanistically distinct catalysts. PMID:26695342

  10. 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

  11. 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. PMID:25976506

  12. 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. PMID:26080275

  13. Quantitative proteomic profiling identifies protein correlates to EGFR kinase inhibition.

    PubMed

    Kani, Kian; Faca, Vitor M; Hughes, Lindsey D; Zhang, Wenxuan; Fang, Qiaojun; Shahbaba, Babak; Luethy, Roland; Erde, Jonathan; Schmidt, Joanna; Pitteri, Sharon J; Zhang, Qing; Katz, Jonathan E; Gross, Mitchell E; Plevritis, Sylvia K; McIntosh, Martin W; Jain, Anjali; Hanash, Samir; Agus, David B; Mallick, Parag

    2012-05-01

    Clinical oncology is hampered by lack of tools to accurately assess a patient's response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not responding to a therapy could be usefully incorporated into tools for monitoring response. Here, we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study, we use stable isotope labeling of amino acids in culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGF receptor (EGFR)-targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information, and a subset consisting of 400 proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and showed that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response. PMID:22411897

  14. Quantitative Proteomic profiling identifies protein correlates to EGFR kinase inhibition

    PubMed Central

    Kani, Kian; Faca, Vitor M.; Hughes, Lindsey D.; Zhang, Wenxuan; Fang, Qiaojun; Shahbaba, Babak; Luethy, Roland; Erde, Jonathan; Schmidt, Joanna; Pitteri, Sharon J.; Zhang, Qing; Katz, Jonathan E.; Gross, Mitchell E.; Plevritis, Sylvia K.; McIntosh, Martin W.; Jain, Anjali; Hanash, Sam; Agus, David B.; Mallick, Parag

    2014-01-01

    Clinical oncology is hampered by a lack of tools to accurately assess a patient’s response to pathway-targeted therapies. Serum and tumor cell surface proteins whose abundance, or change in abundance in response to therapy, differentiates patients responding to a therapy from patients not-responding to a therapy could be usefully incorporated into tools for monitoring response. Here we posit and then verify that proteomic discovery in in vitro tissue culture models can identify proteins with concordant in vivo behavior and further, can be a valuable approach for identifying tumor-derived serum proteins. In this study we use Stable Isotope Labeling of Amino acids in Culture (SILAC) with proteomic technologies to quantitatively analyze the gefitinib-related protein changes in a model system for sensitivity to EGFR targeted tyrosine kinase inhibitors. We identified 3,707 intracellular proteins, 1,276 cell surface proteins, and 879 shed proteins. More than 75% of the proteins identified had quantitative information and a subset consisting of [400] proteins showed a statistically significant change in abundance following gefitinib treatment. We validated the change in expression profile in vitro and screened our panel of response markers in an in vivo isogenic resistant model and demonstrated that these were markers of gefitinib response and not simply markers of phospho-EGFR downregulation. In doing so, we also were able to identify which proteins might be useful as markers for monitoring response and which proteins might be useful as markers for a priori prediction of response. PMID:22411897

  15. 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.

  16. Resolution-enhanced native acidic gel electrophoresis: a method for resolving, sizing, and quantifying prion protein oligomers.

    PubMed

    Ladner, Carol L; Wishart, David S

    2012-07-01

    The formation of β-sheet-rich prion protein (PrP(β)) oligomers from native or cellular PrP(c) is thought to be a key step in the development of prion diseases. To assist in this characterization process we have developed a rapid and remarkably high resolution gel electrophoresis technique called RENAGE (resolution-enhanced native acidic gel electrophoresis) for separating, sizing, and quantifying oligomeric PrP(β) complexes. PrP(β) oligomers formed via either urea/salt or acid conversion can be resolved by RENAGE into a clear set of oligomeric bands differing by just one subunit. Calibration of the size of the PrP(β) oligomer bands was made possible with a cross-linked mouse PrP(90-232) ladder (1- to 11-mer) generated using ruthenium bipyridyl-based photoinduced cross-linking of unmodified proteins (PICUP). This PrP PICUP ladder allowed the size and abundance of PrP(β) oligomers formed from urea/salt and acid conversion to be determined. This distribution consists of 7-, 8-, 9-, 10-, and 11-mers, with the most abundant species being the 8-mer. The high-resolution separation afforded by RENAGE has allowed us to investigate distinctive size and population changes in PrP(β) oligomers formed under various conversion conditions, with various construct lengths, from various species or in the presence of anti-prion compounds. PMID:22490465

  17. Proteomic analysis of surface proteins of Trichinella spiralis muscle larvae by two-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    2013-01-01

    Background Trichinella spiralis is a zoonotic tissue-dwelling parasitic nematode that infects humans and other mammals. Its surface proteins are recognized as antigenic in many infected hosts, being directly exposed to the host’s immune system and are the main target antigens that induce the immune responses. The larval surface proteins may also interact with intestinal epithelial cells and may play an important role in the invasion and development process of T. spiralis. The purpose of this study was to analyze and characterize the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. Methods The surface proteins of T. spiralis muscle larvae were stripped from the cuticle of live larvae by the cetyltrimethylammonium bromide (CTAB) and sodium deoxycholate. The surface protein stripping was examined by an immunofluorescent test (IFT). The surface proteins were analyzed by SDS-PAGE and Western blotting, and then identified by 2-DE and MALDI-TOF/TOF mass spectrometry analysis. Results The IFT results showed that the surface proteins-stripped larvae were not recognized by sera of mice immunized with surface antigens. Western blotting showed 7 of 12 protein bands of the surface proteins were recognized by mouse infection sera at 18 dpi and at 42 dpi. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66 kDa and isoelectric point (pI) from 4 to 7. Twenty-seven of 33 protein spots were identified and characterized to correlate with 15 different proteins. Out of the 14 proteins identified as T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity. All of these 5 proteins were also associated with metabolic processes and 2 of the five proteins were associated with cellular processes. Conclusions In this study, T

  18. 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 ...

  19. Grain protein variability among species of Triticum and Aegilops: quantitative SDS-PAGE studies.

    PubMed

    Cole, E W; Fullington, J G; Kasarda, D D

    1981-01-01

    Total proteins were extracted from degermed seeds of various species of Triticum and Aegilops with solutions containing sodium dodecyl sulfate (SDS) and mercaptoethanol. The reduced, dissociated proteins were fractionated according to molecular weight (MW) by high-resolution polyacrylamide gel electrophoresis in buffers containing SDS (SDS-PAGE). Stained SDS-PAGE patterns were measured by densitometric scanning over a suitable range of optical density. The data were normalized to equivalent total areas for each of the densitometric scans by means of a computer program that also permitted the construction of patterns of hypothetical amphiploids by averaging patterns of two or three diploid species. The grain proteins of most species examined had distinctive qualitative and quantitative aspects that were characteristic of the species even though nearly every accession or cultivar of a species exhibited at least minor differences in pattern from other accessions or cultivars. The main protein components (probably prolamins) of Triticum monococcum ssp. monococcum, T. monococcum ssp. boeoticum, T. urartu, and Aegilops squarrosa had MW's in the range 29-36 X 10(3) whereas the most important components of Ae. speltoides, Ae. longissima, and Ae. searsii had MW's in the range 37-55 × 10(3). Changes in the quantitative expression of particular genes, especially those coding for storage protein components, may have been associated with speciation. The strong predominance of proteins with MW's in the range 29-36 × 10(3) in some accessions of AB genome tetraploids, such as T. turgidum ssp. dicoccoides, may indicate contributions to the B genome of these tetraploids by T. monococcum ssp. boeoticum, T. urartu, or Ae. squarrosa. PMID:24276584

  20. 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

  1. Isobaric Labeling and Data Normalization without Requiring Protein Quantitation

    PubMed Central

    Kim, Phillip D.; Patel, Bhavinkumar B.; Yeung, Anthony T.

    2012-01-01

    Isobaric multiplexed quantitative proteomics can complement high-resolution sample isolation techniques. Here, we report a simple workflow exponentially modified protein abundance index (emPAI)-MW deconvolution (EMMOL) for normalizing isobaric reporter ratios within and between experiments, where small or unknown amounts of protein are used. EMMOL deconvolutes the isobaric tags for relative and absolute quantification (iTRAQ) data to yield the quantity of each protein of each sample in the pool, a new approach that enables the comparison of many samples without including a channel of reference standard. Moreover, EMMOL allows using a sufficient quantity of control sample to facilitate the peptide fractionation (isoelectric-focusing was used in this report), and mass spectrometry MS/MS sequencing yet relies on the broad dynamic range of iTRAQ quantitation to compare relative protein abundance. We demonstrated EMMOL by comparing four pooled samples with 20-fold range differences in protein abundance and performed data normalization without using prior knowledge of the amounts of proteins in each sample, simulating an iTRAQ experiment without protein quantitation prior to labeling. We used emPAI,1 the target protein MW, and the iTRAQ reporter ratios to calculate the amount of each protein in each of the four channels. Importantly, the EMMOL-delineated proteomes from separate iTRAQ experiments can be assorted for comparison without using a reference sample. We observed no compression of expression in iTRAQ ratios over a 20-fold range for all protein abundances. To complement this ability to analyze minute samples, we report an optimized iTRAQ labeling protocol for using 5 μg protein as the starting material. PMID:22468137

  2. 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. PMID:23483553

  3. Target protein separation and preparation by free-flow electrophoresis coupled with charge-to-mass ratio analysis.

    PubMed

    Shen, Qiao-Yi; Guo, Chen-Gang; Yan, Jian; Zhang, Qiang; Xie, Hai-Yang; Jahan, Sharmin; Fan, Liu-Yin; Xiao, Hua; Cao, Cheng-Xi

    2015-06-01

    Herein, a novel strategy was developed to separate and prepare target protein from complex sample by free-flow electrophoresis (FFE), which mainly based on the charge-to-mass ratio (C/M) analysis of proteins. The C/M values of three model proteins, namely Cytochrome C (Cyt C), myoglobin (Mb) and bovine serum albumin (BSA) were analyzed under different pH and the separation of these proteins was predicted by CLC Protein Workbench software. Series of experiments were performed to validate the proposed method. The obtained data showed high accordance with our prediction. In addition, the chamber buffer (CB) of FFE system was optimized to improve the resolution of separation. Meanwhile, in order to evaluate the analytical performance of the proposed method, Cyt C was extracted from swine heart and further separated by FFE based on C/M analysis. Results showed that Cyt C was completely separated from the crude sample and a purity of 96.9% was achieved. The activity of prepared Cyt C was 98.3%, which indicate that the proposed method is promising in a wide variety of research areas where the native properties of proteins should be maintained for downstream analysis. PMID:25890440

  4. Quantitative and qualitative analyses of DNA fragments based on electrical charge detection on a portable electrophoresis device.

    PubMed

    Chen, Gin-Shin; Chen, Sheng-Fu; Lu, Chih-Cheng

    2004-01-01

    A concept regarding DNA fragments electrophoretic analyses by directly detecting electrical charges is proposed. The arrival time and voltage of charged DNA fragments with different charge-to-mass ratio could be detected using the custom-made micro electronic circuits. These time and voltage information imply the size and intensity information acquired from the conventional slab gel image by fluorescent labeling. A prototype of the portable electrophoresis device consists of a flow channel with the dimension of 35 mm (length) x 0.5 mm (width) x 0.2 mm (depth) on an acrylic substrate, and the detection circuit with amplification gain of 10,000 and analogous filter bandwidth between 0.1 Hz and 10 Hz has been developed. A simple experiment was carried out to demonstrate the feasibility of proposed idea. The volume of 2mul of the DNA ladder (1 Kb Plus DNA ladder, Invitrogen, U.S.A.) with the diluted concentration of 0.1mug/mul was loaded into the reservoir when applying the electrical field of 12.5 V/cm to both end of the flow channel, which was only filled with TBE solution. The preliminary results showed that the developed electrophoresis device can pick up the electrical signals of un-separated DNA fragments with total mass of 0.2 mug , and the magnitude is 0.6 V . Micro flow channels fabricated by an excimer-laser machine and low-noise amplifier with high gain, e.g. 100,000 are being processed. Moreover, HEC (hydroxyethylcellulose) solution will be utilized as the media in the micro channels for DNA fragments separation. PMID:17270819

  5. 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

  6. 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

  7. Assessing association between protein truncating variants and quantitative traits

    PubMed Central

    Rivas, Manuel A.; Pirinen, Matti; Neville, Matthew J.; Gaulton, Kyle J.; Moutsianas, Loukas; Lindgren, Cecilia M.; Karpe, Fredrik; McCarthy, Mark I.; Donnelly, Peter

    2013-01-01

    Motivation: In sequencing studies of common diseases and quantitative traits, power to test rare and low frequency variants individually is weak. To improve power, a common approach is to combine statistical evidence from several genetic variants in a region. Major challenges are how to do the combining and which statistical framework to use. General approaches for testing association between rare variants and quantitative traits include aggregating genotypes and trait values, referred to as ‘collapsing’, or using a score-based variance component test. However, little attention has been paid to alternative models tailored for protein truncating variants. Recent studies have highlighted the important role that protein truncating variants, commonly referred to as ‘loss of function’ variants, may have on disease susceptibility and quantitative levels of biomarkers. We propose a Bayesian modelling framework for the analysis of protein truncating variants and quantitative traits. Results: Our simulation results show that our models have an advantage over the commonly used methods. We apply our models to sequence and exome-array data and discover strong evidence of association between low plasma triglyceride levels and protein truncating variants at APOC3 (Apolipoprotein C3). Availability: Software is available from http://www.well.ox.ac.uk/~rivas/mamba Contact: donnelly@well.ox.ac.uk PMID:23860716

  8. Electrolytic Reduction: Modification of Proteins Occurring in Isoelectric Focusing Electrophoresis and in Electrolytic Reactions in the Presence of High Salts

    PubMed Central

    2009-01-01

    Artifacts in two-dimensional electrophoresis (2-DE) caused by the presence of salts in isoelectric focusing (IEF) have been previously described as a result of increasing conductivity and inducing electroosmosis. However, electrolysis induced by the presence of salts should not be disregarded. In this study, electrolytic reduction−oxidation reaction (redox) was found to be enhanced in the presence of salts in IEF. The consequence of the electrolytic redox leads to acidification of the low-pH region and alkalization of the high-pH region within the immobilized pH gradient (IPG) strip. As a result, a breakdown of immobilized pH buffer near the high pH region of IPG strips along with reduction of basic proteins resulted in uncharacterized artifacts in 2-DE. Electrolytic reduction in the presence of alkali and alkaline metal ions was demonstrated to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), protein disulfide bonds, and protein carboxylic acids. Importantly, semipreparative electrolytic reduction of proteins can be carried out in the presence of sodium ions in a homemade electrolytic apparatus. These findings give additional explanations to the observed artifacts in 2-DE and reveal the unknown effects of salts in IEF. Moreover, we have provided a method with the potential to convert proteins or peptides to corresponding modified products containing aldehyde groups that can be used for conjugation with amine-containing compounds. PMID:19438264

  9. Buffer optimization for high resolution of human lung cancer tissue proteins by two-dimensional gel electrophoresis.

    PubMed

    Lee, Kibeom; Pi, Kyungbae; Lee, Keeman

    2009-01-01

    A problem in proteomic analysis of lung cancer tissue is the presence of complex components of different histological backgrounds (squamous cell carcinoma, small cell lung carcinoma, and adenocarcinoma). The efficient solubilization of protein components before two-dimensional electrophoresis (2-DE) is a very critical. Poor solubilization has been associated with a failure to detect proteins and diffuse, streaked and/or trailing protein spots. Here, we have optimized the solubilization of human lung cancer tissue to increase protein resolution. Isoelectric focusing (IEF) rehydration buffer containing a thiourea-urea mixture provided superior resolution, whereas a buffer without thiourea yielded consistently poor results. In addition, IEF rehydration buffers containing CHAPS and DTT gave superior resolution, whereas buffers containing Nonidet P-40 (NP-40) and/or Triton X-100 did not. A tributylphosphine-containing buffer gave consistently poor results. Using optimized conditions, we used 2-D gel analysis of human lung cancer tissue to identify 11 differentially-expressed protein spots by MALDI-mass spectrometry. This study provides a methodological tool to study the complex mammalian proteomes. PMID:18800191

  10. Quantitating Metabolites in Protein Precipitated Serum Using NMR Spectroscopy

    PubMed Central

    2015-01-01

    Quantitative NMR-based metabolite profiling is challenged by the deleterious effects of abundant proteins in the intact blood plasma/serum, which underscores the need for alternative approaches. Protein removal by ultrafiltration using low molecular weight cutoff filters thus represents an important step. However, protein precipitation, an alternative and simple approach for protein removal, lacks detailed quantitative assessment for use in NMR based metabolomics. In this study, we have comprehensively evaluated the performance of protein precipitation using methanol, acetonitrile, perchloric acid, and trichloroacetic acid and ultrafiltration approaches using 1D and 2D NMR, based on the identification and absolute quantitation of 44 human blood metabolites, including a few identified for the first time in the NMR spectra of human serum. We also investigated the use of a “smart isotope tag,” 15N-cholamine for further resolution enhancement, which resulted in the detection of a number of additional metabolites. 1H NMR of both protein precipitated and ultrafiltered serum detected all 44 metabolites with comparable reproducibility (average CV, 3.7% for precipitation; 3.6% for filtration). However, nearly half of the quantified metabolites in ultrafiltered serum exhibited 10–74% lower concentrations; specifically, tryptophan, benzoate, and 2-oxoisocaproate showed much lower concentrations compared to protein precipitated serum. These results indicate that protein precipitation using methanol offers a reliable approach for routine NMR-based metabolomics of human blood serum/plasma and should be considered as an alternative to ultrafiltration. Importantly, protein precipitation, which is commonly used by mass spectrometry (MS), promises avenues for direct comparison and correlation of metabolite data obtained from the two analytical platforms to exploit their combined strength in the metabolomics of blood. PMID:24796490

  11. 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.

  12. 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.

  13. Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser-induced fluorescence detection.

    PubMed

    Kaneta, Takashi; Ogura, Takehito; Imasaka, Totaro

    2011-04-01

    Previously, we have demonstrated postcolumn derivatization of proteins separated by capillary sieving electrophoresis (CSE), in which naphthalene-2,3-dicarbaldehyde was employed as a fluorogenic labeling reagent. Standard proteins separated by CSE were reacted with naphthalene-2,3-dicarbaldehyde in the presence of 2-mercaptoethanol (2-ME) which plays a role of a reducing agent in the derivatization reaction. To improve the sensitivity, we attempted the use of ethanethiol instead of 2-ME. Ethanethiol showed 1.4- to 4.5-fold lower limits of detection for proteins than 2-ME. Furthermore, we found that 8-aminopyrene-1,3,6-trisulfonate (APTS) is a good marker for relative electrophoretic mobilities of proteins in CSE. Since APTS is a fluorescent and trivalent anion, it generates strong fluorescence and migrates faster than any of the proteins. Therefore, we employed APTS as a marker to obtain the relative electrophoretic mobilities of proteins. The present method was applied to the analyses of proteins in biological samples. Human Ewing's family tumor cell line 'RDES' was used as a sample. The cultured cells were lysed with a buffer containing Tris-HCl, NaCl, sodium dodecyl sulfate, and 2-ME. After denaturation, the lysate was directly introduced into the capillary. Several peaks, which would correspond to proteins with molecular mass ranging from 10 to 93 kDa, were found in the cell lysate. In addition, we measured a milk sample by the CSE with postcolumn derivatization. The electropherogram showed five major peaks which corresponded to α-lactalbumin, β-lactoglobulin, κ-casein, bovine serum albumin, and mixture of α- and β-casein. PMID:21449073

  14. Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry*

    PubMed Central

    Kaake, Robyn M.; Wang, Xiaorong; Huang, Lan

    2010-01-01

    Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis. Affinity purification coupled with quantitative mass spectrometry has become the primary method for studying in vivo protein interactions of protein complexes and whole organism proteomes. Recent developments in sample preparation and affinity purification strategies allow the capture, identification, and quantification of protein interactions of protein complexes that are stable, dynamic, transient, and/or weak. Current efforts have mainly focused on generating reliable, reproducible, and high confidence protein interaction data sets for functional characterization. The availability of increasing amounts of information on protein interactions in eukaryotic systems and new bioinformatics tools allow functional analysis of quantitative protein interaction data to unravel the biological significance of the identified protein interactions. Existing studies in this area have laid a solid foundation toward generating a complete map of in vivo protein interaction networks of protein complexes in cells or tissues. PMID:20445003

  15. 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...

  16. A novel, post-column micro-membrane reactor for fluorescent analysis of protein in capillary electrophoresis.

    PubMed

    Liu, Fan; Zhang, Lingyi; Qian, Junhong; Ren, Jun; Gao, Fangyuan; Zhang, Weibing

    2013-11-01

    Based on the semipermeability of hollow fiber membranes, a post-column membrane reactor was developed for capillary electrophoresis (CE)-laser induced fluorescence (LIF) analysis of proteins by using a hollow fiber membrane to connect the separation and detection capillaries. The membrane length between the separation and detection capillaries was 1 mm. Driven by the chemical potential difference between the separation buffer inside the membrane and the fluorescence derivatization solution outside the membrane, the derivatization reagent can be easily drawn into hollow fiber membrane to react with proteins. Also, the separation buffer can be adjusted by the derivatization solution to match the conditions of derivatization without sample loss. The effect of the separation buffer on the derivatization reaction was investigated and the results showed that even a strong acidic solution and multiple additives can be adopted in the separation buffer without destroying the post-column derivatization of proteins. Under the optimized conditions, the highly sensitive detection of BSA was achieved with a detection limit of 3.3 nmol L(-1) and a linear calibration range from 0.007 to 0.1 mg mL(-1). The proposed CE-LIF system with a post-column membrane reactor was also successfully applied to the separation and detection of proteins in rat liver and loach muscle. PMID:24015400

  17. 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...

  18. 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

  19. 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

  20. Protein quantitation using various modes of high performance liquid chromatography.

    PubMed

    Grotefend, Sandra; Kaminski, Lukas; Wroblewitz, Stefanie; Deeb, Sami El; Kühn, Nancy; Reichl, Stephan; Limberger, Markus; Watt, Steven; Wätzig, Hermann

    2012-12-01

    Pharmaceuticals based on proteins (biologicals), such as monoclonal antibodies (mAb), attain more and more relevance since they were established as potent drugs in anticancer therapy or for the treatment of autoimmune based diseases. Due to their high efficiency it is essential to have accurate and precise methods for protein quantitation and the detection of protein aggregates, which in some cases may lead to adverse effects after application. Selectivity and precision of traditional protein quantification methods such as the Bradford assay or SDS-PAGE are insufficient for quality control (QC) purposes. In this work several HPLC separation modes, which can significantly improve these important parameters, were compared for their application in this field. High performance size exclusion (HP-SEC), strong anion exchange (SAX), weak cation exchange (WCX) as well as reversed phase chromatography are all already successfully applied in protein analysis. Good precision (SEC: <1.9%, SAX: <5%, RP: <2% and WCX: <3.5% - RSD% for peak areas day-to-day), high selectivity and low quantitation limits (<15μg/ml) for the model proteins ovalbumin, myoglobin and bovine serum albumin (BSA), respectively cytochrome c and lysozyme in the cation exchange mode, could be achieved. Consecutively, the four separation modes were compared to each other and to electrophoretic techniques in terms of precision, selectivity, analysis time, effort of sample and mobile phase preparation as well as separating capacity. Moreover, the analysis of an IgG1-type antibody was included in this study. PMID:22980318

  1. 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

  2. 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%). PMID:23567122

  3. 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.

  4. 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

  5. Evaluation of a novel hydrophilic derivatized capillary for protein analysis by capillary electrophoresis-electrospray mass spectrometry.

    PubMed

    Cole, R B; Varghese, J; McCormick, R M; Kadlecek, D

    1994-10-01

    A new type of hydrophilic derivatized capillary has been used to enable the on-line capillary electrophoresis separation and electrospray mass spectrometric detection of a mixture of proteins containing bovine cytochrome c, tuna cytochrome c and horse heart myoglobin. Less than 40 fmol of each compound were loaded into the capillary. Baseline resolution of components was achieved, as were accurate assignments of molecular masses. The hydrophilic derivatized capillaries were taken through extensive testing procedures to characterize their performance and capabilities for protein analysis. A mixture of six proteins (cytochrome c, ribonuclease A, alpha-chymotrypsinogen, myoglobin, carbonic anhydrase II and alpha-lactalbumin) in acetic acid-sodium acetate buffer was used to delineate the relationships between migration time and pH, along with migration time and buffer concentration for each protein. The variations in capillary efficiency as a function of pH and as a function of buffer concentration were also characterized for the same six proteins in the acetic acid-sodium acetate system. A pH of 4.8 was found to offer an excellent compromise between separation efficiency (up to 500,000 theoretical plates) and analysis time. Capillary efficiencies were also found to be very good when employing a Tris.HCl electrolyte adjusted to pH 4.8. Lastly, electropherogram reproducibility and capillary durability were examined with the finding that little deterioration of the capillary occurred over the course of 400 injections (200 h run time). This represents a notable improvement over previously documented derivatization procedures designed to reduce protein adsorption to fused-silica capillary walls. PMID:7981821

  6. New capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines.

    PubMed

    van Tricht, Ewoud; Geurink, Lars; Pajic, Bojana; Nijenhuis, Johan; Backus, Harold; Germano, Marta; Somsen, Govert W; Sänger-van de Griend, Cari E

    2015-11-01

    Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID. PMID:26452923

  7. 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.

  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. 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

  10. Microfabricated channel array electrophoresis for characterization and screening of enzymes using RGS-G protein interactions as a model system.

    PubMed

    Pei, Jian; Dishinger, John F; Roman, David L; Rungwanitcha, Chetwana; Neubig, Richard R; Kennedy, Robert T

    2008-07-01

    A microfluidic chip consisting of parallel channels designed for rapid electrophoretic enzyme assays was developed. Radial arrangement of channels and a common waste channel allowed chips with 16 and 36 electrophoresis units to be fabricated on a 7.62 x 7.62 cm(2) glass substrate. Fluorescence detection was achieved using a Xe arc lamp source and commercial charge-coupled device (CCD) camera to image migrating analyte zones in individual channels. Chip performance was evaluated by performing electrophoretic assays for G protein GTPase activity on chip using BODIPY-GTP as enzyme substrate. A 16-channel design proved to be useful in extracting kinetic information by allowing serial electrophoretic assays from 16 different enzyme reaction mixtures at 20 s intervals in parallel. This system was used to rapidly determine enzyme concentrations, optimal enzymatic reaction conditions, and Michaelis-Menten constants. A chip with 36 channels was used for screening for modulators of the G protein-RGS protein interaction by assaying the amount of product formed in enzyme reaction mixtures that contained test compounds. Thirty-six electrophoretic assays were performed in 30 s suggesting the potential throughput up to 4320 assays/h with appropriate sample handling procedures. Both designs showed excellent reproducibility of peak migration time and peak area. Relative standard deviations of normalized peak area of enzymatic product BODIPY-GDP were 5% and 11%, respectively, in the 16- and 36-channel designs. PMID:18465881

  11. Capillary electrophoresis-mass spectrometry of basic proteins using a new physically adsorbed polymer coating. Some applications in food analysis.

    PubMed

    Simó, Carolina; Elvira, Carlos; González, Nieves; San Román, J; Barbas, Coral; Cifuentes, Alejandro

    2004-07-01

    A new physically adsorbed capillary coating for capillary electrophoresis-mass spectrometry (CE-MS) of basic proteins is presented, which is easily obtained by flushing the capillary with a polymer aqueous solution for two min. This coating significantly reduces the electrostatic adsorption of a group of basic proteins (i.e., cytochrome c, lysozyme, and ribonuclease A) onto the capillary wall allowing their analysis by CE-MS. The coating protocol is compatible with electrospray inonization (ESI)-MS via the reproducible separation of the standard basic proteins (%RSD values (n = 5) < 1% for analysis time reproducibility and < 5% for peak heights, measured from the total ion electropherograms (TIEs) within the same day). The LODs determined using cytochrome c with total ion current and extracted ion current defection were 24.5 and 2.9 fmol, respectively. Using this new coating lysozymes from chicken and turkey egg white could be easily distinguished by CE-MS, demonstrating the usefulness of this method to differentiate animal species. Even after sterilization at 120 degrees C for 30 min, lysozyme could be detected, as well as in wines at concentrations much lower than the limit marked by the EC Commission Regulation. Adulteration of minced meat with 5% of egg-white could also be analysed by our CE-MS protocol. PMID:15237406

  12. 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.

  13. 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

  14. 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.

  15. Global Subcellular Characterization of Protein Degradation Using Quantitative Proteomics*

    PubMed Central

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J.; Ly, Tony; Lamond, Angus I.

    2013-01-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ∼5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution. PMID:23242552

  16. 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

  17. Isolation, identification and characterisation of starch-interacting proteins by 2-D affinity electrophoresis.

    PubMed

    Kosar-Hashemi, Behjat; Irwin, Jennifer A; Higgins, Jody; Rahman, Sadequr; Morell, Matthew K

    2006-05-01

    A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function. PMID:16645949

  18. Determination of total sulfite in wine. Zone electrophoresis-isotachophoresis quantitation of sulfate on a chip after an in-sample oxidation of total sulfite.

    PubMed

    Masár, Marián; Danková, Mariana; Olvecká, Eva; Stachurová, Adela; Kaniansky, Dusan; Stanislawski, Bernd

    2005-08-19

    This work deals with the determination of total sulfite in wine. The determination combines an in-sample hydrogen peroxide oxidation of total sulfite in alkalized wine to sulfate with the separation and quantitation of the latter anion by zone electrophoresis (ZE) on-line coupled with isotachophoresis (ITP) on a column-coupling chip. Sample clean up, integrated into the ITP-ZE separation, eliminated wine matrix in an extent comparable to that provided by a highly selective distillation isolation of sulfite. At the same time, conductivity detection, employed to the detection of sulfate in the ZE stage of the ITP-ZE combination, provided for sulfate the concentration limit of detection corresponding to a 90 microg/l concentration of sulfite in the loaded sample (0.9 microl). Such a detectability allowed a reproducible quantitation of total sulfite when its concentration in wine was 15 mg/l. Formaldehyde binding of free sulfite in wine, included into the pre-column sample preparation, prevented an uncontrolled oxidation of this sulfite form. This step contributed to an unbiased determination of sulfate present in the original wine sample (this determination corrected for the concentration of sulfate determined in the sample after the peroxide oxidation of sulfite to the value equivalent to the total sulfite). The 99-101% recoveries of sulfite, determined for appropriately spiked wine samples, indicate a very good accuracy of the present method. Such a statement also supports excellent agreements of the results of quantitation based on the in-sample peroxide oxidation of the total sulfite (bound sulfite released at a high pH) with those in which this analyte was isolated from wine by distillation (bound sulfite released at a very low pH). PMID:16114242

  19. 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. PMID:23857606

  20. 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

  1. Analysis of alpha-synuclein-associated proteins by quantitative proteomics.

    PubMed

    Zhou, Yong; Gu, Guangyu; Goodlett, David R; Zhang, Terry; Pan, Catherine; Montine, Thomas J; Montine, Kathleen S; Aebersold, Ruedi H; Zhang, Jing

    2004-09-10

    To identify the proteins associated with soluble alpha-synuclein (AS) that might promote AS aggregation, a key event leading to neurodegeneration, we quantitatively compared protein profiles of AS-associated protein complexes in MES cells exposed to rotenone, a pesticide that produces parkinsonism in animals and induces Lewy body (LB)-like inclusions in the remaining dopaminergic neurons, and to vehicle. We identified more than 250 proteins associated with Nonidet P-40 soluble AS, and demonstrated that at least 51 of these proteins displayed significant differences in their relative abundance in AS complexes under conditions where rotenone was cytotoxic and induced formation of cytoplasmic inclusions immunoreactive to anti-AS. Overexpressing one of these proteins, heat shock protein (hsp) 70, not only protected cells from rotenone-mediated cytotoxicity but also decreased soluble AS aggregation. Furthermore, the protection afforded by hsp70 transfection appeared to be related to suppression of rotenone-induced oxidative stress as well as mitochondrial and proteasomal dysfunction. PMID:15234983

  2. 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. PMID:26851341

  3. Separation of proteins and peptides by capillary electrophoresis in acid buffers containing high concentrations of surfactants.

    PubMed

    Miksík, I; Deyl, Z

    1999-08-01

    Separations of proteins at acid pH in the presence of a high concentration of surfactant [sodium laurylsulfate (SDS), 50 mmol/l] was investigated. The purpose of using high concentrations of SDS as background electrolyte modifier was threefold: First, the surfactant exerts a washing effect upon the capillary wall thus preventing binding of analytes and possible clogging of the capillary. Second, it was revealed that even under very acid conditions (below pH 3) the surfactant is capable of forming associates with protein analytes which still bear considerable negative charge and can be separated on this basis. Third, the system can be applied not only for protein mixtures sufficiently soluble in neutral to alkaline media (leukocyte lysates, standard proteins), but it can be used also with proteins, that are under such conditions virtually insoluble and their solubilization is possible in acid buffers only (eggshell proteins or collagen CNBr fragments). The result was that adsorption to the capillary wall was minimized and the analytes were separated as negatively charged associates with high efficiency. With collagen fragments partition was possible on the affinity differences of the peptides to the surfactant micelles and inner wall of the capillary. Theoretical plate counts approaching 100,000 were easily achieved even with proteins which under the more conventional operation conditions exhibit considerable sticking to the capillary wall. The other feature of this system is that the associates move very rapidly to the anode. Owing to the low pH, endoosmotic flow is negligible, and therefore the system has to be operated at reversed polarity. PMID:10480258

  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. A preparation of Alzheimer paired helical filaments that displays distinct tau proteins by polyacrylamide gel electrophoresis.

    PubMed Central

    Greenberg, S G; Davies, P

    1990-01-01

    Paired helical filaments (PHFs) are prominent components of Alzheimer disease (AD) neurofibrillary tangles (NFTs). Rather than isolating NFTs, we selected for PHF populations that can be extracted from AD brain homogenates. About 50% of PHF immunoreactivity can be obtained in 27,200 x g supernatants following homogenization in buffers containing 0.8 M NaCl. We further enriched for PHFs by taking advantage of their insolubility in the presence of zwitterionic detergents and 2-mercaptoethanol, removal of aggregates by filtration through 0.45-microns filters, and sucrose density centrifugation. PHF-enriched fractions contained two to five proteins of 57-68 kDa that displayed the same antigenic properties as PHFs. Since the 57- to 68-kDa PHF proteins are antigenically related to tau proteins, they are similar to the tau proteins previously observed in NFTs. However, further analysis revealed that PHF-associated tau can be distinguished from normal, soluble tau by PHF antibodies that do not recognize human adult tau and by one- and two-dimensional PAGE. Images PMID:2116006

  6. 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.

  7. Separation of Teff Eragrostis tef (Zucc.) Trotter seed proteins by capillary electrophoresis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Teff (Eragrostis tef (Zucc.) Trotter) is an important food grain in Ethiopia where it is used in the preparation of the tradional flatbread injera. Teff is also used in celiac-safe food products due to its gluten-free status. Limited research has been reported on protein properties of this interesti...

  8. 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. PMID:27120584

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

    PubMed

    Finney, Lydia; Chishti, Yasmin; Khare, Tripti; Giometti, Carol; Levina, Aviva; Lay, Peter A; Vogt, Stefan

    2010-06-18

    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. PMID:20392082

  10. Two-dimensional polyacrylamide gel electrophoresis of equine seminal plasma proteins and their relation with semen freezability.

    PubMed

    Jobim, M I M; Trein, C; Zirkler, H; Gregory, R M; Sieme, H; Mattos, R C

    2011-09-01

    The objective was to evaluate protein profiles of equine seminal plasma using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and to determine whether any of these proteins were related to semen freezability. Seminal plasma was collected from 10 stallions, of high and low semen freezability, housed at the State Stud of Lower Saxony, and routinely used in AI programs. Twenty-five protein spots were identified from the two-dimensional gel (12%), seven of which were present in all samples (all proteins were identified by MALDI-MS). Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. Of the 25 proteins identified, two spots had greater relative content (P < 0.05) in seminal plasma samples collected from stallions with high semen freezability: spot 5 (80-85 kDa, isoelectric point [pI] 7.54), identified as CRISP-3; and spot 45 (18.2 kDa, pI 5.0-5.2), identified as HSP-2. Conversely, protein content was greater (P < 0.05) in seminal plasma samples from stallions with low semen freezability: spot 7 (75.4 kDa, pI 6.9-7.4), identified as lactoferrin; spot 15 (26.7 kDa, pI 5.51), identified as kallikrein; spot 25 (25 kDa, pI 7.54), identified as CRISP-3; and spot 35 (13.9 kDa, pI 3.8-4.2), identified as HSP-1. In conclusion, there were differences in the seminal plasma protein profile from stallions with high and low semen freezability. Furthermore, CRISP-3 and HSP-2 were potential seminal plasma markers of high semen freezability. PMID:21601917

  11. Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers

    SciTech Connect

    Freeman, S.E.; Blackett, A.D.; Monteleone, D.C.; Setlow, R.B.; Sutherland, B.M.; Sutherland, J.C.

    1986-10-01

    The authors have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controller scanner. The medium lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single stand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single stand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determined pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single stand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amendable to radioactive labeling.

  12. Free-Flow Zone Electrophoresis of Peptides and Proteins in PDMS Microchip for Narrow pI Range Sample Prefractionation Coupled with Mass Spectrometry

    PubMed Central

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

    2010-01-01

    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. PMID:20163146

  13. Electrophoresis of tear proteins as a new diagnostic tool for two high risk groups for dry eye: computer users and contact lens wearers

    PubMed Central

    2011-01-01

    Rationale: Dry eye is the most prevalent condition seen by the ophthalmologist, in particular in elderly. The identification of new common risk factors (computer use and contact lens wear) extends the disease among the young people. The early diagnosis of dry eye is essential, but difficult, because the biochemical changes in tear film usually occur before any detectable signs. Due its advantages, electrophoresis of tear proteins could be an important tool for diagnosis of tear film impairment in high risk groups for dry eye. Objective: The role of tear proteins electrophoresis in early diagnosis of dry eye related to computer use and contact lens wear, as well as the biochemical changes in these high risk groups are presented. Methods: This review will summarize the actual data concerning the electrophoretic changes of tear proteins in computer users and contact lens wearers, two common high risk groups for dry eye. Discussion: Electrophoresis of tear proteins using automated system Hyrys–Hydrasys SEBIA France is an important tool for early diagnosis of tear film alterations and monitoring of therapy. The quantification of many proteins in a single analysis using a small quantity of unconcentrated reflex tears is the main advantage of this technique. Electrophoresis of tear proteins should became a prerequisite, in particular for computer users less than 3h/day, as well as at prescribing contact lenses. Abbreviations: DED– dry eye disease, EGF–epidermal growth factor, IL interleukins, MMP–metalloproteinase, ELISA– Enzyme–linked immunosorbent assay, SDS– sodium dodecyl sulfate, CVS– computer vision syndrome, CLRDE– contact lens– related dry eye PMID:22567044

  14. 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

  15. 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

  16. Polyacrylamide gel plugs enabling 2-D microfluidic protein separations via isoelectric focusing and multiplexed sodium dodecyl sulfate gel electrophoresis.

    PubMed

    Liu, Jikun; Yang, Shuang; Lee, Cheng S; DeVoe, Don L

    2008-06-01

    In situ photopolymerized polyacrylamide (PAAm) gel plugs are used as hydrodynamic flow control elements in a multidimensional microfluidic system combining IEF and parallel SDS gel electrophoresis for protein separations. The PAAm gel plugs offer a simple method to reduce undesirable bulk flow and limit reagent/sample crosstalk without placing unwanted constraints on the selection of separation media, and without hindering electrokinetic ion migration in the complex microchannel network. In addition to improving separation reproducibility, the discrete gel plugs integrated into critical regions of the chip enable the use of a simple pressure-driven sample injection method which avoids electrokinetic injection bias. The gel plugs also serve to greatly simplify operation of the spatially multiplexed system by eliminating the need for complex external fluidic interfaces. Using an FITC-labeled Escherichia coli cell lysate as a model system, the use of gel plugs is shown to significantly enhance separation reproducibility in a chip containing five parallel CGE channels, with an average variance in peak elution time of only 4.1%. PMID:18449857

  17. 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.

  18. Studies on protein poly(ADP-ribosylation) using high resolution gel electrophoresis.

    PubMed

    Boulikas, T

    1990-08-25

    Analysis of poly(ADP-ribose) synthesized in cellular lysates or in isolated nuclei on 100-cm-long thin gels of 20% polyacrylamide, 2.5 M urea permits determination of the exact size of poly(ADP-ribose) molecules using labeled oligonucleotides as molecular weight markers. The size and concentration of poly(ADP-ribose) molecules increase at time intervals during its synthesis. Differences in the concentration of poly(ADP-ribose) size classes among cell lines are also shown. Inhibition of poly(ADP-ribose) degradation by ethacridine that directly interacts with the polymer and inhibits its hydrolysis by poly(ADP-ribose) glycohydrolase shows a dramatic increase in both polymer size and concentration. Use of alkaline conditions for the hydrolysis of poly(ADP-ribose)-protein linkages reveals a specific shortening of all size classes of poly(ADP-ribose) compared with its size in preparations obtained by extensive digestion of nuclei with nucleases, RNases, and proteases. PMID:2167322

  19. Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent.

    PubMed

    Herbert, B R; Molloy, M P; Gooley, A A; Walsh, B J; Bryson, W G; Williams, K L

    1998-05-01

    In this study, dithiothreitol was replaced by tributyl phosphine as the reducing agent in both the sample solution for the first-dimensional isoelectric focusing and during the immobilised pH gradient (IPG) equilibration procedure. Tributyl phosphine improves protein solubility during isoelectric focusing, which results in shorter run times and increased resolution. Tributyl phosphine is nonionic and thus does not migrate in the IPG, therefore maintaining reducing conditions during the course of the first-dimensional separation. The increased solubility provided by the maintenance of reducing conditions gives improved focusing and decreased horizontal streaking on the subsequent second-dimension gel. The use of tributyl phosphine in the equilibration step allows the procedure to be simplified, incorporating reduction and alkylation in a single step. This is possible because, in direct contrast to dithiothreitol (DTT), tributyl phosphine does not contain a free thiol and therefore does not react with thiol-specific alkylating reagents. PMID:9629925

  20. Sub-visible particle quantitation in protein therapeutics.

    PubMed

    Cao, S; Jiao, N; Jiang, Y; Mire-Sluis, A; Narhi, L O

    2009-10-01

    Biologics represent a large and growing segment of the therapeutic medicinal market. Sub-visible particles present in these products are a product quality attribute and a potential patient safety concern yet to be fully explored. Early and consistent particle quantitation and control throughout the product life cycle of these drugs from development to commercial lot release is critical in mitigating any concerns. This requires appropriate analytical methods which can be applied to biopharmaceuticals across a large variety of protein concentrations and modes of administration. The compendial light obscuration method for quantitating sub-visible particles in small volume parenterals is not ideally suited for therapeutic biologics. Approaches to modify the current compendial method so that it is applicable to biologics, including appropriate sample preparation, reduced assay sample volume, increased sizing information, and development of an appropriate sampling plan, are presented in this article. Successful applications of a modified light obscuration method to therapeutic protein products are demonstrated, and a strategy to utilise complimentary methods and techniques at different phases of product development is discussed. PMID:20144454

  1. 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

  2. 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.

  3. Segmented field OFFGEL® electrophoresis.

    PubMed

    Tobolkina, Elena; Cortés-Salazar, Fernando; Momotenko, Dmitry; Maillard, Julien; Girault, Hubert H

    2012-11-01

    A multielectrode setup for protein OFFGEL electrophoresis that significantly improves protein separation efficiency has been developed. Here, the electric field is applied by segments between seven electrodes connected in series to six independent power supplies. The aim of this strategy is to distribute evenly the electric field along the multiwell system, and as a consequence to enhance electrophoresis in terms of separation time, resolution, and protein collection efficiency, while minimizing the overall potential difference and therefore the Joule heating. The performances were compared to a standard two-electrode setup for OFFGEL fractionation of a protein mixture, using UV-Vis spectroscopy for quantification and MALDI-MS for identification. The electrophoretic separation process was simulated, and optimized by solving the time-dependent Nernst-Planck differential equation. PMID:23086720

  4. Measurement of Protein Tyrosine Phosphatase Activity in Single Cells by Capillary Electrophoresis

    PubMed Central

    Phillips, Ryan M.; Bair, Eric; Lawrence, David S.; Sims, Christopher E.; Allbritton, Nancy L.

    2013-01-01

    A fluorescent peptide substrate was used to measure dephosphorylation by protein tyrosine phosphatases (PTP) in cell lysates, and single cells and to investigate the effect of environmental toxins on PTP activity in these systems. Dephosphorylation of the substrate by PTPN1 and PTPN2 obeyed Michaelis-Menten kinetics, with KM values of 770 ± 250 nM and 290 ± 54 nM, respectively. Dose-response curves and IC50 values were determined for the inhibition of these two enzymes by the environmental toxins Zn2+ and 1,2-naphthoquinone, as well as pervanadate. In A431 cell lysates, the reporter was a poor substrate for peptidases (degradation rate of 100 ± 8.2 fmol min−1 mg−1) but an excellent substrate for phosphatases (dephosphorylation rate of 1.4 ± 0.3 nmol min−1 mg−1). Zn2+, 1,2-naphthoquinone and pervanadate inhibited dephosphorylation of the reporter in cell lysates with IC50 values of 470 nM, 35 μM, and 100 nM, respectively. Dephosphorylation of the reporter following loading into living single cells occurred at rates of at least 2 pmol min−1 mg−1. When single cells were exposed to 1,2-naphthoquinone (50 μM), Zn2+ (100 μM), and pervandate (1 mM), dephosphorylation was inhibited with median values and first and third quartile values of 41 (Q1 = 0%, Q3 = 96%), 50 (Q1 = 46%, Q3 = 74%), and 53% (Q1 = 36%, Q3 = 77%), respectively, demonstrating both the impact of these toxic exposures on cell signaling and the heterogeneity of response between cells. This approach will provide a valuable tool for the study of PTP dynamics, particularly in small, heterogeneous populations such as human biopsy specimens. PMID:23682679

  5. 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)

  6. Differences in alcohol-soluble protein from genetically altered wheat using capillary zone electrophoresis, one- and two-dimensional electrophoresis and a novel gluten matrix association factor analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wheat protein composition and organization play interrelated roles in determining physical properties for technological purposes. In prior research, a number of isogenic wheat lines of Bobwhite that have high levels of expression of the native Dx5 and/or Dy10 high molecular weight subunits (HMW-GS)...

  7. Quantitative analysis of protein dynamics during asymmetric cell division.

    PubMed

    Mayer, Bernd; Emery, Gregory; Berdnik, Daniela; Wirtz-Peitz, Frederik; Knoblich, Juergen A

    2005-10-25

    In dividing Drosophila sensory organ precursor (SOP) cells, the fate determinant Numb and its associated adaptor protein Pon localize asymmetrically and segregate into the anterior daughter cell, where Numb influences cell fate by repressing Notch signaling. Asymmetric localization of both proteins requires the protein kinase aPKC and its substrate Lethal (2) giant larvae (Lgl). Because both Numb and Pon localization require actin and myosin, lateral transport along the cell cortex has been proposed as a possible mechanism for their asymmetric distribution. Here, we use quantitative live analysis of GFP-Pon and Numb-GFP fluorescence and fluorescence recovery after photobleaching (FRAP) to characterize the dynamics of Numb and Pon localization during SOP division. We demonstrate that Numb and Pon rapidly exchange between a cytoplasmic pool and the cell cortex and that preferential recruitment from the cytoplasm is responsible for their asymmetric distribution during mitosis. Expression of a constitutively active form of aPKC impairs membrane recruitment of GFP-Pon. This defect can be rescued by coexpression of nonphosphorylatable Lgl, indicating that Lgl is the main target of aPKC. We propose that a high-affinity binding site is asymmetrically distributed by aPKC and Lgl and is responsible for asymmetric localization of cell-fate determinants during mitosis. PMID:16243032

  8. 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

  9. A single-label fluorescent derivatization method for quantitative determination of neurotoxin in vivo by capillary electrophoresis coupled with laser-induced fluorescence detection.

    PubMed

    Chen, Cuiwei; Hu, Yiqin; Shi, Xiaowei; Tao, Chenghao; Zheng, Hongyue; Fei, Weidong; Han, Shunping; Zhu, Jiazhen; Wei, Yinghui; Li, Fanzhu

    2016-07-21

    Neurotoxin (NT), a short-chain α-neurotoxin, is the main neurotoxic protein identified from the venom of Naja naja atra. As an effective drug for the analgesis of advanced cancer patients, NT lasts longer than morphine and does not cause addiction. However, achieving a sensitive and high-resolution measurement of NT is difficult because of the extra-low content of NT in vivo. Therefore, developing a novel method to quantify NT is essential to study its pharmacokinetics in vivo. Although NT contains four primary amine groups that could react with the thiourea in fluorescein isothiocyanate (FITC), we developed a simple and reproducible single-label fluorescent derivatization method for NT which is related to the reaction of N-terminal α-amino of NT alone under optimized derivatization conditions. Furthermore, neurotoxin labelled with fluorescein isothiocyanate (NT-FITC) was prepared by high-performance liquid chromatography (HPLC) with a purity value higher than 99.29% and identified by MALDI-TOF/TOF-MS. Finally, NT-FITC could be detected at 0.8 nmol L(-1) in rat plasma using capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). In this paper, the established method robustly and reliably quantified NT labelled with FITC via intravenous and intramuscular administrations in vivo. In addition, this work fully demonstrated the pharmacokinetic characteristics of NT in vivo, which could reduce the risk of drug accumulation, optimize therapies, and provide sufficient evidence for the rational use of NT in clinical and research laboratories. PMID:27175860

  10. 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

  11. 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

  12. The development of simple and sensitive small-molecule fluorescent probes for the detection of serum proteins after native polyacrylamide gel electrophoresis.

    PubMed

    Wang, Fangfang; Huang, Lingyun; Na, Na; He, Dacheng; Sun, Dezhi; Ouyang, Jin

    2012-05-21

    In this paper, a simple and sensitive small-molecule fluorescent probe, 2,5-dihydroxy-4'-dimethylaminochalcone (DHDMAC), was designed and synthesized for the detection of human serum proteins via hydrophobic interactions after polyacrylamide gel electrophoresis (PAGE). This probe produced lower fluorescence emission in the absence of proteins, and the emission intensity was significantly increased after the interaction with serum proteins. To demonstrate the imaging performance of this probe as a fluorescent dye, a series of experiments was conducted that included sensitivity comparison and 2D-PAGE. The results indicated that the sensitivity of DHDMAC staining is comparable to that of the most widely used fluorescent dye, SYPRO Ruby, and more protein spots (including thyroxine-binding globulin, angiotensinogen, afamin, zinc-α-2-glycoprotein and α-1-antichymotrypsin) were detected after 2D-PAGE. Therefore, DHDMAC is a good protein reporter due to its fast staining procedure, low detection limits and high resolution. PMID:22475746

  13. Imaging Beads-Retained Prey Assay for Rapid and Quantitative Protein-Protein Interaction

    PubMed Central

    Zhou, Yan; Hong, Wanjin; Lu, Lei

    2013-01-01

    Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP) assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications. PMID:23555762

  14. [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

  15. Quantitation of protein-protein interactions by thermal stability shift analysis.

    PubMed

    Layton, Curtis J; Hellinga, Homme W

    2011-08-01

    Thermal stability shift analysis is a powerful method for examining binding interactions in proteins. We demonstrate that under certain circumstances, protein-protein interactions can be quantitated by monitoring shifts in thermal stability using thermodynamic models and data analysis methods presented in this work. This method relies on the determination of protein stabilities from thermal unfolding experiments using fluorescent dyes such as SYPRO Orange that report on protein denaturation. Data collection is rapid and straightforward using readily available real-time polymerase chain reaction instrumentation. We present an approach for the analysis of the unfolding transitions corresponding to each partner to extract the affinity of the interaction between the proteins. This method does not require the construction of a titration series that brackets the dissociation constant. In thermal shift experiments, protein stability data are obtained at different temperatures according to the affinity- and concentration-dependent shifts in unfolding transition midpoints. Treatment of the temperature dependence of affinity is, therefore, intrinsic to this method and is developed in this study. We used the interaction between maltose-binding protein (MBP) and a thermostable synthetic ankyrin repeat protein (Off7) as an experimental test case because their unfolding transitions overlap minimally. We found that MBP is significantly stabilized by Off7. High experimental throughput is enabled by sample parallelization, and the ability to extract quantitative binding information at a single partner concentration. In a single experiment, we were able to quantify the affinities of a series of alanine mutants, covering a wide range of affinities (∼ 100 nM to ∼ 100 μM). PMID:21674662

  16. 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

  17. On-line preconcentration of sodium dodecyl sulfate-protein complexes using electrokinetic supercharging method with a prefilled water plug in capillary sieving electrophoresis.

    PubMed

    Liu, Jing; Kang, Mingchao; Liu, Zhen

    2011-09-01

    An electrokinetic supercharging (EKS) method with a prefilled water plug at the head column of capillary was developed for on-line preconcentration of sodium dodecyl sulfate (SDS)-protein complexes in capillary sieving electrophoresis (CSE). Conventional EKS is a combination of electrokinetic injection with transient isotachophoresis (tr-ITP). The capillary is first filled with background electrolyte, then an appropriate amount of a leading electrolyte is filled and electro-injection is carried out for certain duration. After that, terminating electrolyte is filled, and tr-ITP is subsequently initiated, followed by capillary electrophoresis (CE) separation. In this work, the performance of EKS was evaluated by integrating multiple sub-methods step by step, and a water plug containing polymer was introduced before electrokinetic injection in order to further improve the concentration effect. The positive effects of the sub-methods were verified, including molecular sieving effect of polymer, field enhanced sample injection (FESI) with and without a water plug, and transient isotachophoretic electrophoresis-based FESI. It was observed that analyte discrimination usually encountered in conventional electrokinetic injection was eliminated due to the similar charge to mass ratios of SDS-protein complexes. Based on these results, a hybrid on-line preconcentration method, EKS with injecting a water plug containing polymer before sample electrokinetic injection, was proposed and used to indiscriminately preconcentrate SDS-protein complexes, which provided a sensitivity enhancement factor of more than 1000. It was very suitable for the analysis of low-abundance proteins, providing the information of their molecular mass. PMID:22233073

  18. In-Line Separation by Capillary Electrophoresis Prior to Analysis by Top-Down Mass Spectrometry Enables Sensitive Characterization of Protein Complexes

    PubMed Central

    2015-01-01

    Intact protein analysis via top-down mass spectrometry (MS) provides a bird’s eye view over the protein complexes and complex protein mixtures with the unique capability of characterizing protein variants, splice isoforms, and combinatorial post-translational modifications (PTMs). Here we applied capillary electrophoresis (CE) through a sheathless CE–electrospray ionization interface coupled to an LTQ Velos Orbitrap Elite mass spectrometer to analyze the Dam1 complex from Saccharomyces cerevisiae. We achieved a 100-fold increase in sensitivity compared to a reversed-phase liquid chromatography coupled MS analysis of recombinant Dam1 complex with a total loading of 2.5 ng (12 amol). N-terminal processing forms of individual subunits of the Dam1 complex were observed as well as their phosphorylation stoichiometry upon Mps1p kinase treatment. PMID:25382489

  19. Label-free Quantification of Proteins in Single Embryonic Cells with Neural Fate in the Cleavage-Stage Frog (Xenopus laevis) Embryo using Capillary Electrophoresis Electrospray Ionization High-Resolution Mass Spectrometry (CE-ESI-HRMS).

    PubMed

    Lombard-Banek, Camille; Reddy, Sushma; Moody, Sally A; Nemes, Peter

    2016-08-01

    Quantification of protein expression in single cells promises to advance a systems-level understanding of normal development. Using a bottom-up proteomic workflow and multiplexing quantification by tandem mass tags, we recently demonstrated relative quantification between single embryonic cells (blastomeres) in the frog (Xenopus laevis) embryo. In this study, we minimize derivatization steps to enhance analytical sensitivity and use label-free quantification (LFQ) for single Xenopus cells. The technology builds on a custom-designed capillary electrophoresis microflow-electrospray ionization high-resolution mass spectrometry platform and LFQ by MaxLFQ (MaxQuant). By judiciously tailoring performance to peptide separation, ionization, and data-dependent acquisition, we demonstrate an ∼75-amol (∼11 nm) lower limit of detection and quantification for proteins in complex cell digests. The platform enabled the identification of 438 nonredundant protein groups by measuring 16 ng of protein digest, or <0.2% of the total protein contained in a blastomere in the 16-cell embryo. LFQ intensity was validated as a quantitative proxy for protein abundance. Correlation analysis was performed to compare protein quantities between the embryo and n = 3 different single D11 blastomeres, which are fated to develop into the nervous system. A total of 335 nonredundant protein groups were quantified in union between the single D11 cells spanning a 4 log-order concentration range. LFQ and correlation analysis detected expected proteomic differences between the whole embryo and blastomeres, and also found translational differences between individual D11 cells. LFQ on single cells raises exciting possibilities to study gene expression in other cells and models to help better understand cell processes on a systems biology level. PMID:27317400

  20. 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

  1. Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis.

    PubMed

    Sogame, Yoichiro; Kojima, Katsuhiko; Takeshita, Toshikazu; Kinoshita, Eiji; Matsuoka, Tatsuomi

    2014-01-01

    In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; β-tubulin) was enhanced. PMID:24134620

  2. 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

  3. 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

  4. 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

  5. Extracting gene function from protein-protein interactions using Quantitative BAC InteraCtomics (QUBIC).

    PubMed

    Hubner, Nina C; Mann, Matthias

    2011-04-01

    Large-scale proteomic screens are increasingly employed for placing genes into specific pathways. Therefore generic methods providing a physiological context for protein-protein interaction studies are of great interest. In recent years many protein-protein interactions have been determined by affinity purification followed by mass spectrometry (AP-MS). Among many different AP-MS approaches, the recently developed Quantitative BAC InteraCtomics (QUBIC) approach is particularly attractive as it uses tagged, full-length baits that are expressed under endogenous control. For QUBIC large cell line collections expressing tagged proteins from BAC transgenes or gene trap loci have been developed and are freely available. Here we describe detailed workflows on how to obtain specific protein binding partners with high confidence under physiological conditions. The methods are based on fast, streamlined and generic purification procedures followed by single run liquid chromatography-mass spectrometric analysis. Quantification is achieved either by the stable isotope labeling of amino acids in cell culture (SILAC) method or by a 'label-free' procedure. In either case data analysis is performed by using the freely available MaxQuant environment. The QUBIC approach enables biologists with access to high resolution mass spectrometry to perform small and large-scale protein interactome mappings. PMID:21184827

  6. Profiling of myelin proteins by 2D-gel electrophoresis and multidimensional liquid chromatography coupled to MALDI TOF-TOF mass spectrometry.

    PubMed

    Vanrobaeys, Frank; Van Coster, Rudy; Dhondt, Goedele; Devreese, Bart; Van Beeumen, Jozef

    2005-01-01

    The myelin sheath is an electrically insulating layer that consists of lipids and proteins. It plays a key role in the functioning of the nervous system by allowing fast saltatory conduction of nerve pulses. Profiling of the proteins present in myelin is an indispensable prerequisite to better understand the molecular aspects of this dynamic, functionally active membrane. Two types of protein, the myelin basic protein and the proteolipid protein, account for nearly 85% of the protein content in myelin. Identification and characterization of the other "minor" proteins is, in this respect, a real challenge. In the present work, two proteomic strategies were applied in order to study the protein composition of myelin from the murine central nervous system. First, the protein mixture was separated by 2D-gel electrophoresis and, after spot excision and in-gel digestion, samples were analyzed by mass spectrometry. Via this approach, we identified 57 protein spots, corresponding to 38 unique proteins. Alternatively, the myelin sample was digested by trypsin and the resulting peptide mixture was further analyzed by off-line 2D-liquid chromatography. After the second-dimension separation (nanoLC), the peptides were spotted "on-line" onto a MALDI target and analyzed by MALDI TOF-TOF mass spectrometry. We identified 812 peptides by MALDI MS/MS, representing 93 proteins. Membrane proteins, low abundant proteins, and highly basic proteins were all represented in this shotgun proteomic approach. By combining the results of both approaches, we can present a comprehensive proteomic map of myelin, comprising a total of 103 protein identifications, which is of utmost importance for the molecular understanding of white matter and its disorders. PMID:16335977

  7. 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

  8. 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.

  9. Uncovering Quantitative Protein Interaction Networks for Mouse PDZ Domains using Protein Microarrays

    PubMed Central

    Stiffler, Michael A.; Grantcharova, Viara P.; Sevecka, Mark; MacBeath, Gavin

    2008-01-01

    One of the principle challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach – compatible with genome-wide investigations – that enables us to measure the equilibrium dissociation constant (KD) of recombinant PDZ domains for fluorescently-labeled peptides that represent physiologically-relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the KD for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (KD ≤ 10 μM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network. PMID:16637659

  10. Affinity chromatography, two-dimensional electrophoresis, adapted immunodepletion and mass spectrometry used for detection of porcine and piscine heparin-binding human plasma proteins.

    PubMed

    Bjarnadóttir, Stefanía Guðrún; Flengsrud, Ragnar

    2014-01-01

    Heparin-binding proteins in human plasma were studied using affinity chromatography columns with porcine (2mL, 10.7mg capacity) and piscine heparin (5mL, 2.7mg capacity). Two-dimensional electrophoresis (Bio-Rad Protean II gel system with 16cm×16cm gels using isoelectric focusing (IEF) and nonequilibrium pH-gradient gel electrophoresis (NEPHGE)), Bruker Ultraflex MALDI-TOF mass spectrometry and immunoblotting (NovaBlot semidry discontinuous blotting) were used for unfractionated plasma. This revealed electropherograms with differences between porcine and piscine heparin-binding and totally 17 different fibrinogen variants from all 3 chains. Immunodepletion was used to remove fibrinogen (42.1mg anti-human fibrinogen in 8.4mL resin) and serum albumin (0.42mg binding capacity in 14mL resin) and porcine and piscine heparin-binding proteins were identified using liquid chromatography-mass spectrometry (Ultimate 3000 NanoLC with Acclaim PepMap 100 column (50cm×75μm)-LTQ Orbitrap Mass XL). In total, the binding of 76 putative or acknowledged biomarkers are shown. Of the identified proteins, 14 are not previously shown to be heparin-binding, such as the low concentration proteins lipocalin-1 and tropomyosin and a hitherto not detected protein in plasma, zinc finger protein 483. The putative heparin-binding sequences were analyzed. The results suggest that the combination of group specific affinity and adapted immunodepletion chromatography could be useful in the study of the plasma proteome. PMID:24316520

  11. Use of Two-Dimensional Electrophoresis To Study Differential Protein Expression in Divercin V41-Resistant and Wild-Type Strains of Listeria monocytogenes

    PubMed Central

    Duffes, Frederique; Jenoe, Paul; Boyaval, Patrick

    2000-01-01

    The use of bacteriocins from food-grade lactic acid bacteria to fight against the food-borne pathogen Listeria monocytogenes has been gaining interest. However, the emergence of resistant cells is frequently reported when Listeria is exposed to such antibacterials. A two-dimensional electrophoresis study of whole-cell protein expression of Listeria monocytogenes variants sensitive or resistant to the action of a bacteriocin produced by Carnobacterium divergens V41, divercin V41, is reported in this paper. The resistant variant obtained from the sensitive strain of L. monocytogenes P was also resistant to piscicocins V1 and SF668, but remained sensitive to nisin. Its growth rate was 50% less than the sensitive strain, and the MIC for it was 104 times higher. No reversion of the resistance was observed after 20 successive cultures in the absence of divercin V41. Comparison of the protein patterns by two-dimensional gel electrophoresis analysis showed clear differences. In the resistant variant pattern, at least nine spots had disappeared and eight new ones were observed. One of the newly synthesized proteins was identified as a flagellin of L. monocytogenes. Direct interaction between flagellin and divercin V41 was not evidenced. Intracellular synthesis of flagellin is probably an indirect effect of a modification in transcriptional regulation with widespread effects through a sigma factor. An intense protein, only present in the sensitive strain, was identified as a non-heme iron-binding ferritin displaying strong similarities to Dps proteins. Common modifications in the transcriptional regulation for these two proteins are discussed. PMID:11010876

  12. 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. PMID:19921852

  13. 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.

  14. Fluorescence detection for gel and capillary electrophoresis

    SciTech Connect

    Hogan, B.

    1992-07-21

    First, an indirect fluorescence detection system for the separation of proteins via gel electrophoresis. Quantities as low as 50 nanograms of bovine serum albumin and soybean trypsin inhibitor are separated and detected visually without the need for staining of the analytes. This is very similar to levels of protein commonly separated with gel electrophoresis.

  15. High Resolution CZE-MS Quantitative Characterization of Intact Biopharmaceutical Proteins: Proteoforms of Interferon-β1.

    PubMed

    Bush, David R; Zang, Li; Belov, Arseniy M; Ivanov, Alexander R; Karger, Barry L

    2016-01-19

    New and improved methods are required for the enhanced characterization of complex biopharmaceuticals, especially those with charge and glycan heterogeneity. High resolution separation and mass spectrometry (MS) analysis of intact proteoforms can contribute significantly to the characterization of such proteins, many of which are glycoproteins. Here, we report on capillary zone electrophoresis (CZE) coupled via a commercial CESI sheathless interface to an Orbitrap ELITE MS for the intact analysis of recombinant human interferon-β1 (Avonex, rhIFN-β1), a biopharmaceutical with complex glycosylation at a single N-linked site. Using a cross-linked polyethylenimine coating, column efficiencies between 350,000 and 450,000 plates were produced, allowing separation based on charge and subtle hydrodynamic volume differences. A total of 138 proteoforms were found, and 55 were quantitated. Charge species due to deamidation and sialylation were separated by CZE. Given the high column efficiency, isobaric positional isomers of a single sialic acid on biantennary glycan antennae were resolved. Further, triantennary isomers (antenna on α(1-3) or α(1-6) arms) were separated and confirmed by exoglycosidase digestion. Proteoforms of the N-terminal cleavage of methionine were detected by precursor molecular weight and top-down ETD and HCD analysis of the reduced protein. Quantitative analysis suggested potential correlations between the methionine loss with the relative amount of the deamidation, as well as the level of deamidation with glycan structure. We demonstrate that high resolution CZE separation of intact glycoprotein species coupled to MS has significant potential for the in-depth characterization and quantitative analysis of biopharmaceutical proteoforms. PMID:26641950

  16. Psoriasin, one of several new proteins identified in nasal lavage fluid from allergic and non-allergic individuals using 2-dimensional gel electrophoresis and mass spectrometry

    PubMed Central

    Bryborn, Malin; Adner, Mikael; Cardell, Lars-Olaf

    2005-01-01

    Background Extravasation and luminal entry of plasma occurs continuously in the nose. This process is markedly facilitated in patients with symptomatic allergic rhinitis, resulting in an increased secretion of proteins. Identification of these proteins is an important step in the understanding of the pathological mechanisms in allergic diseases. DNA microarrays have recently made it possible to compare mRNA profiles of lavage fluids from healthy and diseased patients, whereas information on the protein level is still lacking. Methods Nasal lavage fluid was collected from 11 patients with symptomatic allergic rhinitis and 11 healthy volunteers. 2-dimensional gel electrophoresis was used to separate proteins in the lavage fluids. Protein spots were picked from the gels and identified using mass spectrometry and database search. Selected proteins were confirmed with western blot. Results 61 spots were identified, of which 21 were separate proteins. 6 of these proteins (psoriasin, galectin-3, alpha enolase, intersectin-2, Wnt-2B and hypothetical protein MGC33648) had not previously been described in nasal lavage fluids. The levels of psoriasin were markedly down-regulated in allergic individuals. Prolactin-inducible protein was also found to be down-regulated, whereas different fragments of albumin together with Ig gamma 2 chain c region, transthyretin and splice isoform 1 of Wnt-2B were up-regulated among the allergic patients. Conclusion The identification of proteins in nasal lavage fluid with 2-dimensional gelelectrophoresis in combination with mass spectrometry is a novel tool to profile protein expression in allergic rhinitis and it might prove useful in the hunt for new therapeutic targets or diagnostic markers for allergic diseases. Psoriasin is a potent chemotactic factor and its down-regulation during inflammation might be of importance for the outcome of the disease. PMID:16236163

  17. 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.

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

    PubMed

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

    2016-04-01

    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

  19. 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

  20. Comparison of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and antigenic relatedness among outer membrane proteins of 49 Brucella abortus strains.

    PubMed Central

    Verstreate, D R; Winter, A J

    1984-01-01

    Outer membrane proteins were solubilized from 49 strains of Brucella abortus by sequential extraction of physically disrupted cells with N-lauroylsarcosinate and a dipolar ionic detergent (Verstreate et al., Infect. Immun. 35:979-989, 1982). The strains tested included standard agglutination test strain 1119, virulent strain 2308, and eight reference strains representing each of the biotypes; the remainder were isolates from cattle in North America with natural infections and included biotypes 1, 2, and 4. Three principal protein groups with apparent molecular weights of 88,000 to 94,000 (group 1), 35,000 to 40,000 (group 2, now established as porins [Douglas et al., Infect. Immun. 44:16-21, 1984]), and 25,000 to 30,000 (group 3) were observed in every strain. Some variability in banding patterns occurred among strains, but intrastrain variation was sufficient to preclude the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of outer membrane proteins for differentiating among strains of B. abortus. One antigen ([b]) was shared among the porin proteins, and three others ([c], ([d], and ([ e]) were shared among the group 3 proteins of all of the strains tested, indicating that these relationships are probably species wide. These results suggest that it may be possible to use outer membrane proteins from a representative strain of B. abortus in a vaccine for species-wide immunization. Images PMID:6434426

  1. Quantitative analysis of acrylamide labeled serum proteins by LC-MS/MS.

    PubMed

    Faca, Vitor; Coram, Marc; Phanstiel, Doug; Glukhova, Veronika; Zhang, Qing; Fitzgibbon, Matthew; McIntosh, Martin; Hanash, Samir

    2006-08-01

    Isotopic labeling of cysteine residues with acrylamide was previously utilized for relative quantitation of proteins by MALDI-TOF. Here, we explored and compared the application of deuterated and (13)C isotopes of acrylamide for quantitative proteomic analysis using LC-MS/MS and high-resolution FTICR mass spectrometry. The method was applied to human serum samples that were immunodepleted of abundant proteins. Our results show reliable quantitation of proteins across an abundance range that spans 5 orders of magnitude based on ion intensities and known protein concentration in plasma. The use of (13)C isotope of acrylamide had a slightly greater advantage relative to deuterated acrylamide, because of shifts in elution of deuterated acrylamide relative to its corresponding nondeuterated compound by reversed-phase chromatography. Overall, the use of acrylamide for differentially labeling intact proteins in complex mixtures, in combination with LC-MS/MS provides a robust method for quantitative analysis of complex proteomes. PMID:16889424

  2. Quantitative determination of proteins based on strong fluorescence enhancement in curcumin-chitosan-proteins system.

    PubMed

    Wang, Feng; Huang, Wei; Jiang, Lingyan; Tang, Bo

    2012-03-01

    We found that the fluorescence intensity of curcumin (CU) can be highly enhanced by protein bovine serum albumin (BSA) and human serum albumin (HSA) in the presence of chitosan (CTS). Based on this finding, a new fluorimetric method to determine the concentration of protein was developed. Under optimized conditions, the enhanced intensities of fluorescence are quantitatively in proportion to the concentrations of protein in range of 0.007-100 μg·mL(-1) for BSA and 0.004-100 μg·mL(-1) for HSA at 426 nm excitation, and 0.007-100 μg·mL(-1) for BSA and 0.01-100 μg·mL(-1)for HSA at 280 nm excitation, while corresponding qualitative detection limits (S/N = 3) can lower to 3.96, 2.46, 4.56, 9.20 ng·mL(-1), respectively. The method has been successfully used for the determination of HSA in real samples. Based on resonance light scattering and UV-visible absorption spectroscopic analysis, mechanism studies suggested that the highly enhanced fluorescence of CU was resulted from synergic effects of favorable hydrophobic microenvironment provided by BSA and CTS and efficient intermolecular energy transfer between BSA and CU. Protein BSA may bind to CTS through hydrogen bonds, which causes the protein conformation to convert from β-fold to α-helix. CU can combine with the BSA-CTS complex through its center carbonyl carbon, and CTS plays a key role in promoting the energy transfer process by shortening the distance between BSA and CU. PMID:22271351

  3. A Microfluidic Platform for High-Throughput Multiplexed Protein Quantitation

    PubMed Central

    Volpetti, Francesca; Garcia-Cordero, Jose; Maerkl, Sebastian J.

    2015-01-01

    We present a high-throughput microfluidic platform capable of quantitating up to 384 biomarkers in 4 distinct samples by immunoassay. The microfluidic device contains 384 unit cells, which can be individually programmed with pairs of capture and detection antibody. Samples are quantitated in each unit cell by four independent MITOMI detection areas, allowing four samples to be analyzed in parallel for a total of 1,536 assays per device. We show that the device can be pre-assembled and stored for weeks at elevated temperature and we performed proof-of-concept experiments simultaneously quantitating IL-6, IL-1β, TNF-α, PSA, and GFP. Finally, we show that the platform can be used to identify functional antibody combinations by screening 64 antibody combinations requiring up to 384 unique assays per device. PMID:25680117

  4. Fabrication of micro free-flow electrophoresis chip by photocurable monomer binding microfabrication technique for continuous separation of proteins and their numerical simulation.

    PubMed

    Ding, Hui; Li, Xiaoqiong; Lv, Xuefei; Xu, Jiandong; Sun, Xin; Zhang, Zhimeng; Wang, Hailong; Deng, Yulin

    2012-10-01

    In this study, a simple, fast, and reliable method to fabricate a micro free-flow electrophoresis (μFFE) device on glass is presented. The two-dimensional depth channel in the chip was easily achieved by using a photocurable monomer (NOA 81) that served as the bonding material. In such a geometrical structure (two-dimensional depth channel), the effect of fluid behavior on the separation efficiency of micro free-flow zone electrophoresis (μFFZE) was simulated. The results of numerical simulation indicate that the pressure at the inlets may play an important role in the separation performance. Under the optimum separation conditions, four FITC-labeled amino acids were well separated, indicating the validity of the performance of the chip. Since the chip was fabricated by organic polymer bonding, it was easily recyclable through a simple re-fabrication process. The reproducibility of results from these recycling re-fabrication chips was investigated. The RSD of the resolution between FITC-L-glycine and FITC-L-phenylalanine was 5.3%. Furthermore, three FITC-labeled proteins were successfully separated with the resolution of 2.2 and 5.46, respectively, by using the coating of neutral liposome. PMID:22874968

  5. 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. PMID:25319243

  6. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

    PubMed

    Conti, C J; Giménez, I B; Cabrini, R L

    1976-03-01

    Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. PMID:1258094

  7. 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. PMID:23264567

  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. 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.

  10. Plasma Biomarker Discovery Using 3D Protein Profiling Coupled with Label-Free Quantitation

    PubMed Central

    Beer, Lynn A.; Tang, Hsin-Yao; Barnhart, Kurt T.; Speicher, David W.

    2011-01-01

    In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC–MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC–MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as “apparent” biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC–MS/MS with label-free quantitation of candidate biomarkers. PMID:21468938

  11. 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

  12. Identification of dengue RNA binding proteins using RNA chromatography and quantitative mass spectrometry.

    PubMed

    Ward, Alex M; Gunaratne, J; Garcia-Blanco, Mariano A

    2014-01-01

    A major challenge in dengue virus (DENV) research has been to understand the interaction of the viral RNA with host cell proteins during infection. Until recently, there were no comprehensive studies identifying host RNA binding proteins that interact with DENV RNA (Ward et al. RNA Biol 8 (6):1173-1186, 2011). Here, we describe a method for identifying proteins that associate with DENV RNA using RNA chromatography and quantitative mass spectrometry. The method utilizes a tobramycin RNA aptamer incorporated into an RNA containing the dengue 5' and 3' untranslated regions (UTRs) in order to reversibly bind RNA to a tobramycin matrix. The RNA-tobramycin matrix is incubated with SILAC-labeled cell lysates, and bound proteins are eluted using an excess of tobramycin. The eluate is analyzed using quantitative mass spectrometry, which allows direct and quantitative comparison of proteins bound to DENV UTRs and a control RNA-tobramycin matrix. This technique has the advantage of allowing one to distinguish between specific and nonspecific binding proteins based on the ratio of protein preferentially bound to the DENV UTRs versus the control RNA. This methodology can also be used for validation of quantitative mass spectrometry results using conventional Western blotting for specific proteins. Furthermore, though it was specifically developed to identify DENV RNA binding proteins, the RNA chromatography method described here can be applied to a broad range of viral and cellular RNAs for identification of interacting proteins. PMID:24696342

  13. USE OF QUANTITATIVE TWO-DIMENSIONAL GEL ELECTROPHORESIS TO ANALYZE CHANGES IN ALVEOLAR MACROPHAGE PROTEINS IN HUMANS EXPOSED TO OZONE

    EPA Science Inventory

    Acute exposure of humans to 0.4 ppm ozone is known to cause production of components which mediate inflammation and damage in the lung. he contribution of alveolar macrophages to this process is not well understood. n addition, ozone may cause more extensive cellular changes than...

  14. Identification of Tuber borchii Vittad. mycelium proteins separated by two-dimensional polyacrylamide gel electrophoresis using amino acid analysis and sequence tagging.

    PubMed

    Vallorani, L; Bernardini, F; Sacconi, C; Pierleoni, R; Pieretti, B; Piccoli, G; Buffalini, M; Stocchi, V

    2000-11-01

    This paper reports the first results in the proteome analysis of Tuber borchii Vittad. mycelium, an ectomycorrhizal fungus poorly defined genetically, but known for its generation of edible fruit bodies known as white truffles. Employing isoelectric focusing on immobilized pH gradients, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we obtained an electropherogram presenting over 800 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Different reducing agents were tested in the sample preparation buffers, and the standard lysis buffer plus 2% w/v polyvinylpolypyrrolidone allowed the best solubilization and resolution of the proteins. The T. borchii proteins separated in micropreparative gels were electroblotted onto polyvinylidene difluoride membranes and visualized by Coomassie staining. Twenty-three proteins were excised and analyzed by the combination of amino acid and N-terminal analysis. One protein was identified by matching its amino acid composition, estimated isoelectric point and molecular mass against the SWISS-PROT and EMBL databases. Four spots were successfully tagged by Edman microsequencing but no homologous sequences were found in databases. PMID:11271490

  15. High throughput and rapid screening of marine protein hydrolysates enriched in peptides with angiotensin-I-converting enzyme inhibitory activity by capillary electrophoresis.

    PubMed

    He, Hai-Lun; Chen, Xiu-Lan; Wu, Hao; Sun, Cai-Yun; Zhang, Yu-Zhong; Zhou, Bai-Cheng

    2007-12-01

    Twelve kinds of marine protein materials, including fish, shrimp, seashell, algae and seafood wastes were selected for the hydrolysis using four different proteases. The IC(50) values for angiotensin-converting enzyme (ACE) inhibitory activity of 48 hydrolysates were rapidly determined by capillary electrophoresis (CE). The values ranged from 0.17 to 501.7mg/ml, and were affected by both the marine protein resources and the selected proteases. Hydrolysates of the lowest IC(50) values were from shrimp (Acetes chinensis), shark meat, mackerel bone, Polysiphonia urceolata and Spirulina platensis, indicating these five kinds of marine food proteins contained beneficial materials for the production of ACE inhibitory peptides by proteolysis. The hydrolysates obtained using proteases Protamex and SM98011 had lower IC(50) values, showing these two proteases were superior to others. The CE method achieved the same sensitivity as the high performance liquid chromatography (HPLC) method. However, the CE method was faster and, as a result, more economical. Therefore, CE had potential for rapid screening of marine protein hydrolysates enriched in ACE inhibitory peptides. PMID:17317156

  16. 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

  17. 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.

  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. 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…

  20. 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

  1. 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.

  2. 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

  3. 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

  4. A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

    PubMed

    Bohler, Sacha; Sergeant, Kjell; Hoffmann, Lucien; Dizengremel, Pierre; Hausman, Jean-Francois; Renaut, Jenny; Jolivet, Yves

    2011-07-01

    Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity. PMID:21520910

  5. Conformational stability of dimeric proteins: quantitative studies by equilibrium denaturation.

    PubMed Central

    Neet, K. E.; Timm, D. E.

    1994-01-01

    The conformational stability of dimeric globular proteins can be measured by equilibrium denaturation studies in solvents such as guanidine hydrochloride or urea. Many dimeric proteins denature with a 2-state equilibrium transition, whereas others have stable intermediates in the process. For those proteins showing a single transition of native dimer to denatured monomer, the conformational stabilities, delta Gu (H2O), range from 10 to 27 kcal/mol, which is significantly greater than the conformational stability found for monomeric proteins. The relative contribution of quaternary interactions to the overall stability of the dimer can be estimated by comparing delta Gu (H2O) from equilibrium denaturation studies to the free energy associated with simple dissociation in the absence of denaturant. In many cases the large stabilization energy of dimers is primarily due to the intersubunit interactions and thus gives a rationale for the formation of oligomers. The magnitude of the conformational stability is related to the size of the polypeptide in the subunit and depends upon the type of structure in the subunit interface. The practical use, interpretation, and utility of estimation of conformational stability of dimers by equilibrium denaturation methods are discussed. PMID:7756976

  6. 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

  7. Quantitation of sunitinib, an oral multitarget tyrosine kinase inhibitor, and its metabolite in urine samples by nonaqueous capillary electrophoresis time of flight mass spectrometry.

    PubMed

    Rodríguez, Juana; Castañeda, Gregorio; Muñoz, Lorena; Villa, Jose C

    2015-07-01

    A rapid, sensitive, and specific method was developed and validated using a nonaqueous-capillary electrophoresis method with TOF-MS for determination of sunitinib and N-desethyl sunitinib in human urine. In order to avoid ionic suppression a urine samples dilution with methanol 1:10 previous step was used. This was the only treatment step to urine samples before the injection. Despite this dilution of the urine, the detection limit was as low as 0.07 mg/L for sunitinib and 0.15 mg/L for N-desethyl sunitinib. Separation of compounds was achieved with a mixture of 5 mM ammonium formate in methanol. The calibration curves were linear over the range of 0.5-50.0 mg/L for the two analyzed compounds. The within-run and between-run precisions were within 5%, while the accuracy ranged from 96.0 to 100.4%. This method can be used in routine clinical practice to monitor sunitinib and N-desethyl sunitinib drugs in the urine of cancer patients treated with once daily administration. PMID:25873554

  8. 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.

  9. 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.

  10. 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

  11. 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. PMID:27049463

  12. 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.

  13. Self-assembled and covalently linked capillary coating of diazoresin and cyclodextrin-derived dendrimer for analysis of proteins by capillary electrophoresis.

    PubMed

    Yu, Bing; Chi, Ming; Han, Yuxing; Cong, Hailin; Tang, Jianbin; Peng, Qiaohong

    2016-05-15

    Self-assembled and covalently linked capillary coatings of cyclodextrin-derived (CD) dendrimer were prepared using photosensitive diazoresin (DR) as a coupling agent. Layer by layer (LBL) self-assembled DR/CD-dendrimer coatings based on ionic bonding was fabricated first on the inner surface of capillary, and subsequently converted into covalent bonding after treatment with UV light through a unique photochemistry reaction of DR. Protein adsorption on the inner surface of capillary was suppressed by the DR/CD-dendrimer coating, and thus a baseline separation of lysozyme (Lys), myoglobin (Mb), bovine serum albumin (BSA) and ribonuclease A (RNase A) was achieved using capillary electrophoresis (CE). Compared with the bare capillary, the DR/CD-dendrimer covalently linked capillary coatings showed excellent protein separation performance with good stability and repeatability. 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. PMID:26992496

  14. 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. PMID:26875118

  15. Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer.

    PubMed

    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.Graphical Abstract. PMID:26956437

  16. 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.

    2016-03-01

    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.

  17. 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.

  18. 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. PMID:26300480

  19. Evaluation of protein extraction methods suitable for two-dimensional gel electrophoresis of the soybean cyst nematode (Heterodera glycines)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean cyst nematode (Heterodera glycines, SCN) is the most destructive pathogen of soybean (Glycine max (L.) Merr.) worldwide. In this study, three different protein extraction methods including phenol/ammonium acetate (phenol method), thiourea/urea solublization (lysis method) and trichloroaceti...

  20. A fluorescent derivatization method of proteins for the detection of low-level impurities by microchip capillary gel electrophoresis.

    PubMed

    Wenz, Christian; Marchetti-Deschmann, Martina; Herwig, Ela; Schröttner, Evita; Allmaier, Günter; Trojer, Lukas; Vollmer, Martin; Rüfer, Andreas

    2010-01-01

    A novel pre-chip fluorescent derivatization method is presented for protein sizing and quantification by microchip CGE. The derivatization reaction employed a water-soluble and stable fluorescent dye and was performed under conditions that favored the formation of homogeneous reaction products. The method delivered in terms of protein sizing similar results as microchip CGE with on-chip staining but showed an extended linear dynamic range for protein quantification encompassing four orders of magnitude. The sensitivity of the method was similar to standard silver-stained planar gels. The characterization of derivatization reaction products by MS and preparative isoelectric focusing indicated that a constant degree of dye molecule tagging was obtained over a broad range of protein/dye ratios. The method allowed detecting and quantifying an impurity spiked into an antibody preparation down to a level of 0.05%. Advantages of this method compared with CGE approaches with pre-column derivatization include a shorter analysis time and an increased robustness and ease of use. PMID:20162586

  1. Protein expression profile of celiac disease patient with aberrant T cell by two-dimensional difference gel electrophoresis.

    PubMed

    De Re, Valli; Simula, Maria Paola; Caggiari, Laura; Ortz, Nicoletta; Spina, Michele; Da Ponte, Alessandro; De Appolonia, Leandro; Dolcetti, Riccardo; Canzonieri, Vincenzo; Cannizzaro, Renato

    2007-08-01

    One complication of celiac disease (CD) is refractory CD. These patients frequently show aberrant intraepithelial T cell clones and an increasing risk of evolution into enteropathy-associated T cell lymphoma (EATL). There is debate in the literature whether these cases are actually a smoldering lymphoma from the outset. The mechanism inducing T cell proliferation and prognosis remains unknown. Recently, alemtuzumab has been proposed as a promising new approach to treat these patients. Only few single cases have been tested presently, nevertheless, in all of them a clinical improvement has been observed, while intraepithelial lymphocytes (IELs) effectively targeted by alemtuzumab are still a debated issue. Using 2D-DIGE, we found hyperexpressed proteins specifically associated with aberrant T cell in a patient with CD by comparing the protein expression with that of patients with CD and polyclonal T cell or with that of control subjects (patients with polyclonal T cell and no CD). Proteins with a higher expression in duodenal biopsy of the patient with aberrant T cell were identified as IgM, apolipoprotein C-III, and Charcot-Leyden crystal proteins. These preliminary data allow hypothesizing different clinical effects of alemtuzumab in patients with CD, since besides the probable effect of alemtuzumab on T cell, it could effect inflammatory-associated CD52(+) IgM(+)B cell and eosinophils cells, known to produce IgM and Charcot-Leyden crystal proteins, which we demonstrated to be altered in this patient. Results also emphasize the possible association of apolipoprotein with aberrant T cell proliferation. PMID:17785332

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  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. Quantitative chemogenomics: machine-learning models of protein-ligand interaction.

    PubMed

    Andersson, Claes R; Gustafsson, Mats G; Strömbergsson, Helena

    2011-01-01

    Chemogenomics is an emerging interdisciplinary field that lies in the interface of biology, chemistry, and informatics. Most of the currently used drugs are small molecules that interact with proteins. Understanding protein-ligand interaction is therefore central to drug discovery and design. In the subfield of chemogenomics known as proteochemometrics, protein-ligand-interaction models are induced from data matrices that consist of both protein and ligand information along with some experimentally measured variable. The two general aims of this quantitative multi-structure-property-relationship modeling (QMSPR) approach are to exploit sparse/incomplete information sources and to obtain more general models covering larger parts of the protein-ligand space, than traditional approaches that focuses mainly on specific targets or ligands. The data matrices, usually obtained from multiple sparse/incomplete sources, typically contain series of proteins and ligands together with quantitative information about their interactions. A useful model should ideally be easy to interpret and generalize well to new unseen protein-ligand combinations. Resolving this requires sophisticated machine-learning methods for model induction, combined with adequate validation. This review is intended to provide a guide to methods and data sources suitable for this kind of protein-ligand-interaction modeling. An overview of the modeling process is presented including data collection, protein and ligand descriptor computation, data preprocessing, machine-learning-model induction and validation. Concerns and issues specific for each step in this kind of data-driven modeling will be discussed. PMID:21470169

  10. Polyelectrolyte multilayer coatings for the separation of proteins by capillary electrophoresis: Influence of polyelectrolyte nature and multilayer crosslinking.

    PubMed

    Bekri, Samya; Leclercq, Laurent; Cottet, Hervé

    2015-06-19

    The present work aims at studying the influence of the nature of the polyelectrolytes used in successive multiple ionic polymers on the performances of protein separation in acetic acid volatile background electrolyte. A broad library of polyelectrolyte multilayers was compared on the basis of 9 different weak/strong polyanions and 8 different weak/strong polycations. More than 20 couples of different polyelectrolytes were investigated. The separation efficiencies (expressed as the N/l ratio, where N is the plate number and l is the capillary effective length) were systematically compared for the separation of a protein test mixture. The coating stability was evaluated by the relative standard deviation of the migration times. For weak polyelectrolyte multilayers, the influence of the polymer crosslinking on the coating stability and separation efficiency has been studied. Intra-day repeatability of 100 successive runs, and capillary-to-capillary reproducibility were tested on coatings of each category (crosslinked and non crosslinked). The main (not obvious) result rising from this study is that the nature of the polyanion constituting the multilayers is of primary importance for the performance in terms of separation efficiency and stability, even when the mulilayers finish with a polycation. PMID:25976124

  11. Comparison of three modifications of fused-silica capillaries and untreated capillaries for protein profiling of maize extracts by capillary electrophoresis.

    PubMed

    Pobozy, Ewa; Sentkowska, Aleksandra; Piskor, Anna

    2014-09-01

    In this work, capillary electrophoresis was applied to protein profiling of fractionated extracts of maize. A comparative study on the application of uncoated fused-silica capillaries and capillaries modified with hydroxypropylmethylcellulose, ω-iodoalkylammonium salt and a commercially available neutral capillary covalently coated with polyacrylamide is presented. The coating stability, background electrolyte composition, and separation efficiency were investigated. It was found that for zeins separation, the most stable and efficient was the capillary coated with polyacrylamide. Finally, the usefulness of these methods was studied for the differentiation of zein fraction in transgenic and nontransgenic maize. Zeins extracted from maize standards containing 0 and 5% m/m genetic modification were successfully separated, but slight differences were observed in terms of the zein content. Albumin and globulin fractions were analyzed with the use of unmodified fused-silica capillary with borate buffer pH 9 and the capillary coated with polyacrylamide with phosphate buffer pH 3. In the albumin fraction, additional peaks were found in genetically modified samples. PMID:24931305

  12. 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

  13. 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

  14. Time-domain fluorescence lifetime imaging microscopy: a quantitative method to follow transient protein-protein interactions in living cells.

    PubMed

    Padilla-Parra, Sergi; Audugé, Nicolas; Tramier, Marc; Coppey-Moisan, Maïté

    2015-06-01

    Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells. PMID:26034312

  15. 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

  16. 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. PMID:25268018

  17. Multiplexed MRM with Internal Standards for Cerebrospinal Fluid Candidate Protein Biomarker Quantitation.

    PubMed

    Percy, Andrew J; Yang, Juncong; Chambers, Andrew G; Simon, Romain; Hardie, Darryl B; Borchers, Christoph H

    2014-06-30

    Multiplexed quantitation is essential for discovering, verifying, and validating biomarkers for risk stratification, disease prognostication, and therapeutic monitoring. The most promising strategy for quantifying unverified protein biomarkers in biofluids relies on selected/multiple reaction monitoring (SRM or MRM) technology with isotopically labeled standards employed within a bottom-up proteomic workflow. Since cerebrospinal fluid (CSF) is an important fluid for studying central nervous system (CNS) related diseases, we sought to develop a rapid, antibody- and fractionation-free MRM-based approach with a complex mixture of peptide standards to quantify a highly multiplexed panel of candidate protein biomarkers in human CSF. Development involved peptide transition optimization, denaturation/digestion protocol evaluation, transition interference screening, and protein quantitation via peptide standard curves. The final method exhibited excellent reproducibility (average coefficient of variation of <1% for retention time and <6% for signal) and breadth of quantitation (130 proteins from 311 interference-free peptides) in a single 43-min run. These proteins are of high-to-low abundance with determined concentrations from 118 μg/mL (serum albumin) to 550 pg/mL (apolipoprotein C-I). Overall, the method consists of the most highly multiplexed and broadest panel of candidate protein biomarkers in human CSF reported thus far and is well suited for subsequent verification studies on patient samples. PMID:24911472

  18. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    PubMed Central

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    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. PMID:23268931

  19. 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. PMID:23268931

  20. Fluorescence Detection In Electrophoresis

    NASA Astrophysics Data System (ADS)

    Swarner, Susan

    1988-04-01

    Fluorescence detection is in common usage in forensic science laboratories for the visualization of three enzyme markers. The fluorogenic substrates, 4-methylumbelliferyl phosphate, 4-methylutbel-liveryl acetate, and fluorecein diacetate, are acted upon by the enzymes Erythrocyte Acid Phospha, tase, Esterase-D, and Carbonic Anhydrase-III, respectively, to produce compounds visible to the analyst when viewed with transmitted UV light at 365 nm. Additionally, the choice of fluorogenic corn, pounds may help detect a specific enzyme from a related enzyme. One of the responsibilities of a forensic science laboratory may be the analysis of blood for genetically controlled polymorphic enzymes and protein markers. The genetic markers are said to be polymorphic because each exhibits types which can be differentiated and allows for the inclusion or exclusion of possible-donors of the blood. Each genetic marker can be separated into these recognizable types by electrophoresis, a technique which separates compounds based on electrical charges. Electrophoresis is conducted by placing a portion or extract of each bloodstain into a support medium which will conduct electricity. This is known as a plate or membrane. By controlling the pH of the buffer and the potential that is applied to the plate, the analyst can achieve separation of the types within an enzyme marker. The types appear as differing patterns of bands. Once the bloodstain has been subjected to electrophoresis, the enzymes must be visualized. This is generally best accomplished by using the specific activity of the enzyme. For the enzymes described in the present work, the visualization is performed by over-layering the plate with a piece of filter paper that 'has been saturated with the appropriate non-fluorescent substrate and buffer. The bands of enzyme, which is now in discrete patterns, will act upon the non-fluorescent substrate to create a fluorescent compound. The plate is then viewed with transmitted UV

  1. An efficient and rapid method for enrichment of lipophilic proteins from Mycobacterium tuberculosis H37Rv for two-dimensional gel electrophoresis.

    PubMed

    Sharma, Divakar; Bisht, Deepa

    2016-05-01

    Lipophilic proteome profiling is crucial because they have an anticipated role in biological processes and pathogenesis of Mycobacterium tuberculosis. These lipophilic proteins might be used as potential targets for the development of newer diagnostic markers and drug targets due to their association with membranes and drugs. We developed an efficient and rapid method to enrich the lipophilic proteins extraction from M. tuberculosis H37Rv for 2DE. In the extraction of lipophilic proteins, nonionic detergent (Triton X-100) was added in sonication buffer that augmented the solubilization of the proteins at the time of sonication. Enriched whole cell lysate was subjected to direct phase separation using Triton X-114, without the need for preisolation of membranes. In this study, we report that our optimized extraction buffer increased the lipophilic proteins extraction and their improved resolution on 2D gel up to two- to threefolds (quantitatively and qualitatively) as compared to standard extraction buffer. Some proteins were identified by MALDI-TOF/MS. PMID:26935602

  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. PMID:26492574

  3. 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

  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.

  5. Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

    PubMed Central

    Emmott, Edward; Goodfellow, Ian

    2014-01-01

    Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII. PMID:25046639

  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. 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.

  8. 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

  9. A CAPS-based binding assay provides semi-quantitative validation of protein-DNA interactions

    PubMed Central

    Xie, Yongyao; Zhang, Yaling; Zhao, Xiucai; Liu, Yao-Guang; Chen, Letian

    2016-01-01

    Investigation of protein-DNA interactions provides crucial information for understanding the mechanisms of gene regulation. Current methods for studying protein-DNA interactions, such as DNaseI footprinting or gel shift assays, involve labeling DNA with radioactive or fluorescent tags, making these methods costly, laborious, and potentially damaging to the environment. Here, we describe a novel cleaved amplified polymorphic sequence (CAPS)-based binding assay (CBA), which is a label-free method that can simplify the semi-quantitative validation of protein-DNA interactions. The CBA tests the interaction between a protein and its target DNA, based on the CAPS pattern produced due to differences in the accessibility of a restriction endonuclease site (intrinsic or artificial) in amplified DNA in the presence and absence of the protein of interest. Thus, the CBA can produce a semi-quantitative readout of the interaction strength based on the dose of the binding protein. We demonstrate the principle and feasibility of CBA using B3, MADS3 proteins and the corresponding RY or CArG-box containing DNAs. PMID:26877240

  10. Multiplexed Quantitation of Endogenous Proteins in Dried Blood Spots by Multiple Reaction Monitoring - Mass Spectrometry

    PubMed Central

    Chambers, Andrew G.; Percy, Andrew J.; Yang, Juncong; Camenzind, Alexander G.; Borchers, Christoph H.

    2013-01-01

    Dried blood spot (DBS) sampling, coupled with multiple reaction monitoring mass spectrometry (MRM-MS), is a well-established approach for quantifying a wide range of small molecule biomarkers and drugs. This sampling procedure is simpler and less-invasive than those required for traditional plasma or serum samples enabling collection by minimally trained personnel. Many analytes are stable in the DBS format without refrigeration, which reduces the cost and logistical challenges of sample collection in remote locations. These advantages make DBS sample collection desirable for advancing personalized medicine through population-wide biomarker screening. Here we expand this technology by demonstrating the first multiplexed method for the quantitation of endogenous proteins in DBS samples. A panel of 60 abundant proteins in human blood was targeted by monitoring proteotypic tryptic peptides and their stable isotope-labeled analogs by MRM. Linear calibration curves were obtained for 40 of the 65 peptide targets demonstrating multiple proteins can be quantitatively extracted from DBS collection cards. The method was also highly reproducible with a coefficient of variation of <15% for all 40 peptides. Overall, this assay quantified 37 proteins spanning a range of more than four orders of magnitude in concentration within a single 25 min LC/MRM-MS analysis. The protein abundances of the 33 proteins quantified in matching DBS and whole blood samples showed an excellent correlation, with a slope of 0.96 and an R2 value of 0.97. Furthermore, the measured concentrations for 80% of the proteins were stable for at least 10 days when stored at −20 °C, 4 °C and 37 °C. This work represents an important first step in evaluating the integration of DBS sampling with highly-multiplexed MRM for quantitation of endogenous proteins. PMID:23221968

  11. 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

  12. 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

  13. Quantitative surface studies of protein adsorption by infrared spectroscopy. II. Quantification of adsorbed and bulk proteins

    SciTech Connect

    Fink, D.J.; Hutson, T.B.; Chittur, K.K.; Gendreau, R.M.

    1987-08-15

    Attenuated total reflectance Fourier transform infrared spectra of surface-adsorbed proteins are correlated with concentration measurements determined by /sup 125/I-labeled proteins. This paper demonstrates that linear correlations between the intensity of the major bands of proteins and the quantity of proteins can be obtained for human albumin and immunoglobulin G up to surface concentrations of approximately 0.25 microgram/cm2. A poorer correlation was observed for human fibrinogen. A linear correlation was also observed between the concentration in the bulk solution and the major bands of albumin up to a concentration of 60 mg/ml.

  14. Advances in multiplexed MRM-based protein biomarker quantitation toward clinical utility.

    PubMed

    Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Hardie, Darryl B; Borchers, Christoph H

    2014-05-01

    Accurate and rapid protein quantitation is essential for screening biomarkers for disease stratification and monitoring, and to validate the hundreds of putative markers in human biofluids, including blood plasma. An analytical method that utilizes stable isotope-labeled standard (SIS) peptides and selected/multiple reaction monitoring-mass spectrometry (SRM/MRM-MS) has emerged as a promising technique for determining protein concentrations. This targeted approach has analytical merit, but its true potential (in terms of sensitivity and multiplexing) has yet to be realized. Described herein is a method that extends the multiplexing ability of the MRM method to enable the quantitation 142 high-to-moderate abundance proteins (from 31mg/mL to 44ng/mL) in undepleted and non-enriched human plasma in a single run. The proteins have been reported to be associated to a wide variety of non-communicable diseases (NCDs), from cardiovascular disease (CVD) to diabetes. The concentrations of these proteins in human plasma are inferred from interference-free peptides functioning as molecular surrogates (2 peptides per protein, on average). A revised data analysis strategy, involving the linear regression equation of normal control plasma, has been instituted to enable the facile application to patient samples, as demonstrated in separate nutrigenomics and CVD studies. The exceptional robustness of the LC/MS platform and the quantitative method, as well as its high throughput, makes the assay suitable for application to patient samples for the verification of a condensed or complete protein panel. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. PMID:23806606

  15. Functional and Complementary Phosphorylation State Attributes of Human Insulin-like Growth Factor-Binding Protein-1 (IGFBP-1) Isoforms Resolved by Free Flow Electrophoresis

    PubMed Central

    Nissum, Mikkel; Shehab, Majida Abu; Sukop, Ute; Khosravi, Javad M.; Wildgruber, Robert; Eckerskorn, Christoph; Han, Victor K. M.; Gupta, Madhulika B.

    2009-01-01

    Fetal growth restriction (FGR) is a common disorder in which a fetus is unable to achieve its genetically determined potential size. High concentrations of insulin-like growth factor-binding protein-1 (IGFBP-1) have been associated with FGR. Phosphorylation of IGFBP-1 is a mechanism by which insulin-like growth factor-I (IGF-I) bioavailability can be modulated in FGR. In this study a novel strategy was designed to determine a link between IGF-I affinity and the concomitant phosphorylation state characteristics of IGFBP-1 phosphoisoforms. Using free flow electrophoresis (FFE), multiple IGFBP-1 phosphoisoforms in amniotic fluid were resolved within pH 4.43–5.09. The binding of IGFBP-1 for IGF-I in each FFE fraction was determined with BIAcore biosensor analysis. The IGF-I affinity (K) for different IGFBP-1 isoforms ranged between 1.12e−08 and 4.59e−07. LC-MS/MS characterization revealed four phosphorylation sites, Ser(P)98, Ser(P)101, Ser(P)119, and Ser(P)169, of which Ser(P)98 was new. Although the IGF-I binding affinity for IGFBP-1 phosphoisoforms across the FFE fractions did not correlate with phosphopeptide intensities for Ser(P)101, Ser(P)98, and Ser(P)169 sites, a clear association was recorded with Ser(P)119. Our data demonstrate that phosphorylation at Ser119 plays a significant role in modulating affinity of IGFBP-1 for IGF-I. In addition, an altered profile of IGFBP-1 phosphoisoforms was revealed between FGR and healthy pregnancies that may result from potential site-specific phosphorylation. This study provides a strong basis for use of this novel approach in establishing the linkage between phosphorylation of IGFBP-1 and FGR. This overall strategy will also be broadly applicable to other phosphoproteins with clinical and functional significance. PMID:19193607

  16. 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

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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. PMID:27166738

  3. Quantitative Assessment of Protein Interaction with Methyl-Lysine Analogues by Hybrid Computational and Experimental Approaches

    PubMed Central

    2011-01-01

    In cases where binding ligands of proteins are not easily available, structural analogues are often used. For example, in the analysis of proteins recognizing different methyl-lysine residues in histones, methyl-lysine analogues based on methyl-amino-alkylated cysteine residues have been introduced. Whether these are close enough to justify quantitative interpretation of binding experiments is however questionable. To systematically address this issue, we developed, applied, and assessed a hybrid computational/experimental approach that extracts the binding free energy difference between the native ligand (methyl-lysine) and the analogue (methyl-amino-alkylated cysteine) from a thermodynamic cycle. Our results indicate that measured and calculated binding differences are in very good agreement and therefore allow the correction of measured affinities of the analogues. We suggest that quantitative binding parameters for defined ligands in general can be derived by this method with remarkable accuracy. PMID:21991995

  4. Quantitative time-resolved measurement of membrane protein-ligand interactions using microcantilever array sensors

    NASA Astrophysics Data System (ADS)

    Braun, Thomas; Ghatkesar, Murali Krishna; Backmann, Natalija; Grange, Wilfried; Boulanger, Pascale; Letellier, Lucienne; Lang, Hans-Peter; Bietsch, Alex; Gerber, Christoph; Hegner, Martin

    2009-03-01

    Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor-the FhuA receptor of Escherichia coli-is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

  5. Pitfalls in protein quantitation using acid-catalyzed O18 labeling: hydrolysis-driven deamidation

    PubMed Central

    Wang, Shunhai; Bobst, Cedric E.; Kaltashov, Igor A.

    2011-01-01

    Proteolysis combined with O18 labeling emerged recently as a powerful tool for quantitation of proteins for which suitable internal standards cannot be produced using molecular biology methods. Several recent reports suggested that acid-catalyzed O18 labeling may be superior to the commonly accepted enzymatic protocol, as it may allow more significant spacing between the isotopic clusters of labeled and unlabeled peptides, thereby eliminating signal interference and enhancing the quality of quantitation. However, careful examination of this procedure reveals that the results of protein quantitation assisted by acid-catalyzed O18 labeling are highly peptide-dependent. The inconsistency was found to be caused by deamidation of Asn, Gln and carbamidomethylated Cys residues during prolonged exposure of the proteolytic fragments to the acidic environment of the labeling reaction, which translates into a loss in signal for theses peptides. Taking deamidation into account leads to a significant improvement in the consistency of quantitation across a range of different proteolytic fragments. PMID:21819098

  6. 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

  7. 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

  8. 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

  9. Quantitation of proteinuria in nephrotic syndrome by spot urine protein creatinine ratio estimation in children.

    PubMed

    Biswas, A; Kumar, R; Chaterjee, A; Ghosh, J K; Basu, K

    2009-01-01

    In Nephrotic Syndrome the amount of protein excretion is a reflection of activity of disease. Quantitative measurement of proteinuria by a 24-hour urine collection has been the accepted method of evaluation. Recent studies have shown that calculation of protein/creatinine ratio in a spot urine sample correlates well with the 24-hour urine protein (24-HUP) excretion. A study was conducted to compare the accuracy of a spot urinary protein/creatinine ratio (P/C ratio) and urinary dipstick with the 24-hour urine protein. Fifty two samples from 26 patients of nephrotic syndrome were collected. This included a 24-hour urine sample followed by the next voided random spot sample. The protein/creatinine ratio was calculated and dipstick was performed on the spot sample. This was compared with the 24-hour urine protein excretion. The correlation between the three samples was statistically highly significant (p<0.001) for all levels of proteinuria. The normal value of protein/creatinine ratio in Indian children was also estimated on 50 normal children admitted in the ward without any renal diseases calculated to be 0.053 (SE of mean+/-0.003). PMID:19182753

  10. Identification of hypoxia-regulated proteins using MALDI-mass spectrometry imaging combined with quantitative proteomics.

    PubMed

    Djidja, Marie-Claude; Chang, Joan; Hadjiprocopis, Andreas; Schmich, Fabian; Sinclair, John; Mršnik, Martina; Schoof, Erwin M; Barker, Holly E; Linding, Rune; Jørgensen, Claus; Erler, Janine T

    2014-05-01

    Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed. PMID:24702160

  11. A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics.

    PubMed

    Wang, Xiaoshi; Yuan, Zuo-Fei; Fan, Jing; Karch, Kelly R; Ball, Lauren E; Denu, John M; Garcia, Benjamin A

    2016-07-01

    Over the past decades, protein O-GlcNAcylation has been found to play a fundamental role in cell cycle control, metabolism, transcriptional regulation, and cellular signaling. Nevertheless, quantitative approaches to determine in vivo GlcNAc dynamics at a large-scale are still not readily available. Here, we have developed an approach to isotopically label O-GlcNAc modifications on proteins by producing (13)C-labeled UDP-GlcNAc from (13)C6-glucose via the hexosamine biosynthetic pathway. This metabolic labeling was combined with quantitative mass spectrometry-based proteomics to determine protein O-GlcNAcylation turnover rates. First, an efficient enrichment method for O-GlcNAc peptides was developed with the use of phenylboronic acid solid-phase extraction and anhydrous DMSO. The near stoichiometry reaction between the diol of GlcNAc and boronic acid dramatically improved the enrichment efficiency. Additionally, our kinetic model for turnover rates integrates both metabolomic and proteomic data, which increase the accuracy of the turnover rate estimation. Other advantages of this metabolic labeling method include in vivo application, direct labeling of the O-GlcNAc sites and higher confidence for site identification. Concentrating only on nuclear localized GlcNAc modified proteins, we are able to identify 105 O-GlcNAc peptides on 42 proteins and determine turnover rates of 20 O-GlcNAc peptides from 14 proteins extracted from HeLa nuclei. In general, we found O-GlcNAcylation turnover rates are slower than those published for phosphorylation or acetylation. Nevertheless, the rates widely varied depending on both the protein and the residue modified. We believe this methodology can be broadly applied to reveal turnovers/dynamics of protein O-GlcNAcylation from different biological states and will provide more information on the significance of O-GlcNAcylation, enabling us to study the temporal dynamics of this critical modification for the first time. PMID:27114449

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Quantitative evaluation of interaction force between functional groups in protein and polymer brush surfaces.

    PubMed

    Sakata, Sho; Inoue, Yuuki; Ishihara, Kazuhiko

    2014-03-18

    To understand interactions between polymer surfaces and different functional groups in proteins, interaction forces were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Various polymer brush surfaces were systematically prepared by surface-initiated atom transfer radical polymerization as well-defined model surfaces to understand protein adsorption behavior. The polymer brush layers consisted of phosphorylcholine groups (zwitterionic/hydrophilic), trimethylammonium groups (cationic/hydrophilic), sulfonate groups (anionic/hydrophilic), hydroxyl groups (nonionic/hydrophilic), and n-butyl groups (nonionic/hydrophobic) in their side chains. The interaction forces between these polymer brush surfaces and different functional groups (carboxyl groups, amino groups, and methyl groups, which are typical functional groups existing in proteins) were quantitatively evaluated by force-versus-distance curve measurements using atomic force microscopy with a functional-group-functionalized cantilever. Furthermore, the amount of adsorbed protein on the polymer brush surfaces was quantified by surface plasmon resonance using albumin with a negative net charge and lysozyme with a positive net charge under physiological conditions. The amount of proteins adsorbed on the polymer brush surfaces corresponded to the interaction forces generated between the functional groups on the cantilever and the polymer brush surfaces. The weakest interaction force and least amount of protein adsorbed were observed in the case of the polymer brush surface with phosphorylcholine groups in the side chain. On the other hand, positive and negative surfaces generated strong forces against the oppositely charged functional groups. In addition, they showed significant adsorption with albumin and lysozyme, respectively. These results indicated that the interaction force at the functional group level might be

  17. Genome-scale quantitative characterization of bacterial protein localization dynamics throughout the cell cycle

    PubMed Central

    Kuwada, Nathan J; Traxler, Beth; Wiggins, Paul A

    2015-01-01

    Bacterial cells display both spatial and temporal organization, and this complex structure is known to play a central role in cellular function. Although nearly one-fifth of all proteins in Escherichia coli localize to specific subcellular locations, fundamental questions remain about how cellular-scale structure is encoded at the level of molecular-scale interactions. One significant limitation to our understanding is that the localization behavior of only a small subset of proteins has been characterized in detail. As an essential step toward a global model of protein localization in bacteria, we capture and quantitatively analyze spatial and temporal protein localization patterns throughout the cell cycle for nearly every protein in E. coli that exhibits nondiffuse localization. This genome-scale analysis reveals significant complexity in patterning, notably in the behavior of DNA-binding proteins. Complete cell-cycle imaging also facilitates analysis of protein partitioning to daughter cells at division, revealing a broad and robust assortment of asymmetric partitioning behaviors. PMID:25353361

  18. Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

    PubMed

    Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun

    2013-04-01

    To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species. PMID:23464874

  19. 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

  20. Quantitative reduction of the TCR adapter protein SLP-76 unbalances immunity and immune regulation.

    PubMed

    Siggs, Owen M; Miosge, Lisa A; Daley, Stephen R; Asquith, Kelly; Foster, Paul S; Liston, Adrian; Goodnow, Christopher C

    2015-03-15

    Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies. PMID:25662996

  1. 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.

  2. 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.

  3. 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 ...

  4. Multi-Q: a fully automated tool for multiplexed protein quantitation.

    PubMed

    Lin, Wen-Ting; Hung, Wei-Neng; Yian, Yi-Hwa; Wu, Kun-Pin; Han, Chia-Li; Chen, Yet-Ran; Chen, Yu-Ju; Sung, Ting-Yi; Hsu, Wen-Lian

    2006-09-01

    The iTRAQ labeling method combined with shotgun proteomic techniques represents a new dimension in multiplexed quantitation for relative protein expression measurement in different cell states. To expedite the analysis of vast amounts of spectral data, we present a fully automated software package, called Multi-Q, for multiplexed iTRAQ-based quantitation in protein profiling. Multi-Q is designed as a generic platform that can accommodate various input data formats from search engines and mass spectrometer manufacturers. To calculate peptide ratios, the software automatically processes iTRAQ's signature peaks, including peak detection, background subtraction, isotope correction, and normalization to remove systematic errors. Furthermore, Multi-Q allows users to define their own data-filtering thresholds based on semiempirical values or statistical models so that the computed results of fold changes in peptide ratios are statistically significant. This feature facilitates the use of Multi-Q with various instrument types with different dynamic ranges, which is an important aspect of iTRAQ analysis. The performance of Multi-Q is evaluated with a mixture of 10 standard proteins and human Jurkat T cells. The results are consistent with expected protein ratios and thus demonstrate the high accuracy, full automation, and high-throughput capability of Multi-Q as a large-scale quantitation proteomics tool. These features allow rapid interpretation of output from large proteomic datasets without the need for manual validation. Executable Multi-Q files are available on Windows platform at http://ms.iis.sinica.edu.tw/Multi-Q/. PMID:16944945

  5. 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

  6. 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

  7. Protein profiling of human lung telocytes and microvascular endothelial cells using iTRAQ quantitative proteomics

    PubMed Central

    Zheng, Yonghua; Cretoiu, Dragos; Yan, Guoquan; Cretoiu, Sanda Maria; Popescu, Laurentiu M; Fang, Hao; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) are described as a particular type of cells of the interstitial space (www.telocytes.com). Their main characteristics are the very long telopodes with alternating podoms and podomers. Recently, we performed a comparative proteomic analysis of human lung TCs with fibroblasts, demonstrating that TCs are clearly a distinct cell type. Therefore, the present study aims to reinforce this idea by comparing lung TCs with endothelial cells (ECs), since TCs and ECs share immunopositivity for CD34. We applied isobaric tag for relative and absolute quantification (iTRAQ) combined with automated 2-D nano-ESI LC-MS/MS to analyse proteins extracted from TCs and ECs in primary cell cultures. In total, 1609 proteins were identified in cell cultures. 98 proteins (the 5th day), and 82 proteins (10th day) were confidently quantified (screened by two-sample t-test, P < 0.05) as up- or down-regulated (fold change >2). We found that in TCs there are 38 up-regulated proteins at the 5th day and 26 up-regulated proteins at the 10th day. Bioinformatics analysis using Panther revealed that the 38 proteins associated with TCs represented cellular functions such as intercellular communication (via vesicle mediated transport) and structure morphogenesis, being mainly cytoskeletal proteins and oxidoreductases. In addition, we found 60 up-regulated proteins in ECs e.g.: cell surface glycoprotein MUC18 (15.54-fold) and von Willebrand factor (5.74-fold). The 26 up-regulated proteins in TCs at 10th day, were also analysed and confirmed the same major cellular functions, while the 56 down-regulated proteins confirmed again their specificity for ECs. In conclusion, we report here the first extensive comparison of proteins from TCs and ECs using a quantitative proteomics approach. Our data show that TCs are completely different from ECs. Protein expression profile showed that TCs play specific roles in intercellular communication and intercellular signalling. Moreover, they might

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans

    PubMed Central

    Fredens, Julius; Færgeman, Nils J.

    2012-01-01

    Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology to quantitatively examine metabolic and signaling pathways in yeast, fruit flies, plants, cell cultures and mice. However, only metabolic labeling using 15N has been applied to examine such events in the nematode Caenorhabditis elegans. We have recently shown that C. elegans can be completely labeled with heavy-labeled lysine by feeding worms on prelabeled lysine auxotroph Escherichia coli for just one generation. We applied this methodology to examine the organismal response to functional loss or RNAi mediated knock down of the transcription factor NHR-49, and found numerous proteins involved in lipid metabolism to be downregulated, which is consistent with its previously proposed function as a transcriptional regulator of fatty acid metabolism. The combined use of quantitative proteomics and selective gene knockdown by RNAi provides a powerful tool with broad implications for C. elegans biology. PMID:24058826

  13. Autoradiographic method for quantitation of radiolabeled proteins in tissues using indium-111

    SciTech Connect

    Morrell, E.M.; Tompkins, R.G.; Fischman, A.J.; Wilkinson, R.A.; Burke, J.F.; Rubin, R.H.; Strauss, H.W.; Yarmush, M.L. )

    1989-09-01

    A quantitative autoradiographic method was developed to measure 111In-labeled proteins in extravascular tissues with a spatial resolution sufficient to associate these proteins with tissue morphology. A linear relationship between measured grain density and isotope concentration was demonstrated with uniformly-labeled standard sources of epoxy-embedded gelatin containing (111In)albumin; half-distance of spatial resolution was 0.6 micron. The technique was illustrated by measuring 24-hr accumulation of diethylenetriaminepentaacetic acid-coupled 111In-labeled human polyclonal IgG and human serum albumin (HSA) in a thigh infection model in the rat. Gamma camera images localized the infection and showed target-to-background ratios of 2.5 {plus minus} 0.3 for IgG and 1.4 {plus minus} 0.02 for human serum albumin (mean {plus minus} s.d., n = 3). Using quantitative autoradiography, significantly higher average tissue concentrations were found in the infected thighs at 4 to 4.5% of the initial plasma concentrations as compared to 0.2 to 0.3% of initial plasma concentrations in the noninfected thigh (p less than 0.05); these radiolabeled proteins were not inflammatory cell associated and localized primarily within the edematous interstitial spaces of the infection.

  14. Quantitative Liver-Specific Protein Fingerprint in Blood: A Signature for Hepatotoxicity

    PubMed Central

    Hu, Zhiyuan; Lausted, Christopher; Yoo, Hyuntae; Yan, Xiaowei; Brightman, Amy; Chen, Jiankui; Wang, Weizhi; Bu, Xiangli; Hood, Leroy

    2014-01-01

    We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. PMID:24465277

  15. 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. PMID:25874902

  16. 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...

  17. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways.

    PubMed

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-07-17

    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 cofactors (cochaperones) that regulate their specificity and function. However, how these cochaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone-cochaperone-client interaction network in human cells. We uncover hundreds of chaperone clients, delineate their participation in specific cochaperone complexes, and establish a surprisingly distinct network of protein-protein interactions for cochaperones. As a salient example of the power of such analysis, we establish that NUDC family cochaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network and its regulation in development and disease and expand the use of chaperones as sensors for drug-target engagement. PMID:25036637

  18. 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

  19. A Chip-Capillary Hybrid Device for Automated Transfer of Sample Pre-Separated by Capillary Isoelectric Focusing to Parallel Capillary Gel Electrophoresis for Two-Dimensional Protein Separation

    PubMed Central

    Lu, Joann J.; Wang, Shili; Li, Guanbin; Wang, Wei; Pu, Qiaosheng; Liu, Shaorong

    2012-01-01

    In this report, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to re-route the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed. PMID:22830584

  20. 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

  1. Proteomic analysis of rice after different seed space flights by two-dimensional difference electrophoresis

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Liang, Shujian; Sun, Yeqing

    To investigate the biological effects of space environment in rice plants, proteomic profiles of six rice cultivars growing after twice different seed space flights were analyzed by two-dimensional difference gel electrophoresis (2-D DIGE) coupled with mass spectrometry (MS). Over 1500 protein spots were detected in each paired space/ground-control comparison and more than 800 protein spots were reproducible across all the samples. Six proteins including peroxiredoxin and rubisco were found significantly changed in most of the six cultivars after both of the seed space flights, indicating they might be associated with the responses of rice cells to the space environment. Cluster analyses were also applied using the quantitative protein expression data: cultivar hierarchical clustering and principal component analysis both indicated that the rice proteome changed its expression profiles after seed space environment exposures while protein hierarchical clustering revealed that there might be a decrease of protein expression in rice plants after seed space flights.

  2. 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

  3. Polyacrylamide gel electrophoretic methods in the separation of structural muscle proteins.

    SciTech Connect

    Barany, K.; Barany, M.; Giometti, C. S.; Center for Mechanistic Biology and Biotechnology; Univ. of Illinois at Chicago

    1995-04-28

    Polyacrylamide gel electrophoresis plays a major role in analyzing the function of muscle structural proteins. This review describes one- and two-dimensional gel electrophoretic methods for qualitative and quantitative investigation of the muscle proteins, with special emphasis on determination of protein phosphorylation. The electrophoretic studies established the subunit structures of the muscle proteins, characterized their multiple forms, revealed changes in subunit composition or shifts in isoform distribution of specific proteins during development, upon stimulation or denervation of the muscle. Protein phosphorylation during muscle contraction is preferentially studied by two-dimensional gel electrophoresis. The same method demonstrated protein alterations in human neuromuscular diseases.

  4. 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…

  5. Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum*

    PubMed Central

    Foth, Bernardo Javier; Zhang, Neng; Chaal, Balbir Kaur; Sze, Siu Kwan; Preiser, Peter Rainer; Bozdech, Zbynek

    2011-01-01

    Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein

  6. 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.

  7. Quantitative investigations of quantum coherence for a light-harvesting protein at conditions simulating photosynthesis.

    PubMed

    Turner, Daniel B; Dinshaw, Rayomond; Lee, Kyung-Koo; Belsley, Michael S; Wilk, Krystyna E; Curmi, Paul M G; Scholes, Gregory D

    2012-04-14

    Recent measurements using two-dimensional electronic spectroscopy (2D ES) have shown that the initial dynamic response of photosynthetic proteins can involve quantum coherence. We show how electronic coherence can be differentiated from vibrational coherence in 2D ES. On that basis we conclude that both electronic and vibrational coherences are observed in the phycobiliprotein light-harvesting complex PC645 from Chroomonas sp. CCMP270 at ambient temperature. These light-harvesting antenna proteins of the cryptophyte algae are suspended in the lumen, where the pH drops significantly under sustained illumination by sunlight. Here we measured 2D ES of PC645 at increasing levels of acidity to determine if the change in pH affects the quantum coherence; quantitative analysis reveals that the dynamics are insensitive to the pH change. PMID:22374579

  8. 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

  9. 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

  10. Quantitative fluorescence loss in photobleaching for analysis of protein transport and aggregation

    PubMed Central

    2012-01-01

    Background Fluorescence loss in photobleaching (FLIP) is a widely used imaging technique, which provides information about protein dynamics in various cellular regions. In FLIP, a small cellular region is repeatedly illuminated by an intense laser pulse, while images are taken with reduced laser power with a time lag between the bleaches. Despite its popularity, tools are lacking for quantitative analysis of FLIP experiments. Typically, the user defines regions of interest (ROIs) for further analysis which is subjective and does not allow for comparing different cells and experimental settings. Results We present two complementary methods to detect and quantify protein transport and aggregation in living cells from FLIP image series. In the first approach, a stretched exponential (StrExp) function is fitted to fluorescence loss (FL) inside and outside the bleached region. We show by reaction–diffusion simulations, that the StrExp function can describe both, binding/barrier–limited and diffusion-limited FL kinetics. By pixel-wise regression of that function to FL kinetics of enhanced green fluorescent protein (eGFP), we determined in a user-unbiased manner from which cellular regions eGFP can be replenished in the bleached area. Spatial variation in the parameters calculated from the StrExp function allow for detecting diffusion barriers for eGFP in the nucleus and cytoplasm of living cells. Polyglutamine (polyQ) disease proteins like mutant huntingtin (mtHtt) can form large aggregates called inclusion bodies (IB’s). The second method combines single particle tracking with multi-compartment modelling of FL kinetics in moving IB’s to determine exchange rates of eGFP-tagged mtHtt protein (eGFP-mtHtt) between aggregates and the cytoplasm. This method is self-calibrating since it relates the FL inside and outside the bleached regions. It makes it therefore possible to compare release kinetics of eGFP-mtHtt between different cells and experiments. Conclusions We

  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. PMID:25749303

  12. Quantitative Interpretation of Multifrequency Multimode EPR Spectra of Metal Containing Proteins, Enzymes, and Biomimetic Complexes.

    PubMed

    Petasis, Doros T; Hendrich, Michael P

    2015-01-01

    Electron paramagnetic resonance (EPR) spectroscopy has long been a primary method for characterization of paramagnetic centers in materials and biological complexes. Transition metals in biological complexes have valence d-orbitals that largely define the chemistry of the metal centers. EPR spectra are distinctive for metal type, oxidation state, protein environment, substrates, and inhibitors. The study of many metal centers in proteins, enzymes, and biomimetic complexes has led to the development of a systematic methodology for quantitative interpretation of EPR spectra from a wide array of metal containing complexes. The methodology is now contained in the computer program SpinCount. SpinCount allows simulation of EPR spectra from any sample containing multiple species composed of one or two metals in any spin state. The simulations are quantitative, thus allowing determination of all species concentrations in a sample directly from spectra. This chapter will focus on applications to transition metals in biological systems using EPR spectra from multiple microwave frequencies and modes. PMID:26478486

  13. Quantitative prediction of peptide binding to HLA-DP1 protein.

    PubMed

    Ivanov, Stefan; Dimitrov, Ivan; Doytchinova, Irini

    2013-01-01

    The exogenous proteins are processed by the host antigen-processing cells. Peptidic fragments of them are presented on the cell surface bound to the major hystocompatibility complex (MHC) molecules class II and recognized by the CD4+ T lymphocytes. The MHC binding is considered as the crucial prerequisite for T-cell recognition. Only peptides able to form stable complexes with the MHC proteins are recognized by the T-cells. These peptides are known as T-cell epitopes. All T-cell epitopes are MHC binders, but not all MHC binders are T-cell epitopes. The T-cell epitope prediction is one of the main priorities of immunoinformatics. In the present study, three chemometric techniques are combined to derive a model for in silico prediction of peptide binding to the human MHC class II protein HLA-DP1. The structures of a set of known peptide binders are described by amino acid z-descriptors. Data are processed by an iterative self-consisted algorithm using the method of partial least squares, and a quantitative matrix (QM) for peptide binding prediction to HLA-DP1 is derived. The QM is validated by two sets of proteins and showed an average accuracy of 86 percent. PMID:24091413

  14. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

    PubMed

    Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  15. Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine

    PubMed Central

    Dick Jr, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

    2015-01-01

    ABSTRACT The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90–110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques. PMID:25997113

  16. 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

  17. 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

  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 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

  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

    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. PMID:26957635

  20. 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...

  1. 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...

  2. 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...

  3. Identification of quantitative trait loci (QTL) controlling protein, oil, and five major fatty acids’ contents in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improved seed composition in soybean (Glycine max L. Merr.) for protein and oil quality is one of the major goals of soybean breeders. A group of genes that act as quantitative traits with their effects can alter protein, oil, palmitic, stearic, oleic, linoleic, and linolenic acids percentage in soy...

  4. Quantitative immunological determination of 12 plasma proteins excreted in human urine collected before and after exercise

    PubMed Central

    Poortmans, Jacques; Jeanloz, Roger W.

    1968-01-01

    Urine was collected from 6 healthy male adults at rest and from 20 male adults after a marathon race (25 miles). The concentrated urines were quantitatively analyzed, by single radial immunodiffusion, for their content in 12 different plasma proteins: tryptophan-rich prealbumin, albumin, α1-acid glycoprotein, α1-antitrypsin, ceruloplasmin, haptoglobin, Gc-globulin, transferrin, hemopexin, β2-glycoprotein I, γA-globulin, and γG-globulin. Albumin, γA-globulin, and γG-globulin represent the major part of the plasma proteins detected in normal urine excreted by humans at rest (12, 0.5, and 2.5 mg respectively, out of a total excretion of 17.5 mg of plasma proteins per 24 hr). The other plasma proteins were excreted at a lower rate (< 0.4 mg/24 hr). The relative content of tryptophan-rich prealbumin, α1-antitrypsin, Gc-globulin, transferrin, and γG-globulin was lower in normal urine than in normal serum, whereas that of α1-acid glycoprotein, β2-glycoprotein I, and γA-globulin was higher. The ratio of γG-globulin to γA-globulin was 4.9:1. When plotted on a logarithmic scale, no direct relationship between the molecular weight of a protein and the value of its renal clearance could be observed. Strenuous exercise increased (up to 50-fold) the excretion of plasma proteins which represent 82% of the total proteins found in urine, instead of 57% in urine collected from humans at rest. There was particularly a significant rise of tryptophan-rich albumin, albumin, α1-acid glycoprotein, transferrin, γA-globulin, and γG-globulin (0.26, 127, 11.8, 3.3, 1.2, and 2.0 μg respectively, out of a total excretion of 167 μg of plasma proteins per min). The ratio of γG-globulin to γA-globulin was 16:1. After exercise, the renal clearance of proteins increased from 2 to 40 times, but, as for the urine of normal subjects at rest, no direct relationship between molecular weight and renal clearance could be observed. Images PMID:4170390

  5. 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.

  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. Comparative proteomics and difference gel electrophoresis.

    PubMed

    Minden, Jonathan

    2007-12-01

    The goal of comparative proteomics is to analyze proteome changes in response to development, disease, or environment. This is a two-step process in which proteins within cellular extracts are first fractionated to reduce sample complexity, and then the proteins are identified by mass spectrometry. Two-dimensional electrophoresis (2DE) is the long-time standard for protein separation, but it has suffered from poor reproducibility and limited sensitivity. Difference gel electrophoresis (DIGE), in which two protein samples are separately labeled with different fluorescent dyes and then co-electrophoresed on the same 2DE gel, was developed to overcome the reproducibility and sensitivity limitations. In this essay, I discuss the principles of comparative proteomics and the development of DIGE. PMID:18251249

  8. 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.

  9. Fraction collector for electrophoresis

    NASA Technical Reports Server (NTRS)

    Bier, M.

    1977-01-01

    Rotating-tube electrophoresis apparatus employs rotating jet of eluting buffer to reduce effects of convection during separation. Designed for separation of microorganisms and biological species, system combines gravity/gradient compensating of lumen with buffer flush at fraction outlet to increase separation efficiency.

  10. Assessment of Protein Binding of 5-Hydroxythalidomide Bioactivated in Humanized Mice with Human P450 3A-Chromosome or Hepatocytes by Two-Dimensional Electrophoresis/Accelerator Mass Spectrometry.

    PubMed

    Yamazaki, Hiroshi; Suemizu, Hiroshi; Kazuki, Yasuhiro; Oofusa, Ken; Kuribayashi, Shunji; Shimizu, Makiko; Ninomiya, Shinichi; Horie, Toru; Shibata, Norio; Guengerich, F Peter

    2016-08-15

    Bioactivation of 5-hydroxy-[carbonyl-(14)C]thalidomide, a known metabolite of thalidomide, by human artificial or native cytochrome P450 3A enzymes, and nonspecific binding in livers of mice was assessed using two-dimensional electrophoresis combined with accelerator mass spectrometry. The apparent major target proteins were liver microsomal cytochrome c oxidase subunit 6B1 and ATP synthase subunit α in mice containing humanized P450 3A genes or transplanted humanized liver. Liver cytosolic retinal dehydrogenase 1 and glutathione transferase A1 were targets in humanized mice with P450 3A and hepatocytes, respectively. 5-Hydroxythalidomide is bioactivated by human P450 3A enzymes and trapped with proteins nonspecifically in humanized mice. PMID:27464947

  11. A Genome-Wide Association Study Identifies Protein Quantitative Trait Loci (pQTLs)

    PubMed Central

    Corsi, Anna-Maria; Stevens, Kara; Rafferty, Ian; Lauretani, Fulvio; Murray, Anna; Gibbs, J. Raphael; Paolisso, Giuseppe; Rafiq, Sajjad; Simon-Sanchez, Javier; Lango, Hana; Scholz, Sonja; Weedon, Michael N.; Arepalli, Sampath; Rice, Neil; Washecka, Nicole; Hurst, Alison; Britton, Angela; Henley, William; van de Leemput, Joyce; Li, Rongling; Newman, Anne B.; Tranah, Greg; Harris, Tamara; Panicker, Vijay; Dayan, Colin; Bennett, Amanda; McCarthy, Mark I.; Ruokonen, Aimo; Jarvelin, Marjo-Riitta; Guralnik, Jack; Bandinelli, Stefania; Frayling, Timothy M.; Singleton, Andrew; Ferrucci, Luigi

    2008-01-01

    include the presence of strong genetic effects in cis locations. The identification of protein quantitative trait loci (pQTLs) may be a powerful complementary method of improving our understanding of disease pathways. PMID:18464913

  12. Dynamics of natural killer cell receptor revealed by quantitative analysis of photoswitchable protein.

    PubMed

    Pageon, Sophie V; Aquino, Gerardo; Lagrue, Kathryn; Köhler, Karsten; Endres, Robert G; Davis, Daniel M

    2013-11-01

    Natural Killer (NK) cell activation is dynamically regulated by numerous activating and inhibitory surface receptors that accumulate at the immune synapse. Quantitative analysis of receptor dynamics has been limited by methodologies that rely on indirect measurements such as fluorescence recovery after photobleaching. Here, we report an apparently novel approach to study how proteins traffic to and from the immune synapse using NK cell receptors tagged with the photoswitchable fluorescent protein tdEosFP, which can be irreversibly photoswitched from a green to red fluorescent state by ultraviolet light. Thus, after a localized switching event, the movement of the photoswitched molecules can be temporally and spatially resolved by monitoring fluorescence in two regions of interest. By comparing images with mathematical models, we evaluated the diffusion coefficient of the receptor KIR2DL1 (0.23 ± 0.06 μm(2) s(-1)) and assessed how synapse formation affects receptor dynamics. Our data conclude that the inhibitory NK cell receptor KIR2DL1 is continually trafficked into the synapse, and remains surprisingly stable there. Unexpectedly, however, in NK cells forming synapses with multiple target cells simultaneously, KIR2DL1 at one synapse can relocate to another synapse. Thus, our results reveal a previously undetected intersynaptic exchange of protein. PMID:24209843

  13. 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

  14. 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

  15. 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

  16. A new fusion protein platform for quantitatively measuring activity of multiple proteases

    PubMed Central

    2014-01-01

    Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with

  17. 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

  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. Absolute quantitative autoradiography of low concentrations of (/sup 125/I)-labeled proteins in arterial tissue

    SciTech Connect

    Schnitzer, J.J.; Morrel, E.M.; Colton, C.K.; Smith, K.A.; Stemerman, M.B.

    1987-12-01

    We developed a method for absolute quantitative autoradiographic measurement of very low concentrations of (/sup 125/I)-labeled proteins in arterial tissue using Kodak NTB-2 nuclear emulsion. A precise linear relationship between measured silver grain density and isotope concentration was obtained with uniformly labeled standard sources composed of epoxy-embedded gelatin containing glutaraldehyde-fixed (/sup 125/I)-albumin. For up to 308-day exposures of 1 micron-thick tissue sections, background grain densities ranged from about two to eight grains/1000 micron 2, and the technique was sensitive to as little as about one grain/1000 micron 2 above background, which correspond to a radioactivity concentration of about 2 x 10(4) cpm/ml. A detailed statistical analysis of variability was performed and the sum of all sources of variation quantified. The half distance for spatial resolution was 1.7 micron. Both visual and automated techniques were employed for quantitative grain density analysis. The method was illustrated by measurement of in vivo transmural (/sup 125/I)-low-density lipoprotein (( /sup 125/I)-LDL) concentration profiles in de-endothelialized rabbit thoracic aortic wall.

  2. Thin-layer immunoaffinity chromatography with bar code quantitation of C-reactive protein.

    PubMed

    Nilsson, S; Lager, C; Laurell, T; Birnbaum, S

    1995-09-01

    A rapid thin-layer immunoaffinity chromatographic method for quantitation in serum of an acute phase reactant, C-reactive protein (CRP), which can differentiate between viral and bacteria] infections, is described, where material and reagent costs are minimal. The analysis is based on the "sandwich" assay format using monoclonal antibodies directed against two sites of CRP. One of the antibodies is covalently bound to defined zones on a thin-layer immunoaffinity chromatography membrane, while the other antibody is covalently bound to deeply dyed blue latex particles. After incubation (CRP sample and latex particles), the CRP-latex immunocomplex is allowed to migrate along the immunoaffinity chromatography membrane. In the presence of antigen, a sandwich is formed between the CRP-latex immunocomplex and membrane-bound antibodies, which results in the appearance of blue lines on the membrane. Antibody immobilization on the TLC membrane is made with a redesigned piezoelectric-driven ink-jet printer. The time required for the analysis is less than 10 min. Quantitation is achieved either by counting the lines visually, with scanning reflectometry, or with a modified bar code reader. The limit of detection was estimated in the low femtomolar range using the naked eye as detector. PMID:8779423

  3. Quantitative Profiling of the Activity of Protein Lysine Methyltransferase SMYD2 Using SILAC-Based Proteomics.

    PubMed

    Olsen, Jonathan B; Cao, Xing-Jun; Han, Bomie; Chen, Lisa Hong; Horvath, Alexander; Richardson, Timothy I; Campbell, Robert M; Garcia, Benjamin A; Nguyen, Hannah

    2016-03-01

    The significance of non-histone lysine methylation in cell biology and human disease is an emerging area of research exploration. The development of small molecule inhibitors that selectively and potently target enzymes that catalyze the addition of methyl-groups to lysine residues, such as the protein lysine mono-methyltransferase SMYD2, is an active area of drug discovery. Critical to the accurate assessment of biological function is the ability to identify target enzyme substrates and to define enzyme substrate specificity within the context of the cell. Here, using stable isotopic labeling with amino acids in cell culture (SILAC) coupled with immunoaffinity enrichment of mono-methyl-lysine (Kme1) peptides and mass spectrometry, we report a comprehensive, large-scale proteomic study of lysine mono-methylation, comprising a total of 1032 Kme1 sites in esophageal squamous cell carcinoma (ESCC) cells and 1861 Kme1 sites in ESCC cells overexpressing SMYD2. Among these Kme1 sites is a subset of 35 found to be potently down-regulated by both shRNA-mediated knockdown of SMYD2 and LLY-507, a selective small molecule inhibitor of SMYD2. In addition, we report specific protein sequence motifs enriched in Kme1 sites that are directly regulated by endogenous SMYD2 activity, revealing that SMYD2 substrate specificity is more diverse than expected. We further show direct activity of SMYD2 toward BTF3-K2, PDAP1-K126 as well as numerous sites within the repetitive units of two unique and exceptionally large proteins, AHNAK and AHNAK2. Collectively, our findings provide quantitative insights into the cellular activity and substrate recognition of SMYD2 as well as the global landscape and regulation of protein mono-methylation. PMID:26750096

  4. 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.

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. A Slot Blot Immunoassay for Quantitative Detection of Plasmodium falciparum Circumsporozoite Protein in Mosquito Midgut Oocyst

    PubMed Central

    Kumar, Sanjai; Zheng, Hong; Deng, Bingbing; Mahajan, Babita; Grabias, Bryan; Kozakai, Yukiko; Morin, Merribeth J.; Locke, Emily; Birkett, Ashley; Miura, Kazutoyo; Long, Carole

    2014-01-01

    There is still a need for sensitive and reproducible immunoassays for quantitative detection of malarial antigens in preclinical and clinical phases of vaccine development and in epidemiology and surveillance studies, particularly in the vector host. Here we report the results of sensitivity and reproducibility studies for a research-grade, quantitative enhanced chemiluminescent-based slot blot assay (ECL-SB) for detection of both recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP) and native PfCSP from Oocysts (Pf Oocyst) developing in the midguts of Anopheles stephensi mosquitoes. The ECL-SB detects as little as 1.25 pg of rPfCSP (linear range of quantitation 2.5–20 pg; R2 = 0.9505). We also find the earliest detectable expression of native PfCSP in Pf Oocyst by ECL-SB occurs on day 7 post feeding with infected blood meal. The ECL-SB was able to detect approximately as few as 0.5 day 8 Pf Oocysts (linear quantitation range 1–4, R2 = 0.9795) and determined that one Pf Oocyst expressed approximately 2.0 pg (0.5–3 pg) of native PfCSP, suggesting a similar range of detection for recombinant and native forms of Pf CSP. The ECL-SB is highly reproducible; the Coefficient of Variation (CV) for inter-assay variability for rPf CSP and native PfCSP were 1.74% and 1.32%, respectively. The CVs for intra-assay variability performed on three days for rPf CSP were 2.41%, 0.82% and 2% and for native Pf CSP 1.52%, 0.57%, and 1.86%, respectively. In addition, the ECL-SB was comparable to microscopy in determining the P. falciparum prevalence in mosquito populations that distinctly contained either high and low midgut Pf Oocyst burden. In whole mosquito samples, estimations of positivity for P. falciparum in the high and low burden groups were 83.3% and 23.3% by ECL-SB and 85.7% and 27.6% by microscopy. Based on its performance characteristics, ECL-SB could be valuable in vaccine development and to measure the parasite prevalence in mosquitoes and

  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. PMID:25770625

  12. 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.

  13. 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

  14. 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

  15. 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. PMID:23659366

  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. 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.

  18. Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools

    PubMed Central

    Cui, Yi; Choudhury, Samrat Roy; Irudayaraj, Joseph

    2015-01-01

    In this work we evaluate the interaction of two optogenetic protein variants (CIB1, CIBN) with their complementary protein CRY2 by single-molecule tools in cell-free extracts. After validating the blue light induced co-localization of CRY2 and CIB1/N by Förster resonance energy transfer (FRET) in live cells, a fluorescence correlation spectroscopy (FCS) based method was developed to quantitatively determine the in vitro association of the extracted proteins. Our experiments suggest that CIB1, in comparison with CIBN, possesses a better coupling efficiency with CRY2 due to its intact protein structure and lower diffusion rate within 300 s detection window. PMID:24780222

  19. 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

  20. Quantitative Proteomics Reveals the Temperature-Dependent Proteins Encoded by a Series of Cluster Genes in Thermoanaerobacter Tengcongensis*

    PubMed Central

    Chen, Zhen; Wen, Bo; Wang, Quanhui; Tong, Wei; Guo, Jiao; Bai, Xue; Zhao, Jingjing; Sun, Yao; Tang, Qi; Lin, Zhilong; Lin, Liang; Liu, Siqi

    2013-01-01

    Comprehensive and quantitative information of the thermophile proteome is an important source for understanding of the survival mechanism under high growth temperature. Thermoanaerobacter tengcongensis (T. tengcongensis), a typical anaerobic thermophilic eubacterium, was selected to quantitatively evaluate its protein abundance changes in response to four different temperatures. With optimized procedures of isobaric tags for relative and absolute quantitation quantitative proteomics (iTRAQ), such as peptide fractionation with high-pH reverse phase (RP) high performance liquid chromatography (HPLC), tandem MS acquisition mode in LTQ Orbitrap Velos MS, and evaluation of the quantification algorithms, high quality of the quantitative information of the peptides identified were acquired. In total, 1589 unique proteins were identified and defined 251 as the temperature-dependent proteins. Analysis of genomic locations toward the correspondent genes of these temperature-dependent proteins revealed that more than 30% were contiguous units with relevant biological functions, which are likely to form the operon structures in T. tengcongensis. The RNA sequencing (RNA-seq) data further demonstrated that these cluster genes were cotranscribed, and their mRNA abundance changes responding to temperature exhibited the similar trends as the proteomic results, suggesting that the temperature-dependent proteins are highly associated with the correspondent transcription status. Hence, the operon regulation is likely an energy-efficient mode for T. tengcongensis survival. In addition, evaluation to the functions of differential proteomes indicated that the abundance of the proteins participating in sulfur-respiration on the plasma membrane was decreased as the temperature increased, whereas the glycolysis-related protein abundance was increased. The energy supply in T. tengcongensis at high temperature is, therefore, speculated not mainly through the respiration chain reactions. PMID

  1. Streptococcus mutans protein synthesis during mixed-species biofilm development by high-throughput quantitative proteomics.

    PubMed

    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. 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...

  3. Analysis of protein complexes through model-based biclustering of label-free quantitative AP-MS data.

    PubMed

    Choi, Hyungwon; Kim, Sinae; Gingras, Anne-Claude; Nesvizhskii, Alexey I

    2010-06-22

    Affinity purification followed by mass spectrometry (AP-MS) has become a common approach for identifying protein-protein interactions (PPIs) and complexes. However, data analysis and visualization often rely on generic approaches that do not take advantage of the quantitative nature of AP-MS. We present a novel computational method, nested clustering, for biclustering of label-free quantitative AP-MS data. Our approach forms bait clusters based on the similarity of quantitative interaction profiles and identifies submatrices of prey proteins showing consistent quantitative association within bait clusters. In doing so, nested clustering effectively addresses the problem of overrepresentation of interactions involving baits proteins as compared with proteins only identified as preys. The method does not require specification of the number of bait clusters, which is an advantage against existing model-based clustering methods. We illustrate the performance of the algorithm using two published intermediate scale human PPI data sets, which are representative of the AP-MS data generated from mammalian cells. We also discuss general challenges of analyzing and interpreting clustering results in the context of AP-MS data. PMID:20571534

  4. Evidence for the spontaneous formation of disulfide crosslinked aggregates of tubulin during nondenaturing electrophoresis.

    PubMed

    Correia, J J; Welch, M K; Williams, R C

    1987-06-01

    Phosphocellulose-purified tubulin has been shown to form a characteristic "ladder" of nonmicrotubular aggregates during nondenaturing gel electrophoresis (J. J. Correia and R. C. Williams, Jr. (1985) Arch. Biochem. Biophys. 239, 120-129). In this paper we describe evidence that the intersubunit bonds responsible for formation of these oligomeric particles are disulfides. Two-dimensional nondenaturing-denaturing gel electrophoresis demonstrates that each aggregate zone is composed of alpha- and beta-subunits of tubulin. Omission of beta-mercaptoethanol during the sodium dodecyl sulfate (SDS)-electrophoresis step causes a pattern of aggregates to appear and implicates disulfide linkages in their stabilization. Molecular weights, estimated from mobilities in the second (SDS) dimension of two-dimensional gels, suggest that the aggregates are crosslinked in units of monomers, not heterodimers. Consistent with this conclusion, alpha- or beta-subunits alone (isolated by isoelectric focusing) will form the same ladder of aggregates. The disulfide crosslinking of tubulin is also achievable in solution. It is favored by high concentrations of alcohol, the presence of oxidizing agents, high pH, and high temperature, conditions that denature tubulin and cause rapid noncovalent aggregation or precipitation. When aggregate formation was monitored as a function of time by SDS-gel electrophoresis in the absence of beta-mercaptoethanol and by quantitative sulfhydryl and disulfide titrations, the most effective conditions for the crosslinking reaction included greater than 75% alcohol, excess H2O2, or excess iodine. These results suggest that proximity of a hydrophobic gel matrix, high pH, the presence of oxidizing agents, high protein concentration, tubulin's propensity to aggregate nonspecifically, and the availability of as many as 20 sulfhydryls in alpha beta-tubulin contribute, during nondenaturing gel electrophoresis, to the spontaneous formation of disulfide

  5. High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington’s Disease Model

    PubMed Central

    Titus, Steven A; Southall, Noel; Marugan, Juan; Austin, Christopher P; Zheng, Wei

    2012-01-01

    A hallmark of Huntington’s disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson’s, Alzheimer’s, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear. Identification of small molecule probes that modulate protein aggregation and cytotoxicity caused by aggregated proteins may greatly facilitate the studies on pathogenesis of these diseases and potentially lead to development of new therapies. Based on a detergent insoluble property of the Huntingtin protein aggregates, we have developed a homogenous assay to rapidly quantitate the levels of protein aggregates in a cellular model of Huntington’s disease. The protein aggregation assay has also been multiplexed with a protease release assay for the measurement of cytotoxicity resulting from aggregated proteins in the same cells. Through a testing screen of a compound library, we have demonstrated that this multiplexed cytotoxicity and protein aggregation assay has ability to identify active compounds that prevent cell death and/or modulate protein aggregation in cells of the Huntington’s disease model. Therefore, this multiplexed screening approach is also useful for development of high-throughput screening assays for other neurodegenerative diseases involving protein aggregation. PMID:23346268

  6. Shotgun Quantitative Proteomic Analysis of Proteins Responding to Drought Stress in Brassica rapa L. (Inbred Line “Chiifu”)

    PubMed Central

    Kwon, Soon-Wook

    2016-01-01

    Through a comparative shotgun quantitative proteomics analysis in Brassica rapa (inbred line Chiifu), total of 3,009 nonredundant proteins were identified with a false discovery rate of 0.01 in 3-week-old plants subjected to dehydration treatment for 0, 24, and 48 h, plants subjected to drought stress. Ribulose-bisphosphate carboxylases, chlorophyll a/b-binding protein, and light harvesting complex in photosystem II were highly abundant proteins in the leaves and accounted for 9%, 2%, and 4%, respectively, of the total identified proteins. Comparative analysis of the treatments enabled detection of 440 differentially expressed proteins during dehydration. The results of clustering analysis, gene ontology (GO) enrichment analysis, and analysis of composite expression profiles of functional categories for the differentially expressed proteins indicated that drought stress reduced the levels of proteins associated with photosynthesis and increased the levels of proteins involved in catabolic processes and stress responses. We observed enhanced expression of many proteins involved in osmotic stress responses and proteins with antioxidant activities. Based on previously reported molecular functions, we propose that the following five differentially expressed proteins could provide target genes for engineering drought resistance in plants: annexin, phospholipase D delta, sDNA-binding transcriptional regulator, auxin-responsive GH3 family protein, and TRAF-like family protein. PMID:27419125

  7. Shotgun Quantitative Proteomic Analysis of Proteins Responding to Drought Stress in Brassica rapa L. (Inbred Line "Chiifu").

    PubMed

    Kwon, Soon-Wook; Kim, Mijeong; Kim, Hijin; Lee, Joohyun

    2016-01-01

    Through a comparative shotgun quantitative proteomics analysis in Brassica rapa (inbred line Chiifu), total of 3,009 nonredundant proteins were identified with a false discovery rate of 0.01 in 3-week-old plants subjected to dehydration treatment for 0, 24, and 48 h, plants subjected to drought stress. Ribulose-bisphosphate carboxylases, chlorophyll a/b-binding protein, and light harvesting complex in photosystem II were highly abundant proteins in the leaves and accounted for 9%, 2%, and 4%, respectively, of the total identified proteins. Comparative analysis of the treatments enabled detection of 440 differentially expressed proteins during dehydration. The results of clustering analysis, gene ontology (GO) enrichment analysis, and analysis of composite expression profiles of functional categories for the differentially expressed proteins indicated that drought stress reduced the levels of proteins associated with photosynthesis and increased the levels of proteins involved in catabolic processes and stress responses. We observed enhanced expression of many proteins involved in osmotic stress responses and proteins with antioxidant activities. Based on previously reported molecular functions, we propose that the following five differentially expressed proteins could provide target genes for engineering drought resistance in plants: annexin, phospholipase D delta, sDNA-binding transcriptional regulator, auxin-responsive GH3 family protein, and TRAF-like family protein. PMID:27419125

  8. 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. PMID:26670623

  9. Quantitative analysis of cell surface membrane proteins using membrane-impermeable chemical probe coupled with 18O labeling

    SciTech Connect

    Zhang, Haizhen; Brown, Roslyn N.; Qian, Weijun; Monroe, Matthew E.; Purvine, Samuel O.; Moore, Ronald J.; Gritsenko, Marina A.; Shi, Liang; Romine, Margaret F.; Fredrickson, Jim K.; Pasa-Tolic, Ljiljana; Smith, Richard D.; Lipton, Mary S.

    2010-05-03

    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 environmental electron receptors. LC/MS/MS analysis resulted in the identification of about 79% membrane proteins among all proteins identified from the enriched sample. To illustrate the quantification of membrane proteome changes, enriched membrane protein samples from wild-type and mutant cells (generated from deletion of a type II secretion protein, GspD) were further labeled with 16O and 18O at the peptide level prior to LC-MS analysis. A chemical-probe-labeled pure protein has also been used as an internal standard for normalization purpose. The quantitative data revealed reduced abundances of many outer membrane proteins such as OmcA and MtrC in ΔgspD mutant cells, which agreed well with previously published studies.

  10. Identification of Protein Network Alterations upon Retinal Ischemia-Reperfusion Injury by Quantitative Proteomics Using a Rattus norvegicus Model

    PubMed Central

    Tian, Han; Wang, Leilei; Cai, Ruiqi; Zheng, Ling; Guo, Lin

    2014-01-01

    Retinal ischemia is a common feature associated with several ocular diseases, including diabetic retinopathy. In this study, we investigated the effect of a retinal ischemia and reperfusion (I/R) injury on protein levels via a quantitative shotgun strategy using stable isotope dimethyl labeling combined with LC-MS/MS analysis. Based on the relative quantitation data of 1088 proteins, 234 proteins showed a greater than 1.5-fold change following I/R injury, 194 of which were up-regulated and 40 were down-regulated. Gene ontology analysis revealed that after I/R injury, there was an increase in the metabolic-process related proteins but a decline in cell communication, system process and transport-related proteins. A ribosome protein network and a secreted protein network consisting of many protease inhibitors were identified among the up-regulated proteins, despite a suppression of the mammalian target of rapamycin (mTOR) pathway following the I/R injury. A synaptic-related protein network was found to be significantly down-regulated, implicating a functional reduction of neurons following a retinal I/R injury. Our results provide new systems-biology clues for the study of retinal ischemia. PMID:25549249

  11. Relative, label-free protein quantitation: spectral counting error statistics from nine replicate MudPIT samples.

    PubMed

    Cooper, Bret; Feng, Jian; Garrett, Wesley M

    2010-09-01

    Nine replicate samples of peptides from soybean leaves, each spiked with a different concentration of bovine apotransferrin peptides, were analyzed on a mass spectrometer using multidimensional protein identification technology (MudPIT). Proteins were detected from the peptide tandem mass spectra, and the numbers of spectra were statistically evaluated for variation between samples. The results corroborate prior knowledge that combining spectra from replicate samples increases the number of identifiable proteins and that a summed spectral count for a protein increases linearly with increasing molar amounts of protein. Furthermore, statistical analysis of spectral counts for proteins in two- and three-way comparisons between replicates and combined replicates revealed little significant variation arising from run-to-run differences or data-dependent instrument ion sampling that might falsely suggest differential protein accumulation. In these experiments, spectral counting was enabled by PANORAMICS, probability-based software that predicts proteins detected by sets of observed peptides. Three alternative approaches to counting spectra were also evaluated by comparison. As the counting thresholds were changed from weaker to more stringent, the accuracy of ratio determination also changed. These results suggest that thresholds for counting can be empirically set to improve relative quantitation. All together, the data confirm the accuracy and reliability of label-free spectral counting in the relative, quantitative analysis of proteins between samples. PMID:20541435

  12. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    NASA Astrophysics Data System (ADS)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  13. Targeted quantitative proteomic investigation employing multiple reaction monitoring on quantitative changes in proteins that regulate volatile biosynthesis of strawberry fruit at different ripening stages.

    PubMed

    Song, Jun; Du, Lina; Li, Li; Palmer, Leslie Campbell; Forney, Charles F; Fillmore, Sherry; Zhang, ZhaoQi; Li, XiHong

    2015-08-01

    A targeted quantitative proteomic investigation employing the multiple reaction monitoring (MRM, SRM) technique was conducted on strawberry fruit at different development stages. We investigated 22 proteins and isoforms from 32 peptides with 111 peptide transitions, which may be involved in the volatile aroma biosynthesis pathway. The normalized protein abundance was significantly changed in coincidence with increased volatile production and advanced fruit maturities. Among them, alcohol acyltransferase (AAT), quinone oxidoreductase (QR), malonyl Co-A decarboxylase, (MLYCD), pyruvate decarboxylase (PDC), acetyl Co-A carboxylase (ACCase), and acyl Co-A synthetase (ACAs) were increased significantly. Several alcohol dehydrogenases (ADHs), and 3-oxoacyl-ACP synthase were significantly decreased. Furthermore, the expression of seven genes related to strawberry volatile production was also investigated using real-time qPCR. Among the tested genes, QR, AAT, ACCase, OMT, PDC and ADH showed increased up-regulation during fruit ripening, while 3-isopropylmalate dehydrogenase (IMD) decreased. Strong correlation between quantitative proteomic data and gene expression suggested that AAT, QR, ACCase, and PDC played critical roles in volatile biosynthesis of strawberry during fruit ripening. Poor correlation between protein abundance and gene expression of ADH was found. PMID:26087350

  14. Characterization of a Highly Conserved Histone Related Protein, Ydl156w, and Its Functional Associations Using Quantitative Proteomic Analyses*

    PubMed Central

    Gilmore, Joshua M.; Sardiu, Mihaela E.; Venkatesh, Swaminathan; Stutzman, Brent; Peak, Allison; Seidel, Chris W.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

    2012-01-01

    A significant challenge in biology is to functionally annotate novel and uncharacterized proteins. Several approaches are available for deducing the function of proteins in silico based upon sequence homology and physical or genetic interaction, yet this approach is limited to proteins with well-characterized domains, paralogs and/or orthologs in other species, as well as on the availability of suitable large-scale data sets. Here, we present a quantitative proteomics approach extending the protein network of core histones H2A, H2B, H3, and H4 in Saccharomyces cerevisiae, among which a novel associated protein, the previously uncharacterized Ydl156w, was identified. In order to predict the role of Ydl156w, we designed and applied integrative bioinformatics, quantitative proteomics and biochemistry approaches aiming to infer its function. Reciprocal analysis of Ydl156w protein interactions demonstrated a strong association with all four histones and also to proteins strongly associated with histones including Rim1, Rfa2 and 3, Yku70, and Yku80. Through a subsequent combination of the focused quantitative proteomics experiments with available large-scale genetic interaction data and Gene Ontology functional associations, we provided sufficient evidence to associate Ydl156w with multiple processes including chromatin remodeling, transcription and DNA repair/replication. To gain deeper insights into the role of Ydl156w in histone biology we investigated the effect of the genetic deletion of ydl156w on H4 associated proteins, which lead to a dramatic decrease in the association of H4 with RNA polymerase III proteins. The implication of a role for Ydl156w in RNA Polymerase III mediated transcription was consequently verified by RNA-Seq experiments. Finally, using these approaches we generated a refined network of Ydl156w-associated proteins. PMID:22199229

  15. Protein alterations associated with pancreatic cancer and chronic pancreatitis found in human plasma using global quantitative proteomics profiling

    PubMed Central

    Pan, Sheng; Chen, Ru; Crispin, David A.; May, Damon; Stevens, Tyler; McIntosh, Martin; Bronner, Mary P.; Ziogas, Argyrios; Anton-Culver, Hoda; Brentnall, Teresa A.

    2011-01-01

    Pancreatic cancer is a lethal disease that is difficult to diagnose at early stages when curable treatments are effective. Biomarkers that can improve current pancreatic cancer detection would have great value in improving patient management and survival rate. A large scale quantitative proteomics study was performed to search for the plasma protein alterations associated with pancreatic cancer. The enormous complexity of the plasma proteome and the vast dynamic range of protein concentration therein present major challenges for quantitative global profiling of plasma. To address these challenges, multi-dimensional fractionation at both protein and peptide levels was applied to enhance the depth of proteomics analysis. Employing stringent criteria, more than thirteen hundred proteins total were identified in plasma across 8-orders of magnitude in protein concentration. Differential proteins associated with pancreatic cancer were identified, and their relationship with the proteome of pancreatic tissue and pancreatic juice from our previous studies was discussed. A subgroup of differentially expressed proteins was selected for biomarker testing using an independent cohort of plasma and serum samples from well-diagnosed patients with pancreatic cancer, chronic pancreatitis and non-pancreatic disease controls. Using ELISA methodology, the performance of each of these protein candidates was benchmarked against CA19-9, the current gold standard for a pancreatic cancer blood test. A composite marker of TIMP1 and ICAM1 demonstrate significantly better performance than CA19-9 in distinguishing pancreatic cancer from the non-pancreatic disease controls and chronic pancreatitis controls. In addition, protein AZGP1 was identified as a biomarker candidate for chronic pancreatitis. The discovery and technical challenges associated with plasma-based quantitative proteomics are discussed and may benefit the development of plasma proteomics technology in general. The protein

  16. Analysis of protein complexes through model-based biclustering of label-free quantitative AP-MS data

    PubMed Central

    Choi, Hyungwon; Kim, Sinae; Gingras, Anne-Claude; Nesvizhskii, Alexey I

    2010-01-01

    Affinity purification followed by mass spectrometry (AP-MS) has become a common approach for identifying protein–protein interactions (PPIs) and complexes. However, data analysis and visualization often rely on generic approaches that do not take advantage of the quantitative nature of AP-MS. We present a novel computational method, nested clustering, for biclustering of label-free quantitative AP-MS data. Our approach forms bait clusters based on the similarity of quantitative interaction profiles and identifies submatrices of prey proteins showing consistent quantitative association within bait clusters. In doing so, nested clustering effectively addresses the problem of overrepresentation of interactions involving baits proteins as compared with proteins only identified as preys. The method does not require specification of the number of bait clusters, which is an advantage against existing model-based clustering methods. We illustrate the performance of the algorithm using two published intermediate scale human PPI data sets, which are representative of the AP-MS data generated from mammalian cells. We also discuss general challenges of analyzing and interpreting clustering results in the context of AP-MS data. PMID:20571534

  17. Quantitative evaluation of proteins with bicinchoninic acid (BCA): resonance Raman and surface-enhanced resonance Raman scattering-based methods.

    PubMed

    Chen, Lei; Yu, Zhi; Lee, Youngju; Wang, Xu; Zhao, Bing; Jung, Young Mee

    2012-12-21

    A rapid and highly sensitive bicinchoninic acid (BCA) reagent-based protein quantitation tool was developed using competitive resonance Raman (RR) and surface-enhanced resonance Raman scattering (SERRS) methods. A chelation reaction between BCA and Cu(+), which is reduced by protein in an alkaline environment, is exploited to create a BCA-Cu(+) complex that has strong RR and SERRS activities. Using these methods, protein concentrations in solutions can be quantitatively measured at concentrations as low as 50 μg mL(-1) and 10 pg mL(-1). There are many advantages of using RR and SERRS-based assays. These assays exhibit a much wider linear concentration range and provide an additional one (RR method) to four (SERRS method) orders of magnitude increase in detection limits relative to UV-based methods. Protein-to-protein variation is determined using a reference to a standard curve at concentrations of BSA that exhibits excellent recoveries. These novel methods are extremely accurate in detecting total protein concentrations in solution. This improvement in protein detection sensitivity could yield advances in the biological sciences and medical diagnostic field and extend the applications of reagent-based protein assay techniques. PMID:23099478

  18. Trading accuracy for speed: A quantitative comparison of search algorithms in protein sequence design.

    PubMed

    Voigt, C A; Gordon, D B; Mayo, S L

    2000-06-01

    Finding the minimum energy amino acid side-chain conformation is a fundamental problem in both homology modeling and protein design. To address this issue, numerous computational algorithms have been proposed. However, there have been few quantitative comparisons between methods and there is very little general understanding of the types of problems that are appropriate for each algorithm. Here, we study four common search techniques: Monte Carlo (MC) and Monte Carlo plus quench (MCQ); genetic algorithms (GA); self-consistent mean field (SCMF); and dead-end elimination (DEE). Both SCMF and DEE are deterministic, and if DEE converges, it is guaranteed that its solution is the global minimum energy conformation (GMEC). This provides a means to compare the accuracy of SCMF and the stochastic methods. For the side-chain placement calculations, we find that DEE rapidly converges to the GMEC in all the test cases. The other algorithms converge on significantly incorrect solutions; the average fraction of incorrect rotamers for SCMF is 0.12, GA 0.09, and MCQ 0.05. For the protein design calculations, design positions are progressively added to the side-chain placement calculation until the time required for DEE diverges sharply. As the complexity of the problem increases, the accuracy of each method is determined so that the results can be extrapolated into the region where DEE is no longer tractable. We find that both SCMF and MCQ perform reasonably well on core calculations (fraction amino acids incorrect is SCMF 0.07, MCQ 0.04), but fail considerably on the boundary (SCMF 0.28, MCQ 0.32) and surface calculations (SCMF 0.37, MCQ 0.44). PMID:10835284

  19. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

    1998-04-21

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  20. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

  1. 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 system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  2. Multiplexed capillary electrophoresis system

    DOEpatents

    Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

    1996-12-10

    The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

  3. Comparative Study of Early Cold-Regulated Proteins by Two-Dimensional Difference Gel Electrophoresis Reveals a Key Role for Phospholipase Dα1 in Mediating Cold Acclimation Signaling Pathway in Rice.

    PubMed

    Huo, Chenmin; Zhang, Baowen; Wang, Hui; Wang, Fawei; Liu, Meng; Gao, Yingjie; Zhang, Wenhua; Deng, Zhiping; Sun, Daye; Tang, Wenqiang

    2016-04-01

    To understand the early signaling steps that regulate cold responses in rice, two-dimensional difference gel electrophoresis (2-D DIGE)(1)was used to study early cold-regulated proteins in rice seedlings. Using mass spectrometry, 32 spots, which represent 26 unique proteins that showed an altered expression level within 5 min of cold treatment were identified. Among these proteins, Western blot analyses confirmed that the cellular phospholipase D α1 (OsPLDα1) protein level was increased as early as 1 min after cold treatment. Genetic studies showed that reducing the expression ofOsPLDα1makes rice plants more sensitive to chilling stress as well as cold acclimation increased freezing tolerance. Correspondingly, cold-regulated proteomic changes and the expression of the cold-responsive C repeat/dehydration-responsive element binding 1 (OsDREB1) family of transcription factors were inhibited in thepldα1mutant. We also found that the expression ofOsPLDα1is directly regulated by OsDREB1A. This transcriptional regulation ofOsPLDα1could provide positive feedback regulation of the cold signal transduction pathway in rice. OsPLDα1 hydrolyzes phosphatidylcholine to produce the signal molecule phosphatidic acid (PA). By lipid-overlay assay, we demonstrated that the rice cold signaling proteins, MAP kinase 6 (OsMPK6) and OsSIZ1, bind directly to PA. Taken together, our results suggest that OsPLDα1 plays a key role in transducing cold signaling in rice by producing PA and regulatingOsDREB1s' expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins. PMID:26747563

  4. Quantitative analysis of aberrant protein glycosylation in liver cancer plasma by AAL-enrichment and MRM mass spectrometry.

    PubMed

    Ahn, Yeong Hee; Shin, Park Min; Kim, Yong-Sam; Oh, Na Ree; Ji, Eun Sun; Kim, Kwang Hoe; Lee, Yeon Jung; Kim, Sung Ho; Yoo, Jong Shin

    2013-11-01

    A lectin-coupled mass spectrometry (MS) approach was employed to quantitatively monitor aberrant protein glycosylation in liver cancer plasma. To do this, we compared the difference in the total protein abundance of a target glycoprotein between hepatocellular carcinoma (HCC) plasmas and hepatitis B virus (HBV) plasmas, as well as the difference in lectin-specific protein glycoform abundance of the target glycoprotein. Capturing the lectin-specific protein glycoforms from a plasma sample was accomplished by using a fucose-specific aleuria aurantia lectin (AAL) immobilized onto magnetic beads via a biotin-streptavidin conjugate. Following tryptic digestion of both the total plasma and its AAL-captured fraction of each HCC and HBV sample, targeted proteomic mass spectrometry was conducted quantitatively by a multiple reaction monitoring (MRM) technique. From the MRM-based analysis of the total plasmas and AAL-captured fractions, differences between HCC and HBV plasma groups in fucosylated glycoform levels of target glycoproteins were confirmed to arise from both the change in the total protein abundance of the target proteins and the change incurred by aberrant fucosylation on target glycoproteins in HCC plasma, even when no significant change occurs in the total protein abundance level. Combining the MRM-based analysis method with the lectin-capturing technique proved to be a successful means of quantitatively investigating aberrant protein glycosylation in cancer plasma samples. Additionally, it was elucidated that the differences between HCC and control groups in fucosylated biomarker candidates A1AT and FETUA mainly originated from an increase in fucosylation levels on these target glycoproteins, rather than an increase in the total protein abundance of the target glycoproteins. PMID:24027776

  5. Quantitative assessment of the multivalent protein-carbohydrate interactions on silicon.

    PubMed

    Yang, Jie; Chazalviel, Jean-Noël; Siriwardena, Aloysius; Boukherroub, Rabah; Ozanam, François; Szunerits, Sabine; Gouget-Laemmel, Anne Chantal

    2014-10-21

    A key challenge in the development of glycan arrays is that the sensing interface be fabricated reliably so as to ensure the sensitive and accurate analysis of the protein-carbohydrate interaction of interest, reproducibly. These goals are complicated in the case of glycan arrays as surface sugar density can influence dramatically the strength and mode of interaction of the sugar ligand at any interface with lectin partners. In this Article, we describe the preparation of carboxydecyl-terminated crystalline silicon (111) surfaces onto which are grafted either mannosyl moieties or a mixture of mannose and spacer alcohol molecules to provide "diluted" surfaces. The fabrication of the silicon surfaces was achieved efficiently through a strategy implicating a "click" coupling step. The interactions of these newly fabricated glycan interfaces with the lectin, Lens culinaris, have been characterized using quantitative infrared (IR) spectroscopy in the attenuated total geometry (ATR). The density of mannose probes and lectin targets was precisely determined for the first time by the aid of special IR calibration experiments, thus allowing for the interpretation of the distribution of mannose and its multivalent binding with lectins. These experimental findings were accounted for by numerical simulations of lectin adsorption. PMID:25216376

  6. A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

    PubMed Central

    Helka, Blake-Joseph; Brennan, John D.

    2013-01-01

    Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays. PMID:24022739

  7. Non Linear Programming (NLP) formulation for quantitative modeling of protein signal transduction pathways.

    PubMed

    Mitsos, Alexander; Melas, Ioannis N; Morris, Melody K; Saez-Rodriguez, Julio; Lauffenburger, Douglas A; Alexopoulos, Leonidas G

    2012-01-01

    Modeling of signal transduction pathways plays a major role in understanding cells' function and predicting cellular response. Mathematical formalisms based on a logic formalism are relatively simple but can describe how signals propagate from one protein to the next and have led to the construction of models that simulate the cells response to environmental or other perturbations. Constrained fuzzy logic was recently introduced to train models to cell specific data to result in quantitative pathway models of the specific cellular behavior. There are two major issues in this pathway optimization: i) excessive CPU time requirements and ii) loosely constrained optimization problem due to lack of data with respect to large signaling pathways. Herein, we address both issues: the former by reformulating the pathway optimization as a regular nonlinear optimization problem; and the latter by enhanced algorithms to pre/post-process the signaling network to remove parts that cannot be identified given the experimental conditions. As a case study, we tackle the construction of cell type specific pathways in normal and transformed hepatocytes using medium and large-scale functional phosphoproteomic datasets. The proposed Non Linear Programming (NLP) formulation allows for fast optimization of signaling topologies by combining the versatile nature of logic modeling with state of the art optimization algorithms. PMID:23226239

  8. Application of capillary electrophoresis-inductively coupled plasma mass spectrometry to comparative studying of the reactivity of antitumor ruthenium(III) complexes differing in the nature of counter-ion toward human serum proteins.

    PubMed

    Połeć-Pawlak, Kasia; Abramski, Jan K; Ferenc, Julia; Foteeva, Lidia S; Timerbaev, Andrei R; Keppler, Bernhard K; Jarosz, Maciej

    2008-05-30

    Varying the counter-ion is a highly supportive practice in tackling the problem of poor water-solubility of metal complexes of pharmaceutical importance. As a matter of fact, the relevant structural modification may alter the metabolic pathways and possibly the mode of action of a drug. To prove that this does not take place for one of the lead anticancer metal-based developmental compounds, indazolium trans-[RuCl(4)(1H-indazole)(2)] (KP1019), its reactivity toward human serum proteins was assessed under simulated physiological conditions and compared to that of a much more soluble analogue, sodium trans-[RuCl(4)(1H-indazole)(2)] (KP1339). For such kinetic assaying, capillary electrophoresis (CE) interfaced online with inductively coupled plasma mass spectrometry (ICP-MS) to specifically monitor changes in the metal speciation following the formation of ruthenium-protein adducts was applied. The rate constants of interaction with albumin and transferrin were determined at pharmacologically fitting drug-to-protein ratios as on average 0.0319+/-0.0021 min(-1) and 0.0931+/-0.0019 min(-1) (KP1019) and 0.0316+/-0.0018 min(-1) and 0.0935+/-0.0053 min(-1) (KP1339), respectively. The results of this brief study showed that changing from organic to inorganic counter-ion at the stage of formulation could commonly be recommended for improving ruthenium-based drug solubility and bioavailability. PMID:18433763

  9. Quantitation of proteinuria with urinary protein/creatinine ratios and random testing with dipsticks in nephrotic children.

    PubMed

    Abitbol, C; Zilleruelo, G; Freundlich, M; Strauss, J

    1990-02-01

    We examined the relative feasibility of using random urinary dipstick testing and urinary protein/creatinine ratios in the quantitation of proteinuria. Sixty-four children with relapsing nephrotic syndrome, ranging in age from 1 1/2 to 16 years, contributed 145 timed urine collections and 150 random specimens, which were analyzed by urinary protein dipstick, quantitation of protein and creatinine, or both. Total protein excretion