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Sample records for electrophoresis protein profiles

  1. 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.

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

    PubMed

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

    2014-07-23

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

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

    PubMed

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

    2014-07-23

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

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

    PubMed

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

    2015-01-01

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

  5. Protein electrophoresis - serum

    MedlinePlus

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

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

    PubMed

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

    2013-11-01

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

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Stage-specific analysis of plasma protein profiles in ovarian cancer: Difference in-gel electrophoresis analysis of pooled clinical samples

    PubMed Central

    Bailey, Mark J.; Shield-Artin, Kristy L.; Oliva, Karen; Ayhan, Mustafa; Reisman, Simone; Rice, Gregory E.

    2013-01-01

    Introduction: Ovarian cancer is the leading cause of death from gynecological cancer. Non-specific symptoms early in disease and the lack of specific biomarkers hinder early diagnosis. Multi-marker blood screening tests have shown promise for improving identification of early stage disease; however, available tests lack sensitivity, and specificity. Materials and Methods: In this study, pooled deeply-depleted plasma from women with Stage 1, 2 or 3 ovarian cancer and healthy controls were used to compare the 2-dimensional gel electrophoresis (2-DE) protein profiles and identify potential novel markers of ovarian cancer progression. Results/Discussion: Stage-specific variation in biomarker expression was observed. For example, apolipoprotein A1 expression is relatively low in control and Stage 1, but shows a substantial increase in Stage 2 and 3, thus, potential of utility for disease confirmation rather than early detection. A better marker for early stage disease was tropomyosin 4 (TPM4). The expression of TPM4 increased by 2-fold in Stage 2 before returning to “normal” levels in Stage 3 disease. Multiple isoforms were also identified for some proteins and in some cases, displayed stage-specific expression. An interesting example was fibrinogen alpha, for which 8 isoforms were identified. Four displayed a moderate increase at Stage 1 and a substantial increase for Stages 2 and 3 while the other 4 showed only moderate increases. Conclusion: Herein is provided an improved summary of blood protein profiles for women with ovarian cancer stratified by stage. PMID:23858298

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

    PubMed

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

    2011-11-18

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

  13. Analysis of Protein Oligomerization by Electrophoresis.

    PubMed

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

    2016-01-01

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

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

    ERIC Educational Resources Information Center

    Browning, Mark; Vanable, Joseph

    2002-01-01

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

  15. Proteomic Profiling of Macrophages by 2D Electrophoresis

    PubMed Central

    Bouvet, Marion; Turkieh, Annie; Acosta-Martin, Adelina E.; Chwastyniak, Maggy; Beseme, Olivia; Amouyel, Philippe; Pinet, Florence

    2014-01-01

    The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins. PMID:25408153

  16. Muscle protein analysis by two-dimensional gel electrophoresis

    SciTech Connect

    Giometti, C.S.

    1982-01-01

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

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

    PubMed

    Ma, S

    2005-01-01

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

  18. Proteomic profiling of hempseed proteins from Cheungsam.

    PubMed

    Park, Seul-Ki; Seo, Jong-Bok; Lee, Mi-Young

    2012-02-01

    Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.

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

    PubMed

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

    1999-11-01

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

  20. Proteomic Profiling of Aging in Glomeruli of Mice by using Two-Dimensional Differential Gel Electrophoresis

    PubMed Central

    Liu, Xiaodan; Fan, Qiuling; Yang, Gang; Wang, Lining

    2015-01-01

    Background Glomerular proteins were analyzed by proteomics to screen proteins participating in maturation of glomeruli before senescence and to find key proteins involved in the aging process. Material/Methods Glomeruli of C57BL/6 mice at 8 and 20 weeks were separated by kidney perfusion. Proteomic profiles of glomeruli were investigated by using two-dimensional differential gel electrophoresis and MALDI-TOF mass spectrometry. Results We identified 22 differentially expressed proteins. Among them, 3 proteins were significantly up-regulated and 19 proteins were significantly down-regulated in mature mice. Out of these 22 proteins, 18% take part in protein transport, protein targeting, and proteolysis; 5% in glycolysis; 14% in transcription; 9% in electron transport; 9% were chaperones; and 9% were hydrolases. Conclusions Our results provide insights into the glomerular differentially expressed proteins correlated with renal aging. In this study we found that aging altered the expression of ATP synthase subunit beta. Further studies on this protein might help to understand the mechanism of renal aging. PMID:25659849

  1. 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.

  2. 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

  3. Procedures for two-dimensional electrophoresis of proteins

    SciTech Connect

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

    1996-10-01

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

  4. 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.

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

    PubMed

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

    2010-04-01

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

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

    PubMed Central

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

    2009-01-01

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

  7. 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.

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

    PubMed

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

    2016-01-01

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

  9. Outer membrane protein profiles of Haemophilus pleuropneumoniae.

    PubMed Central

    Rapp, V J; Munson, R S; Ross, R F

    1986-01-01

    Outer membrane protein profiles of Haemophilus pleuropneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells were disrupted by sonication, and outer membrane-enriched fractions were prepared by differential centrifugation and selective solubilization of the inner membrane with sodium N-lauroyl sarcosinate. Colony type, growth medium, time of harvest, and in vitro or in vivo passage had no appreciable effect on the protein profiles of the strains examined. Seven patterns were distinguished among the reference strains of the nine capsular serotypes. These patterns were based on the mobility of the major outer membrane proteins migrating in the 39,000- to 44,000-molecular-weight region of the gel, a 16K to 16.5K protein, and a heat-modifiable 29K protein. Strains of serotypes 1 and 9 had identical outer membrane protein profiles, as did strains of serotypes 2 and 6. The reference strains of the remaining five serotypes each had a distinct pattern. The outer membrane protein profiles of 95 field isolates belonging to serotypes 1, 5, 7, and 9 from swine in the midwestern United States were determined and compared with the reference patterns. The results indicate that the population of H. pleuropneumoniae is clonal, with three predominant clones distinguished by both serotype and outer membrane protein profile responsible for the majority of H. pleuropneumoniae disease occurring in swine in the United States. Images PMID:3699889

  10. 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.

  11. Sheathless capillary electrophoresis-mass spectrometry for anionic metabolic profiling.

    PubMed

    Gulersonmez, Mehmet Can; Lock, Stephen; Hankemeier, Thomas; Ramautar, Rawi

    2016-04-01

    The performance of CE coupled on-line to MS via a sheathless porous tip sprayer was evaluated for anionic metabolic profiling. A representative metabolite mixture and biological samples were used for the evaluation of various analytical parameters, such as peak efficiency (plate numbers), migration time and peak area repeatability, and LODs. The BGE, i.e. 10% acetic acid (pH 2.2), previously used for cationic metabolic profiling was now assessed for anionic metabolic profiling by using MS detection in negative ion mode. For test compounds, RSDs for migration times and peak areas were below 2 and 11%, respectively, and plate numbers ranged from 60 000 to 40 0000 demonstrating a high separation efficiency. Critical metabolites with low or no retention on reversed-phase LC could be efficiently separated and selectively analyzed by the sheathless CE-MS method. An injection volume of only circa 20 nL resulted in LODs between 10 and 200 nM (corresponding to an amount of 0.4-4 fmol), which was an at least tenfold improvement as compared to LODs obtained by conventional CE-MS approaches for these analytes. The methodology was applied to anionic metabolic profiling of glioblastoma cell line extracts. Overall, a sheathless CE-MS method has been developed for highly efficient and sensitive anionic metabolic profiling studies, which can also be used for cationic metabolic profiling studies by only switching the MS detection and separation voltage polarity.

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

    PubMed

    McMaster, Mary L; Csako, Gyorgy

    2008-03-01

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

  13. Metabolomic profiling of anionic metabolites by capillary electrophoresis mass spectrometry.

    PubMed

    Soga, Tomoyoshi; Igarashi, Kaori; Ito, Chiharu; Mizobuchi, Katsuo; Zimmermann, Hans-Peter; Tomita, Masaru

    2009-08-01

    We describe a sheath flow capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) method in the negative mode using a platinum electrospray ionization (ESI) spray needle, which allows the comprehensive analysis of anionic metabolites. The material of the spray needle had significant effect on the measurement of anions. A stainless steel spray needle was oxidized and corroded at the anodic electrode due to electrolysis. The precipitation of iron oxides (rust) plugged the capillary outlet, resulting in shortened capillary lifetime. Many anionic metabolites also formed complexes with the iron oxides or migrating nickel ion, which was also generated by electrolysis and moved toward the cathode (the capillary inlet). The metal-anion complex formation significantly reduced detection sensitivity of the anionic compounds. The use of a platinum ESI needle prevented both oxidation of the metals and needle corrosion. Sensitivity using the platinum needle increased from several- to 63-fold, with the largest improvements for anions exhibiting high metal chelating properties such as carboxylic acids, nucleotides, and coenzyme A compounds. The detection limits for most anions were between 0.03 and 0.87 micromol/L (0.8 and 24 fmol) at a signal-to-noise ratio of 3. This method is quantitative, sensitive, and robust, and its utility was demonstrated by the analysis of the metabolites in the central metabolic pathways extracted from mouse liver. PMID:19522513

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

    PubMed

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

    2007-07-01

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

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

    PubMed

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

    1997-06-27

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

  16. 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

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

    PubMed

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

    2015-12-01

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

  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. Establishment of two-dimensional gel electrophoresis profiles of the human acute promyelocytic leukemia cell line NB4.

    PubMed

    He, Pengcheng; Liu, Yanfeng; Zhang, Mei; Wang, Xiaoning; Wang, Huaiyu; Xi, Jieying; Wei, Kaihua; Wang, Hongli; Zhao, Jing

    2012-09-01

    To explore optimum conditions for establishing a two‑dimensional gel electrophoresis (2-DE) map of the human acute promyelocytic leukemia (APL) cell line NB4 and to analyze its protein profiles, we extracted total proteins from NB4 cells using cell disruption, liquid nitrogen freeze-thawing and fracturing by ultrasound, and quantified the extracted protein samples using Bradford's method. 2-DE was applied to separate the proteins, which were silver-stained in the gel. Well‑separated protein spots were selected from the gel using the ImageMaster™ 2D Platinum analysis system. Moreover, the effects of various protein sample sizes (140, 160 and 180 µg) on the 2-DE maps of the NB4 cells were determined and compared. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), peptide mass fingerprinting (PMF) and database searching were used to identify the proteins. When the quantity of loading proteins was 160 µg, clear, well-resolved, reproducible 2-DE proteomic profiles of the NB4 cells were obtained. The average number of protein spots in 3 gels was 1160±51 with an average matching rate of 81%. A total of 10 proteins were identified by mass spectrometry and database queries, certain proteins were products of oncogenes and others were involved in cell cycle regulation and signal transduction. In summary, 2-DE profiles of the proteome of NB4 cells were established and certain proteins were identified by MALDI-TOF-MS and PMF which lay the foundation of further proteomic research of NB4 cells. These data should be useful for establishing a human APL proteome database. PMID:22736039

  20. 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.

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

    PubMed

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

    2013-12-27

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

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

    PubMed

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

    2000-06-01

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

  3. 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.

  4. 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.

  5. 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

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

    PubMed Central

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

    2015-01-01

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

  7. DNA profiling by capillary array electrophoresis with non-covalent fluorescent labeling.

    PubMed

    Olson, Nels A; Khandurina, Julia; Guttman, Andras

    2004-10-01

    Increasing need for large-scale DNA profiling necessitated the development of automated electrophoresis based methods enabling rapid, high performance analysis of nucleic acids in a wide molecular-mass range. In this paper, we report on the adaptation of a commercial 96-capillary array electrophoresis (CAE) instrument for high-throughput DNA fragment analysis and the evaluation of the effects of different non-covalent DNA staining dyes on separation efficiency. The applicability of different color internal fluorescent standards is shown with mathematical spectral overlap correction algorithms. Large-scale quality control assessment of oligonucleotide probes using non-covalent fluorophore labeling is also demonstrated. The method requires small sample amounts, offers automation and quantification capabilities to accommodate modern biotechnology industry needs.

  8. 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...

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

    PubMed

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

    2016-01-01

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

  10. Distinguishing ichthyoses by protein profiling.

    PubMed

    Rice, Robert H; Bradshaw, Katie M; Durbin-Johnson, Blythe P; Rocke, David M; Eigenheer, Richard A; Phinney, Brett S; Schmuth, Matthias; Gruber, Robert

    2013-01-01

    To explore the usefulness of protein profiling for characterization of ichthyoses, we here determined the profile of human epidermal stratum corneum by shotgun proteomics. Samples were analyzed after collection on tape circles from six anatomic sites (forearm, palm, lower leg, forehead, abdomen, upper back), demonstrating site-specific differences in profiles. Additional samples were collected from the forearms of subjects with ichthyosis vulgaris (filaggrin (FLG) deficiency), recessive X-linked ichthyosis (steroid sulfatase (STS) deficiency) and autosomal recessive congenital ichthyosis type lamellar ichthyosis (transglutaminase 1 (TGM1) deficiency). The ichthyosis protein expression patterns were readily distinguishable from each other and from phenotypically normal epidermis. In general, the degree of departure from normal was lower from ichthyosis vulgaris than from lamellar ichthyosis, parallel to the severity of the phenotype. Analysis of samples from families with ichthyosis vulgaris and concomitant modifying gene mutations (STS deficiency, GJB2 deficiency) permitted correlation of alterations in protein profile with more complex genetic constellations.

  11. 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

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

    PubMed

    Hiratsuka, Atsunori; Yokoyama, Kenji

    2009-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Corradini, D

    1997-10-10

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

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

    PubMed

    Dolnik, Vladislav; Gurske, William A

    2011-10-01

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

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

    PubMed

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

    2000-12-01

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

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

    PubMed

    Jenkins, M A; Guerin, M D

    1997-10-10

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

  19. Pulsed-field gel electrophoresis and ribotype profiles of clinical and environmental Vibrio vulnificus isolates.

    PubMed Central

    Tamplin, M L; Jackson, J K; Buchrieser, C; Murphree, R L; Portier, K M; Gangar, V; Miller, L G; Kaspar, C W

    1996-01-01

    Vibrio vulnificus belongs to the autochthonous bacterial flora of warm estuarine waters. It can cause life-threatening extraintestinal disease in persons who have underlying illness and who consume raw shellfish or contact wounds with estuarine water. Currently, very little is known about genetic diversity within this species. In this report, we describe high-level variation in restriction fragment length polymorphism profiles among 53 clinical and 78 environmental isolates, as determined by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. When combined ribotype profiles of clinical and environmental isolates were analyzed, four predominant clusters were observed. Interestingly, a low number (16%) of clinical isolates were found in cluster C, compared with clusters A, B, and D (range, 50 to 83%). In addition, 83% of all Hawaiian isolates were located in a single cluster, indicating a possible relationship between geography and genotype. We also report that spontaneous translucent colonial morphotypes were distinct by both restriction fragment length polymorphism and biochemical profiles, compared with opaque parent strains. PMID:8837412

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

    PubMed

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

    2011-06-01

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

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

    PubMed

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

    2016-08-16

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

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

    ERIC Educational Resources Information Center

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

    2003-01-01

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

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

    ERIC Educational Resources Information Center

    Kim, Thomas D.; Craig, Paul A.

    2010-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    1993-12-01

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

  6. 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.

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

    PubMed

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

    2004-08-01

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

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

    PubMed

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

    2008-07-01

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

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

    PubMed

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

    1995-05-12

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

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

    PubMed

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

    2011-05-01

    Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities. PMID:21277175

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

    PubMed

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

    2015-04-01

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

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

    PubMed

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

    2006-03-01

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

  13. Changes in Predominance of Pulsed-Field Gel Electrophoresis Profiles of Bordetella pertussis Isolates, United States, 2000-2012.

    PubMed

    Cassiday, Pamela K; Skoff, Tami H; Jawahir, Selina; Tondella, M Lucia

    2016-03-01

    To clarify the characteristics of circulating Bordetella pertussis isolates, we used pulsed-field gel electrophoresis (PFGE) to analyze 5,262 isolates collected in the United States during 2000-2012. We found 199 PFGE profiles; 5 profiles accounted for 72% of isolates. The most common profile, CDC013, accounted for 35%-46% of isolates tested from 2000-2009; however, the proportion of isolates of this profile rapidly decreased in 2010. Profile CDC237, first seen in 2009, increased rapidly and accounted for 29% of 2012 isolates. No location bias was observed among profiles during 2000-2010, but differences were observed among isolates from different states during 2012. Predominant profiles match those observed in recent European PFGE studies. PFGE profile changes are concurrent with other recent molecular changes in B. pertussis and may be contributing to the reemergence of pertussis in the United States. Continued PFGE monitoring is critical for understanding the changing epidemiology of pertussis.

  14. Changes in Predominance of Pulsed-Field Gel Electrophoresis Profiles of Bordetella pertussis Isolates, United States, 2000-2012.

    PubMed

    Cassiday, Pamela K; Skoff, Tami H; Jawahir, Selina; Tondella, M Lucia

    2016-03-01

    To clarify the characteristics of circulating Bordetella pertussis isolates, we used pulsed-field gel electrophoresis (PFGE) to analyze 5,262 isolates collected in the United States during 2000-2012. We found 199 PFGE profiles; 5 profiles accounted for 72% of isolates. The most common profile, CDC013, accounted for 35%-46% of isolates tested from 2000-2009; however, the proportion of isolates of this profile rapidly decreased in 2010. Profile CDC237, first seen in 2009, increased rapidly and accounted for 29% of 2012 isolates. No location bias was observed among profiles during 2000-2010, but differences were observed among isolates from different states during 2012. Predominant profiles match those observed in recent European PFGE studies. PFGE profile changes are concurrent with other recent molecular changes in B. pertussis and may be contributing to the reemergence of pertussis in the United States. Continued PFGE monitoring is critical for understanding the changing epidemiology of pertussis. PMID:26886905

  15. Changes in Predominance of Pulsed-Field Gel Electrophoresis Profiles of Bordetella pertussis Isolates, United States, 2000–2012

    PubMed Central

    Skoff, Tami H.; Jawahir, Selina; Tondella, M. Lucia

    2016-01-01

    To clarify the characteristics of circulating Bordetella pertussis isolates, we used pulsed-field gel electrophoresis (PFGE) to analyze 5,262 isolates collected in the United States during 2000–2012. We found 199 PFGE profiles; 5 profiles accounted for 72% of isolates. The most common profile, CDC013, accounted for 35%–46% of isolates tested from 2000–2009; however, the proportion of isolates of this profile rapidly decreased in 2010. Profile CDC237, first seen in 2009, increased rapidly and accounted for 29% of 2012 isolates. No location bias was observed among profiles during 2000–2010, but differences were observed among isolates from different states during 2012. Predominant profiles match those observed in recent European PFGE studies. PFGE profile changes are concurrent with other recent molecular changes in B. pertussis and may be contributing to the reemergence of pertussis in the United States. Continued PFGE monitoring is critical for understanding the changing epidemiology of pertussis. PMID:26886905

  16. 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

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

    PubMed

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

    2007-08-01

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

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

    PubMed

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

    2002-05-01

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

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

    PubMed

    Lupinovich, V L; Kopylova, N V

    1980-01-01

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

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

    PubMed

    Smith, A C

    1988-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2014-07-01

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

  3. 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

  4. 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.

  5. 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

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

    PubMed

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-27

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

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

    PubMed Central

    Mao, Xiaoying; Hua, Yufei; Chen, Guogang

    2014-01-01

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

  8. Analysis of 953 Human Proteins from a Mitochondrial HEK293 Fraction by Complexome Profiling

    PubMed Central

    Wessels, Hans J. C. T.; Vogel, Rutger O.; Lightowlers, Robert N.; Spelbrink, Johannes N.; Rodenburg, Richard J.; van den Heuvel, Lambert P.; van Gool, Alain J.; Gloerich, Jolein; Smeitink, Jan A. M.; Nijtmans, Leo G.

    2013-01-01

    Complexome profiling is a novel technique which uses shotgun proteomics to establish protein migration profiles from fractionated blue native electrophoresis gels. Here we present a dataset of blue native electrophoresis migration profiles for 953 proteins by complexome profiling. By analysis of mitochondrial ribosomal complexes we demonstrate its potential to verify putative protein-protein interactions identified by affinity purification – mass spectrometry studies. Protein complexes were extracted in their native state from a HEK293 mitochondrial fraction and separated by blue native gel electrophoresis. Gel lanes were cut into gel slices of even size and analyzed by shotgun proteomics. Subsequently, the acquired protein migration profiles were analyzed for co-migration via hierarchical cluster analysis. This dataset holds great promise as a comprehensive resource for de novo identification of protein-protein interactions or to underpin and prioritize candidate protein interactions from other studies. To demonstrate the potential use of our dataset we focussed on the mitochondrial translation machinery. Our results show that mitoribosomal complexes can be analyzed by blue native gel electrophoresis, as at least four distinct complexes. Analysis of these complexes confirmed that 24 proteins that had previously been reported to co-purify with mitoribosomes indeed co-migrated with subunits of the mitochondrial ribosome. Co-migration of several proteins involved in biogenesis of inner mitochondrial membrane complexes together with mitoribosomal complexes suggested the possibility of co-translational assembly in human cells. Our data also highlighted a putative ribonucleotide complex that potentially contains MRPL10, MRPL12 and MRPL53 together with LRPPRC and SLIRP. PMID:23935861

  9. Capillary Electrophoresis in Wine Science.

    PubMed

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

    2016-01-01

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

  10. 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

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    2014-09-01

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

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

    PubMed

    Ko, Y G; Thompson, G A

    1995-01-01

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

  14. Microchip Electrophoresis with Amperometric Detection Method for Profiling Cellular Nitrosative Stress Markers

    PubMed Central

    Gunasekara, Dulan B.; Siegel, Joseph M.; Caruso, Giuseppe; Hulvey, Matthew K.; Lunte, Susan M.

    2014-01-01

    Summary The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2−) in cell lysates. There was a 2.5- to 4-fold increase in NO2− production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2− inside a single unstimulated macrophage cell was estimatedto be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions. PMID:24728039

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

    PubMed

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

    2016-07-01

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

  16. Capillary Electrophoresis Profiles and Fluorophore Components of Humic Acids in Nebraska Corn and Philippine Rice Soils

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As humic substances represent relatively high molecular mass polyelectrolytes containing aromatic, aliphatic and heterocyclic subunits, capillary electrophoresis (CE) has become an attractive method for “finger-print” characterization of humic acids. In addition, fluorescence excitation-emission ma...

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

    PubMed

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

    2005-01-01

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

  18. 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.

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

    PubMed

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

    2014-11-01

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

  20. Surface modified capillary electrophoresis combined with in solution isoelectric focusing and MALDI-TOF/TOF MS: a gel-free multidimensional electrophoresis approach for proteomic profiling--exemplified on human follicular fluid.

    PubMed

    Hanrieder, Jörg; Zuberovic, Aida; Bergquist, Jonas

    2009-04-24

    Development of miniaturized analytical tools continues to be of great interest to face the challenges in proteomic analysis of complex biological samples such as human body fluids. In the light of these challenges, special emphasis is put on the speed and simplicity of newly designed technological approaches as well as the need for cost efficiency and low sample consumption. In this study, we present an alternative multidimensional bottom-up approach for proteomic profiling for fast, efficient and sensitive protein analysis in complex biological matrices. The presented setup was based on sample pre-fractionation using microscale in solution isoelectric focusing (IEF) followed by tryptic digestion and subsequent capillary electrophoresis (CE) coupled off-line to matrix assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI TOF MS/MS). For high performance CE-separation, PolyE-323 modified capillaries were applied to minimize analyte-wall interactions. The potential of the analytical setup was demonstrated on human follicular fluid (hFF) representing a typical complex human body fluid with clinical implication. The obtained results show significant identification of 73 unique proteins (identified at 95% significance level), including mostly acute phase proteins but also protein identities that are well known to be extensively involved in follicular development.

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

    PubMed

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

    2005-05-15

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

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    1994-06-01

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

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

    PubMed Central

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

    2013-01-01

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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. SERUM PROTEIN PROFILES IN COCCIDIOIDOMYCOSIS

    PubMed Central

    Reed, William B.; Heiskell, Charles L.; Holeman, Charles W.; Carpenter, Charles

    1962-01-01

    Serum protein analysis is a valuable addition to the present methods for evaluating the status of the individual patient with coccidioidomycosis. The albumin protein and albumin glycoprotein decrease and gamma protein increases in relation to severity of infection. In 40 patients with coccidioidomycosis, changes in individual protein fractions could be significantly correlated with conventional laboratory tests, such as the complement fixation test, erythrocyte sedimentation rate and hematocrit. Changes in the alpha, glycoprotein concentration, the erythrocyte sedimentation rate and the hematocrit value appear to be related to the degree of inflammation, while the changes in the gamma protein and the beta, glycoprotein appear to be related to the specific antibody response. PMID:13973566

  10. Serum protein profiles in coccidioidomycosis.

    PubMed

    REED, W B; HEISKELL, C L; HOLEMAN, C W; CARPENTER, C

    1962-12-01

    Serum protein analysis is a valuable addition to the present methods for evaluating the status of the individual patient with coccidioidomycosis. The albumin protein and albumin glycoprotein decrease and gamma protein increases in relation to severity of infection. In 40 patients with coccidioidomycosis, changes in individual protein fractions could be significantly correlated with conventional laboratory tests, such as the complement fixation test, erythrocyte sedimentation rate and hematocrit. Changes in the alpha, glycoprotein concentration, the erythrocyte sedimentation rate and the hematocrit value appear to be related to the degree of inflammation, while the changes in the gamma protein and the beta, glycoprotein appear to be related to the specific antibody response.

  11. 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.

  12. 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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2012-01-20

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

  15. Profiling of urinary proteins in Karan Fries cows reveals more than 1550 proteins.

    PubMed

    Bathla, Shveta; Rawat, Preeti; Baithalu, Rubina; Yadav, Munna Lal; Naru, Jasmine; Tiwari, Anurag; Kumar, Sudarshan; Balhara, Ashok K; Singh, Surender; Chaudhary, Suman; Kumar, Rajesh; Lotfan, Masoud; Behare, Pradip; Phulia, Sushil K; Mohanty, Tushar K; Kaushik, Jai K; Nallapeta, Shivramaiah; Singh, Inderjeet; Ambatipudi, Srinivas K; Mohanty, Ashok K

    2015-09-01

    Urine is a non-invasive source of biological fluid, which reflects the physiological status of the mammals. We have profiled the cow urinary proteome and analyzed its functional significance. The urine collected from three healthy cows was concentrated by diafiltration (DF) followed by protein extraction using three methods, namely methanol, acetone, and ammonium sulphate (AS) precipitation and Proteo Spin urine concentration kit (PS). The quality of the protein was assessed by two-dimensional gel electrophoresis (2DE). In-gel digestion method revealed more proteins (1191) in comparison to in-solution digestion method (541). Collectively, 938, 606 and 444 proteins were identified in LC-MS/MS after in-gel and in-solution tryptic digestion of proteins prepared by AS, PS and DF methods, respectively resulting in identification of a total of 1564 proteins. Gene ontology (GO) using Panther7.0 grouped the majority of the proteins into cytoplasmic (location), catalytic activity (function), and metabolism (biological processes), while Cytoscape grouped proteins into complement and coagulation cascades; protease inhibitor activity and wound healing. Functional significance of few selected proteins seems to play important role in their physiology. Comparative analysis with human urine revealed 315 overlapping proteins. This study reports for the first time evidence of more than 1550 proteins in urine of healthy cow donors. This article is part of a Special Issue entitled: Proteomics in India.

  16. 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

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

    PubMed

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

    1976-03-01

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

  18. Alteration of sperm protein profile induced by cigarette smoking.

    PubMed

    Chen, Xiaohui; Xu, Wangjie; Miao, Maohua; Zhu, Zijue; Dai, Jingbo; Chen, Zhong; Fang, Peng; Wu, Junqing; Nie, Dongsheng; Wang, Lianyun; Wang, Zhaoxia; Qiao, Zhongdong; Shi, Huijuan

    2015-07-01

    Cigarette smoking is associated with lower semen quality, but how cigarette smoking changes the semen quality remains unclear. The aim of this study was to screen the differentially expressed proteins in the sperm of mice with daily exposure to cigarette smoke. The 2D gel electrophoresis (2DE) and mass spectrometry (MS) analyses results showed that the mouse sperm protein profile was altered by cigarette smoking. And 22 of the most abundant proteins that correspond to differentially expressed spots in 2DE gels of the sperm samples were identified. These proteins were classified into different groups based on their functions, such as energy metabolism, reproduction, and structural molecules. Furthermore, the 2DE and MS results of five proteins (Aldoa, ATP5a1, Gpx4, Cs, and Spatc1) were validated by western blot analysis and reverse transcriptase-polymerase chain reaction. Results showed that except Spatc1 the other four proteins showed statistically significant different protein levels between the smoking group and the control group (P < 0.05). The expressions of three genes (Aldoa, Gpx4, and Spatc1) were significantly different (P < 0.05) at transcription level between the smoking group and the control group. In addition, five proteins (Aldoa, ATP5a1, Spatc1, Cs, and Gpx4) in human sperm samples from 30 male smokers and 30 non-smokers were detected by western blot analysis. Two proteins (Aldoa and Cs) that are associated with energy production were found to be significantly altered, suggesting that these proteins may be potential diagnostic markers for evaluation of smoking risk in sperm. Further study of these proteins may provide insight into the pathogenic mechanisms underlying infertility in smoking persons.

  19. 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

  20. 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.

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

    PubMed

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

    2015-01-15

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

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

    PubMed

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

    2016-09-01

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

  3. 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

  4. 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.

  5. Serum Protein Profile Alterations in Hemodialysis Patients

    SciTech Connect

    Murphy, G A; Davies, R W; Choi, M W; Perkins, J; Turteltaub, K W; McCutchen-Maloney, S L; Langlois, R G; Curzi, M P; Trebes, J E; Fitch, J P; Dalmasso, E A; Colston, B W; Ying, Y; Chromy, B A

    2003-11-18

    Background: Serum protein profiling patterns can reflect the pathological state of a patient and therefore may be useful for clinical diagnostics. Here, we present results from a pilot study of proteomic expression patterns in hemodialysis patients designed to evaluate the range of serum proteomic alterations in this population. Methods: Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOFMS) was used to analyze serum obtained from patients on periodic hemodialysis treatment and healthy controls. Serum samples from patients and controls were first fractionated into six eluants on a strong anion exchange column, followed by application to four array chemistries representing cation exchange, anion exchange, metal affinity and hydrophobic surfaces. A total of 144 SELDI-TOF-MS spectra were obtained from each serum sample. Results: The overall profiles of the patient and control samples were consistent and reproducible. However, 30 well-defined protein differences were observed; 15 proteins were elevated and 15 were decreased in patients compared to controls. Serum from one patient exhibited novel protein peaks suggesting possible additional changes due to a secondary disease process. Conclusion: SELDI-TOF-MS demonstrated dramatic serum protein profile differences between patients and controls. Similarity in protein profiles among dialysis patients suggests that patient physiological responses to end-stage renal disease and/or dialysis therapy have a major effect on serum protein profiles.

  6. Capillary electrophoresis: Imaging of electroosmotic and pressure driven flow profiles in fused silica capillaries

    NASA Technical Reports Server (NTRS)

    Williams, George O., Jr.

    1996-01-01

    This study is a continuation of the summer of 1994 NASA/ASEE Summer Faculty Fellowship Program. This effort is a portion of the ongoing work by the Biophysics Branch of the Marshall Space Flight Center. The work has focused recently on the separation of macromolecules using capillary electrophoresis (CE). Two primary goals were established for the effort this summer. First, we wanted to use capillary electrophoresis to study the electrohydrodynamics of a sample stream. Secondly, there was a need to develop a methodology for using CE for separation of DNA molecules of various sizes. In order to achieve these goals we needed to establish a procedure for detection of a sample plug under the influence of an electric field Detection of the sample with the microscope and image analysis system would be helpful in studying the electrohydrodynamics of this stream under load. Videotaping this process under the influence of an electric field in real time would also be useful. Imaging and photography of the sample/background electrolyte interface would be vital to this study. Finally, detection and imaging of electroosmotic flow and pressure driven flow must be accomplished.

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

    PubMed

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

    2015-03-25

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

  8. 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

  9. 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.

  10. Profiling protein function with small molecule microarrays

    PubMed Central

    Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

    2002-01-01

    The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675

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

    PubMed

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

    2012-10-21

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

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

    PubMed

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

    2001-01-01

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

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

    PubMed

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

    2014-10-01

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

  14. High-Throughput Profiling of FFPE Tissue Using Parallel Electrophoresis and MALDI MS

    PubMed Central

    Aerni, Hans-Rudolf; Cornett, Dale S.

    2009-01-01

    Analysis of formalin-fixed and paraffin-embedded tissues (FFPE) is increasingly recognized as a strategy for the discovery and validation of clinically useful biomarker candidates. Large tissue collections including tissue microarrays (TMA) are available but current analytical strategies for their characterization have limited throughput. In this report, we describe a workflow for rapid analysis of hundreds of FFPE tissue specimens. The strategy combines parallel sample processing and on-chip electrophoresis with automated MALDI MS analysis. The method is optimized for small quantities of clinically valuable tissues allowing detection of hundreds of peptides from a single core in a TMA section. We describe results from the optimization of the method and apply it for the analysis of tissue microarrays containing formalin fixed tissue specimens from human kidney. PMID:19650658

  15. 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.

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

    PubMed

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

    2013-11-01

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

  17. Reconstruction of SAXS Profiles from Protein Structures

    PubMed Central

    Putnam, Daniel K.; Lowe, Edward W.

    2013-01-01

    Small angle X-ray scattering (SAXS) is used for low resolution structural characterization of proteins often in combination with other experimental techniques. After briefly reviewing the theory of SAXS we discuss computational methods based on 1) the Debye equation and 2) Spherical Harmonics to compute intensity profiles from a particular macromolecular structure. Further, we review how these formulas are parameterized for solvent density and hydration shell adjustment. Finally we introduce our solution to compute SAXS profiles utilizing GPU acceleration. PMID:24688746

  18. Activity-Based Protein Profiling of Microbes

    SciTech Connect

    Sadler, Natalie C.; Wright, Aaron T.

    2015-02-01

    Activity-Based Protein Profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems include: enhancing protein annotation, characterizing protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

  19. 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.

  20. 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.

  1. Denaturing gradient gel electrophoresis profiling of bacterial communities composition in Arabian Sea.

    PubMed

    Singh, Sanjay Kumar; Ramaiah, Nagappa

    2011-05-01

    Denaturing gradient gel electrophoresis (DGGE) was used to elucidate spatial and temporal variations in bacterial community composition (BCC) from four locations along the central west coast of India. DNA extracts from 36 water samples collected from surface, mid-depth (-10 m) and dose to bottom (-20 m) during premonsoon, postmonsoon, monsoon were analyzed by PCRfor amplifying variable region of 16S rRNAgene and subsequently through DGGE. Prominent bands were excised, cloned and sequenced indicated the preponderance of gammaproteobacteria, bacteroidetes and cyanobacteria. Non-metric dimensional scaling of the DGGE gels indicated that the spatial variations in BCC were prominent among the sampling locations. Temporal variations in the BCC appear to be influenced by monsoonal processes. The canonical correspondence analyses suggest that the concentration of chlorophyll a and nitrate are two important environmental factors for both spatial and temporal variations in BCC. Chlorophyll a seems to be impart a top-down control of BCC while nitrate, the bottom-up control. Our results also suggest that BCC can vary over a small geographic distance in highly dynamic, seasonally predisposed tropical coastal waters.

  2. Developmental changes in the protein profiles of human cardiac and skeletal muscle.

    PubMed

    Tipler, T D; Edwards, Y H; Hopkinson, D A

    1978-05-01

    1. The use of SDS electrophoresis as a tool for the analysis of development processes in man has been evaluated. 2. The protein profiles of cardiac and skeletal muscle from foetal (10--24 weeks gestation) infant and adult specimens have been analysed and striking developmental changes were found which involved all the major proteins. 3. Before 20 weeks gestation the soluble protein profile of skeletal muscle appears to consist largely of extracellular proteins. 4. Myoglobin was found in foetal cardiac muscle from 20 weeks gestation but was not demonstrable in foetal (greater than 24 weeks) skeletal muscle. Foetal and adult myoglobin were indistinguishable. 5. A limited survey of the protein patterns of brain, liver and kidney was carried out. In general these tissues show less developmental change than skeletal or cardiac muscle.

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

    PubMed Central

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

    2014-01-01

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

  4. 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) ...

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

    PubMed

    Sun, Yao; Bao, Pengju; Zhang, Genbao

    2013-01-01

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

  6. Assigning protein functions by comparative genome analysis protein phylogenetic profiles

    DOEpatents

    Pellegrini, Matteo; Marcotte, Edward M.; Thompson, Michael J.; Eisenberg, David; Grothe, Robert; Yeates, Todd O.

    2003-05-13

    A computational method system, and computer program are provided for inferring functional links from genome sequences. One method is based on the observation that some pairs of proteins A' and B' have homologs in another organism fused into a single protein chain AB. A trans-genome comparison of sequences can reveal these AB sequences, which are Rosetta Stone sequences because they decipher an interaction between A' and B. Another method compares the genomic sequence of two or more organisms to create a phylogenetic profile for each protein indicating its presence or absence across all the genomes. The profile provides information regarding functional links between different families of proteins. In yet another method a combination of the above two methods is used to predict functional links.

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    2000-06-01

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

  9. 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

  10. Comparative root protein profiles of Korean ginseng (Panax ginseng) and Indian ginseng (Withania somnifera).

    PubMed

    Nagappan, Arulkumar; Karunanithi, Nithya; Sentrayaperumal, Sundareswaran; Park, Kwang-Ii; Park, Hyeon-Soo; Lee, Do Hoon; Kang, Sang-Rim; Kim, Jin-A; Senthil, Kalaiselvi; Natesan, Senthil; Muthurajan, Raveendran; Kim, Gon Sup

    2012-01-01

    Ginsenosides and withanolides are the secondary metabolites from Panax ginseng and Withania somnifera, respectively. These compounds have similar biological properties. Two-dimensional electrophoresis (2-DE) analysis was utilized to reveal the protein profile in the roots of both plants, with the aim of clarifying similarly- and differentially-expressed proteins. Total proteins of Korea ginseng (P. ginseng) and Indian ginseng (W. somnifera) roots were separated by 2-DE using a pH 4-7 immobilized pH gradient strip in the first dimension and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis in the second dimension. The protein spots were visualized by silver staining. Twenty-one P. ginseng proteins and 35 W. somnifera proteins were chosen for identification by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry; of these, functions were ascribed to 14 and 22 of the P. ginseng and W. somnifera proteins, respectively. Functions mainly included general cell metabolism, defense and secondary metabolism. ATPase and alcohol dehydrogenase proteins were expressed in both plants. The results of this study, to our knowledge, are the first to provide a reference 2-DE map for the W. somnifera root proteome, and will aid in the understanding of the expression and functions of proteins in the roots of Korean ginseng and Indian ginseng.

  11. CCProf: exploring conformational change profile of proteins

    PubMed Central

    Chang, Che-Wei; Chou, Chai-Wei; Chang, Darby Tien-Hao

    2016-01-01

    In many biological processes, proteins have important interactions with various molecules such as proteins, ions or ligands. Many proteins undergo conformational changes upon these interactions, where regions with large conformational changes are critical to the interactions. This work presents the CCProf platform, which provides conformational changes of entire proteins, named conformational change profile (CCP) in the context. CCProf aims to be a platform where users can study potential causes of novel conformational changes. It provides 10 biological features, including conformational change, potential binding target site, secondary structure, conservation, disorder propensity, hydropathy propensity, sequence domain, structural domain, phosphorylation site and catalytic site. All these information are integrated into a well-aligned view, so that researchers can capture important relevance between different biological features visually. The CCProf contains 986 187 protein structure pairs for 3123 proteins. In addition, CCProf provides a 3D view in which users can see the protein structures before and after conformational changes as well as binding targets that induce conformational changes. All information (e.g. CCP, binding targets and protein structures) shown in CCProf, including intermediate data are available for download to expedite further analyses. Database URL: http://zoro.ee.ncku.edu.tw/ccprof/ PMID:27016699

  12. 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

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

    PubMed

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

    2013-12-19

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

  14. 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.

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

    PubMed

    Urzainqui, A; Tabarés, E; Carrasco, L

    1987-09-01

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

  16. Impact of Profiling Technologies in the Understanding of Recombinant Protein Production

    NASA Astrophysics Data System (ADS)

    Vijayendran, Chandran; Flaschel, Erwin

    Since expression profiling methods have been available in a high throughput fashion, the implication of these technologies in the field of biotechnology has increased dramatically. Microarray technology is one such unique and efficient methodology for simultaneous exploration of expression levels of numerous genes. Likewise, two-dimensional gel electrophoresis or multidimensional liquid chromatography coupled with mass spectrometry are extensively utilised for studying expression levels of numerous proteins. In the field of biotechnology these highly parallel analytical methods have paved the way to study and understand various biological phenomena depending on expression patterns. The next phenomenological level is represented by the metabolome and the (metabolic) fluxome. However, this chapter reviews gene and protein profiling and their impact on understanding recombinant protein production. We focus on the computational methods utilised for the analyses of data obtained from these profiling technologies as well as prominent results focusing on recombinant protein expression with Escherichia coli. Owing to the knowledge accumulated with respect to cellular signals triggered during recombinant protein production, this field is on the way to design strategies for developing improved processes. Both gene and protein profiling have exhibited a handful of functional categories to concentrate on in order to identify target genes and proteins, respectively, involved in the signalling network with major impact on recombinant protein production.

  17. Application of capillary electrophoretic chips in protein profiling of plant extracts for identification of genetic modifications of maize.

    PubMed

    Poboży, Ewa; Filaber, Monika; Koc, Anna; Garcia-Reyes, Juan F

    2013-09-01

    In this study, the chip gel electrophoresis with LIF detection was applied in protein profiling of fractionated and total extracts of maize standards. The sensitivity of such determinations can be enhanced by lyophilization of extracts or employing filtering and preconcentration with cutoff filters. Combinatorial peptide ligand library applied for sample processing prior to the electrophoretic analysis was, especially, an effective pretreatment step in the determination of low-abundance proteins. Several repeatable differences were observed for protein profiles between maize standards not containing the genetically modified organisms (GMOs) and those containing GMO, which can be potentially employed for identification of GMO in maize samples and foods of maize origin. PMID:23856913

  18. Application of capillary electrophoretic chips in protein profiling of plant extracts for identification of genetic modifications of maize.

    PubMed

    Poboży, Ewa; Filaber, Monika; Koc, Anna; Garcia-Reyes, Juan F

    2013-09-01

    In this study, the chip gel electrophoresis with LIF detection was applied in protein profiling of fractionated and total extracts of maize standards. The sensitivity of such determinations can be enhanced by lyophilization of extracts or employing filtering and preconcentration with cutoff filters. Combinatorial peptide ligand library applied for sample processing prior to the electrophoretic analysis was, especially, an effective pretreatment step in the determination of low-abundance proteins. Several repeatable differences were observed for protein profiles between maize standards not containing the genetically modified organisms (GMOs) and those containing GMO, which can be potentially employed for identification of GMO in maize samples and foods of maize origin.

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

    PubMed

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

    2013-06-21

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

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

    PubMed

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

    2002-08-01

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

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

    PubMed

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

    1994-02-01

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

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

    PubMed

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

    2015-02-01

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

  3. Multiplexed protein profiling by sequential affinity capture

    PubMed Central

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter

    2016-01-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off‐target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi‐automated sequential capture assay. This novel bead‐based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read‐out by a secondary capture bead array. We demonstrate in a proof‐of‐concept setting that target detection via two sequential affinity interactions reduced off‐target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA‐based signal amplification, and demonstrate the applicability of the dual capture bead‐based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

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

    PubMed

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

    2006-01-01

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

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

    PubMed Central

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

    2008-01-01

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

  6. Maize IgE binding proteins: each plant a different profile?

    PubMed Central

    2014-01-01

    Background Allergies are nearly always triggered by protein molecules and the majority of individuals with documented immunologic reactions to foods exhibit IgE hypersensitivity reactions. In this study we aimed to understand if natural differences, at proteomic level, between maize populations, may induce different IgE binding proteins profiles among maize-allergic individuals. We also intended to deepen our knowledge on maize IgE binding proteins. Results In order to accomplish this goal we have used proteomic tools (SDS-PAGE and 2-D gel electrophoresis followed by western blot) and tested plasma IgE reactivity from four maize-allergic individuals against four different protein fractions (albumins, globulins, glutelins and prolamins) of three different maize cultivars. We have observed that maize cultivars have different proteomes that result in different IgE binding proteins profiles when tested against plasma from maize-allergic individuals. We could identify 19 different maize IgE binding proteins, 11 of which were unknown to date. Moreover, we found that most (89.5%) of the 19 identified potential maize allergens could be related to plant stress. Conclusions These results lead us to conclude that, within each species, plant allergenic potential varies with genotype. Moreover, considering the stress-related IgE binding proteins identified, we hypothesise that the environment, particularly stress conditions, may alter IgE binding protein profiles of plant components. PMID:24650160

  7. Comparative profiling of N-glycans isolated from serum samples of ovarian cancer patients and analyzed by microchip electrophoresis.

    PubMed

    Mitra, Indranil; Alley, William R; Goetz, John A; Vasseur, Jacqueline A; Novotny, Milos V; Jacobson, Stephen C

    2013-10-01

    Ovarian cancer is the fifth leading cause of cancer-related mortalities for women in the United States and the most lethal gynecological cancer. Aberrant glycosylation has been linked to several human diseases, including ovarian cancer, and accurate measurement of changes in glycosylation may provide relevant diagnostic and prognostic information. In this work, we used microchip electrophoresis coupled with laser-induced fluorescence detection to determine quantitative differences among the N-glycan profiles of control individuals and late-stage recurrent ovarian cancer patients prior to and after an experimental drug treatment that combined docetaxel and imatinib mesylate. N-Glycans were enzymatically released from 5-μL aliquots of serum samples, labeled with the anionic fluorescent tag, 8-aminopyrene-1,3,6-trisulfonic acid, and analyzed on microfluidic devices. A 22-cm long separation channel, operated at 1250 V/cm, generated analysis times less than 100 s, separation efficiencies up to 8 × 10(5) plates (3.6 × 10(6) plates/m), and migration time reproducibilities better than 0.1% relative standard deviation after peak alignment. Principal component analysis (PCA) and analysis of variance (ANOVA) tests showed significant differences between the control and both pre- and post-treatment cancer samples and subtle differences between the pre- and post-treatment cancer samples. Area-under-the-curve (AUC) values from receiver operating characteristics (ROC) tests were used to evaluate the diagnostic merit of N-glycan peaks, and specific N-glycan peaks used in combination provided AUCs > 0.90 (highly accurate test) when the control and pretreatment cancer samples and control and post-treatment samples were compared.

  8. Protein kinase profiling assays: a technology review.

    PubMed

    Wang, Yuren; Ma, Haiching

    2015-11-01

    Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.

  9. 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.

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

    PubMed

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

    2000-10-01

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

  11. 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.

  12. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    1999-01-01

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

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

    PubMed

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

    1994-01-01

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

  16. 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.

  17. 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

  18. PROCAIN: protein profile comparison with assisting information

    PubMed Central

    Wang, Yong; Sadreyev, Ruslan I.; Grishin, Nick V.

    2009-01-01

    Detection of remote sequence homology is essential for the accurate inference of protein structure, function and evolution. The most sensitive detection methods involve the comparison of evolutionary patterns reflected in multiple sequence alignments (MSAs) of protein families. We present PROCAIN, a new method for MSA comparison based on the combination of ‘vertical’ MSA context (substitution constraints at individual sequence positions) and ‘horizontal’ context (patterns of residue content at multiple positions). Based on a simple and tractable profile methodology and primitive measures for the similarity of horizontal MSA patterns, the method achieves the quality of homology detection comparable to a more complex advanced method employing hidden Markov models (HMMs) and secondary structure (SS) prediction. Adding SS information further improves PROCAIN performance beyond the capabilities of current state-of-the-art tools. The potential value of the method for structure/function predictions is illustrated by the detection of subtle homology between evolutionary distant yet structurally similar protein domains. ProCAIn, relevant databases and tools can be downloaded from: http://prodata.swmed.edu/procain/download. The web server can be accessed at http://prodata.swmed.edu/procain/procain.php. PMID:19357092

  19. PROCAIN: protein profile comparison with assisting information.

    PubMed

    Wang, Yong; Sadreyev, Ruslan I; Grishin, Nick V

    2009-06-01

    Detection of remote sequence homology is essential for the accurate inference of protein structure, function and evolution. The most sensitive detection methods involve the comparison of evolutionary patterns reflected in multiple sequence alignments (MSAs) of protein families. We present PROCAIN, a new method for MSA comparison based on the combination of 'vertical' MSA context (substitution constraints at individual sequence positions) and 'horizontal' context (patterns of residue content at multiple positions). Based on a simple and tractable profile methodology and primitive measures for the similarity of horizontal MSA patterns, the method achieves the quality of homology detection comparable to a more complex advanced method employing hidden Markov models (HMMs) and secondary structure (SS) prediction. Adding SS information further improves PROCAIN performance beyond the capabilities of current state-of-the-art tools. The potential value of the method for structure/function predictions is illustrated by the detection of subtle homology between evolutionary distant yet structurally similar protein domains. ProCAIn, relevant databases and tools can be downloaded from: http://prodata.swmed.edu/procain/download. The web server can be accessed at http://prodata.swmed.edu/procain/procain.php. PMID:19357092

  20. PROCAIN: protein profile comparison with assisting information.

    PubMed

    Wang, Yong; Sadreyev, Ruslan I; Grishin, Nick V

    2009-06-01

    Detection of remote sequence homology is essential for the accurate inference of protein structure, function and evolution. The most sensitive detection methods involve the comparison of evolutionary patterns reflected in multiple sequence alignments (MSAs) of protein families. We present PROCAIN, a new method for MSA comparison based on the combination of 'vertical' MSA context (substitution constraints at individual sequence positions) and 'horizontal' context (patterns of residue content at multiple positions). Based on a simple and tractable profile methodology and primitive measures for the similarity of horizontal MSA patterns, the method achieves the quality of homology detection comparable to a more complex advanced method employing hidden Markov models (HMMs) and secondary structure (SS) prediction. Adding SS information further improves PROCAIN performance beyond the capabilities of current state-of-the-art tools. The potential value of the method for structure/function predictions is illustrated by the detection of subtle homology between evolutionary distant yet structurally similar protein domains. ProCAIn, relevant databases and tools can be downloaded from: http://prodata.swmed.edu/procain/download. The web server can be accessed at http://prodata.swmed.edu/procain/procain.php.

  1. 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.

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

    PubMed Central

    2014-01-01

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

  3. 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

  4. 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.

  5. 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.

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

    PubMed

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

    1985-02-01

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

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

    PubMed

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

    2014-07-04

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

  8. Binary Oscillatory Crossflow Electrophoresis

    NASA Technical Reports Server (NTRS)

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

    1996-01-01

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

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

    PubMed

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

    2012-02-01

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

  10. Denaturing gradient gel electrophoresis analysis of bacterial community profiles in the rhizosphere of cry1AC-carrying Brassica rapa subsp. pekinensis.

    PubMed

    Jung, Sera; Park, Semi; Kim, Daeha; Kim, Seung Bum

    2008-02-01

    The effect of genetically modified (GM) Brassica rapa subsp. pekinensis (Chinese cabbage) expressing Bt toxin gene (cry1AC) to the rhizosphere bacterial community was examined using the denaturing gradient gel electrophoresis (DGGE) fingerprinting method. From the visual comparison of the DGGE profiles, there were no significant differences between the profiles of Bt and control rhizosphere in both Suwon and Yesan samples. From the sequence analysis of the individual bands, Sphingomonas sp. of Alphaproteobacteria and several actinobacterial members were identified as the main bacterial taxa in both Suwon and Yesan samples. In the multiple correspondence analysis, no clear separation between Bt and control rhizosphere was seen in both Suwon and Yesan datasets. The profiles of bulk soils were separated from those of rhizosphere. The DGGE fingerprinting analyses indicated that Bt crops did not significantly alter the genetic composition of rhizosphere bacterial communities.

  11. 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...

  12. Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves.

    PubMed

    Huang, Hong-Lei; Cendan, Cruz-Miguel; Roza, Carolina; Okuse, Kenji; Cramer, Rainer; Timms, John F; Wood, John N

    2008-01-01

    Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel alpha2delta-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability. PMID:18700027

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

    PubMed

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

    2013-06-18

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

  14. 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

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

    PubMed

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

    2000-06-01

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

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

    PubMed

    Hack, N; Crawford, N

    1984-08-15

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

  17. 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%).

  18. 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.

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

    PubMed

    Han, Aishan; Hosokawa, Kazuo; Maeda, Mizuo

    2009-10-01

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

  20. 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.

  1. 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.

  2. Identification of chemical-specific protein profiles in Daphnia magna using neural networks

    SciTech Connect

    Iamonte, T.; Broadt, T.; Bradley, B.

    1995-12-31

    One dimensional gel electrophoresis was performed on whole-animal homogenates of 10 Daphnia magna exposed for 48 hours to one toxic and one non-toxic concentration of 2,4-dinitrophenol and sodium pentachlorophenate, two uncouplers of oxidative phosphorylation; malathion, an organophosphate; and permethrine, a pyrethroid, along with culture water and solvent controls, as appropriate. Ten randomized complete block exposures were conducted to minimize among-cohort variability. The 10-animal samples were gel electrophoresed, visualized using neutral silver staining and digitized with a Molecular Dynamics personal laser densitometer equipped with ImageQuant software. Densitometric data were used in a commercial neural network software package to construct a learning set, or database, of the protein profiles induced by the known chemical treatments. Novel data sets were then presented to the neural network program for assignment to treatment categories. Although no differences in protein profile between controls and chemical treatments and among chemical treatments could be detected visually in one dimensional gels, the neural network was able to correctly assign each sample to the appropriate learned treatment category about 70 percent of the time. Key proteins used by the neural network software to learn the protein profile of each chemical were identified by molecular weight and assigned a relative importance for identification of that chemical.

  3. 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

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

    PubMed

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

    1994-10-01

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

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

    PubMed

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

    2016-02-11

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

  6. 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

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

    PubMed

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

    2013-05-29

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

  8. 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.

  9. Optimized sequence retrieval from single bands of temperature gradient gel electrophoresis profiles of the amplified 16S rDNA fragments from an activated sludge system.

    PubMed

    Zhang, Xueli; Yan, Xing; Gao, Pingping; Wang, Linghua; Zhou, Zhihua; Zhao, Liping

    2005-01-01

    Sequence retrieval from single bands of polymerase chain reaction (PCR)-denaturing gel electrophoresis (DGE) profiles is an important but often difficult step for molecular diversity analysis of complex microbial communities such as activated sludge systems. We analyzed the temperature gradient gel electrophoresis (TGGE) profiles of PCR-amplified 16S rDNA fragments from an activated sludge sample of a coking wastewater treatment plant. Single bands were excised, and a clone library was constructed for each. Sequence heterogeneity in each single band was found to be significantly overestimated due to single-stranded DNA (ssDNA) contamination formed during the PCR amplification, since only 10-60% of library clones of each single TGGE band had identical migration behavior compared with the parent band. Three methods, digestion with mung bean nuclease, optimization of PCR amplification, and purification via denatured polyacrylamide gel electrophoresis (d-PAGE), were compared for their ability to minimize ssDNA contamination, with the last one being the most efficient. After using d-PAGE to minimize ssDNA to a nearly nondetectable level, 70-100% of library clones for each single TGGE band had identical migration compared with the parent band. Several sequences were found in each of six single bands, and this co-migration could be predicted with the Poland software. The predominant bacteria of the activated sludge were assessed via a combination of sequence retrieval from each single TGGE band and band intensity analysis. Only beta and alpha subclasses of the Proteobacteria were detected, 93.8% and 6.2%, respectively. Our work suggests that prior to constructing a clone library to retrieve the actual sequence diversity of a single DGE band, it is advisable to minimize ssDNA contamination to a nondetectable level.

  10. 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.

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

    PubMed

    Apoga, D; Ek, B; Tunlid, A

    2001-04-13

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

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

    PubMed

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

    1994-10-01

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

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

    PubMed

    Okajima, T; Tanabe, T; Yasuda, T

    1993-06-01

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

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

    PubMed

    Dalluge, J J; Sander, L C

    1998-12-15

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

  15. 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.

  16. 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.

  17. 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.

  18. Proteome Profile of Functional Mitochondria from Human Skeletal Muscle Using One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

    PubMed Central

    Lefort, Natalie; Yi, Zhengping; Bowen, Benjamin; Glancy, Brian; De Filippis, Eleanna A.; Mapes, Rebekka; Hwang, Hyonson; Flynn, Charles R.; Willis, Wayne T.; Civitarese, Anthony; Højlund, Kurt; Mandarino, Lawrence J.

    2009-01-01

    Mitochondria can be isolated from skeletal muscle in a manner that preserves tightly coupled bioenergetic function in vitro. The purpose of this study was to characterize the composition of such preparations using a proteomics approach. Mitochondria isolated from human vastus lateralis biopsies were functional as evidenced by their response to carbohydrate and fat-derived fuels. Using one-dimensional gel electrophoresis and HPLC-ESI-MS/MS, 823 unique proteins were detected, and 487 of these were assigned to the mitochondrion, including the newly characterized SIRT5, MitoNEET and RDH13. Proteins detected included 9 of the 13 mitochondrial DNA-encoded proteins and 86 of 104 electron transport chain (ETC) and ETC-related proteins. In addition, 59 of 78 proteins of the 55S mitoribosome, several TIM and TOM proteins and cell death proteins were present. This study presents an efficient method for future qualitative assessments of proteins from functional isolated mitochondria from small samples of healthy and diseased skeletal muscle. PMID:19567276

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

    PubMed

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

    2013-08-23

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

  20. Differential expression profile of membrane proteins in L-02 cells exposed to trichloroethylene.

    PubMed

    Hong, Wen-Xu; Huang, Aibo; Lin, Sheng; Yang, Xifei; Yang, Linqing; Zhou, Li; Huang, Haiyan; Wu, Desheng; Huang, Xinfeng; Xu, Hua; Liu, Jianjun

    2016-10-01

    Trichloroethylene (TCE), a halogenated organic solvent widely used in industries, is known to cause severe hepatotoxicity. However, the mechanisms underlying TCE hepatotoxicity are still not well understood. It is predicted that membrane proteins are responsible for key biological functions, and recent studies have revealed that TCE exposure can induce abnormal levels of membrane proteins in body fluids and cultured cells. The aim of this study is to investigate the TCE-induced alterations of membrane proteins profiles in human hepatic L-02 liver cells. A comparative membrane proteomics analysis was performed in combination with two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 15 proteins were identified as differentially expressed (4 upregulated and 11 downregulated) between TCE-treated cells and normal controls. Among this, 14 of them are suggested as membrane-associated proteins by their transmembrane domain and/or subcellular location. Furthermore, the differential expression of β subunit of adenosine triphosphate synthase (ATP5B) and prolyl 4-hydroxylase, β polypeptide (P4HB) were verified by Western blot analysis in TCE-treated L-02 cells. Our work not only reveals the association between TCE exposure and altered expression of membrane proteins but also provides a novel strategy to discover membrane biomarkers and elucidate the potential mechanisms involving with membrane proteins response to chemical-induced toxic effect.

  1. Evaluation and Comparison of Four Protein Extraction Protocols for Mono- and Two-Dimensional Electrophoresis in Mytilus Galloprovincialis

    PubMed Central

    Chirollo, Claudia; Boccia, Federica; Smaldone, Giorgio; Marrone, Raffaele; Pepe, Tiziana

    2015-01-01

    In this study, four protein extraction protocols from Mytilus galloprovincialis were evaluated with the aim to identify the most practical, efficient and reproducible method. Four extraction protocols frequently used for mussels and organic matrices were selected and compared. The methods were based on the use of: i) TRIzol reagent; ii) Lysis buffer; iii) phenylmethanesulfonyl fluoride; iv) trichloroacetic acid-acetone. Protein concentration was measured by the Bradford method. Three specimens of mussels were studied and the analysis was conducted in triplicate for each of the four protocols. Results indicated that the four methods could extract significantly different protein profiles. The highest number of protein spots resolved in 2DE gels and the best reproducibility was obtained using trichloroacetic acid-acetone protocol. Results afforded the selection of a suitable extraction protocol to be used for ecotoxicoproteomics studies from mussels and for other proteomic studies conducted by particularly complex tissues such as Mytilus galloprovincialis. PMID:27800405

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

    PubMed

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

    1999-01-01

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

  3. Liver protein profiling in chronic hepatitis C: identification of potential predictive markers for interferon therapy outcome.

    PubMed

    Perdomo, Ariel Basulto; Ciccosanti, Fabiola; Iacono, Oreste Lo; Angeletti, Claudio; Corazzari, Marco; Daniele, Nicola; Testa, Angela; Pisa, Roberto; Ippolito, Giuseppe; Antonucci, Giorgio; Fimia, Gian Maria; Piacentini, Mauro

    2012-02-01

    The current anti-hepatitis C virus (HCV) therapy, based on pegylated-interferon alpha and ribavirin, has limited success rate and is accompanied by several side effects. The aim of this study was to identify protein profiles in pretreatment liver biopsies of HCV patients correlating with the outcome of antiviral therapy. Cytosolic or membrane/organelle-enriched protein extracts from liver biopsies of eight HCV patients were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. Overall, this analysis identified 21 proteins whose expression levels correlate with therapy response. These factors are involved in interferon-mediated antiviral activity, stress response, and energy metabolism. Moreover, we found that post-translational modifications of dihydroxyacetone kinase were also associated with therapy outcome. Differential expression of the five best performing markers (STAT1, Mx1, DD4, DAK, and PD-ECGF) was confirmed by immunoblotting assays in an independent group of HCV patients. Finally, we showed that a prediction model based on the expression levels of these markers classifies responder and nonresponder patients with an accuracy of 85.7%. These results provide evidence that the analysis of pretreatment liver protein profiles is valuable for discriminating between responder and nonresponder HCV patients, and may contribute to reduce the number of nonresponder patients exposed to therapy-associated risks.

  4. 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.

  5. Recent advances in protein profiling of tissues and tissue fluids.

    PubMed

    Yang, Shi; Huang, Chun-Ming

    2007-08-01

    Creating protein profiles of tissues and tissue fluids, which contain secreted proteins and peptides released from various cells, is critical for biomarker discovery as well as drug and vaccine target selection. It is extremely difficult to obtain pure samples from tissues or tissue fluids, however, and identification of complex protein mixtures is still a challenge for mass spectrometry analysis. Here, we summarize recent advances in techniques for extracting proteins from tissues for mass spectrometry profiling and imaging. We also introduce a novel technique using a capillary ultrafiltration (CUF) probe to enable in vivo collection of proteins from the tissue microenvironment. The CUF probe technique is compared with existing sampling techniques, including perfusion, saline wash, fine-needle aspiration and microdialysis. In this review, we also highlight quantitative mass spectrometric proteomic approaches with, and without, stable-isotope labels. Advances in quantitative proteomics will significantly improve protein profiling of tissue and tissue fluid samples collected by CUF probes.

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

    PubMed

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

    2010-03-15

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

  7. Rapid differentiation of commercial juices and blends by using sugar profiles obtained by capillary zone electrophoresis with indirect UV detection.

    PubMed

    Navarro-Pascual-Ahuir, María; Lerma-García, María Jesús; Simó-Alfonso, Ernesto F; Herrero-Martínez, José Manuel

    2015-03-18

    A method for the determination of sugars in several fruit juices and nectars by capillary zone electrophoresis with indirect UV-vis detection has been developed. Under optimal conditions, commercial fruit juices and nectars from several fruits were analyzed, and the sugar and cyclamate contents were quantified in less than 6 min. A study for the detection of blends of high-value juices (orange and pineapple) with cheaper alternatives was also developed. For this purpose, different chemometric techniques, based on sugar content ratios, were applied. Linear discriminant analysis showed that fruit juices can be distinguished according to the fruit type, juice blends also being differentiated. Multiple linear regression models were also constructed to predict the adulteration of orange and pineapple juices with grape juice. This simple and reliable methodology provides a rapid analysis of fruit juices of economic importance, which is relevant for quality control purposes in food industries and regulatory agencies.

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

    PubMed

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

    2000-11-01

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

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

    PubMed

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

    2004-01-01

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

  10. Pathogen Induced Changes in the Protein Profile of Human Tears from Fusarium Keratitis Patients

    PubMed Central

    Ananthi, Sivagnanam; Venkatesh Prajna, Namperumalsamy; Lalitha, Prajna; Valarnila, Murugesan; Dharmalingam, Kuppamuthu

    2013-01-01

    Fusarium is the major causative agent of fungal infections leading to corneal ulcer (keratitis) in Southern India and other tropical countries. Keratitis caused by Fusarium is a difficult disease to treat unless antifungal therapy is initiated during the early stages of infection. In this study tear proteins were prepared from keratitis patients classified based on the duration of infection. Among the patients recruited, early infection (n = 35), intermediate (n = 20), late (n = 11), samples from five patients in each group were pooled for analysis. Control samples were a pool of samples from 20 patients. Proteins were separated on difference gel electrophoresis (DIGE) and the differentially expressed proteins were quantified using DeCyder software analysis. The following differentially expressed proteins namely alpha-1-antitrypsin, haptoglobin α2 chain, zinc-alpha-2-glycoprotein, apolipoprotein, albumin, haptoglobin precursor - β chain, lactoferrin, lacrimal lipocalin precursor, cystatin SA III precursor, lacritin precursor were identified using mass spectrometry. Variation in the expression level of some of the proteins was confirmed using western blot analysis. This is the first report to show stage specific tear protein profile in fungal keratitis patients. Validation of this data using a much larger sample set could lead to clinical application of these findings. PMID:23308132

  11. Proteomic profiling reveals a catalogue of new candidate proteins for human skin aging.

    PubMed

    Laimer, Martin; Kocher, Thomas; Chiocchetti, Andreas; Trost, Andrea; Lottspeich, Friedrich; Richter, Klaus; Hintner, Helmut; Bauer, Johann W; Onder, Kamil

    2010-10-01

    Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).

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

    PubMed

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

    2013-08-01

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

  13. 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

  14. Characterization of fruit products by capillary zone electrophoresis and liquid chromatography using the compositional profiles of polyphenols: application to authentication of natural extracts.

    PubMed

    Navarro, Meritxell; Núñez, Oscar; Saurina, Javier; Hernández-Cassou, Santiago; Puignou, Lluis

    2014-02-01

    Capillary zone electrophoresis (CZE) and high performance liquid chromatography (HPLC) were applied to the authentication of fruit products based on the compositional profiles of polyphenols. Various sample treatments were used to maximize the overall recovery of polyphenols or specific fractions, such as phenolic acids or anthocyanins. The resulting CZE and HPLC data were treated with Principal Component Analysis (PCA) showing that samples were mainly clustered according to the fruit of origin, with cranberry- and grape-based products clearly separated in groups. A possible adulterated cranberry extract was analyzed more deeply by high resolution mass spectrometry (HRMS) in order to identify the presence of A-type proanthocyanidins, which are characteristic and more abundant in cranberry-based products. In accordance with PCA interpretation, HRMS results indicated that the suspicious sample was not a cranberry-based product, allowing us to validate and demonstrate the suitability of both CZE- and HPLC-proposed methods for the characterization of fruit-based products.

  15. Profiling of sperm proteins and association of sperm PDC-109 with bull fertility.

    PubMed

    Somashekar, Lakshminarayana; Selvaraju, Sellappan; Parthipan, Sivashanmugam; Ravindra, Janivara Parameswaraiah

    2015-01-01

    The composition of sperm proteins influences the fertilizing ability of sperm and hence the present study was conducted (i) to profile sperm proteins expression patterns in bulls of differing fertility index and (ii) to identify and relate the abundant sperm proteins with bull fertility. The semen samples were collected from Holstein-Friesian bulls (n = 12) varying in conception rate (CR) (high/low). The frozen semen straws (three ejaculates, from each bull) were used to study (a) sperm kinetic parameters, (b) plasmalemma integrity, (c) mitochondrial membrane potential, and (d) chromatin distribution. Three bulls were randomly selected from each group (n = 3) and the neat sperm pellets were subjected to percoll purification, followed by protein isolation using 0.1% Triton X100. The sperm kinetic parameters, plasmalemma integrity, mitochondrial membrane potential, and the chromatin distribution did not differ significantly between groups. The number of acidic (pI; 3.1-5.6, 37%) and basic (pI; 7.9-10.0, 27%) proteins and their pattern of expression varied significantly (p < 0.05) between high and low fertile bulls. The abundant sperm protein spots in 2D-gel electrophoresis (2DE) were identified as seminal plasma protein PDC-109 (i.e., protein with N-terminus aspartic acid, D and carboxy terminus cystine, having 109 amino acids) and its isoform and spermadhesin-1 (SPADH1). The western blot analysis confirmed the presence of PDC-109 isoform proteins at 15.4 kDa (pI 5.3 and 5.5). The seminal plasma protein PDC-109 was abundant in the low fertile when compared to the high fertile group (p < 0.05). This study suggests that the imbalance in acidic and basic sperm proteins may influence sperm fertility and sperm PDC-109 levels above a certain threshold affects bull fertility.

  16. 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)

  17. Rapid and simple profiling of lipoproteins by polyacrylamide-gel disc electrophoresis to determine the heterogeneity of low-density lipoproteins (LDLs) including small, dense LDL.

    PubMed

    Nakano, Takanari; Inoue, Ikuo; Seo, Makoto; Takahashi, Seiichiro; Awata, Takuya; Komoda, Tsugikazu; Katayama, Shigehiro

    2009-01-01

    This study aimed to explore the potential of polyacrylamide-gel disc electrophoresis (PAGE) for lipoprotein profiling in clinical practice. Blood samples were collected from 146 patients with type 2 diabetes mellitus and lipid parameters were assayed by PAGE, including small, dense low-density lipoprotein (LDL) (n = 41), and triglyceride-rich lipoprotein remnant cholesterol (n = 37). We also used a commercial kit to measure small, dense LDL (n = 41). By PAGE, we obtained the percentage of the area under the curve (AUC %) of each peaks and calculated respective AUC% x total cholesterol (AUC%xTC) values. The calculated values of LDL-AUC%xTC, small LDL-AUC%xTC, and HDL-AUC%xTC values were correlated well with values from homogeneous assay for LDL-cholesterol, small, dense LDL-cholesterol, and HDL-cholesterol assays (r = 0.94, 0.81, and 0.89, respectively). PAGE combined with measurement of total cholesterol and triglycerides provides a rapid evaluation of anti- or pro-atherogenic lipoproteins and a simple profiling system for both the "quantity" and "quality" of lipoproteins, allowing a better assessment of the risk of coronary artery diseases. This article discusses several methods for simple and rapid lipid profiling and outlines some recent patents relevant to the methods.

  18. Quantitative peptide and protein profiling by mass spectrometry.

    PubMed

    Schmidt, Alexander; Bisle, Birgit; Kislinger, Thomas

    2009-01-01

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

  19. 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.

  20. Protein Profiling of Bladder Urothelial Cell Carcinoma

    PubMed Central

    Hu, Jinghai; Ye, Fei; Cui, Miao; Lee, Peng; Wei, Chengguo; Hao, Yuanyuan; Wang, Xiaoqing; Wang, Yanbo; Lu, Zhihua; Galsky, Matthew; McBride, Russell; Wang, Li; Wang, Dongwen; Cordon-Cardo, Carlos; Wang, Chunxi; Zhang, David Y.

    2016-01-01

    This study aimed to detect protein changes that can assist to understand the underlying biology of bladder cancer. The data showed forty five proteins were found to be differentially expressed comparing tumors vs non-tumor tissues, of which EGFR and cdc2p34 were correlated with muscle invasion and histological grade. Ten proteins (ß-catenin, HSP70, autotaxin, Notch4, PSTPIP1, DPYD, ODC, cyclinB1, calretinin and EPO) were able to classify muscle invasive BCa (MIBC) into 2 distinct groups, with group 2 associated with poorer survival. Finally, 3 proteins (P2X7, cdc25B and TFIIH p89) were independent factors for favorable overall survival. PMID:27626805

  1. Protein Profiling of Bladder Urothelial Cell Carcinoma.

    PubMed

    Hu, Jinghai; Ye, Fei; Cui, Miao; Lee, Peng; Wei, Chengguo; Hao, Yuanyuan; Wang, Xiaoqing; Wang, Yanbo; Lu, Zhihua; Galsky, Matthew; McBride, Russell; Wang, Li; Wang, Dongwen; Cordon-Cardo, Carlos; Wang, Chunxi; Zhang, David Y

    2016-01-01

    This study aimed to detect protein changes that can assist to understand the underlying biology of bladder cancer. The data showed forty five proteins were found to be differentially expressed comparing tumors vs non-tumor tissues, of which EGFR and cdc2p34 were correlated with muscle invasion and histological grade. Ten proteins (ß-catenin, HSP70, autotaxin, Notch4, PSTPIP1, DPYD, ODC, cyclinB1, calretinin and EPO) were able to classify muscle invasive BCa (MIBC) into 2 distinct groups, with group 2 associated with poorer survival. Finally, 3 proteins (P2X7, cdc25B and TFIIH p89) were independent factors for favorable overall survival. PMID:27626805

  2. Chromosomal assignment of human genomic NotI restriction fragments in a two-dimensional electrophoresis profile

    SciTech Connect

    Yoshikawa, Hirohide; Nagai, Hisaki; Matsubara, Kenichi

    1996-01-01

    Using DNA from sorted human chromosomes and two-dimensional gel electrophoresis, we assigned 2295 NotI sites, 43% of the total, to specific chromosomes and designated the procedure CA-RLGS (chromosome-assigned restriction landmark genomic scanning). Although the NotI enzyme is sensitive to DNA methylation, our results suggested that the majority of the spots did not seem to be affected by this modification. The NotI sites were distributed at higher levels in chromosomes 17, 19, and 22, suggesting higher gene content in these chromosomes. Most spots were assigned to unique chromosomes, but some spots were found on two or more chromosomes. Quantitative analysis revealed the intensity of the DNA spots on the sex chromosomes to be haploid and that of the chromosome 21 spots in DNA from a male with Down syndrome to be trisomic, although there were exceptions. We report here the first-generation CA-RLGS map of the human genome. 23 refs., 4 figs.

  3. Profiling of a microbial community under confined conditions in a fed-batch garbage decomposer by denaturing gradient gel electrophoresis.

    PubMed

    Horisawa, Sakae; Sakuma, Yoh; Nakamura, Yasunori; Doi, Shuichi

    2008-05-01

    In order to determine the conditions for the maximum performance of a fed-batch composting (FBC) reactor, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial communities established under the confined conditions of moisture content and environmental temperature. To evaluate the effects of microbial community structures on the performance of FBC reactors, degradation experiments using small-scale reactors and model waste were conducted under confined environmental conditions. A high degradation rate was observed under a wide range of MC conditions (30-60%) and at higher than usual temperatures (30-50 degrees C). The microbial communities that formed in the experimental FBC reactors were analyzed by DGGE of PCR-amplified 16S rRNA genes. The DGGE banding patterns at the same level as the degradation rates were similar even if the environmental conditions were different. Sequence analysis of the DGGE bands revealed the primary microbes which act in the reactor.

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

    PubMed

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

    2013-01-01

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

  5. Protein profiling of hemocytes from the terrestrial crustacean Armadillidium vulgare.

    PubMed

    Herbinière, Juline; Grève, Pierre; Strub, Jean-Marc; Thiersé, Danièle; Raimond, Maryline; van Dorsselaer, Alain; Martin, Gilbert; Braquart-Varnier, Christine

    2008-01-01

    To establish and maintain a successful infection, microbial pathogens have evolved various strategies to infect the host in the face of a functional immune system. In this context, the alpha-proteobacteria Wolbachia capacities to infect new host species have been greatly evidenced. Indeed, in terrestrial isopods, experimentally transferred Wolbachia invade all host tissues, including immune cells such as hemocytes. To investigate mechanisms that have to be avoided by bacteria to maintain themselves in hemocytes, we characterized the hemocyte proteome of Armadillidium vulgare by a 2D gel electrophoresis approach. Fifty-six proteins were identified and classified into functional groups (stress and immunity, glucose metabolisms, cytoskeleton, others). We focused on immune response and cytoskeleton proteins often exploited by bacteria to invade their host. From the microsequences obtained by mass spectrometry, PCR primers were designed to amplify seven partial cDNAs encoding masquerade, alpha2-macroglobulin, transglutaminase, MnSOD, calreticulin, cyclophilin, and vinculin, confirming their expression in hemocytes. PMID:18329099

  6. Electrophoresis in space.

    PubMed

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

    1999-01-01

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

  7. Kidney cell electrophoresis, continuing task

    NASA Technical Reports Server (NTRS)

    Todd, P. W.

    1985-01-01

    Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated to provide ground support in the form of analytical cell electrophoresis and flow cytometry. 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. Cells were prepared in suspension prior to flight in electrophoresis buffer and 10% calf serum. Electrophoretic separation proceeded in electrophoresis buffer without serum in the Continuous Flow Electrophoretic Separator, and fractions were collected into sample bags containing culture medium and concentrated serum. Fractions that yielded enough progeny cells were analyzed for morphology and electrophoretic mobility distributions. It is noted that the lowest mobility fraction studied produced higher mobility progeny while the other fractions produced progeny cells with mobilities related to the fractions from which they were collected.

  8. Changes in green coffee protein profiles during roasting.

    PubMed

    Montavon, Philippe; Mauron, Anne-France; Duruz, Eliane

    2003-04-01

    To reveal its flavor, coffee has to be roasted. In fact, the green coffee bean contains all ingredients necessary for the later development of coffee flavor. It is now widely accepted that free amino acids and peptides are required for the generation of coffee aroma. However, the mechanisms leading to defined mixtures of free amino acids and peptides remain unknown. Information pertaining to the identification of precursor proteins is also lacking. To answer some of these questions, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was used to follow the fate of green coffee proteins. Two conditions were considered: roasting and incubation of green coffee suspensions at 37 degrees C. Coffee beans were observed to acquire the potential to spontaneously release H(2)O(2) upon polymerization of their proteins during roasting. Fragmentation of proteins was also observed. Conversely, H(2)O(2) was found to control polymerization and fragmentation of green coffee proteins in solution at 37 degrees C. Polymerization and fragmentation patterns under the two conditions were comparable. These observations suggest that the two conditions under study triggered, at least to some extent, similar biochemical mechanisms involving autoxidation. Throughout this study, a unique fragmentation cascade involving the 11S coffee storage protein was identified. Generated fragments shared an atypical staining behavior linked to their sensitivity to redox conditions.

  9. Reverse Phase Protein Arrays for Compound Profiling.

    PubMed

    Moerke, Nathan; Fallahi-Sichani, Mohammad

    2016-01-01

    Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies. This article describes a procedure for treating cells and preparing cell lysates, as well as a procedure for generating RPPAs using these lysates. A method for probing, imaging, and analyzing RPPAs is also described. These procedures are readily adaptable to a wide range of studies of cell signaling in response to drugs and other perturbations. © 2016 by John Wiley & Sons, Inc. PMID:27622568

  10. Comparative protein profiles of Butea superba tubers under seasonal changes.

    PubMed

    Leelahawong, Chonchanok; Srisomsap, Chantragan; Cherdshewasart, Wichai; Chokchaichamnankit, Daranee; Vinayavekhin, Nawaporn; Sangvanich, Polkit

    2016-07-01

    Seasonal changes are major factors affecting environmental conditions which induce multiple stresses in plants, leading to changes in protein relative abundance in the complex cellular plant metabolic pathways. Proteomics was applied to study variations in proteome composition of Butea. superba tubers during winter, summer and rainy season throughout the year using two-dimensional polyacrylamide gel electrophoresis coupled with a nanoflow liquid chromatography coupled to electrospray ionization quadrupole-time-of-flight tandem mass spectrometry. A total of 191 protein spots were identified and also classified into 12 functional groups. The majority of these were mainly involved in carbohydrate and energy metabolism (30.37 %) and defense and stress (18.32 %). The results exhibited the highest numbers of identified proteins in winter-harvested samples. Forty-five differential proteins were found in different seasons, involving important metabolic pathways. Further analysis indicated that changes in the protein levels were due mainly to temperature stress during summer and to water stress during winter, which affected cellular structure, photosynthesis, signal transduction and homeostasis, amino-acid biosynthesis, protein destination and storage, protein biosynthesis and stimulated defense and stress mechanisms involving glycolytic enzymes and relative oxygen species catabolizing enzymes. The proteins with differential relative abundances might induce an altered physiological status within plant tubers for survival. The work provided new insights into the better understanding of the molecular basis of plant proteomes and stress tolerance mechanisms, especially during seasonal changes. The finding suggested proteins that might potentially be used as protein markers in differing seasons in other plants and aid in selecting B. superba tubers with the most suitable medicinal properties in the future. PMID:27198528

  11. Diversity of pulsed-field gel electrophoresis profiles of Campylobacter jejuni and Campylobacter coli from broiler chickens in France.

    PubMed

    Denis, M; Rose, V; Huneau-Salaün, A; Balaine, L; Salvat, G

    2008-08-01

    In 2003 to 2004, 26 free-range broilers flocks excreting Campylobacter were studied for identification of Campylobacter species and genotype diversity. Seventeen flocks were sampled at the end of the indoor rearing period and 9 before departure to the slaughterhouse after access to an open area. Out of 513 isolates, 315 were identified as Campylobacter jejuni and 198 as Campylobacter coli. Pulsed-field gel electrophoresis analysis revealed 35 genotypes for C. jejuni and 43 genotypes for C. coli; 38.4% of the isolates were clustered into 16 genetic groups. This kind of poultry production system is characterized by a large number of Campylobacter coli isolates. Flocks sampled during the indoor phase were predominantly contaminated by C. jejuni, whereas those sampled during warm months were predominantly contaminated by C. coli. The Campylobacter population was genetically highly diverse: multiple genotypes were detected in a single flock. Six flocks were each found to harbor a mixture of genotypes; these isolates were genetically closely related and were grouped into clusters of at least 80% genetic similarity. Isolates with genotypes found in different flocks and strains, but sharing the same genetic clusters, were detected in different farms and at different times in the year. Flocks sampled during the indoor rearing period and when farmers use fresh farm-made litter were associated with a small number of clusters. In conclusion, Campylobacter species were genetically highly diverse. Our findings suggest the presence of genomic rearrangements in Campylobacter colonizing the chick intestine and that some Campylobacter strains are adapted to poultry. In addition, the species diversity in the flocks was affected by season and cloistering measures. Litter and exposure to an open area were likely sources of flock Campylobacter contamination.

  12. Canola proteins for human consumption: extraction, profile, and functional properties.

    PubMed

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies.

  13. Canola Proteins for Human Consumption: Extraction, Profile, and Functional Properties

    PubMed Central

    Tan, Siong H; Mailer, Rodney J; Blanchard, Christopher L; Agboola, Samson O

    2011-01-01

    Canola protein isolate has been suggested as an alternative to other proteins for human food use due to a balanced amino acid profile and potential functional properties such as emulsifying, foaming, and gelling abilities. This is, therefore, a review of the studies on the utilization of canola protein in human food, comprising the extraction processes for protein isolates and fractions, the molecular character of the extracted proteins, as well as their food functional properties. A majority of studies were based on proteins extracted from the meal using alkaline solution, presumably due to its high nitrogen yield, followed by those utilizing salt extraction combined with ultrafiltration. Characteristics of canola and its predecessor rapeseed protein fractions such as nitrogen yield, molecular weight profile, isoelectric point, solubility, and thermal properties have been reported and were found to be largely related to the extraction methods. However, very little research has been carried out on the hydrophobicity and structure profiles of the protein extracts that are highly relevant to a proper understanding of food functional properties. Alkaline extracts were generally not very suitable as functional ingredients and contradictory results about many of the measured properties of canola proteins, especially their emulsification tendencies, have also been documented. Further research into improved extraction methods is recommended, as is a more systematic approach to the measurement of desired food functional properties for valid comparison between studies. PMID:21535703

  14. A study on the vertical profile of bacterial DNA structure in the Puruogangri (Tibetan Plateau) ice core using denaturing gradient gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Zhang, Xinfang; Yao, Tandong; An, Lizhe; Tian, Lide; Xu, Shijian

    The bacterial DNA structures at different depths in the Puruogangri (Tibetan Plateau) ice core (83.45 m) were investigated by the denaturing gradient gel electrophoresis (DGGE) DNA fingerprinting technique. DGGE profiles indicated that the bacterial species diversity in glacial ice is high, and indigenous species represented by common bands in all samples may grow on the glacial surface. Bacterial diversity, as estimated by Shannon indices (mean 2.91; SD 0.25; n = 13), was comparable to that of soil habitats and had a positive correlation with Ca2+ concentration (R = 0.71; P < 0.01), a good proxy of dust. This suggested that the soil ecosystem was the main source of bacteria in this glacier. The low similarity indices (0-43%) were found between the ice-core samples, which corresponded to the episodic deposition under defined climatic conditions and low activity of microorganisms in glacial ice. The profiles of bacterial species composition in glacial ice may be a bioindicator of climatic changes or dating.

  15. Active community profiling via capillary electrophoresis single-strand conformation polymorphism analysis of amplified 16S rRNA and 16S rRNA genes.

    PubMed

    Hiibel, Sage R; Pruden, Amy; Crimi, Barbara; Reardon, Kenneth F

    2010-12-01

    Here, we report the validation and advancement of a high-throughput method for fingerprinting the active members of a microbial community. This method, termed active community profiling (ACP), provides information about both the composition and the activity of mixed microbial cultures via comparative measurements of amplified 16S rRNA (RNA) and 16S rRNA genes (DNA). Capillary electrophoresis is used to resolve single-strand conformation polymorphisms of polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) products, producing electropherograms representative of the community structure. Active members of the community are distinguished by elevated RNA:DNA peak area ratios. Chemostat experiments with defined populations were conducted to validate the ACP approach. Using a pure culture of Escherichia coli, a direct correlation was found between the growth rate and the RNA:DNA peak ratio. In a second validation experiment, a binary culture of E. coli and Pseudomonas putida was subjected to a controlled environmental change consisting of a shift to anaerobic conditions. ACP revealed the expected cessation of growth of P. putida, an obligate aerobe, while the corresponding DNA-only analysis indicated no change in the culture. Finally, ACP was applied to a complex microbial community, and a novel binning approach was demonstrated for integrating the RNA and DNA electropherograms. ACP thus represents a significant advance from traditional DNA-based profiling techniques, which do not distinguish active from inactive or dead cells, and is well suited for high-throughput community analysis.

  16. Metagenome and Metatranscriptome Analyses Using Protein Family Profiles

    PubMed Central

    Zhong, Cuncong; Yooseph, Shibu

    2016-01-01

    Analyses of metagenome data (MG) and metatranscriptome data (MT) are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM) framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR) gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE) genes. Finally, HMM-GRASPx was used to

  17. Metagenome and Metatranscriptome Analyses Using Protein Family Profiles.

    PubMed

    Zhong, Cuncong; Edlund, Anna; Yang, Youngik; McLean, Jeffrey S; Yooseph, Shibu

    2016-07-01

    Analyses of metagenome data (MG) and metatranscriptome data (MT) are often challenged by a paucity of complete reference genome sequences and the uneven/low sequencing depth of the constituent organisms in the microbial community, which respectively limit the power of reference-based alignment and de novo sequence assembly. These limitations make accurate protein family classification and abundance estimation challenging, which in turn hamper downstream analyses such as abundance profiling of metabolic pathways, identification of differentially encoded/expressed genes, and de novo reconstruction of complete gene and protein sequences from the protein family of interest. The profile hidden Markov model (HMM) framework enables the construction of very useful probabilistic models for protein families that allow for accurate modeling of position specific matches, insertions, and deletions. We present a novel homology detection algorithm that integrates banded Viterbi algorithm for profile HMM parsing with an iterative simultaneous alignment and assembly computational framework. The algorithm searches a given profile HMM of a protein family against a database of fragmentary MG/MT sequencing data and simultaneously assembles complete or near-complete gene and protein sequences of the protein family. The resulting program, HMM-GRASPx, demonstrates superior performance in aligning and assembling homologs when benchmarked on both simulated marine MG and real human saliva MG datasets. On real supragingival plaque and stool MG datasets that were generated from healthy individuals, HMM-GRASPx accurately estimates the abundances of the antimicrobial resistance (AMR) gene families and enables accurate characterization of the resistome profiles of these microbial communities. For real human oral microbiome MT datasets, using the HMM-GRASPx estimated transcript abundances significantly improves detection of differentially expressed (DE) genes. Finally, HMM-GRASPx was used to

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

    NASA Astrophysics Data System (ADS)

    Erijman, Leonardo; Clegg, Robert M.

    1996-03-01

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

  19. Comparison of the outer membrane protein and lipopolysaccharide profiles of mucoid and nonmucoid Pseudomonas aeruginosa.

    PubMed Central

    Kelly, N M; MacDonald, M H; Martin, N; Nicas, T; Hancock, R E

    1990-01-01

    Laboratory-derived mucoid variants of Pseudomonas aeruginosa were selected by plating the standard PAO1 laboratory strain with bacteriophage. These mucoid variants formed two distinct groups of strains on the basis of phage typing. The first group had the same phage-typing pattern as the parent PAO1 strain, while the second group had a distinctly different phage-typing pattern. One strain from each group was assessed along with the parent PAO1 strain for its outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles by sodium dodecyl sulfate-gel electrophoresis followed by appropriate staining. The mucoid derivatives were found to differ from the parent PAO1 nonmucoid strain in having lost a high-molecular-weight LPS species. Furthermore, the reversion of the mucoid strains to the nonmucoid phenotype was accompanied by a return of the missing high-molecular-weight LPS species. No observable difference between the mucoid derivatives and the parent nonmucoid strain was noted in the OMP profiles. The opposite was found in the case of four isolates of mucoid P. aeruginosa from patients with cystic fibrosis. Two OMP bands (of approximately 55 and 25 kilodaltons) were present in the mucoid isolates but missing in their sister nonmucoid strains. In the case of the cystic fibrosis isolates, no difference in the LPS profiles within mucoid-nonmucoid pairs was noted. Images PMID:2121789

  20. Optimizing Human Bile Preparation for Two-Dimensional Gel Electrophoresis

    PubMed Central

    Cheng, Hao-Tsai; Sung, Chang-Mu; Pai, Betty Chien-Jung; Liu, Nai-Jen; Chen, Carl PC

    2016-01-01

    Aims. Bile is an important body fluid which assists in the digestion of fat and excretion of endogenous and exogenous compounds. In the present study, an improved sample preparation for human bile was established. Methods and Material. The method involved acetone precipitation followed by protein extraction using commercially available 2D Clean-Up kit. The effectiveness was evaluated by 2-dimensional electrophoresis (2DE) profiling quality, including number of protein spots and spot distribution. Results. The total protein of bile fluid in benign biliary disorders was 0.797 ± 0.465 μg/μL. The sample preparation method using acetone precipitation first followed by 2D Clean-Up kit protein extraction resulted in better quality of 2DE gel images in terms of resolution as compared with other sample preparation methods. Using this protocol, we obtained approximately 558 protein spots on the gel images and with better protein spots presentation of haptoglobin, serum albumin, serotransferrin, and transthyretin. Conclusions. Protein samples of bile prepared using acetone precipitation followed by 2D Clean-Up kit exhibited high protein resolution and significant protein profile. This optimized protein preparation protocol can effectively concentrate bile proteins, remove abundant proteins and debris, and yield clear presentation of nonabundant proteins and its isoforms on 2-dimensional electrophoresis gel images. PMID:26966686

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

    PubMed

    Rabilloud, T

    1994-02-01

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

  2. Distinct Lysosomal Network Protein Profiles in Parkinsonian Syndrome Cerebrospinal Fluid

    PubMed Central

    Boman, Andrea; Svensson, Samuel; Boxer, Adam; Rojas, Julio C.; Seeley, William W.; Karydas, Anna; Miller, Bruce; Kågedal, Katarina; Svenningsson, Per

    2016-01-01

    Background: Clinical diagnosis of parkinsonian syndromes like Parkinson’s disease (PD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) is hampered by overlapping symptomatology and lack of diagnostic biomarkers, and definitive diagnosis is only possible post-mortem. Objective: Since impaired protein degradation plays an important role in many neurodegenerative disorders, we hypothesized that profiles of select lysosomal network proteins in cerebrospinal fluid could be differentially expressed in these parkinsonian syndromes. Methods: Cerebrospinal fluid samples were collected from PD patients (n = 18), clinically diagnosed 4-repeat tauopathy patients; corticobasal syndrome (CBS) (n = 3) and PSP (n = 8); and pathologically diagnosed PSP (n = 8) and CBD patients (n = 7). Each patient set was compared to its appropriate control group consisting of age and gender matched individuals. Select lysosomal network protein levels were detected via Western blotting. Factor analysis was used to test the diagnostic sensitivity, specificity and accuracy of the select lysosomal network protein expression profiles. Results: PD, CBD and PSP were markedly different in their cerebrospinal fluid lysosomal network protein profiles. Lysosomal-associated membrane proteins 1 and 2 were significantly decreased in PD; early endosomal antigen 1 was decreased and lysozyme increased in PSP; and lysosomal-associated membrane proteins 1 and 2, microtubule-associated protein 1 light chain 3 and lysozyme were increased in CBD. A panel of lysosomal-associated membrane protein 2, lysozyme and microtubule-associated protein 1 light chain discriminated between controls, PD and 4-repeat tauopathies. Conclusions: This study offers proof of concept that select lysosomal network proteins are differentially expressed in cerebrospinal fluid of Parkinson’s disease, corticobasal syndrome and progressive supranuclear palsy. Lysosomal network protein analysis

  3. Comparison of Metalloproteinase Protein and Activity Profiling

    PubMed Central

    Giricz, Orsi; Lauer, Janelle L.; Fields, Gregg B.

    2010-01-01

    Proteolytic enzymes play fundamental roles in many biological processes. Members of the matrix metalloproteinase (MMP) family have been shown to take part in processes crucial in disease progression. The present study used the ExcelArray Human MMP/TIMP Array to quantify MMP and tissue inhibitor of metalloproteinase (TIMP) production in the lysates and media of 14 cancer and one normal cell line. The overall patterns were very similar in terms of which MMPs and TIMPs were secreted in the media versus associated with the cells in the individual samples. However, more MMP was found in the media, both in amount and in variety. TIMP-1 was produced in all cell lines. MMP activity assays with three different FRET substrates were then utilized to determine if protein production correlated with function for the WM-266-4 and BJ cell lines. Metalloproteinase activity was observed for both cell lines with a general MMP substrate (Knight SSP), consistent with protein production data. However, although both cell lines promoted the hydrolysis of a more selective MMP substrate (NFF-3), metalloproteinase activity was only confirmed in the BJ cell line. The use of inhibitors to confirm metalloproteinase activities pointed to the strengths and weaknesses of in situ FRET substrate assays. PMID:20920458

  4. Electrophoresis experiments in microgravity

    NASA Technical Reports Server (NTRS)

    Snyder, Robert S.; Rhodes, Percy H.

    1991-01-01

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

  5. Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors.

    SciTech Connect

    Beliaev, A. S.; Thompson, D. K.; Khare, T.; Lim, H.; Brandt, C. C.; Li, G.; Murray, A. E.; Heidelberg, J. F.; Giometti, C. S.; Yates, J., III; Nealson, K. H.; Tiedje, J. M.; Zhou, J.; Biosciences Division; ORNL; Scripps Research Inst.; Michigan State Univ.; The Inst. for Genomic Research; Jet Propulsion Laboratory; California Inst. of Tech.

    2002-01-01

    Changes in mRNA and protein expression profiles of Shewanella oneidenesis MR-1 during switch from aerobic to fumarate-, Fe(III)-, or nitrate-reducing conditions were examined using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In response to changes in growth conditions, 121 of the 691 arrayed genes displayed at least a two-fold difference in transcript abundance as determined by microarray analysis. Genes involved in aerobic respiration encoding cytochrome c and d oxidases and TCA cycle enzymes were repressed under anaerobic conditions. Genes induced during anaerobic respiration included those involved in cofactor biosynthesis and assembly (moaACE, ccmHF, nosD, cysG), substrate transport (cysUP, cysTWA, dcuB), and anaerobic energy metabolism (dmsAB, psrC, pshA, hyaABC, hydA). Transcription of genes encoding a periplasmic nitrate reductase (napBHGA), cytochrome c{sub 552}, and prismane was elevated 8- to 56-fold in response to the presence of nitrate, while cymA, ifcA, and frdA were specifically induced three- to eightfold under fumarate-reducing conditions. The mRNA levels for two oxidoreductase-like genes of unknown function and several cell envelope genes involved in multidrug resistance increased two- to fivefold specifically under Fe(III)-reducing conditions. Analysis of protein expression profiles under aerobic and anaerobic conditions revealed 14 protein spots that showed significant differences in abundance on 2-D gels. Protein identification by mass spectrometry indicated that the expression of prismane, dihydrolipoamide succinyltransferase, and alcaligin siderophore biosynthesis protein correlated with the microarray data.

  6. Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors.

    PubMed

    Beliaev, Alex S; Thompson, Dorothea K; Khare, Tripti; Lim, Hanjo; Brandt, Craig C; Li, Guangshan; Murray, Alison E; Heidelberg, John F; Giometti, Carol S; Yates, John; Nealson, Kenneth H; Tiedje, James M; Zhoui, Jizhong

    2002-01-01

    Changes in mRNA and protein expression profiles of Shewanella oneidenesis MR-1 during switch from aerobic to fumarate-, Fe(III)-, or nitrate-reducing conditions were examined using DNA microarrays and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In response to changes in growth conditions, 121 of the 691 arrayed genes displayed at least a two-fold difference in transcript abundance as determined by microarray analysis. Genes involved in aerobic respiration encoding cytochrome c and d oxidases and TCA cycle enzymes were repressed under anaerobic conditions. Genes induced during anaerobic respiration included those involved in cofactor biosynthesis and assembly (moaACE, ccmHF, nosD, cysG), substrate transport (cysUP, cysTWA, dcuB), and anaerobic energy metabolism (dmsAB, psrC, pshA, hyaABC, hydA). Transcription of genes encoding a periplasmic nitrate reductase (napBHGA), cytochrome c552, and prismane was elevated 8- to 56-fold in response to the presence of nitrate, while cymA, ifcA, and frdA were specifically induced three- to eightfold under fumarate-reducing conditions. The mRNA levels for two oxidoreductase-like genes of unknown function and several cell envelope genes involved in multidrug resistance increased two- to fivefold specifically under Fe(III)-reducing conditions. Analysis of protein expression profiles under aerobic and anaerobic conditions revealed 14 protein spots that showed significant differences in abundance on 2-D gels. Protein identification by mass spectrometry indicated that the expression of prismane, dihydrolipoamide succinyltransferase, and alcaligin siderophore biosynthesis protein correlated with the microarray data. PMID:11881834

  7. Focusing on casein gene cluster and protein profile in Garganica goat milk.

    PubMed

    Albenzio, Marzia; Santillo, Antonella; d'Angelo, Francesca; Sevi, Agostino

    2009-02-01

    A survey was carried out in eight goat dairy farms, a total of 71 individual Garganica goat milk samples were collected for genomic DNA extraction. Casein alleles and haplotype frequencies of Garganica population were estimated. Individual milks were also analysed for chemical composition, rheological properties, and protein profile. The strong A* allele of CSN1S1 was predominant in the population investigated, the weak allele F of CSN1S1 showed a relatively high frequency and the null alleles N and 01 were first observed in this breed. At CSN1S2 locus the strong A* allele was the most frequent, followed by the F allele and the null allele. The strong A* allele was predominant at CSN2 locus, and relatively high incidence of null allele 0 was observed. CSN3 locus was monomorphic for B* allele. The exact test of sample differentiation based on haplotype frequencies discriminate the farms into two groups characterized by the highest frequency of strong (S-CSN1S1) or weak (W-CSN1S1) alleles at CSN1S1. Protein and casein contents were higher in the group characterized by strong allele than in the group with weak allele at CSN1S1. The 2D electrophoresis technique was performed to screen goat casein variability at the protein level and to evaluate global casein genotype (alphas1, alphas2, beta and kappa-CN). Gels displayed the protein profile associated with casein genotype, and demonstrated differences in the protein expression deriving from interactions between loci. The variability of goat casein loci in Garganica goat breed could be exploited to differentiate the population on the basis of milk utilization and could represent a strategy to preserve the genotype of this autochthonous breed.

  8. Sensory and protein profiles of Mexican Chihuahua cheese.

    PubMed

    Paul, Moushumi; Nuñez, Alberto; Van Hekken, Diane L; Renye, John A

    2014-11-01

    Native microflora in raw milk cheeses, including the Mexican variety Queso Chihuahua, contribute to flavor development through degradation of milk proteins. The effects of proteolysis were studied in four different brands of Mexican Queso Chihuahua made from raw milk. All of the cheeses were analyzed for chemical and sensory characteristics. Sensory testing revealed that the fresh cheeses elicited flavors of young, basic cheeses, with slight bitter notes. Analysis by gel electrophoresis and reverse phase-high performance liquid chromatography (RP-HPLC) revealed that the Queseria Blumen (X) and Queseria Super Fino (Z) cheeses show little protein degradation over time while the Queseria America (W) and Queseria Lago Grande (Y) samples are degraded extensively when aged at 4 °C for 8 weeks. Analysis of the mixture of water-soluble cheese proteins by mass spectrometry revealed the presence of short, hydrophobic peptides in quantities correlating with bitterness. All cheese samples contained enterococcal strains known to produce enterocins. The W and Y cheese samples had the highest number of bacteria and exhibited greater protein degradation than that observed for the X and Z cheeses. PMID:26396342

  9. [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

  10. Human tissue profiling with multidimensional protein identification technology.

    PubMed

    Cagney, Gerard; Park, Stephen; Chung, Clement; Tong, Bianca; O'Dushlaine, Colm; Shields, Denis C; Emili, Andrew

    2005-01-01

    Profiling of tissues and cell types through systematic characterization of expressed genes or proteins shows promise as a basic research tool, and has potential applications in disease diagnosis and classification. We used multidimensional protein identification protein identification technology (MudPIT) to analyze proteomes for enriched nuclear extracts of eight human tissues: brain, heart, liver, lung, muscle, pancreas, spleen, and testis. We show that the method is approximately 80% reproducible. We address issues of relative abundance, tissue-specificity, and selectivity, and the significance of proteins whose expression does not correlate with that of the corresponding mRNA. Surprisingly, most proteins are detected in a single tissue. These proteins tend to fulfill specialist (and potentially tissue-specific) functions compared to proteins expressed in two or more tissues.

  11. Objective Diagnosis of Cervical Cancer by Tissue Protein Profile Analysis

    NASA Astrophysics Data System (ADS)

    Patil, Ajeetkumar; Bhat, Sujatha; Rai, Lavanya; Kartha, V. B.; Chidangil, Santhosh

    2011-07-01

    Protein profiles of homogenized normal cervical tissue samples from hysterectomy subjects and cancerous cervical tissues from biopsy samples collected from patients with different stages of cervical cancer were recorded using High Performance Liquid Chromatography coupled with Laser Induced Fluorescence (HPLC-LIF). The Protein profiles were subjected to Principle Component Analysis to derive statistically significant parameters. Diagnosis of sample types were carried out by matching three parameters—scores of factors, squared residuals, and Mahalanobis Distance. ROC and Youden's Index curves for calibration standards were used for objective estimation of the optimum threshold for decision making and performance.

  12. Profiling proteins in nutraceutical formulations: characterization of the constituents.

    PubMed

    Bellomaria, Alessia; Nepravishta, Ridvan; Marchetti, Mario; Paci, Maurizio

    2016-03-01

    Several nutraceutical preparations containing proteins, amino acids and other small molecules are nowadays present on the market. In this work we propose NMR spectroscopy such as (1)H NMR, (1)H-(1)H TOCSY and DOSY for their constituents characterization, identification and profiling, comparing these results with those obtained by electrophoretic technique such as SDS-PAGE. The (1)H NMR spectroscopy was applied for measurements of the amino acids and other small compounds added from the manufacturer. Further the autocorrelation function obtained from the one dimensional spectrum was used without the complete assignment of the resonances of the NMR spectrum of proteins for the evaluation of the folding quality and stability. Finally the DOSY NMR technique was performed on the samples for the characterization of the mean molecular weight range of proteins. All this features considered together create an important set of data useful for the evaluation of the protein profiling and the characterization of such formulations.

  13. DSP: a protein shape string and its profile prediction server.

    PubMed

    Sun, Jiangming; Tang, Shengnan; Xiong, Wenwei; Cong, Peisheng; Li, Tonghua

    2012-07-01

    Many studies have demonstrated that shape string is an extremely important structure representation, since it is more complete than the classical secondary structure. The shape string provides detailed information also in the regions denoted random coil. But few services are provided for systematic analysis of protein shape string. To fill this gap, we have developed an accurate shape string predictor based on two innovative technologies: a knowledge-driven sequence alignment and a sequence shape string profile method. The performance on blind test data demonstrates that the proposed method can be used for accurate prediction of protein shape string. The DSP server provides both predicted shape string and sequence shape string profile for each query sequence. Using this information, the users can compare protein structure or display protein evolution in shape string space. The DSP server is available at both http://cheminfo.tongji.edu.cn/dsp/ and its main mirror http://chemcenter.tongji.edu.cn/dsp/.

  14. Proteomic Analysis of the Protein Expression Profile in the Mature Nigella sativa (Black Seed).

    PubMed

    Alanazi, Ibrahim O; Benabdelkamel, Hicham; Alfadda, Assim A; AlYahya, Sami A; Alghamdi, Waleed M; Aljohi, Hasan A; Almalik, Abdulaziz; Masood, Afshan

    2016-08-01

    Nigella sativa (N. sativa) seed has been used as an important nutritional flavoring agent and in traditional medicine for treating many illnesses since ancient times. Understanding the proteomic component of the seed may lead to enhance the understanding of its structural and biological functional complexity. In this study, we have analyzed its proteome profile based on gel-based proteome mapping technique that includes one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry strategy. We have not come across any such studies that have been performed in N. sativa seeds up to date. A total of 277 proteins were identified, and their functional, metabolic, and location-wise annotations were carried out using the UniProt database. The majority of proteins identified in the proteome dataset based on their function were those involved in enzyme catalytic activity, nucleotide binding, and protein binding while the major cellular processes included regulation of biological process followed by regulation of secondary biological process, cell organization and biogenesis, protein metabolism, and transport. The identified proteome was localized mainly to the nucleus then to the cytoplasm, plasma membrane, mitochondria, plastid, and others. A majority of the proteins were involved in biochemical pathways involving carbohydrate metabolism, amino acid and shikimate pathway, lipid metabolism, nucleotide, cell organization and biogenesis, transport, and defense processes. The identified proteins in the dataset help to improve our understanding of the pathways involved in N. sativa seed metabolism and its biochemical features and detail out useful information that may help to utilize these proteins. This study could thus pave a way for future further high-throughput studies using a more targeted proteomic approach. PMID:27020565

  15. Seminal plasma protein profiles of ejaculates obtained by internal artificial vagina and electroejaculation in Brahman bulls.

    PubMed

    Rego, J P A; Moura, A A; Nouwens, A S; McGowan, M R; Boe-Hansen, G B

    2015-09-01

    The present study was conducted to investigate if differences exist in the seminal plasma protein profile from mature Brahman bulls using two methods of semen collection: internal artificial vagina (IAV) and electroejaculation (EEJ). Semen was collected four times from three bulls on the same day and parameters were assessed immediately post-collection. Seminal plasma proteins were evaluated by 2-D fluorescence difference gel electrophoresis and identified by mass spectrometry. Semen volume was greater (P < 0.05) for EEJ (4.6 ± 0.35 mL) than for IAV (1.86 ± 0.24 mL) but sperm concentration was greater in IAV (1505 ± 189 × 10(6) sperm/mL) than in EEJ samples (344 ± 87 × 10(6) sperm/mL). Sperm motility and the percentage of normal sperm were not different between treatments. Total concentration of seminal plasma proteins was greater for samples collected by IAV as compared to EEJ (19.3 ± 0.9 compared with 13.0 ± 1.8 mg/mL, P < 0.05; respectively). Based on 2-D gels, 22 spots had a greater volume (P < 0.05) in gels derived from IAV samples, corresponding to 21 proteins identified as transferrin, albumin, epididymal secretory glutathione peroxidase, among others. Thirty-three spots, corresponding to 26 proteins, had a greater volume (P < 0.05) in gels derived from EEJ samples. These proteins were identified as spermadhesin-1, Bovine Sperm Protin 1, 3 and 5 isoforms, angiogenin-1, alpha-1B-glycoprotein, clusterin, nucleobindin-1, cathepsins, spermadhesin Z13, annexins, among others. Thus, proteins in greater amounts in samples obtained by IAV and EEJ were mainly of epididymal origin and accessory sex glands, respectively. PMID:26282524

  16. Differentiation and numerical analysis of oral yeasts based on SDS-Page profiles. Influence of the culture media on the whole-cell protein extracts.

    PubMed

    Höfling, J F; Rosa, E A; Pereira, C V; Boriollo, M F; Rodrigues, J A

    2001-08-01

    The application of gel electrophoresis and numerical analysis of yeast soluble proteins analysis to the investigation of 12 oral yeast strains belonging to five species is described. It involves one-dimensional electrophoresis of SDS-solubilized whole-cell proteins using different culture media for the cultivation of the cells, integration densitometries in the areas of the gels and percentages of the proteins extraction. These extracts were prepared from four isolates of Candida albicans, two of C. tropicalis, C. guilliermondii, C. parapsilosis and C. krusei. The extracts from whole-cells proteins using different culture media for the cultivation of the cells were fractionated by slab electrophoresis using a discontinuous buffer system. The corresponding patterns showed at least 36 polypeptides in the range of 14.4-200 kDa. Different isolates of each species were clearly different in each of the five species. The data obtained suggest that different nutritional compositions led to the expression of different proteins derived from alternatives metabolic pathways expressed by the electrophoretic profiles. The construction of a database of protein fingerprints and numerical analysis based on such data, may have some implications in the classification and identification of such species with epidemiological, ecological and taxonomic purposes. A well defined or synthetic culture media seems to be much properly.

  17. SDS-PAGE and IR spectroscopy to evaluate modifications in the viral protein profile induced by a cationic porphyrinic photosensitizer.

    PubMed

    Costa, Liliana; Esteves, Ana Cristina; Correia, António; Moreirinha, Catarina; Delgadillo, Ivonne; Cunha, Ângela; Neves, Maria G P S; Faustino, Maria A F; Almeida, Adelaide

    2014-12-01

    Reactive oxygen species can be responsible for microbial photodynamic inactivation due to its toxic effects, which include severe damage to proteins, lipids and nucleic acids. In this study, the photo-oxidative modifications of the proteins of a non-enveloped T4-like bacteriophage, induced by the cationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide were evaluated. Two methods were used: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. SDS-PAGE analysis showed that the phage protein profile was considerably altered after photodynamic treatment. Seven protein bands putatively corresponding to capsid and tail tube proteins were attenuated and two other were enhanced. Infrared spectroscopy confirmed the time-dependent alteration on the phage protein profile detected by SDS-PAGE, indicative of a response to oxidative damage. Infrared analysis showed to be a promising and rapid screening approach for the analysis of the modifications induced on viral proteins by photosensitization. In fact, one single infrared spectrum can highlight the changes induced to all viral molecular structures, overcoming the delays and complex protocols of the conventional methods, in a much simple and cost effective way. PMID:25241141

  18. Serum protein profile of Crohn's disease treated with infliximab.

    PubMed

    Gazouli, Maria; Anagnostopoulos, Athanasios K; Papadopoulou, Aggeliki; Vaiopoulou, Anna; Papamichael, Konstantinos; Mantzaris, Gerassimos; Theodoropoulos, George E; Anagnou, Nicholas P; Tsangaris, George Th

    2013-11-01

    The infliximab (IFX) has dramatically improved the treatment of Crohn's disease (CD). However, the need for predictive factors, indicative of patients' response to IFX, has yet to be met. In the current study, proteomics technologies were employed in order to monitor for differences in protein expression in a cohort of patients following IFX administration, aiming at identifying a panel of candidate protein biomarkers of CD, symptomatic of response to treatment. We enrolled 18 patients, who either had achieved clinical and serological remission (Rm, n=6), or response (Rs, n=6) and/or were PNRs (n=6), to IFX. Serum samples were subjected to two-dimensional Gel Electrophoresis. Following evaluation of densitometrical data, protein spots exhibiting differential expression among the groups, were further characterized by MALDI-TOF-MS. Identified proteins where evaluated by immunoblot analysis while functional network association was carried out to asses significance. Proteins apolipoprotein A-I (APOA1), apolipoprotein E (APOE), complement C4-B (CO4B), plasminogen (PLMN), serotransferrin (TRFE), beta-2-glycoprotein 1 (APOH), and clusterin (CLUS) were found to be up-regulated in the PNR and Rs groups whereas their levels displayed no changes in the Rm group when compared to baseline samples. Additionally, leucine-rich alpha-2-glycoprotein (A2GL), vitamin D-binding protein (VTDB), alpha-1B-glycoprotein (A1BG) and complement C1r subcomponent (C1R) were significantly increased in the serum of the Rm group. Through the incorporation of proteomics technologies, novel serum marker-molecules demonstrating high sensitivity and specificity are introduced, hence offering an innovative approach regarding the evaluation of CD patients' response to IFX therapy.

  19. Classification of Spanish white wines using their electrophoretic profiles obtained by capillary zone electrophoresis with amperometric detection.

    PubMed

    Arribas, Alberto Sánchez; Martínez-Fernández, Marta; Moreno, Mónica; Bermejo, Esperanza; Zapardiel, Antonio; Chicharro, Manuel

    2014-06-01

    A method was developed for the simultaneous detection of eight polyphenols (t-resveratrol, (+)-catechin, quercetin and p-coumaric, caffeic, sinapic, ferulic, and gallic acids) by CZE with electrochemical detection. Separation of these polyphenols was achieved within 25 min using a 200 mM borate buffer (pH 9.4) containing 10% methanol as separation electrolyte. Amperometric detection of polyphenols was carried out with a glassy carbon electrode (GCE) modified with a multiwalled carbon nanotubes (CNT) layer obtained from a dispersion of CNT in polyethylenimine. The excellent electrochemical properties of this modified electrode allowed the detection and quantification of the selected polyphenols in white wines without any pretreatment step, showing remarkable signal stability despite the presence of potential fouling substances in wine. The electrophoretic profiles of white wines, obtained using this methodology, have proven to be useful for the classification of these wines by means of chemometric multivariate techniques. Principal component analysis and discriminant analysis allowed accurate classification of wine samples on the basis of their grape varietal (verdejo and airén) using the information contained in selected zones of the electropherogram. The utility of the proposed CZE methodology based on the electrochemical response of CNT-modified electrodes appears to be promising in the field of wine industry and it is expected to be successfully extended to classification of a wider range of wines made of other grape varietals.

  20. Experimental study of the impact of antimicrobial treatments on Campylobacter, Enterococcus and PCR-capillary electrophoresis single-strand conformation polymorphism profiles of the gut microbiota of chickens.

    PubMed

    Mourand, Gwenaëlle; Jouy, Eric; Bougeard, Stéphanie; Dheilly, Alexandra; Kérouanton, Annaëlle; Zeitouni, Salman; Kempf, Isabelle

    2014-11-01

    An experiment was conducted to compare the impact of antimicrobial treatments on the susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis, and on the diversity of broiler microbiota. Specific-pathogen-free chickens were first orally inoculated with strains of Campylobacter and Enterococcus faecium. Birds were then orally treated with recommended doses of oxytetracycline, sulfadimethoxine/trimethoprim, amoxicillin or enrofloxacin. Faecal samples were collected before, during and after antimicrobial treatment. The susceptibility of Campylobacter, Enterococcus faecium and Enterococcus faecalis strains isolated on supplemented or non-supplemented media was studied and PCR-capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) profiles of the gut microbiota were analysed. Enrofloxacin-resistant Campylobacter were selected in the enrofloxacin-treated group and showed the Thr86Ile mutation in the gyrA gene. Acquisition of the tetO gene in Campylobacter coli isolates was significantly more frequent in birds given oxytetracycline. No impact of amoxicillin treatment on the susceptibility of Campylobacter could be detected. Ampicillin- and sulfadimethoxine/trimethoprim-resistant Enterococcus faecium were selected in amoxicillin-treated broilers, but no selection of the inoculated vancomycin-resistant Enterococcus faecium could be detected, although it was also resistant to tetracycline and sulfadimethoxine/trimethoprim. PCR-CE-SSCP revealed significant variations in a few peaks in treated birds as compared with non-treated chickens. In conclusion, antimicrobial treatments perturbed chicken gut microbiota, and certain antimicrobial treatments selected or co-selected resistant strains of Campylobacter and Enterococcus.

  1. Anionic metabolite profiling by capillary electrophoresis-mass spectrometry using a noncovalent polymeric coating. Orange juice and wine as case studies.

    PubMed

    Acunha, Tanize; Simó, Carolina; Ibáñez, Clara; Gallardo, Alberto; Cifuentes, Alejandro

    2016-01-01

    In several metabolomic studies, it has already been demonstrated that capillary electrophoresis hyphenated to mass spectrometry (CE-MS) can detect an important group of highly polar and ionized metabolites that are overseen by techniques such as NMR, LC-MS and GC-MS, providing complementary information. In this work, we present a strategy for anionic metabolite profiling by CE-MS using a cationic capillary coating. The polymer, abbreviated as PTH, is composed of a poly-(N,N,N',N'-tetraethyldiethylenetriamine, N-(2-hydroxypropyl) methacrylamide, TEDETAMA-co-HPMA (50:50) copolymer. A CE-MS method based on PTH-coating was optimized for the analysis of a group of 16 standard anionic metabolites. Separation was achieved within 12min, with high separation efficiency (up to 92,000 theoretical plates per meter), and good repeatability, namely, relative standard deviation values for migration times and peak areas were below 0.2 and 2.1%, respectively. The optimized method allowed the detection of 87 metabolites in orange juice and 142 metabolites in red wine, demonstrating the good possibilities of this strategy for metabolomic applications.

  2. Anionic metabolite profiling by capillary electrophoresis-mass spectrometry using a noncovalent polymeric coating. Orange juice and wine as case studies.

    PubMed

    Acunha, Tanize; Simó, Carolina; Ibáñez, Clara; Gallardo, Alberto; Cifuentes, Alejandro

    2016-01-01

    In several metabolomic studies, it has already been demonstrated that capillary electrophoresis hyphenated to mass spectrometry (CE-MS) can detect an important group of highly polar and ionized metabolites that are overseen by techniques such as NMR, LC-MS and GC-MS, providing complementary information. In this work, we present a strategy for anionic metabolite profiling by CE-MS using a cationic capillary coating. The polymer, abbreviated as PTH, is composed of a poly-(N,N,N',N'-tetraethyldiethylenetriamine, N-(2-hydroxypropyl) methacrylamide, TEDETAMA-co-HPMA (50:50) copolymer. A CE-MS method based on PTH-coating was optimized for the analysis of a group of 16 standard anionic metabolites. Separation was achieved within 12min, with high separation efficiency (up to 92,000 theoretical plates per meter), and good repeatability, namely, relative standard deviation values for migration times and peak areas were below 0.2 and 2.1%, respectively. The optimized method allowed the detection of 87 metabolites in orange juice and 142 metabolites in red wine, demonstrating the good possibilities of this strategy for metabolomic applications. PMID:26296988

  3. 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

  4. 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

  5. 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

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

    PubMed

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

    2016-05-13

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

  7. Protein and Amino Acid Profiles of Different Whey Protein Supplements.

    PubMed

    Almeida, Cristine C; Alvares, Thiago S; Costa, Marion P; Conte-Junior, Carlos A

    2016-01-01

    Whey protein (WP) supplements have received increasing attention by consumers due to the high nutritional value of the proteins and amino acids they provide. However, some WP supplements may not contain the disclosed amounts of the ingredients listed on the label, compromising the nutritional quality and the effectiveness of these supplements. The aim of this study was to evaluate and compare the contents of total protein (TP), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), free essential amino acids (free EAA), and free branched-chain amino acids (free BCAA), amongst different WP supplements produced by U.S. and Brazilian companies. Twenty commercial brands of WP supplements were selected, ten manufactured in U.S. (WP-USA) and ten in Brazil (WP-BRA). The TP was analyzed using the Kjeldahl method, while α-LA, β-LG, free EAA, and free BCAA were analyzed using HPLC system. There were higher (p < 0.05) concentrations of TP, α-LA, β-LG, and free BCAA in WP-USA supplements, as compared to the WP-BRA supplements; however, there was no difference (p > 0.05) in the content of free EAA between WP-USA and WP-BRA. Amongst the 20 brands evaluated, four WP-USA and seven WP-BRA had lower (p < 0.05) values of TP than those specified on the label. In conclusion, the WP-USA supplements exhibited better nutritional quality, evaluated by TP, α-LA, β-LG, and free BCAA when compared to WP-BRA.

  8. Proteomic analysis of cellular protein expression profiles in response to grass carp reovirus infection.

    PubMed

    Xu, Dan; Song, Lang; Wang, Hao; Xu, Xiaoyan; Wang, Tu; Lu, Liqun

    2015-06-01

    Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. To better understand the molecular responses to GCRV infection, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization tandem mass spectroscopy were performed to investigate altered proteins in C. idella kidney (CIK) cells. Differentially expressed proteins in mock infected CIK cells and GCRV-infected CIK cells were compared. Twenty-three differentially expressed spots were identified (22 upregulated spots and 1 downregulated spot), which included cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism-associated proteins and ubiquitin proteasome pathway-associated proteins. Moreover, 10 of the corresponding genes of the differentially expressed proteins were quantified by real-time reverse transcription polymerase chain reaction to examine their transcriptional profiles. The T cell internal antigen 1 (TIA1) and Ras-GTPase-activating SH3-domain-binding protein1 (G3BP1) of the cellular stress granule pathway from grass carp C. idella (designated as CiTIA1 and CiG3BP1) were upregulated and downregulated during GCRV infection, respectively. The full-length cDNA of CiTIA1 was 2753 bp, with an open reading frame (ORF) of 1155bp, which encodes a putative 385-amino acid protein. The 2271 bp full-length cDNA of CiG3BP1 comprised an ORF of 1455 bp that encodes a putative 485-amino acid protein. Phylogenetic analysis revealed that the complete ORFs of CiTIA1 and CiG3BP1 were very similar to zebrafish and well-characterized mammalian homologs. The expressions of the cellular proteins CiTIA1 and CiG3BP1 in response to GCRV were validated by western blotting, which indicated that the GCRV should unlink TIA1 aggregation and stress granule formation. This study provides useful information on the proteomic

  9. Biomedical applications of capillary electrophoresis

    NASA Astrophysics Data System (ADS)

    Kartsova, L. A.; Bessonova, E. A.

    2015-08-01

    The review deals with modern analytical approaches used in capillary electrophoresis for solving medical and biological problems: search for biomarkers of various diseases and rapid diagnosis based on characteristic profiles of biologically active compounds by capillary electrophoresis with mass spectrometric detection; monitoring of the residual drugs in biological fluids for evaluating the efficiency of drug therapy; testing of the enantiomeric purity of pharmaceutical products; the use of novel materials as components of stationary and pseudo-stationary phases in capillary electrophoresis and capillary electrochromatography to increase the selectivity of separation of components of complex matrices; and identification of various on-line preconcentration techniques to reduce the detection limits of biologically active analytes. A topical trend in capillary electrophoresis required in clinical practice, viz., the design of microfluidic systems, is discussed. The bibliography includes 173 references.

  10. Protein profiles and immunoreactivities of Acanthamoeba morphological groups and genotypes.

    PubMed

    Pumidonming, Wilawan; Koehsler, Martina; Leitsch, David; Walochnik, Julia

    2014-11-01

    Acanthamoeba is a free-living protozoan found in a wide variety of habitats. A classification of Acanthamoeba into currently eighteen genotypes (T1-T18) has been established, however, data on differences between genotypes on the protein level are scarce. The aim of this study was to compare protein and immunoreactivity profiles of Acanthamoeba genotypes. Thirteen strains, both clinical and non-clinical, from genotypes T4, T5, T6, T7, T9, T11 and T12, representing three morphological groups, were investigated for their protein profiles and IgG, IgM and IgA immunoreactivities. It was shown that protein and immunoreactivity profiles of Acanthamoeba genotypes T4, T5, T6, T7, T9, T11 and T12 are clearly distinct from each other, but the banding patterns correlate to the morphological groups. Normal human sera revealed anti-Acanthamoeba antibodies against isolates of all investigated genotypes, interestingly, however only very weak IgM and virtually no IgA immunoreactivity with T7 and T9, both representing morphological group I. The strongest IgG, IgM and IgA immunoreactivities were observed for genotypes T4, T5 and T6. Differences of both, protein and immunological patterns, between cytopathic and non-cytopathic strains, particularly within genotype T4, were not at the level of banding patterns, but rather in expression levels.

  11. 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.

  12. 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

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

    PubMed

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

    2005-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1998-02-01

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

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

    PubMed

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

    2011-04-01

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

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

    PubMed

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

    2009-12-15

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

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

    PubMed

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

    2004-03-01

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

  18. O-GlcNAc profiling: from proteins to proteomes

    PubMed Central

    2014-01-01

    O-linked β-D-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) onto serine and threonine residues of proteins is an important post-translational modification (PTM), which is involved in many crucial biological processes including transcription, translation, proteasomal degradation, and signal transduction. Aberrant protein O-GlcNAcylation is directly linked to the pathological progression of chronic diseases including diabetes, cancer, and neurodegenerative disorders. Identification, site mapping, and quantification of O-GlcNAc proteins are a prerequisite to decipher their functions. In this review, we mainly focus on technological developments regarding O-GlcNAc protein profiling. Specifically, on one hand, we show how these techniques are being used for the comprehensive characterization of certain targeted proteins in which biologists are most interested. On the other hand, we present several newly developed approaches for O-GlcNAcomic profiling as well as how they provide us with a systems perspective to crosstalk amongst different PTMs and complicated biological events. Promising technical trends are also highlighted to evoke more efforts by diverse laboratories, which would further expand our understanding of the physiological and pathological roles of protein O-GlcNAcylation in chronic diseases. PMID:24593906

  19. PLIP: fully automated protein-ligand interaction profiler.

    PubMed

    Salentin, Sebastian; Schreiber, Sven; Haupt, V Joachim; Adasme, Melissa F; Schroeder, Michael

    2015-07-01

    The characterization of interactions in protein-ligand complexes is essential for research in structural bioinformatics, drug discovery and biology. However, comprehensive tools are not freely available to the research community. Here, we present the protein-ligand interaction profiler (PLIP), a novel web service for fully automated detection and visualization of relevant non-covalent protein-ligand contacts in 3D structures, freely available at projects.biotec.tu-dresden.de/plip-web. The input is either a Protein Data Bank structure, a protein or ligand name, or a custom protein-ligand complex (e.g. from docking). In contrast to other tools, the rule-based PLIP algorithm does not require any structure preparation. It returns a list of detected interactions on single atom level, covering seven interaction types (hydrogen bonds, hydrophobic contacts, pi-stacking, pi-cation interactions, salt bridges, water bridges and halogen bonds). PLIP stands out by offering publication-ready images, PyMOL session files to generate custom images and parsable result files to facilitate successive data processing. The full python source code is available for download on the website. PLIP's command-line mode allows for high-throughput interaction profiling.

  20. PLIP: fully automated protein-ligand interaction profiler.

    PubMed

    Salentin, Sebastian; Schreiber, Sven; Haupt, V Joachim; Adasme, Melissa F; Schroeder, Michael

    2015-07-01

    The characterization of interactions in protein-ligand complexes is essential for research in structural bioinformatics, drug discovery and biology. However, comprehensive tools are not freely available to the research community. Here, we present the protein-ligand interaction profiler (PLIP), a novel web service for fully automated detection and visualization of relevant non-covalent protein-ligand contacts in 3D structures, freely available at projects.biotec.tu-dresden.de/plip-web. The input is either a Protein Data Bank structure, a protein or ligand name, or a custom protein-ligand complex (e.g. from docking). In contrast to other tools, the rule-based PLIP algorithm does not require any structure preparation. It returns a list of detected interactions on single atom level, covering seven interaction types (hydrogen bonds, hydrophobic contacts, pi-stacking, pi-cation interactions, salt bridges, water bridges and halogen bonds). PLIP stands out by offering publication-ready images, PyMOL session files to generate custom images and parsable result files to facilitate successive data processing. The full python source code is available for download on the website. PLIP's command-line mode allows for high-throughput interaction profiling. PMID:25873628

  1. Self-organized criticality in proteins: Hydropathic roughening profiles of G-protein-coupled receptors

    NASA Astrophysics Data System (ADS)

    Phillips, J. C.

    2013-03-01

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein conformational functionality is often dominated by long-range hydrophobic or hydrophilic interactions which both drive protein compaction and mediate protein-protein interactions. Superfamily transmembrane G-protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome; their amino acid sequences form the largest database for protein-membrane interactions. While there are now structural data on the heptad transmembrane structures of representatives of several heptad families, here we show how fresh insights into global and some local chemical trends in GPCR properties can be obtained accurately from sequences alone, especially by algebraically separating the extracellular and cytoplasmic loops from transmembrane segments. The global mediation of long-range water-protein interactions occurs in conjunction with modulation of these interactions by roughened interfaces. Hydropathic roughening profiles are defined here solely in terms of amino acid sequences, and knowledge of protein coordinates is not required. Roughening profiles both for GPCR and some simpler protein families display accurate and transparent connections to protein functionality, and identify natural length scales for protein functionality.

  2. Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins

    PubMed Central

    2010-01-01

    Background Transformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest. Methods We compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots. Results Correlation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics. Conclusions Our results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines. PMID:20731849

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

    PubMed Central

    Stockton, Steven D; Devi, Lakshmi A

    2014-01-01

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

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

    PubMed

    Levinson, S S

    1997-06-27

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

  5. The use of a sulfonated capillary on chiral capillary electrophoresis/mass spectrometry of amphetamine-type stimulants for methamphetamine impurity profiling.

    PubMed

    Mikuma, Toshiyasu; Iwata, Yuko T; Miyaguchi, Hajime; Kuwayama, Kenji; Tsujikawa, Kenji; Kanamori, Tatsuyuki; Inoue, Hiroyuki

    2015-04-01

    Chiral capillary electrophoresis/tandem mass spectrometry (CE/MS/MS) using a chemically modified capillary containing sulfonated groups was developed for the following 8 amphetamine-type stimulants (ATS): amphetamine, methamphetamine (MA), norephedrine, norpseudoephedrine, ephedrine (EP), pseudoephedrine (pEP), dimethylamphetamine and methylephedrine. The running buffer was 10 mM formic acid containing 20 mM highly sulfated γ-cyclodextrin (pH 2.5) as the chiral selector. All 16 enantiomers were well resolved within 60 min, and precisely identified due to their characteristic mass spectra. Further, the RSDs of the migration times of the analytes were no more than 0.3% without any standardization. (1R,2S)-(-)-EP and (1S,2S)-(+)-pEP, which are important ATS impurities originating in the precursors, were added to a highly concentrated MA solution (1 mg/mL) and analyzed as mock samples for MA impurity analysis. Acceptable repeatability of the migration times of (-)-EP and (+)-pEP (ca. 0.3% RSDs) was still observed without interference from the large amount of MA. The limits of detection (LOD) of (-)-EP and (+)-pEP were approximately 2 μg/mL, therefore, their LOD as the impurity concentrations were calculated at about 0.2%. Seized MA samples were dissolved in water at a high concentration (1 mg/mL) and analyzed by this method. (-)-EP and (+)-pEP were clearly detected as impurities. Although these compounds had similar migration times and mass spectral patterns, the fine repeatability allowed easy identification of the impurities by a simple comparison of the absolute migration times of the specimens and those of authentic standards. This study is the first to report the use of a chemically modified capillary for the impurity profiling on CE/MS/MS.

  6. [SHIFTS IN URINE PROTEIN PROFILE DURING DRY IMMERSION].

    PubMed

    Pastushkova L Kh; Kononikhin, A S; Tiys, E S; Nosovsky, A M; Dobrokhotov, I V; Ivanisenko, V A; Nikolaev, E N; Novoselova, N M; Custaud, M A; Larina, I M

    2015-01-01

    The study was aimed at tracking the proteomic profile of urine in 8 normal volunteers to 5-day dry immersion (DI). The proteome composition was determined by chromatography-mass spectrometry on high-efficient on-line liquid nano chromatograph Agilent 1100; complementary information about the protein spectra was obtained by dint of mass-spectrometer MaXis Impact 4G and hybrid mass-spectrometer LTQ-FT. Functional associations between proteins and biological functions were analyzed using computer system ANDCell (Associative Networks Discovery in Cells). A total of 256 proteins were identified; for 43 proteins difference in the detection rate during the baseline data collection and on DI day 4 exceeded 20%. PMID:26554129

  7. 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.

  8. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-06-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity.

  9. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed Central

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-01-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity. Images PMID:6286971

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

    PubMed Central

    2013-01-01

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

  11. Interaction of silver nanoparticles with proteins: a characteristic protein concentration dependent profile of SPR signal.

    PubMed

    Banerjee, Victor; Das, K P

    2013-11-01

    Silver nanoparticles are finding increasing applications in biological systems, for example as antimicrobial agents and potential candidates for control drug release systems. In all such applications, silver nanoparticles interact with proteins and other biomolecules. Hence, the study of such interactions is of considerable importance. While BSA has been extensively used as a model protein for the study of interaction with the silver nanoparticles, studies using other proteins are rather limited. The interaction of silver nanoparticles with light leads to collective oscillation of the conducting electrons giving rise to surface plasmon resonance (SPR). Here, we have studied the protein concentration dependence of the SPR band profiles for a number of proteins. We found that for all the proteins, with increase in concentration, the SPR band intensity initially decreased, reaching minima and then increased again leading to a characteristic "dip and rise" pattern. Minimum point of the pattern appeared to be related to the isoelectric point of the proteins. Detailed dynamic light scattering and transmission electron microscopy studies revealed that the consistency of SPR profile was dependent on the average particle size and state of association of the silver nanoparticles with the change in the protein concentration. Fluorescence spectroscopic studies showed the binding constants of the proteins with the silver nanoparticles were in the nano molar range with more than one nanoparticle binding to protein molecule. Structural studies demonstrate that protein retains its native-like structure on the nanoparticle surface unless the molar ratio of silver nanoparticles to protein exceeds 10. Our study reveals that nature of the protein concentration dependent profile of SPR signal is a general phenomena and mostly independent of the size and structure of the proteins.

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

    PubMed

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

    2000-04-01

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

  13. Capillary Electrophoresis in Metabolomics.

    PubMed

    Maier, Tanja Verena; Schmitt-Kopplin, Philippe

    2016-01-01

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

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

    PubMed

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

    1997-02-15

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

  15. 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.

  16. A comparative protein profile of mammalian erythrocyte membranes identified by mass spectrometry.

    PubMed

    Sharma, Savita; Punjabi, Vinny; Zingde, Surekha M; Gokhale, Sadashiv M

    2014-11-01

    A comparative analysis of erythrocyte membrane proteins of economically important animals, goat (Capra aegagrus hircus), buffalo (Bubalus bubalis), pig (Sus scrofa), cow (Bos tauras), and human (Homo sapiens) was performed. Solubilized erythrocyte membrane proteins were separated by sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE), visualized by staining the gels with Commassie Brilliant Blue (CBB), and identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Emerging results show that all major erythrocyte membrane proteins present in human are also seen in all the animals except for band 4.5 which could not be identified. Band 3 is seen as more intense and compact, band 4.1 appears as a doublet in all the animal erythrocyte membranes, band 4.2 exhibits a slightly higher molecular weight (Mr) in buffalo, and cow and band 4.9 has a higher Mr in all the animals relative to the human protein. In addition, there are two new bands in the goat membrane, band G1, identified as HSP 90α, and band G2 identified as HSP 70. A new band C2 identified as HSP 70 is also seen in cow membranes. Peroxiredoxin II is of lower intensity and/or higher Mr in the animals. The difference in size of the proteins possibly indicates the variations in the composition of the amino acids. The difference in intensity of the proteins among these mammalians highlights the presence of less or more number of copies of that protein per cell. This data complement the earlier observations of differences in the sialoglycoprotein profile and effect of proteases and neuraminidase on agglutination among the mammalian erythrocytes. This study provides a platform to understand the molecular architecture of the individual erythrocytes, and in turn the dependent disorders, their phylogenetic relationship and also generates a database of erythrocyte membrane proteins of mammals. The animals selected for this study are of economic importance as

  17. 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.

  18. 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

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

    PubMed

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

    1999-03-01

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

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

    PubMed

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

    2003-12-01

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

  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. Detecting protein complexes from active protein interaction networks constructed with dynamic gene expression profiles

    PubMed Central

    2013-01-01

    Background Protein interaction networks (PINs) are known to be useful to detect protein complexes. However, most available PINs are static, which cannot reflect the dynamic changes in real networks. At present, some researchers have tried to construct dynamic networks by incorporating time-course (dynamic) gene expression data with PINs. However, the inevitable background noise exists in the gene expression array, which could degrade the quality of dynamic networkds. Therefore, it is needed to filter out contaminated gene expression data before further data integration and analysis. Results Firstly, we adopt a dynamic model-based method to filter noisy data from dynamic expression profiles. Then a new method is proposed for identifying active proteins from dynamic gene expression profiles. An active protein at a time point is defined as the protein the expression level of whose corresponding gene at that time point is higher than a threshold determined by a standard variance involved threshold function. Furthermore, a noise-filtered active protein interaction network (NF-APIN) is constructed. To demonstrate the efficiency of our method, we detect protein complexes from the NF-APIN, compared with those from other dynamic PINs. Conclusion A dynamic model based method can effectively filter out noises in dynamic gene expression data. Our method to compute a threshold for determining the active time points of noise-filtered genes can make the dynamic construction more accuracy and provide a high quality framework for network analysis, such as protein complex prediction. PMID:24565281

  3. 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…

  4. 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.

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

    PubMed

    Kaneta, Takashi

    2016-01-01

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

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

    PubMed

    Chang, Chung-Wei; Tseng, Wei-Lung

    2010-04-01

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

  7. Anti-Viral Antibody Profiling by High Density Protein Arrays

    PubMed Central

    Bian, Xiaofang; Wiktor, Peter; Kahn, Peter; Brunner, Al; Khela, Amritpal; Karthikeyan, Kailash; Barker, Kristi; Yu, Xiaobo; Magee, Mitch; Wasserfall, Clive H.; Gibson, David; Rooney, Madeleine E; Qiu, Ji; LaBaer, Joshua

    2015-01-01

    Viral infections elicit anti-viral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection and understanding of the mechanisms of virus associated diseases. In this work, we assayed anti-viral antibodies using a novel high density-nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter- array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal to background (S/B) ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis (JIA) and type 1 diabetes (T1D). Common as well as unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development. PMID:25758251

  8. Protein content and amino acids profile of pseudocereals.

    PubMed

    Mota, Carla; Santos, Mariana; Mauro, Raul; Samman, Norma; Matos, Ana Sofia; Torres, Duarte; Castanheira, Isabel

    2016-02-15

    Quinoa (Chenopodium quinoa), amaranth (Amaranthus caudatus) and buckwheat (Fagopyrum esculentum) represent the main protein source in several diets, although these pseudocereals are not currently present in the FCDB nutrient profile information. The aim of this work is to characterise the AA profile of these pseudocereals and compare them with rice. Total protein content revealed to vary from 16.3g/100g (quinoa Salta) to 13.1g/100g (buckwheat) and lower values were found in rice samples (6.7g/100g). For pseudocereals the most abundant essential AA was leucine. Quinoa-Salta evidences the highest leucine content (1013mg/100g) and the minor methionine content (199mg/100g). Buckwheat was the cereal with the highest phenylalanine content (862mg/100g). Rice (Oryza sativa) presents the lowest content for all AA. Results showed pseudocereals as the best source of AA. EuroFIR guidelines where strictly followed and proved to be a crucial tool to guarantee data interchangeability and comparability. PMID:26433287

  9. ProtPhylo: identification of protein-phenotype and protein-protein functional associations via phylogenetic profiling.

    PubMed

    Cheng, Yiming; Perocchi, Fabiana

    2015-07-01

    ProtPhylo is a web-based tool to identify proteins that are functionally linked to either a phenotype or a protein of interest based on co-evolution. ProtPhylo infers functional associations by comparing protein phylogenetic profiles (co-occurrence patterns of orthology relationships) for more than 9.7 million non-redundant protein sequences from all three domains of life. Users can query any of 2048 fully sequenced organisms, including 1678 bacteria, 255 eukaryotes and 115 archaea. In addition, they can tailor ProtPhylo to a particular kind of biological question by choosing among four main orthology inference methods based either on pair-wise sequence comparisons (One-way Best Hits and Best Reciprocal Hits) or clustering of orthologous proteins across multiple species (OrthoMCL and eggNOG). Next, ProtPhylo ranks phylogenetic neighbors of query proteins or phenotypic properties using the Hamming distance as a measure of similarity between pairs of phylogenetic profiles. Candidate hits can be easily and flexibly prioritized by complementary clues on subcellular localization, known protein-protein interactions, membrane spanning regions and protein domains. The resulting protein list can be quickly exported into a csv text file for further analyses. ProtPhylo is freely available at http://www.protphylo.org.

  10. 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...

  11. 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

  12. Screening method of carbohydrate-binding proteins in biological sources by capillary affinity electrophoresis and its application to determination of Tulipa gesneriana agglutinin in tulip bulbs.

    PubMed

    Nakajima, Kazuki; Kinoshita, Mitsuhiro; Oda, Yasuo; Masuko, Takashi; Kaku, Hanae; Shibuya, Naoto; Kakehi, Kazuaki

    2004-09-01

    We developed capillary affinity electrophoresis (CAE) to analyze the molecular interaction between carbohydrate chains and proteins in solution state. A mixture of oligosaccharides derived from a glycoprotein was labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS), and used as glycan library without isolation. Interaction of a carbohydrate-binding protein with each oligosaccharide in the mixture could be simultaneously observed, and relative affinities of oligosaccharides toward the protein were accurately determined. In this study, we applied CAE to detect the presence of lectins in some plants (Japanese elderberry bark and tulip bulb). In the crude extract of the elderberry bark, binding activity toward sialo-carbohydrate chains could be easily detected. We also examined the presence of lectins in the crude extract of tulip bulbs and determined the detailed carbohydrate-binding specificity of Tulipa gesneriana agglutinin (TGA), one of the lectins from tulip bulbs. Kinetic studies demonstrated that TGA showed novel carbohydrate-binding specificity and preferentially recognized triantennary oligosaccharides with Gal residues at nonreducing termini and a Fuc residue linked through alpha(1-6) linkage at chitobiose portion of the reducing termini but not tetraantennary carbohydrates. The results described here indicate that CAE will be a valuable method for both screening of lectins in natural sources and determination of their detailed carbohydrate-binding specificities.

  13. Phaseolin seed variability in common bean (Phaseolus vulgaris L.) by capillary gel electrophoresis.

    PubMed

    Salmanowicz, B P

    2001-01-01

    Phaseolin, the major seed storage protein of Phaseolus vulgaris from forty-four wild and cultivated accessions, was studied using sodium dodecyl sulphate-capillary gel electrophoresis (SDS-CGE). In total, eleven phaseolin profiles, revealing polypeptide subunit variation in the range from 45.6 kDa to 54.4 kDa, were recorded. The number of polypeptide subunits recorded in particular profiles varied from 3 to 6; in total, eight phaseolin subunits were distinguished in the examined material. Ferguson plot analysis was used to correct non-ideal behaviour of phaseolin polypeptide subunits in capillary gel electrophoresis in the presence of SDS. The obtained results are compared to electrophoretic data received by slab polyacrylamide gel electrophoresis. The SDS-CGE method appears to provide a powerful tool for disclosure of phaseolin subunit variability.

  14. Effect of phosphorus levels on the protein profiles of secreted protein and root surface protein of rice.

    PubMed

    Shinano, Takuro; Yoshimura, Tomoko; Watanabe, Toshihiro; Unno, Yusuke; Osaki, Mitsuru; Nanjo, Yohei; Komatsu, Setsuko

    2013-11-01

    Plant roots are complicated organs that absorb water and nutrients from the soil. Roots also play an essential role in protecting plants from attack by soil pathogens and develop a beneficial role with some soil microorganisms. Plant-derived rhizosphere proteins (e.g., root secretory proteins and root surface binding proteins) are considered to play important roles in developing mutual relationships in the rhizosphere. In the rhizosphere, where plant roots meet the surrounding environment, it has been suggested that root secretory protein and root surface binding protein are important factors. Furthermore, it is not known how the physiological status of the plant affects the profile of these proteins. In this study, rice plants were grown aseptically, with or without phosphorus nutrition, and proteins were obtained from root bathing solution (designated as root secretory proteins) and obtained using 0.2 M CaCl2 solution (designated as root surface binding proteins). The total number of identified proteins in the root bathing solution was 458, and the number of root surface binding proteins was 256. More than half of the proteins were observed in both fractions. Most of the proteins were categorized as either having signal peptides or no membrane transport helix sites. The functional categorization suggested that most of the proteins seemed to have secretory pathways and were involved in defense/disease-related functions. These characteristics seem to be unique to rhizosphere proteins, and the latter might be part of the plants strategy to defeat pathogens in the soil. The low phosphorus treatment significantly increased the number of pathogenesis-related proteins in the root secretory proteins, whereas the change was small in the case of the root surface binding proteins. The results suggested that the roots are actively and selectively secreting protein into the rhizosphere. PMID:24083427

  15. 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.

  16. 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.

  17. An Economical Electrophoresis Apparatus

    ERIC Educational Resources Information Center

    Andrews, I. M.

    1975-01-01

    Describes the production of an electrophoresis apparatus from commonly discarded articles. Outlines paper and gel electrophoresis and its application to the separation of amino acids and intestinal enzymes. (GS)

  18. Zn-Responsive Proteome Profiling and Time-Dependent Expression of Proteins Regulated by MTF-1 in A549 Cells

    PubMed Central

    Zhao, Wen-jie; Song, Qun; Wang, Yan-hong; Li, Ke-jin; Mao, Li; Hu, Xin; Lian, Hong-zhen; Zheng, Wei-juan; Hua, Zi-chun

    2014-01-01

    Zinc plays a critical role in many biological processes. However, it is toxic at high concentrations and its homeostasis is strictly regulated by metal-responsive transcription factor 1 (MTF-1) together with many other proteins to protect cells against metal toxicity and oxidative stresses. In this paper, we used high-resolution two-dimensional gel electrophoresis (2DE) to profile global changes of the whole soluble proteome in human lung adenocarcinoma (A549) cells in response to exogenous zinc treatment for 24 h. Eighteen differentially expressed proteins were identified by MALDI TOF/TOF and MASCOT search. In addition, we used Western blotting and RT-PCR to examine the time-dependent changes in expression of proteins regulated by MTF-1 in response to Zn treatment, including the metal binding protein MT-1, the zinc efflux protein ZnT-1, and the zinc influx regulator ZIP-1. The results indicated that variations in their mRNA and protein levels were consistent with their functions in maintaining the homeostasis of zinc. However, the accumulation of ZIP-1 transcripts was down-regulated while the protein level was up-regulated during the same time period. This may be due to the complex regulatory mechanism of ZIP-1, which is involved in multiple signaling pathways. Maximal changes in protein abundance were observed at 10 h following Zn treatment, but only slight changes in protein or mRNA levels were observed at 24 h, which was the time-point frequently used for 2DE analyses. Therefore, further study of the time-dependent Zn-response of A549 cells would help to understand the dynamic nature of the cellular response to Zn stress. Our findings provide the basis for further study into zinc-regulated cellular signaling pathways. PMID:25162517

  19. 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.

  20. 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

  1. 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.

  2. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    PubMed Central

    Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

  3. Influence of the ionic strength of acidic background electrolytes on the separation of proteins by capillary electrophoresis.

    PubMed

    Bekri, Samya; Leclercq, Laurent; Cottet, Hervé

    2016-02-01

    The ionic strength is one of the key parameters for optimizing CE separations. However, only a few data are available in the literature about the ionic strength effect on the separation of proteins. The effect of ionic strength on separation performances is rather complex since many different parameters are involved: such as the protein effective mobility, the electroosmotic mobility, the separation efficiency via the electromigration dispersion, as well as the viscosity and temperature of the background electrolyte. In the present work, the influence of ionic strength on the electrophoretic separation of five model proteins has been investigated in acidic conditions, on successive multi-ionic layers coated capillary, in counter-electroosmotic mode with anodic electroosmotic flow. The decrease in effective and electroosmotic mobilities with increasing ionic strength were compared using the slope-plot approach, which is very helpful for understanding the observed changes in apparent selectivity and resolution. The relative decrease of the protein effective mobility was about 30-40% of the mobility determined at 5mM ionic strength per ionic strength decade. It was found that relatively low ionic strength (∼5-10mM) was preferable to optimize the overall separation of the five model proteins. PMID:26780847

  4. 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.

  5. 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.

  6. Two-dimensional Gel Electrophoresis (2DE)

    NASA Astrophysics Data System (ADS)

    Kłodzińska, Ewa; Buszewski, Bogusław

    The chemical compounds, which are present in the environment, increasingly cause bad effects on health. The most serious effects are tumors and various mutations at the cellular level. Such compounds, from the analytical point of view, can serve the function of biomarkers, constituting measurable changes in the organism's cells and biochemical processes occurring therein. The challenge of the twenty-first century is therefore searching for effective and reliable methods of identification of biomarkers as well as understanding bodily functions, which occur in living organisms at the molecular level. The irreplaceable tool for these examinations is proteomics, which includes both quality and quantity analysis of proteins composition, and also makes it possible to learn their functions and expressions. The success of proteomics examinations lies in the usage of innovative analytical techniques, such as electromigration technique, two-dimensional electrophoresis in polyacrylamide gel (2D PAGE), liquid chromatography, together with high resolution mass spectrometry and bio-informatical data analysis. Proteomics joins together a number of techniques used for analysis of hundreds or thousands of proteins. Its main task is not the examination of proteins inside the particular tissue but searching for the differences in the proteins' profile between bad and healthy tissues. These differences can tell us a lot regarding the cause of the sickness as well as its consequences. For instance, using the proteomics analysis it is possible to find relatively fast new biomarkers of tumor diseases, which in the future will be used for both screening and foreseeing the course of illness. In this chapter we focus on two-dimensional electrophoresis because as it seems, it may be of enormous importance when searching for biomarkers of cancer diseases.

  7. The effect of oxidation or alkylation on the separation of wool keratin proteins by two-dimensional gel electrophoresis.

    PubMed

    Plowman, Jeffrey E; Flanagan, Leanne M; Paton, Louise N; Fitzgerald, Anna C; Joyce, Nigel I; Bryson, Warren G

    2003-06-01

    Comparative two-dimensional electrophoretic (2-DE) studies were performed over a time-course to examine the effect of oxidation or alkylation on the separation of wool keratin proteins. The effect of oxidation was followed by treating scoured wool fibres with increasing levels of hydrogen peroxide, ranging from 0-12 g/L, using conditions mimicking the industrial wool bleaching process. Peroxide treatment was found to have only a minor effect on the 2-DE separation of the intermediate filament protein (IFP) class. Conversely, peroxide treatment of the 24-28 kDa high sulphur protein (HSP) class, which contains up to 40 cysteine residues per protein, resulted in the gradual disappearance of the major HSP spots correlated with the appearance of a few discrete spots at lower isoelectric point (pI). This suggested that only a few specific cysteine residues were being oxidized to cysteic acid by treatment with hydrogen peroxide. Peroxide treatment also appeared to have affected a discrete number of cysteine residues among proteins in the high glycine-tyrosine protein (HGTP) class, reducing the intensity of the high pI spots, while correspondingly increasing the intensity of those at lower pI. In a separate study, wool proteins were alkylated with iodoacetamide (1 M, pH 8) for periods ranging from 10 min to 48 h. In contrast to treatment with peroxide, the pI values of the HSP spots were unaffected by alkylation, irrespective of the length of this treatment. Alkylation resulted in a shift to lower pI and a loss of resolution of individual spots in the Type I and II IFP trains, to the extent that after 24 h alkylation individual spots in these trains merged. In addition after 1 h the intensity of the high pI Type II IFPs decreased until they were no longer visible on the 2-DE map after 24 h. Similarly as alkylation time increased, the major, high pI HGTP spots decreased in intensity. In unison with their decrease, some of the lower pI spots increased in intensity, while new

  8. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona.

    PubMed

    Hayashi, Yuya; Miclaus, Teodora; Engelmann, Péter; Autrup, Herman; Sutherland, Duncan S; Scott-Fordsmand, Janeck J

    2016-01-01

    Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells' protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.

  9. Supported Molecular Matrix Electrophoresis.

    PubMed

    Matsuno, Yu-Ki; Kameyama, Akihiko

    2015-01-01

    Mucins are difficult to separate using conventional gel electrophoresis methods such as SDS-PAGE and agarose gel electrophoresis, owing to their large size and heterogeneity. On the other hand, cellulose acetate membrane electrophoresis can separate these molecules, but is not compatible with glycan analysis. Here, we describe a novel membrane electrophoresis technique, termed "supported molecular matrix electrophoresis" (SMME), in which a porous polyvinylidene difluoride (PVDF) membrane filter is used to achieve separation. This description includes the separation, visualization, and glycan analysis of mucins with the SMME technique. PMID:26139278

  10. Coparative assessment of irradiated proteins in potato tuber with untreated control by High Performance Liquid Chromatography (HPLC) and gel electrophoresis

    NASA Astrophysics Data System (ADS)

    Ghojaie, M.; Sayhoon, M.

    1995-02-01

    About 2% of the weight of potato tuber is composed of proteins. In spite of their low quantity the proteins play a key role in the physiological activities leading to the break of the dormancy period and start of the cell division. This causes sprouting and also greening due to chlorophyll formation. This in turn is always accompanied by the production of the glycoalkaloid solanine in the flesh of tuber. For evaluation of radiation effect (dose range 50-250 Gy) and probable structural changes (amino acid release), analysis of selected proteins (molecular range 5 × 10^4 - 2 × 10^5 Dalton) of potato tuber in both irradiated and control type by HPLC showed no considerable changes in retention times, but qualitative assessment of amino acids by Pico-Tag^TM Pre-derivatizing method had some changes in quantity of amino acids like lysine which was increased 1 month after irradiation while Glutamic acid had considerable decreasment after the same time of irradiation.

  11. 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

  12. Strain-dependent profile of misfolded prion protein aggregates.

    PubMed

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-01-01

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration. PMID:26877167

  13. Strain-dependent profile of misfolded prion protein aggregates.

    PubMed

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V; Soto, Claudio

    2016-01-01

    Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

  14. Strain-dependent profile of misfolded prion protein aggregates

    PubMed Central

    Morales, Rodrigo; Hu, Ping Ping; Duran-Aniotz, Claudia; Moda, Fabio; Diaz-Espinoza, Rodrigo; Chen, Baian; Bravo-Alegria, Javiera; Makarava, Natallia; Baskakov, Ilia V.; Soto, Claudio

    2016-01-01

    Prions are composed of the misfolded prion protein (PrPSc) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrPSc aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrPSc aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrPSc aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrPSc aggregates and the incubation periods for the strains studied. The relative presence of PrPSc in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrPSc aggregates in prion-induced neurodegeneration. PMID:26877167

  15. DNA binding protein identification by combining pseudo amino acid composition and profile-based protein representation

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

    DNA-binding proteins play an important role in most cellular processes. Therefore, it is necessary to develop an efficient predictor for identifying DNA-binding proteins only based on the sequence information of proteins. The bottleneck for constructing a useful predictor is to find suitable features capturing the characteristics of DNA binding proteins. We applied PseAAC to DNA binding protein identification, and PseAAC was further improved by incorporating the evolutionary information by using profile-based protein representation. Finally, Combined with Support Vector Machines (SVMs), a predictor called iDNAPro-PseAAC was proposed. Experimental results on an updated benchmark dataset showed that iDNAPro-PseAAC outperformed some state-of-the-art approaches, and it can achieve stable performance on an independent dataset. By using an ensemble learning approach to incorporate more negative samples (non-DNA binding proteins) in the training process, the performance of iDNAPro-PseAAC was further improved. The web server of iDNAPro-PseAAC is available at http://bioinformatics.hitsz.edu.cn/iDNAPro-PseAAC/.

  16. A large-scale electrophoresis- and chromatography-based determination of gene expression profiles in bovine brain capillary endothelial cells after the re-induction of blood-brain barrier properties

    PubMed Central

    2010-01-01

    Background Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein). Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the well-established in vitro BBB model developed in our laboratory. Results A total of 215 protein spots (corresponding to 130 distinct proteins) were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin) and constitutes valuable evidence for predictions based on genome annotation. Conclusions Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets. PMID:21078152

  17. Profiling of β-Lactam Selectivity for Penicillin-Binding Proteins in Streptococcus pneumoniae D39

    PubMed Central

    Kocaoglu, Ozden; Tsui, Ho-Ching T.; Winkler, Malcolm E.

    2015-01-01

    Selective fluorescent β-lactam chemical probes enable the visualization of the transpeptidase activity of penicillin-binding proteins (PBPs) at different stages of bacterial cell division. To facilitate the development of new fluorescent probes for PBP imaging, we evaluated 20 commercially available β-lactams for selective PBP inhibition in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae. Live cells were treated with β-lactam antibiotics at different concentrations and subsequently incubated with Bocillin FL (Boc-FL; fluorescent penicillin) to saturate uninhibited PBPs. Fluorophore-labeled PBPs were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence scanning. Among 20 compounds tested, carbapenems (doripenem and meropenem) were coselective for PBP1a, PBP2x, and PBP3, while six of the nine penicillin compounds were coselective for PBP2x and PBP3. In contrast, the seven cephalosporin compounds tested display variability in their PBP-binding profiles. Three cephalosporin compounds (cefoxitin, cephalexin, and cefsulodin) and the monobactam aztreonam exhibited selectivity for PBP3, while only cefuroxime (a cephalosporin) was selective for PBP2x. Treatment of S. pneumoniae cultures with a sublethal concentration of cefuroxime that inhibited 60% of PBP2x activity and less than 20% of the activity of other PBPs resulted in formation of elongated cells. In contrast, treatment of S. pneumoniae cultures with concentrations of aztreonam and cefoxitin that inhibited up to 70% of PBP3 activity and less than 30% of other PBPs resulted in no discernible morphological changes. Additionally, correlation of the MIC and IC50s for each PBP, with the exception of faropenem, amdinocillin (mecillinam), and 6-APA, suggests that pneumococcal growth inhibition is primarily due to the inhibition of PBP2x. PMID:25845878

  18. Profiling of β-lactam selectivity for penicillin-binding proteins in Streptococcus pneumoniae D39.

    PubMed

    Kocaoglu, Ozden; Tsui, Ho-Ching T; Winkler, Malcolm E; Carlson, Erin E

    2015-01-01

    Selective fluorescent β-lactam chemical probes enable the visualization of the transpeptidase activity of penicillin-binding proteins (PBPs) at different stages of bacterial cell division. To facilitate the development of new fluorescent probes for PBP imaging, we evaluated 20 commercially available β-lactams for selective PBP inhibition in an unencapsulated derivative of the D39 strain of Streptococcus pneumoniae. Live cells were treated with β-lactam antibiotics at different concentrations and subsequently incubated with Bocillin FL (Boc-FL; fluorescent penicillin) to saturate uninhibited PBPs. Fluorophore-labeled PBPs were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence scanning. Among 20 compounds tested, carbapenems (doripenem and meropenem) were coselective for PBP1a, PBP2x, and PBP3, while six of the nine penicillin compounds were coselective for PBP2x and PBP3. In contrast, the seven cephalosporin compounds tested display variability in their PBP-binding profiles. Three cephalosporin compounds (cefoxitin, cephalexin, and cefsulodin) and the monobactam aztreonam exhibited selectivity for PBP3, while only cefuroxime (a cephalosporin) was selective for PBP2x. Treatment of S. pneumoniae cultures with a sublethal concentration of cefuroxime that inhibited 60% of PBP2x activity and less than 20% of the activity of other PBPs resulted in formation of elongated cells. In contrast, treatment of S. pneumoniae cultures with concentrations of aztreonam and cefoxitin that inhibited up to 70% of PBP3 activity and less than 30% of other PBPs resulted in no discernible morphological changes. Additionally, correlation of the MIC and IC50s for each PBP, with the exception of faropenem, amdinocillin (mecillinam), and 6-APA, suggests that pneumococcal growth inhibition is primarily due to the inhibition of PBP2x. PMID:25845878

  19. 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

  20. Mid-scale free-flow electrophoresis with gravity-induced uniform flow of background buffer in chamber for the separation of cells and proteins.

    PubMed

    Dong, Yu-Chao; Shao, Jing; Yin, Xiao-Yang; Fan, Liu-yin; Cao, Cheng-Xi

    2011-07-01

    A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.

  1. 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.

  2. Measurement of neonatal equine immunoglobulins for assessment of colostral immunoglobulin transfer: comparison of single radial immunodiffusion with the zinc sulfate turbidity test, serum electrophoresis, refractometry for total serum protein, and the sodium sulfite precipitation test.

    PubMed

    Rumbaugh, G E; Ardans, A A; Ginno, D; Trommershausen-Smith, A

    1978-02-01

    Four procedures for assessment of adequacy of colostral immunoglobulin (Ig) transfer in foals were evaluated. Results of zinc sulfate turbidity test, serum electrophoresis, total serum protein refractometry, and sodium sulfite precipitation test were compared with immunoglobulin G content determined by single radial immunodiffusion. The zinc sulfate turbidity test gave acceptable results for IgG, except that hemolyzed serum samples gave higher than expected values. A correction factor for hemolyzed serum was found to be useful. Serum electrophoresis was a satisfactory method of estimating IgG content. Total serum protein values may not be a valid basis for estimating IgG content, inasmuch as postsuckling total protein values were found to decrease in some foals in which passive transfer of IgG had been adequate. Sodium sulfite precipitation reactions were too unpredictable to be of value for determination of neonatal IgG concentration.

  3. Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply

    PubMed Central

    Ceballos-Laita, Laura; Gutierrez-Carbonell, Elain; Lattanzio, Giuseppe; Vázquez, Saul; Contreras-Moreira, Bruno; Abadía, Anunciación; Abadía, Javier; López-Millán, Ana-Flor

    2015-01-01

    The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III)-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164) were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5%) changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as with Fe

  4. Development of a non-denaturing 2D gel electrophoresis protocol for screening in vivo uranium-protein targets in Procambarus clarkii with laser ablation ICP MS followed by protein identification by HPLC-Orbitrap MS.

    PubMed

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

    2014-10-01

    Limited knowledge about in vivo non-covalent uranium (U)-protein complexes is largely due to the lack of appropriate analytical methodology. Here, a method for screening and identifying the molecular targets of U was developed. The approach was based on non-denaturing 1D and 2D gel electrophoresis (ND-PAGE and ND-2D-PAGE (using ND-IEF as first dimension previously described)) in conjunction with laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for the detection of U-containing proteins. The proteins were then identified by µbore HPLC-Orbitrap MS/MS. The method was applied to the analysis of cytosol of hepatopancreas (HP) of a model U-bioaccumulating organism (Procambarus clarkii). The imaging of uranium in 2D gels revealed the presence of 11 U-containing protein spots. Six protein candidates (i.e. ferritin, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, cytosolic manganese superoxide dismutase (Mn-SOD), glutathione S transferase D1 and H3 histone family protein) were then identified by matching with the data base of crustacea Decapoda species (e.g. crayfish). Among them, ferritin was the most important one. This strategy is expected to provide an insight into U toxicology and metabolism. PMID:25059147

  5. Proteomics reveals differences in protein abundance and highly similar antigenic profiles between Besnoitia besnoiti and Besnoitia tarandi.

    PubMed

    García-Lunar, P; Regidor-Cerrillo, J; Ortega-Mora, L M; Gutiérrez-Expósito, D; Alvarez-García, G

    2014-10-15

    Besnoitia besnoiti and Besnoitia tarandi are two cyst-forming apicomplexan parasites of the genus Besnoitia. B. besnoiti uses cattle as an intermediate host, in which it causes a disease that progresses in two sequential phases: the acute anasarca stage and the chronic scleroderma stage. Reindeer and caribou act as intermediate hosts for B. tarandi, which causes clinical signs similar to those caused by B. besnoiti. Previous studies demonstrated high molecular similarity, as determined by 18S and ITS-1 RNA sequences, between these Besnoitia spp., and strong serological cross-reactivity between these species has recently been demonstrated. Thus, a difference gel electrophoresis approach and mass spectrometry analysis were used to describe the proteomes and explore differences in protein abundance between B. besnoiti and B. tarandi in tachyzoite extracts. Immunoproteomes were also compared using 2-DE immunoblotting with polyclonal sera from experimentally infected rabbits. From approximately 1400 spots detected in DIGE-gels, 28 and 29 spots were differentially abundant in B. besnoiti and B. tarandi tachyzoites, respectively (± 1.5-fold, p<0.05). Four and 13 spots were exclusively detected in B. besnoiti and B. tarandi, respectively. Of the 32 differentially abundant spots analyzed by MALDI-TOF/MS, 6 up-regulated B. besnoiti proteins (LDH; HSP90; purine nucleoside phosphorylase and 3 hypothetical proteins) and 6 up-regulated B. tarandi proteins (G3PDH; LDH; PDI; mRNA decapping protein and 2 hypothetical proteins) were identified. Interestingly, no specific antigen spots were recognized by sera on any of the Besnoitia species studied and a similar antigen profile has been observed for B. tarandi and B. besnoiti sera when cross reactions were studied. This fact corroborates the difficulty in discerning Besnoitia infections using current serological assays. The present study underscores the importance of sequencing the B. besnoiti genome for species diversity studies of

  6. Correlation of protein and gene expression profiles of inflammatory proteins after endotoxin challenge in human subjects.

    PubMed

    Prabhakar, Uma; Conway, Theresa M; Murdock, Paul; Mooney, Jeff L; Clark, Steve; Hedge, Priti; Bond, Brian C; Jazwinska, Elizabeth C; Barnes, Michael R; Tobin, Frank; Damian-Iordachi, Valeriu; Greller, Larry; Hurle, Mark; Stubbs, Andrew P; Li, Zhong; Valoret, Elizabeth I; Erickson-Miller, Connie; Cass, Lisa; Levitt, Blanche; Davis, Hugh M; Jorkasky, Diane K; Williams, William V

    2005-07-01

    Administration of endotoxin (LPS) in humans results in profound physiological responses, including activation of peripheral blood mononuclear cells and the release of inflammatory factors. The time course of the response of selected inflammatory proteins was examined in healthy subjects (n = 6) administered a single intravenous dose of the purified derivative of endotoxin (3.0 ng/kg). Microarray analysis demonstrated changes in the expression of a number of genes, which were confirmed in separate in vitro endotoxin stimulation experiments. Subsequent TaqMan analysis of genes of interest indicated time-dependent changes in the expression of many of these genes. This included pre-B cell enhancing factor, which was identified on microarray analysis as being markedly upregulated following endotoxin stimulation. Protein expression of the genes examined by TaqMan analysis was measured and demonstrated the appearance of tumor necrosis factor (TNF)-alpha and sTNF-R proteins in the plasma beginning within 1 h after dosing, followed by other cytokines/ inflammatory markers (e.g., IL-1ra, G-CSF, IL-6, IL-8, and IL-10) and suppressors of cytokine signaling (SOCS-1 and SOCS-3). In general, cytokine protein expression correlated well with gene expression; however, the temporal profile of expression of some genes did not correlate well with the protein data. For many of these proteins, the lack of correlation was attributable to alternate tissue sources, which were demonstrated on TaqMan analysis. Principal component analysis indicated that cytokines could be grouped according to their temporal pattern of response, with most transcript levels returning to baseline 24 h following endotoxin administration. The combination of cDNA microarray and TaqMan analysis to identify and quantify changes in gene expression, along with the analysis of protein expression, can be useful in investigating inflammatory and other diseases.

  7. Global protein profiling studies of chikungunya virus infection identify different proteins but common biological processes.

    PubMed

    Smith, Duncan R

    2015-01-01

    Chikungunya fever (CHIKF) caused by the mosquito-transmitted chikungunya virus (CHIKV) swept into international prominence from late 2005 as an epidemic of CHIKF spread around countries surrounding the Indian Ocean. Although significant advances have been made in understanding the pathobiology of CHIKF, numerous questions still remain. In the absence of commercially available specific drugs to treat the disease, or a vaccine to prevent the diseases, the questions have particular significance. A number of studies have used global proteome analysis to increase our understanding of the process of CHIKV infection using a number of different experimental techniques and experimental systems. In all, over 700 proteins have been identified in nine different analyses by five different groups as being differentially regulated. Remarkably, only a single protein, eukaryotic elongation factor 2, has been identified by more than two different groups as being differentially regulated during CHIKV infection. This review provides a critical overview of the studies that have used global protein profiling to understand CHIKV infection and shows that while a broad consensus is emerging on which biological processes are altered during CHIKV infection, this consensus is poorly supported in terms of consistent identification of any key proteins mediating those biological processes.

  8. 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.

  9. 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.

  10. Improved Electrophoresis Cell

    NASA Technical Reports Server (NTRS)

    Rhodes, P. H.; Snyder, R. S.

    1982-01-01

    Several proposed modifications are expected to improve performance of a continous-flow electrophoresis cell. Changes would allow better control of buffer flow and would increase resolution by suppressing thermal gradients. Improved electrophoresis device would have high resolution and be easy to operate. Improvements would allow better flow control and heat dissipation.

  11. 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.

  12. Predicting the Effect of Mutations on Protein-Protein Binding Interactions through Structure-Based Interface Profiles

    PubMed Central

    Brender, Jeffrey R.; Zhang, Yang

    2015-01-01

    The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies. PMID:26506533

  13. Predicting the Effect of Mutations on Protein-Protein Binding Interactions through Structure-Based Interface Profiles.

    PubMed

    Brender, Jeffrey R; Zhang, Yang

    2015-10-01

    The formation of protein-protein complexes is essential for proteins to perform their physiological functions in the cell. Mutations that prevent the proper formation of the correct complexes can have serious consequences for the associated cellular processes. Since experimental determination of protein-protein binding affinity remains difficult when performed on a large scale, computational methods for predicting the consequences of mutations on binding affinity are highly desirable. We show that a scoring function based on interface structure profiles collected from analogous protein-protein interactions in the PDB is a powerful predictor of protein binding affinity changes upon mutation. As a standalone feature, the differences between the interface profile score of the mutant and wild-type proteins has an accuracy equivalent to the best all-atom potentials, despite being two orders of magnitude faster once the profile has been constructed. Due to its unique sensitivity in collecting the evolutionary profiles of analogous binding interactions and the high speed of calculation, the interface profile score has additional advantages as a complementary feature to combine with physics-based potentials for improving the accuracy of composite scoring approaches. By incorporating the sequence-derived and residue-level coarse-grained potentials with the interface structure profile score, a composite model was constructed through the random forest training, which generates a Pearson correlation coefficient >0.8 between the predicted and observed binding free-energy changes upon mutation. This accuracy is comparable to, or outperforms in most cases, the current best methods, but does not require high-resolution full-atomic models of the mutant structures. The binding interface profiling approach should find useful application in human-disease mutation recognition and protein interface design studies.

  14. Capillary electrophoresis coupled with end-column electrochemiluminescence for the determination of ephedrine in human urine, and a study of its interactions with three proteins.

    PubMed

    Yang, Ran; Zeng, Hua-Jin; Li, Jian-Jun; Zhang, Ying; Li, Shi-Jun; Qu, Ling-Bo

    2011-01-01

    A tris(2,2-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺)-based electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE) method has been established for the sensitive determination of ephedrine for the first time. Under the optimized conditions [ECL detection at 1.15 V, 25 mmol/L phosphate buffer solution (PBS), pH 8.0, as running buffer, separation voltage 12.5 kV, 5 mmol/L Ru(bpy)₃²⁺ with 60 mmol/L PBS, pH 8.5, in the detection cell] linear correlation (r = 0.9987) between ECL intensity and ephedrine concentration was obtained in the range 6.0 × 10⁻⁸-6.0 × 10⁻⁶ g/mL. The detection limit was 4.5 × 10⁻⁹ g/mL (S:N = 3). The developed method was successfully applied to the analysis of ephedrine in human urine and the investigation of its interactions with three proteins, including bovine serum albumin (BSA), cytochrome C (Cyt-C) and myoglobin (Mb). The number of binding sites and the binding constants between ephedrine and BSA, Cyt-C and Mb were 8.52, 12.60, 10.66 and 1.55 × 10⁴  mol/L, 6.58 × 10³  mol/L and 1.59 × 10⁴  mol/L, respectively.

  15. Investigation of protein expression profiles of erythritol-producing Candida magnoliae in response to glucose perturbation.

    PubMed

    Kim, Hyo Jin; Lee, Hyeong-Rho; Kim, Chang Sup; Jin, Yong-Su; Seo, Jin-Ho

    2013-08-15

    Protein expression patterns of an erythritol-producing yeast, Candida magnoliae, were analyzed to identify differentially expressed proteins in response to glucose perturbation. Specifically, wild type C. magnoliae was grown under high and low glucose conditions and the cells were harvested at both mid-exponential and erythritol production phases for proteomic studies. In order to analyze intracellular protein abundances from the harvested cells quantitatively, total intracellular proteins were extracted and applied to two-dimensional gel electrophoresis for separation and visualization of individual proteins. Among the proteins distributed in the range of pI 4-7 and molecular weight 29-97kDa, five osmo-responsive proteins were drastically changed in response to glucose perturbation. Hsp60 (Heat-shock protein 60), transaldolase and NADH:quinone oxidoreductase were down-regulated under the high glucose condition and Bro1 (BCK1-like Resistance to Osmotic shock) and Eno1 (enolase1) were up-regulated. These proteins are directly or indirectly related with cellular stress response. Importantly, protein expression patterns of Hsp60, Bro1 and Eno1 were strongly correlated with previous studies identifying the proteins perturbed by osmotic stress for other organisms including Saccharomyces cerevisiae.

  16. Fractionation profiling: a fast and versatile approach for mapping vesicle proteomes and protein–protein interactions

    PubMed Central

    Borner, Georg H. H.; Hein, Marco Y.; Hirst, Jennifer; Edgar, James R.; Mann, Matthias; Robinson, Margaret S.

    2014-01-01

    We developed “fractionation profiling,” a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps. PMID:25165137

  17. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

    PubMed Central

    Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

    2016-01-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452

  18. Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling.

    PubMed

    Larance, Mark; Kirkwood, Kathryn J; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A J; Lamond, Angus I

    2016-07-01

    We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).

  19. Effects of the processing methods of corn grain and soybean meal on milk protein expression profiles in dairy cows.

    PubMed

    Li, S S; Shen, J S; Ren, D X; Liu, J X

    2015-02-01

    A proteomic approach was used to investigate the effects of the processing method of corn grain and soybean meal on the milk protein expression profile in lactating dairy cows. A total of 12 multiparous Holstein dairy cows were used in a 4×4 Latin square design with a 2×2 factorial arrangement. The primary factors examined were corn (finely ground (FGC) v. steam-flaked (SFC)) and soybean meal (solvent-extracted (SSBM) v. heat-treated (HSBM)), which were used to formulate four diets with the same basal ingredient: 27% FGC and 9% SSBM; 27% SFC and 9% SSBM; 27% FGC and 9% HSBM; and 27% SFC and 9% HSBM. Each period lasted for 21 days. Milk samples were collected on days 18, 19 and 20 of each period. Changes in the milk proteins were assessed by two-dimensional (2D) electrophoresis and ImageMaster 2D Platinum 6.0 software. A total of 13 spots displayed variations in protein spot abundance according to the statistical analysis. These spots were identified by a matrix-assisted laser desorption/ionization-time of flight/time of flight MS. According to the gels, the relative abundance of α(s2)-casein (CN) fragments was higher in the cows fed the SFC-HSBM than that for SFC-SSBM, whereas β-CN, α-lactalbumin and zinc-alpha-2-glycoprotein fragments were down-regulated in HSBM-fed cows. The relative decrease of β-CN expression was validated by western blot and agreed with the MS data. These results suggested that the method used to process soybean meal modified the synthesis and secretion of milk proteins in lactating dairy cows' mammary glands.

  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.

  1. 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

  2. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation

    PubMed Central

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  3. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

    PubMed

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways.

  4. Phosphoproteome Profiling of SH-SY5y Neuroblastoma Cells Treated with Anesthetics: Sevoflurane and Isoflurane Affect the Phosphorylation of Proteins Involved in Cytoskeletal Regulation.

    PubMed

    Lee, Joomin; Ahn, Eunsook; Park, Wyun Kon; Park, Seyeon

    2016-01-01

    Inhalation anesthetics are used to decrease the spinal cord transmission of painful stimuli. However, the molecular or biochemical processes within cells that regulate anesthetic-induced responses at the cellular level are largely unknown. Here, we report the phosphoproteome profile of SH-SY5y human neuroblastoma cells treated with sevoflurane, a clinically used anesthetic. Phosphoproteins were isolated from cell lysates and analyzed using two-dimensional gel electrophoresis. The phosphorylation of putative anesthetic-responsive marker proteins was validated using western blot analysis in cells treated with both sevoflurane and isoflurane. A total of 25 phosphoproteins were identified as differentially phosphorylated proteins. These included key regulators that signal cytoskeletal remodeling steps in pathways related to vesicle trafficking, axonal growth, and cell migration. These proteins included the Rho GTPase, Ras-GAP SH3 binding protein, Rho GTPase activating protein, actin-related protein, and actin. Sevoflurane and isoflurane also resulted in the dissolution of F-actin fibers in SH-SY5y cells. Our results show that anesthetics affect the phosphorylation of proteins involved in cytoskeletal remodeling pathways. PMID:27611435

  5. Species Differentiation of a Diverse Suite of Bacillus Spores by Mass Spectrometry-Based Protein Profiling

    PubMed Central

    Dickinson, Danielle N.; La Duc, Myron T.; Haskins, William E.; Gornushkin, Igor; Winefordner, James D.; Powell, David H.; Venkateswaran, Kasthuri

    2004-01-01

    In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of Bacillus spores. MALDI-TOFMS protein profiles of 11 different strains of Bacillus spores, encompassing nine different species, were evaluated. Bacillus species selected for MALDI-TOFMS analysis represented the spore-forming bacterial diversity of typical class 100K clean room spacecraft assembly facilities. A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time. A library of MALDI-TOFMS spectra was created from these nine Bacillus species, the most diverse protein profiling study of the genus reported to date. Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study. The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations. The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis. Proteomic studies of Bacillus subtilis 168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum. Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified. The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the Bacillus genus. PMID:14711677

  6. Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

    PubMed

    Lim, Sanghyun; Chisholm, Kenneth; Coffin, Robert H; Peters, Rick D; Al-Mughrabi, Khalil I; Wang-Pruski, Gefu; Pinto, Devanand M

    2012-04-01

    Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

  7. Rapid protein profiling facilitates surveillance of invasive mosquito species

    PubMed Central

    2014-01-01

    Background Invasive aedine mosquito species have become a major issue in many parts of the world as most of them are recognised vectors or potentially involved in transmission of pathogens. Surveillance of these mosquitoes (e.g. Ae. aegypti, Yellow fever mosquito, Aedes albopictus, Asian tiger mosquito) is mainly done by collecting eggs using ovitraps and by identification of the larvae hatched in the laboratory. In order to replace this challenging and laborious procedure, we have evaluated matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) for easy and rapid species identification. Methods Individual protein profiles were generated using five eggs each of nine aedine species (Ae. aegypti, Ae. albopictus, Ae. atropalpus, Ae. cretinus, Ae. geniculatus, Ae. japonicus, Ae. koreicus, Ae. phoeniciae, Ae. triseriatus) from various geographical origins, and species-specific biomarker mass sets could be generated. A blinded validation using our reference data base for automated egg identification was performed. In addition, pools of 10 aedine eggs (132 two-species and 18 three-species pools) in different ratios were evaluated. Results Specific biomarker mass sets comprising 18 marker masses could be generated for eggs of nine container-inhabiting aedine species, including all the major invasive and indigenous species of Europe and North America. Two additional masses shared by all investigated aedine species are used as internal calibrators. Identification of single eggs was highly accurate (100% specificity, 98.75% sensitivity), and this method is also of value for the identification of species in pools of ten eggs. When mixing two or three species, all were identified in all pools in at least 2 or 1 of the 4 loaded replicates, respectively, if the “lesser abundant” species in the pool accounted for three or more eggs. Conclusions MALDI-TOF MS, which is widely applied for routine identification of microorganisms in clinical

  8. 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.

  9. A New Standard-Based Polynomial Interpolation (SBPIn) Method to Address Gel-to-Gel Variability for the Comparison of Multiple Denaturing Gradient Gel Electrophoresis Profile Matrices

    PubMed Central

    Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M.

    2013-01-01

    The Standard-Based Polynomial Interpolation (SBPIn) method is a new simple three-step protocol proposed to address common gel-to-gel variations for the comparison of sample profiles across multiple DGGE gels. The advantages of this method include no requirement for additional software or modification of the standard DGGE protocol. PMID:23234884

  10. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    PubMed

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  11. Correlation between phylogroups and intracellular proteomes of Propionibacterium acnes and differences in the protein expression profiles between anaerobically and aerobically grown cells.

    PubMed

    Dekio, Itaru; Culak, Renata; Fang, Min; Ball, Graham; Gharbia, Saheer; Shah, Haroun N

    2013-01-01

    Propionibacterium acnes is one of the dominant commensals on the human skin and also an opportunistic pathogen in relation to acne, sarcoidosis, prostate cancer, and various infections. Recent investigations using housekeeping and virulence genes have revealed that the species consists of three major evolutionary clades (types I, II, and III). In order to investigate protein expression differences between these phylogroups, proteomic profiles of 21 strains of P. acnes were investigated. The proteins extracted from cells cultured under anaerobic and aerobic conditions were analysed using a SELDI-TOF mass spectrometer, high-resolution capillary gel electrophoresis, and LC-MS/ MS. The SELDI spectral profiles were visualised as a heat map and a dendrogram, which resulted in four proteomic groups. Strains belonging to type I were represented in the proteome Group A, while Group B contained type III strains. Groups C and D contained mixtures of types I and II. Each of these groups was not influenced by differences in culture conditions. Under anoxic growth conditions, a type IB strain yielded high expressions of some proteins, such as methylmalonyl-CoA epimerase and the Christie-Atkins-Munch-Petersen (CAMP) factor. The present study revealed good congruence between genomic and proteomic data suggesting that the microenvironment of each subtype may influence protein expression. PMID:23878795

  12. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Thompson, Vicki S; Lacey, Jeffrey A; Gentillon, Cynthia A; Apel, William A

    2015-03-03

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  13. Identification of discriminant proteins through antibody profiling, methods and apparatus for identifying an individual

    DOEpatents

    Apel, William A.; Thompson, Vicki S; Lacey, Jeffrey A.; Gentillon, Cynthia A.

    2016-08-09

    A method for determining a plurality of proteins for discriminating and positively identifying an individual based from a biological sample. The method may include profiling a biological sample from a plurality of individuals against a protein array including a plurality of proteins. The protein array may include proteins attached to a support in a preselected pattern such that locations of the proteins are known. The biological sample may be contacted with the protein array such that a portion of antibodies in the biological sample reacts with and binds to the proteins forming immune complexes. A statistical analysis method, such as discriminant analysis, may be performed to determine discriminating proteins for distinguishing individuals. Proteins of interest may be used to form a protein array. Such a protein array may be used, for example, to compare a forensic sample from an unknown source with a sample from a known source.

  14. Molecular classification of fatty liver by high-throughput profiling of protein post-translational modifications.

    PubMed

    Urasaki, Yasuyo; Fiscus, Ronald R; Le, Thuc T

    2016-04-01

    We describe an alternative approach to classifying fatty liver by profiling protein post-translational modifications (PTMs) with high-throughput capillary isoelectric focusing (cIEF) immunoassays. Four strains of mice were studied, with fatty livers induced by different causes, such as ageing, genetic mutation, acute drug usage, and high-fat diet. Nutrient-sensitive PTMs of a panel of 12 liver metabolic and signalling proteins were simultaneously evaluated with cIEF immunoassays, using nanograms of total cellular protein per assay. Changes to liver protein acetylation, phosphorylation, and O-N-acetylglucosamine glycosylation were quantified and compared between normal and diseased states. Fatty liver tissues could be distinguished from one another by distinctive protein PTM profiles. Fatty liver is currently classified by morphological assessment of lipid droplets, without identifying the underlying molecular causes. In contrast, high-throughput profiling of protein PTMs has the potential to provide molecular classification of fatty liver.

  15. 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 ...

  16. Electrophoresis operations in space

    NASA Technical Reports Server (NTRS)

    Richman, D. W.

    1982-01-01

    Application of electrophoresis in space processing is described. Spaceborne experiments in areas such as biological products and FDA approved drugs are discussed. These experiments will be carried on shuttle payloads.

  17. Current two-dimensional electrophoresis technology for proteomics.

    PubMed

    Görg, Angelika; Weiss, Walter; Dunn, Michael J

    2004-12-01

    Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where Mr and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037-1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5-12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the

  18. Protein profile of mature soybean seeds and prepared soybean milk.

    PubMed

    Capriotti, Anna Laura; Caruso, Giuseppe; Cavaliere, Chiara; Samperi, Roberto; Stampachiacchiere, Serena; Zenezini Chiozzi, Riccardo; Laganà, Aldo

    2014-10-01

    The soybean (Glycine max (L.) Merrill) is economically the most important bean in the world, providing a wide range of vegetable proteins. Soybean milk is a colloidal solution obtained as water extract from swelled and ground soybean seeds. Soybean proteins represent about 35-40% on a dry weight basis and they are receiving increasing attention with respect to their health effects. However, the soybean is a well-recognized allergenic food, and therefore, it is urgent to define its protein components responsible for the allergenicity in order to develop hypoallergenic soybean products for sensitive people. The main aim of this work was the characterization of seed and milk soybean proteome and their comparison in terms of protein content and specific proteins. Using a shotgun proteomics approach, 243 nonredundant proteins were identified in mature soybean seeds.

  19. Furosemide and Potassium Chloride-induced Alteration in Protein Profile of Left Ventricle and its Associated Risk for Sudden Cardiac Death

    PubMed Central

    Murugan, Ponniah Senthil; Selvam, Govindan Sadasivam

    2014-01-01

    Background: Potassium ion (K+) plays an essential role in maintaining the electrical potential across the plasma membrane of cells. An abnormal serum K+ level is associated with increased risk of ventricular arrhythmia and sudden cardiac death (SCD) and these patients are generally prescribed with furosemide and potassium chloride (KCl). We explored the association between the use of these drugs and the risk of SCD by analyzing biochemical parameters and proteomic changes. Materials and Methods: The rats were administered with furosemide and KCl and their effect was analyzed by studying cardiac and oxidative markers, electrolyte content and histopathology. Two-dimensional gel electrophoresis (2-DE) and electrospray ionization-mass spectrometry were performed to investigate the LV proteomic changes. Results: Furosemide and KCl treatments showed significant effect on physiological and biochemical parameters, and LV histopathology of experimental rats. Proteomic analysis indicated 17 differentially expressed proteins. Among them, eight protein spots were identified using peptide mass fingerprinting. In furosemide-treated group, four proteins were upregulated and two proteins were downregulated when compared to 2-DE proteomic profile of control. While in KCl-treated rats, seven proteins were found downregulated. Conculsion: The present study revealed the differential expression of proteins by furosemide and KCl treatment. Thus, the results suggest that the use of these drugs leads to proteomic alteration, which involve in cardiac conductivity that might increase the risk of SCD. PMID:24748728

  20. Membrane protein stability analyses by means of protein energy profiles in case of nephrogenic diabetes insipidus.

    PubMed

    Heinke, Florian; Labudde, Dirk

    2012-01-01

    Diabetes insipidus (DI) is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000-30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP) is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI). NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R) in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems.

  1. Maternal stress predicts altered biogenesis and the profile of mitochondrial proteins in the frontal cortex and hippocampus of adult offspring rats.

    PubMed

    Głombik, Katarzyna; Stachowicz, Aneta; Ślusarczyk, Joanna; Trojan, Ewa; Budziszewska, Bogusława; Suski, Maciej; Kubera, Marta; Lasoń, Władysław; Wędzony, Krzysztof; Olszanecki, Rafał; Basta-Kaim, Agnieszka

    2015-10-01

    Currently, much attention is focused on the influence of mitochondrial disturbances at the onset of depression. The goal of this study was to investigate the impact of prenatal stress (an animal model of depression) on the mitochondrial biogenesis proteins and mitoproteome profile in the frontal cortex and hippocampus of adult 3-month-old male rats following a prenatal stress procedure. Our results show that rats that were exposed to prenatal stress stimuli displayed depression-like behaviors based on the sucrose preference and elevated plus maze tests. It has been found that the level of the PGC-1α protein was reduced in the frontal cortex and hippocampus of the adult offspring after the prenatal stress procedure. Moreover, in the frontal cortex, the level of the pro-apoptotic protein Bax was up-regulated. Two-dimensional electrophoresis coupled with mass spectrometry showed the statistically significant down-regulation of the mitochondrial ribosomal protein L12 (Mrpl12) and mitochondrial NADH dehydrogenase [ubiquinone] flavoprotein 2 (NDUFV2) as well as the up-regulation of the Tubulin Polymerization Promoting Proteins (Tppp/p25) in the frontal cortex. In contrast, in the hippocampus, the mitochondrial pyruvate dehydrogenase E1 component subunit beta, the voltage-dependent anion-selective channel protein 2 (VDAC2), and the GTP-binding nuclear protein RAN (RAN) were down-regulated and the expression of phosphatidylethanolamine-binding protein 1 (PEBP-1) was enhanced. These findings provide new evidence that stress during pregnancy may lead not only to behavioral deficits, but also to disturbances in the brain mitoproteome profile in adult rat offspring.

  2. 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.

  3. UV-B Radiation Stress Causes Alterations in Whole Cell Protein Profile and Expression of Certain Genes in the Rice Phyllospheric Bacterium Enterobacter cloacae.

    PubMed

    Kumar, Jay; Babele, Piyoosh K; Singh, Divya; Kumar, Ashok

    2016-01-01

    Among the different types of UV radiation, UV-B radiation (280-315 nm) has gained much attention mainly due to its increasing incidence on the Earth's surface leading to imbalances in natural ecosystems. This study deals with the effects of UV-B radiation on the proteome and gene expression in a rice phyllospheric bacterium, Enterobacter cloacae. Of the five bacteria isolated from rice leaves, E. cloacae showed the highest level of resistance to UV-B and total killing occurred after 8 h of continuous exposure to UV-B. Reactive oxygen species were induced by UV-B exposure and increased with increasing duration of exposure. Protein profiling by SDS-PAGE and 2-dimensional gel electrophoresis (2-DE) revealed major changes in the number as well as expression of proteins. Analysis of 2-DE gel spots indicated up/down-regulation of several proteins under the stress of UV-B radiation. Thirteen differentially expressed proteins including two hypothetical proteins were identified by MALDI-TOF MS and assigned to eight functional categories. Both the hypothetical proteins (gi 779821175 and gi 503938301) were over-expressed after UV-B irradiation; gi 503938301 was characterized as a member of FMN reductase superfamily whereas gi 779821175 seems to be a structural protein as it did not show any functional domain. That the expression of certain proteins under UV-B stress is indeed up-regulated was confirmed by qRT-PCR. Transcript analysis of selected gene including genes of hypothetical proteins (cp011650 and cp002886) showed over-expression under UV-B stress as compared to untreated control cultures. Although this study deals with a limited number of proteins, identification of differentially expressed proteins reported herein may prove useful in future studies especially for assessing their significance in the protection mechanism of bacteria against UV-B radiation stress.

  4. UV-B Radiation Stress Causes Alterations in Whole Cell Protein Profile and Expression of Certain Genes in the Rice Phyllospheric Bacterium Enterobacter cloacae

    PubMed Central

    Kumar, Jay; Babele, Piyoosh K.; Singh, Divya; Kumar, Ashok

    2016-01-01

    Among the different types of UV radiation, UV-B radiation (280-315 nm) has gained much attention mainly due to its increasing incidence on the Earth’s surface leading to imbalances in natural ecosystems. This study deals with the effects of UV-B radiation on the proteome and gene expression in a rice phyllospheric bacterium, Enterobacter cloacae. Of the five bacteria isolated from rice leaves, E. cloacae showed the highest level of resistance to UV-B and total killing occurred after 8 h of continuous exposure to UV-B. Reactive oxygen species were induced by UV-B exposure and increased with increasing duration of exposure. Protein profiling by SDS-PAGE and 2-dimensional gel electrophoresis (2-DE) revealed major changes in the number as well as expression of proteins. Analysis of 2-DE gel spots indicated up/down-regulation of several proteins under the stress of UV-B radiation. Thirteen differentially expressed proteins including two hypothetical proteins were identified by MALDI-TOF MS and assigned to eight functional categories. Both the hypothetical proteins (gi 779821175 and gi 503938301) were over-expressed after UV-B irradiation; gi 503938301 was characterized as a member of FMN reductase superfamily whereas gi 779821175 seems to be a structural protein as it did not show any functional domain. That the expression of certain proteins under UV-B stress is indeed up-regulated was confirmed by qRT-PCR. Transcript analysis of selected gene including genes of hypothetical proteins (cp011650 and cp002886) showed over-expression under UV-B stress as compared to untreated control cultures. Although this study deals with a limited number of proteins, identification of differentially expressed proteins reported herein may prove useful in future studies especially for assessing their significance in the protection mechanism of bacteria against UV-B radiation stress. PMID:27672388

  5. UV-B Radiation Stress Causes Alterations in Whole Cell Protein Profile and Expression of Certain Genes in the Rice Phyllospheric Bacterium Enterobacter cloacae.

    PubMed

    Kumar, Jay; Babele, Piyoosh K; Singh, Divya; Kumar, Ashok

    2016-01-01

    Among the different types of UV radiation, UV-B radiation (280-315 nm) has gained much attention mainly due to its increasing incidence on the Earth's surface leading to imbalances in natural ecosystems. This study deals with the effects of UV-B radiation on the proteome and gene expression in a rice phyllospheric bacterium, Enterobacter cloacae. Of the five bacteria isolated from rice leaves, E. cloacae showed the highest level of resistance to UV-B and total killing occurred after 8 h of continuous exposure to UV-B. Reactive oxygen species were induced by UV-B exposure and increased with increasing duration of exposure. Protein profiling by SDS-PAGE and 2-dimensional gel electrophoresis (2-DE) revealed major changes in the number as well as expression of proteins. Analysis of 2-DE gel spots indicated up/down-regulation of several proteins under the stress of UV-B radiation. Thirteen differentially expressed proteins including two hypothetical proteins were identified by MALDI-TOF MS and assigned to eight functional categories. Both the hypothetical proteins (gi 779821175 and gi 503938301) were over-expressed after UV-B irradiation; gi 503938301 was characterized as a member of FMN reductase superfamily whereas gi 779821175 seems to be a structural protein as it did not show any functional domain. That the expression of certain proteins under UV-B stress is indeed up-regulated was confirmed by qRT-PCR. Transcript analysis of selected gene including genes of hypothetical proteins (cp011650 and cp002886) showed over-expression under UV-B stress as compared to untreated control cultures. Although this study deals with a limited number of proteins, identification of differentially expressed proteins reported herein may prove useful in future studies especially for assessing their significance in the protection mechanism of bacteria against UV-B radiation stress. PMID:27672388

  6. UV-B Radiation Stress Causes Alterations in Whole Cell Protein Profile and Expression of Certain Genes in the Rice Phyllospheric Bacterium Enterobacter cloacae

    PubMed Central

    Kumar, Jay; Babele, Piyoosh K.; Singh, Divya; Kumar, Ashok

    2016-01-01

    Among the different types of UV radiation, UV-B radiation (280-315 nm) has gained much attention mainly due to its increasing incidence on the Earth’s surface leading to imbalances in natural ecosystems. This study deals with the effects of UV-B radiation on the proteome and gene expression in a rice phyllospheric bacterium, Enterobacter cloacae. Of the five bacteria isolated from rice leaves, E. cloacae showed the highest level of resistance to UV-B and total killing occurred after 8 h of continuous exposure to UV-B. Reactive oxygen species were induced by UV-B exposure and increased with increasing duration of exposure. Protein profiling by SDS-PAGE and 2-dimensional gel electrophoresis (2-DE) revealed major changes in the number as well as expression of proteins. Analysis of 2-DE gel spots indicated up/down-regulation of several proteins under the stress of UV-B radiation. Thirteen differentially expressed proteins including two hypothetical proteins were identified by MALDI-TOF MS and assigned to eight functional categories. Both the hypothetical proteins (gi 779821175 and gi 503938301) were over-expressed after UV-B irradiation; gi 503938301 was characterized as a member of FMN reductase superfamily whereas gi 779821175 seems to be a structural protein as it did not show any functional domain. That the expression of certain proteins under UV-B stress is indeed up-regulated was confirmed by qRT-PCR. Transcript analysis of selected gene including genes of hypothetical proteins (cp011650 and cp002886) showed over-expression under UV-B stress as compared to untreated control cultures. Although this study deals with a limited number of proteins, identification of differentially expressed proteins reported herein may prove useful in future studies especially for assessing their significance in the protection mechanism of bacteria against UV-B radiation stress.

  7. Simple luminescence detectors using a light-emitting diode or a Xe lamp, optical fiber and charge-coupled device, or photomultiplier for determining proteins in capillary electrophoresis: a critical comparison.

    PubMed

    Casado-Terrones, Silvia; Fernández-Sánchez, Jorge F; Segura-Carretero, Antonio; Fernández-Gutiérrez, Alberto

    2007-06-01

    The performance of two homemade fluorescence-induced capillary electrophoresis detectors, one based on light-emitting diode (LED) as the excitation source and a charge-coupled device (CCD) photodetector and the other based on a commercial luminescence spectrometer (Xe lamp) as the excitation source and a photomultiplier tube as a detector, were compared for the determination of fluorescent proteins R-phycoerythrin and B-phycoerythrin. Both devices use commercially available, reasonably priced optical components that can be used by nonexperts. After fine optimization of several optical and separation parameters in both devices, a zone capillary electrophoresis methodology was achieved with 50mM borate buffer (pH 8.4) and 10mM phytic acid for the determination of two phycobiliproteins. Detection limits of 0.50 and 0.64microg/ml for R-phycoerythrin and B-phycoerythrin, respectively, were achieved by using the LED-induced fluorescence capillary electrophoresis (LED-IF-CE) system, and corresponding detection limits of 2.73 and 2.16microg/ml were achieved by using the Xe lamp-IF-CE system. Analytical performance and other parameters, such as cost and potential to miniaturization, are compared for both devices.

  8. Dry and moist heating-induced changes in protein molecular structure, protein subfraction, and nutrient profiles in soybeans.

    PubMed

    Samadi; Yu, P

    2011-12-01

    Heat processing has been used to improve protein utilization and availability of animal nutrition. However, to date, few studies exist on heat-induced protein molecular structure changes on a molecular basis. The aims of this study were to use molecular spectroscopy as a novel approach to determine heat-induced protein molecular structure changes affected by moist and dry heating and quantify protein molecular structures and nutritive value in the rumen and intestine in dairy cattle. In this study, soybean was used as a model for feed protein and was autoclaved at 120°C for 1h (moist heating) and dry heated at 120°C for 1h. The parameters assessed in this study included protein structure α-helix and β-sheet and their ratio, protein subfractions associated with protein degradation behaviors, intestinal protein availability, and energy values. The results show that heat treatments changed the protein molecular structure. Both dry and moist heating increased the amide I-to-amide II ratio. However, for the protein α-helix-to-β-sheet ratio, moist heating decreased but dry heating increased the ratio. Compared with dry heating, moist heating dramatically changed the chemical and nutrient profiles of soybean seed. It greatly decreased soluble crude protein, nonprotein nitrogen, and increased neutral detergent insoluble protein. Both dry and moist heating treatments did not alter digestible nutrients and energy values. Heating tended to decrease the nonprotein nitrogen fraction (soluble and rapidly degradable protein fraction) and true protein 1 fraction (fast-degradable protein fraction). Conversely, the true protein 3 fraction (slowly degradable fraction) significantly increased. The in situ rumen study showed that moist heating decreased protein rumen degradability and increased intestinal digestibility of rumen-undegradable protein. Compared with the raw soybeans, dry heating did not affect rumen degradability and intestinal digestibility. In conclusion, compared

  9. Using support vector machine and evolutionary profiles to predict antifreeze protein sequences.

    PubMed

    Zhao, Xiaowei; Ma, Zhiqiang; Yin, Minghao

    2012-01-01

    Antifreeze proteins (AFPs) are ice-binding proteins. Accurate identification of new AFPs is important in understanding ice-protein interactions and creating novel ice-binding domains in other proteins. In this paper, an accurate method, called AFP_PSSM, has been developed for predicting antifreeze proteins using a support vector machine (SVM) and position specific scoring matrix (PSSM) profiles. This is the first study in which evolutionary information in the form of PSSM profiles has been successfully used for predicting antifreeze proteins. Tested by 10-fold cross validation and independent test, the accuracy of the proposed method reaches 82.67% for the training dataset and 93.01% for the testing dataset, respectively. These results indicate that our predictor is a useful tool for predicting antifreeze proteins. A web server (AFP_PSSM) that implements the proposed predictor is freely available.

  10. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage

    NASA Astrophysics Data System (ADS)

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-01

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1 ×, DE3 ×, ME3 ×, NEL3 ×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P = 0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P = 0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P = 0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P = 0.09), RD of PB2 (29 vs. 62 g/kg CP, P = 0.02), RD of CB2 (251 vs. 198 g/kg DM, P = 0.06), RD of CB3 (236 vs. 261 g/kg CHO, P = 0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P = 0.06), and metabolic bioenergy (P = 0.095). As to protein molecular structure, there were differences in protein structure 1st

  11. Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage.

    PubMed

    Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

    2016-03-15

    The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1×, DE3×, ME3×, NEL3×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P=0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P=0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P=0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P=0.09), RD of PB2 (29 vs. 62 g/kg CP, P=0.02), RD of CB2 (251 vs. 198 g/kg DM, P=0.06), RD of CB3 (236 vs. 261 g/kg CHO, P=0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P=0.06), and metabolic bioenergy (P=0.095). As to protein molecular structure, there were differences in protein structure 1st and 2nd amide groups (P

  12. Multidimensional profiling of cell surface proteins and nuclear markers

    SciTech Connect

    Han, Ju; Chang, Hang; Andarawewa, Kumari; Yaswen, Paul; Helen Barcellos-Hoff, Mary; Parvin, Bahram

    2009-01-30

    Cell membrane proteins play an important role in tissue architecture and cell-cell communication. We hypothesize that segmentation and multidimensional characterization of the distribution of cell membrane proteins, on a cell-by-cell basis, enable improved classification of treatment groups and identify important characteristics that can otherwise be hidden. We have developed a series of computational steps to (i) delineate cell membrane protein signals and associate them with a specific nucleus; (ii) compute a coupled representation of the multiplexed DNA content with membrane proteins; (iii) rank computed features associated with such a multidimensional representation; (iv) visualize selected features for comparative evaluation through heatmaps; and (v) discriminate between treatment groups in an optimal fashion. The novelty of our method is in the segmentation of the membrane signal and the multidimensional representation of phenotypic signature on a cell-by-cell basis. To test the utility of this method, the proposed computational steps were applied to images of cells that have been irradiated with different radiation qualities in the presence and absence of other small molecules. These samples are labeled for their DNA content and E-cadherin membrane proteins. We demonstrate that multidimensional representations of cell-by-cell phenotypes improve predictive and visualization capabilities among different treatment groups, and identify hidden variables.

  13. Binding profile of spiramycin to oviducal proteins of laying hens.

    PubMed

    Furusawa, N

    2000-12-01

    In vitro protein binding of spiramycin (SP) in the plasma and oviducts of laying hens was studied. The data for SP were compared with those for oxytetracycline (OTC), sulphadimidine (SDD), sulphamonomethoxine (SMM) and sulphaquinoxaline (SQ). The two oviduct segments, magnum (M) and isthmus plus shell gland (IS), were collected. The soluble (cell sap) fractions from the magnum (M-S9) and the isthmus plus shell gland (IS-S9) were used as samples. Plasma protein binding was highest for SQ (81.4%) (P < 0.01), and lowest for SDD (30.9%) (P < 0.01). No M-S9 protein binding of OTC was found. The IS-S9 protein binding of SP (60.4%) was very much higher than those of OTC (0.8%), SDD (4.1%), SMM (4.0%) and SQ (12.3%) (P < 0.01). Biological half-lives of these drugs in egg albumen were directly correlated to the extent of their binding to IS proteins. Of plasma, M-S9 and IS-S9, variation in SP concentration in the ranges from 1 to 20 micrograms/ml did not alter the binding properties of the drug. PMID:11199206

  14. Optimization of translation profiles enhances protein expression and solubility.

    PubMed

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5'-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein.

  15. Optimization of Translation Profiles Enhances Protein Expression and Solubility

    PubMed Central

    Hess, Anne-Katrin; Saffert, Paul; Liebeton, Klaus; Ignatova, Zoya

    2015-01-01

    mRNA is translated with a non-uniform speed that actively coordinates co-translational folding of protein domains. Using structure-based homology we identified the structural domains in epoxide hydrolases (EHs) and introduced slow-translating codons to delineate the translation of single domains. These changes in translation speed dramatically improved the solubility of two EHs of metagenomic origin in Escherichia coli. Conversely, the importance of transient attenuation for the folding, and consequently solubility, of EH was evidenced with a member of the EH family from Agrobacterium radiobacter, which partitions in the soluble fraction when expressed in E. coli. Synonymous substitutions of codons shaping the slow-transiting regions to fast-translating codons render this protein insoluble. Furthermore, we show that low protein yield can be enhanced by decreasing the free folding energy of the initial 5’-coding region, which can disrupt mRNA secondary structure and enhance ribosomal loading. This study provides direct experimental evidence that mRNA is not a mere messenger for translation of codons into amino acids but bears an additional layer of information for folding, solubility and expression level of the encoded protein. Furthermore, it provides a general frame on how to modulate and fine-tune gene expression of a target protein. PMID:25965266

  16. Proteomic profiling of microbial transglutaminase-induced polymerization of milk proteins.

    PubMed

    Hsieh, J F; Pan, P H

    2012-02-01

    Microbial transglutaminase (MTGase)-induced polymerization of individual milk proteins during incubation was investigated using a proteomics-based approach. The addition of MTGase (0.25-2.0 units/mL) caused the milk proteins to polymerize after a 3-h incubation period. Sodium dodecyl sulfate-PAGE analysis showed that the total intensities of the protein bands that corresponded to α(S)-casein, β-casein, and κ-casein decreased from 8,245.6, 6,677.2, and 586.6 arbitrary units to 1,911.7, 0.0, and 66.2 arbitrary units, respectively. Components with higher molecular weights were observed, and the intensity of these proteins increased after 3h of incubation. These results support that inter- or intramolecular crosslinking occurred in the casein proteins of MTGase-treated milk. Two-dimensional electrophoresis analysis indicated that isomers of β-casein, κ-casein, a fraction of serum albumin, α(S1)-casein, α(S2)-casein, β-lactoglobulin, and α-lactalbumin in the milk were polymerized following incubation with MTGase. In addition, MTGase-induced polymerization occurred earlier for β-casein and κ-casein isomers than for other milk proteins.

  17. Proteomic profiling of camel and cow milk proteins under heat treatment.

    PubMed

    Felfoul, Imène; Jardin, Julien; Gaucheron, Frédéric; Attia, Hamadi; Ayadi, M A

    2017-02-01

    Cow and camel milk proteins before and after heat treatment at 80°C for 60min were identified using LC/MS and LC-MS/MS following monodimensional electrophoresis. The database used for the identification of camel and cow proteins was set from http://www.uniprot.org/. The obtained results showed that, after heating, camel milk at 80°C for 60min, camel α-lactalbumin (α-la) and peptidoglycan recognition protein (PGRP) were not detected while camel serum albumin (CSA) was significantly diminished. When heating cow milk at 80°C for 60min, α-lactalbumin (α-la) and β-lactoglobulin (β-lg) were not significantly detected. Moreover, 19 protein bands from SDS-PAGE were analyzed and a total of 45 different proteins were identified by LC-MS/MS. Casein fractions were kept intact under a heat treatment of 80°C during 60min of both camel and cow milks. Camel and bovine whey proteins were affected by a heat treatment of 80°C for 60min. PMID:27596405

  18. The Up-Regulation of Ribosomal Proteins Further Regulates Protein Expression Profile in Female Schistosoma japonicum after Pairing

    PubMed Central

    Sun, Jun; Li, Chen; Wang, Suwen

    2015-01-01

    Background Pairing of Schistosoma males and females leads to and maintains female sexual maturation. However, the mechanism by which pairing facilitates sexual maturation of females is not clear. An increasing body of evidence suggests that ribosomal proteins have regulatory rather than constitutive roles in protein translation. Methodology/Principal Findings To investigate the effect of ribosome regulation on female sex maturation, Solexa and iTRAQ techniques were used to analyze the relationship between ribosomal gene or protein expression and sexual development of Schistosoma females. In the present study, considerably higher number of ribosomal genes or proteins were found to be differentially expressed in paired 23-day-old females. Moreover, mature female-specific proteins associated with egg production, such as ferritin-1 heavy chain and superoxide dismutase, were selectively highly expressed in paired females, rather than higher level of protein synthesis of all transcripts compared with those in unpaired 23-day-old females. Furthermore, other developmental stages were utilized to investigate different expression pattern of ribosomal proteins in females by analysing 18-day-old female schistosomula from single- or double-sex infections to determine the relationship between ribosomal protein expression pattern and development. Results showed that undeveloped 18-day-old females from single- and double-sex infections, as well as 23-day-old unpaired females, possessed similar ribosomal protein expression patterns, which were distinct from those in 23-day-old paired females. Conclusions/Significance Our findings reveal that the pairing of females and males triggers a specialized ribosomal protein expression profile which further regulates the protein profile for sexual maturation in Schistosoma japonicum, based on its gene expression profile. PMID:26070205

  19. 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

  20. Phenomenology of colloidal crystal electrophoresis

    NASA Astrophysics Data System (ADS)

    Medebach, Martin; Palberg, Thomas

    2003-08-01

    We studied the motion of polycrystalline solids comprising of charged sub-micron latex spheres suspended in deionized water. These were subjected to a low frequency alternating square wave electric field in an optical cell of rectangular cross section. Velocity profiles in X and Y direction were determined by Laser Doppler Velocimetry. The observed complex flow profiles are time dependent due to the combined effects of electro-osmosis, electrophoresis, crystal elasticity, and friction of the crystals at the cell wall. On small time scales elastic deformation occurs. On long time scales channel formation is observed. At intermediate times steady state profiles are dominated by a solid plug of polycrystalline material moving in the cell center. At large field strengths the plug shear melts. Mobilities in the shear molten state are on the order of (6.5±0.5) 10-8 m2 V-1 s-1 and connect continuously with those of the equilibrium fluid. The apparent mobility of the plug is much larger than of the fluid and like the mobility of the fluid decreases with increasing particle number density. We qualitatively attribute the accelerated motion of the plug to an incomplete exposure to the electro-osmotic flow profile.

  1. Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

    PubMed

    Walker, John; Acestor, Nathalie; Gongora, Rafael; Quadroni, Manfredo; Segura, Iris; Fasel, Nicolas; Saravia, Nancy G

    2006-02-01

    Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL. PMID:16325936

  2. Hierarchical Partitioning of Metazoan Protein Conservation Profiles Provides New Functional Insights

    PubMed Central

    Witztum, Jonathan; Persi, Erez; Horn, David; Pasmanik-Chor, Metsada; Chor, Benny

    2014-01-01

    The availability of many complete, annotated proteomes enables the systematic study of the relationships between protein conservation and functionality. We explore this question based solely on the presence or absence of protein homologues (a.k.a. conservation profiles). We study 18 metazoans, from two distinct points of view: the human's and the fly's. Using the GOrilla gene ontology (GO) analysis tool, we explore functional enrichment of the “universal proteins”, those with homologues in all 17 other species, and of the “non-universal proteins”. A large number of GO terms are strongly enriched in both human and fly universal proteins. Most of these functions are known to be essential. A smaller number of GO terms, exhibiting markedly different properties, are enriched in both human and fly non-universal proteins. We further explore the non-universal proteins, whose conservation profiles are consistent with the “tree of life” (TOL consistent), as well as the TOL inconsistent proteins. Finally, we applied Quantum Clustering to the conservation profiles of the TOL consistent proteins. Each cluster is strongly associated with one or a small number of specific monophyletic clades in the tree of life. The proteins in many of these clusters exhibit strong functional enrichment associated with the “life style” of the related clades. Most previous approaches for studying function and conservation are “bottom up”, studying protein families one by one, and separately assessing the conservation of each. By way of contrast, our approach is “top down”. We globally partition the set of all proteins hierarchically, as described above, and then identify protein families enriched within different subdivisions. While supporting previous findings, our approach also provides a tool for discovering novel relations between protein conservation profiles, functionality, and evolutionary history as represented by the tree of life. PMID:24594619

  3. 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…

  4. 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.

  5. Proteomic profiling of cellular proteins interacting with the hepatitis C virus core protein.

    PubMed

    Kang, Su-Min; Shin, Min-Jung; Kim, Jung-Hee; Oh, Jong-Won

    2005-05-01

    Hepatitis C virus (HCV) is a causative agent of chronic hepatitis and hepatocellular carcinoma. The core protein of HCV packages the viral RNA genome to form a nucleocapsid. In addition to its function as a structural protein, core protein is involved in regulation of cellular transcription, virus-induced transformation, and pathogenesis. To gain insights into cellular functions of the core protein by identification of cellular proteins interacting with the core protein, we employed a proteomic approach. Hepatocytes soluble cytoplasmic proteins were applied to the core proteins immobilized on Ni-nitrilotriacetic resin and total bound cellular proteins were resolved by 2-DE. Analyses of interacting proteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry allowed identification of 14 cellular proteins binding to the core protein. These proteins include DEAD-box polypeptide 5, similar in function to a known protein identified previously by yeast two-hybrid screening and 13 newly identified cellular proteins. Interestingly, nine protein spots were identified as intermediate microfilament proteins, including cytokeratins (five spots for cytokeratin 8, two for cytokeratin 19, and one for cytokeratin 18) and vimentin. Cytokeratin 8 and vimentin, which were previously shown to be involved in the infection processes of other viruses, were further analyzed to confirm their in vivo interactions with the core protein by immunoblotting and immunofluorescence microscopy. We discuss the functional implications of the interactions of the core protein with newly identified cellular proteins in HCV infection and pathogenesis.

  6. Protein profile of the seminal plasma of collared peccaries (Pecari tajacu Linnaeus, 1758).

    PubMed

    Santos, E A A; Sousa, P C; Martins, J A M; Moreira, R A; Monteiro-Moreira, A C O; Moreno, F B M B; Oliveira, M F; Moura, A A; Silva, A R

    2014-06-01

    This study was conducted to characterize the major proteins of the peccary seminal plasma, based on the semen samples collected from nine adult and reproductively sound animals. Our approach included the use of two-dimensional electrophoresis followed by Coomassie blue staining and analysis of polypeptide maps with PDQuest Software (Bio-Rad). Proteins were identified by tandem mass spectrometry (LC-MS/MS). We detected 179 protein spots per gel and 98 spots were identified by mass spectrometry, corresponding to 23 different proteins. The combined intensity of those spots accounted for 56.2±6% of the intensities of all spots and 60.9% of the intensities of spots presented in every protein map. Protein spots identified as clusterin represented 19.7±8.3% of the integrated optical densities of all spots detected in the seminal plasma maps. There was a negative association (r=-0.87; P<0.05) between the intensity of a clusterin spot and the percentage of sperm with functional membrane. Spermadhesin porcine seminal plasma protein 1 and bodhesin 2 comprised 5.4±1.9 and 8.8±3.9% of the total intensity of all spots respectively. Many proteins appeared in a polymorphic pattern, such as clusterin (27 spots), epididymal secretory glutathione peroxidase (ten spots), inter-α-trypsin inhibitor (12 spots), and IgG-binding protein (ten spots), among others. In conclusion, we presently describe the major seminal plasma proteome of the peccary, which exhibits a distinct high expression of clusterin isoforms. Knowledge of wild species reproductive biology is crucial for an understanding of their survival strategies and adaptation in a changing environment.

  7. Candida albicans PROTEIN PROFILE CHANGES IN RESPONSE TO THE BUTANOLIC EXTRACT OF Sapindus saponariaL.

    PubMed Central

    FIORINI, Adriana; ROSADO, Fabio Rogério; BETTEGA, Eliane Martins da Silva; MELO, Kátia Cristina Sibin; KUKOLJ, Caroline; BONFIM-MENDONÇA, Patrícia de Souza; SHINOBU-MESQUITA, Cristiane Suemi; GHIRALDI, Luciana Dias; CAMPANERUT, Paula Aline Zanetti; CAPOCI, Isis Regina Grenier; GODOY, Janine Silva Ribeiro; FERREIRA, Izabel Cristina Piloto; SVIDZINSKI, Terezinha Inez Estivalet

    2016-01-01

    Candida albicans is an opportunistic human pathogen that is capable of causing superficial and systemic infections in immunocompromised patients. Extracts of Sapindus saponaria have been used as antimicrobial agents against various organisms. In the present study, we used a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify the changes in protein abundance of C. albicans after exposure to the minimal inhibitory concentration (MIC) and sub-minimal inhibitory concentration (sub-MIC) of the butanolic extract (BUTE) of S. saponaria and also to fluconazole. A total of six different proteins with greater than 1.5 fold induction or repression relative to the untreated control cells were identified among the three treatments. In general, proteins/enzymes involved with the glycolysis (GPM1, ENO1, FBA1), amino acid metabolism (ILV5, PDC11) and protein synthesis (ASC1) pathways were detected. In conclusion, our findings reveal antifungal-induced changes in protein abundance of C. albicans. By using the previously identified components of the BUTE of S. saponaria(e.g., saponins and sesquiterpene oligoglycosides), it will be possible to compare the behavior of compounds with unknown mechanisms of action, and this knowledge will help to focus the subsequent biochemical work aimed at defining the effects of these compounds. PMID:27074319

  8. Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling.

    PubMed

    Melle, Christian; Bogumil, Ralf; Ernst, Günther; Schimmel, Bettina; Bleul, Annett; von Eggeling, Ferdinand

    2006-04-01

    Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis. PMID:16502466

  9. Protein profiling of alpha-fetoprotein producing gastric adenocarcinoma

    PubMed Central

    He, Liang; Ye, Fei; Qu, Linlin; Wang, Daguang; Cui, Miao; Wei, Chengguo; Xing, Yanpeng; Lee, Peng; Suo, Jian; Zhang, David Y.

    2016-01-01

    Alpha-fetoprotein (AFP) producing gastric adenocarcinoma is considered as a rare subtype of gastric adenocarcinoma. Compared with AFP non-producing gastric adenocarcinoma, our study and other previous studies showed that AFP producing gastric adenocarcinoma is more aggressive and prone to liver metastasis. Using the Protein Pathway Array, 11 of out of 286 proteins tested were found to be differentially expressed between AFP producing (n=32) and AFP non-producing (n=45) gastric adenocarcinoma tissues. In addition, the high level expression of XIAP and IGF-Irβ in gastric adenocarcinoma tissues was independent factors for poor prognosis in AFP producing gastric adenocarcinoma patients. A risk model based on the XIAP and IGF-Irβ expression levels can separate AFP producing gastric adenocarcinoma patients into 2 subgroups and each subgroup had a distinct set of signaling pathways involved. In conclusion, AFP producing gastric adenocarcinoma is a heterogeneous cancer with different clinical outcomes, biological behaviors and underlying molecular alterations. PMID:27057629

  10. Salivary Protein Profiles among HER2/neu-Receptor-Positive and -Negative Breast Cancer Patients: Support for Using Salivary Protein Profiles for Modeling Breast Cancer Progression

    PubMed Central

    Streckfus, Charles F.; Arreola, Daniel; Edwards, Cynthia; Bigler, Lenora

    2012-01-01

    Purpose. The objective of this study was to compare the salivary protein profiles from individuals diagnosed with breast cancer that were either HER2/neu receptor positive or negative. Methods. Two pooled saliva specimens underwent proteomic analysis. One pooled specimen was from women diagnosed with stage IIa HER2/neu-receptor-positive breast cancer patients (n = 10) and the other was from women diagnosed with stage IIa HER2/neu-receptor-negative cancer patients (n = 10). The pooled samples were trypsinized and the peptides labeled with iTRAQ reagent. Specimens were analyzed using an LC-MS/MS mass spectrometer. Results. The results yielded approximately 71 differentially expressed proteins in the saliva specimens. There were 34 upregulated proteins and 37 downregulated proteins. PMID:22570650

  11. Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling.

    PubMed

    Sjöberg, Ronald; Mattsson, Cecilia; Andersson, Eni; Hellström, Cecilia; Uhlen, Mathias; Schwenk, Jochen M; Ayoglu, Burcu; Nilsson, Peter

    2016-09-25

    High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions. PMID:26417875

  12. Distinct profiles of functional discrimination among G proteins determine the actions of G protein-coupled receptors.

    PubMed

    Masuho, Ikuo; Ostrovskaya, Olga; Kramer, Grant M; Jones, Christopher D; Xie, Keqiang; Martemyanov, Kirill A

    2015-12-01

    Members of the heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor (GPCR) family play key roles in many physiological functions and are extensively exploited pharmacologically to treat diseases. Many of the diverse effects of individual GPCRs on cellular physiology are transduced by heterotrimeric G proteins, which are composed of α, β, and γ subunits. GPCRs interact with and stimulate the binding of guanosine triphosphate (GTP) to the α subunit to initiate signaling. Mammalian genomes encode 16 different G protein α subunits, each one of which has distinct properties. We developed a single-platform, optical strategy to monitor G protein activation in live cells. With this system, we profiled the coupling ability of individual GPCRs for different α subunits, simultaneously quantifying the magnitude of the signal and the rates at which the receptors activated the G proteins. We found that individual receptors engaged multiple G proteins with varying efficacy and kinetics, generating fingerprint-like profiles. Different classes of GPCR ligands, including full and partial agonists, allosteric modulators, and antagonists, distinctly affected these fingerprints to functionally bias GPCR signaling. Finally, we showed that intracellular signaling modulators further altered the G protein-coupling profiles of GPCRs, which suggests that their differential abundance may alter signaling outcomes in a cell-specific manner. These observations suggest that the diversity of the effects of GPCRs on cellular physiology may be determined by their differential engagement of multiple G proteins, coupling to which produces signals with varying signal magnitudes and activation kinetics, properties that may be exploited pharmacologically. PMID:26628681

  13. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    PubMed

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1.54-fold (p

  14. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity.

    PubMed

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G

    2013-12-01

    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001) in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides) subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA) of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly (p < 0.05) more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH) was 1.54-fold (p

  15. 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.

  16. 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.

  17. 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.

  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. Z-scan Fluorescence Profile Deconvolution of Cytosolic and Membrane-associated Protein Populations

    PubMed Central

    Smith, Elizabeth M.; Hennen, Jared; Chen, Yan; Mueller, Joachim D.

    2015-01-01

    This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Further, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C-delta1 labeled with green fluorescent protein upon ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area to volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible. PMID:25862080

  20. Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins

    PubMed Central

    Yu, Yinni; Blair, Steve; Gillespie, David; Jensen, Randy; Myszka, David G.; Badran, Ahmed H.; Ghosh, Indraneel; Chagovetz, Alexander

    2010-01-01

    Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG), and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemi-methylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the KD for 1xMBD-GFP ranged from 106 nM to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The KD values for non-symmetrical methylation motifs were consistently greater (> 2 µM), but the binding selectivity between symmetric and hemi-methylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density. PMID:20507169

  1. Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties

    NASA Technical Reports Server (NTRS)

    Orihuela, C. J.; Janssen, R.; Robb, C. W.; Watson, D. A.; Niesel, D. W.

    2000-01-01

    We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

  2. Identifying dynamic protein complexes based on gene expression profiles and PPI networks.

    PubMed

    Li, Min; Chen, Weijie; Wang, Jianxin; Wu, Fang-Xiang; Pan, Yi

    2014-01-01

    Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of "closeness" and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481

  3. Multiplexed protein profiling on microarrays by rolling-circle amplification

    PubMed Central

    Schweitzer, Barry; Roberts, Scott; Grimwade, Brian; Shao, Weiping; Wang, Minjuan; Fu, Qin; Shu, Quiping; Laroche, Isabelle; Zhou, Zhimin; Tchernev, Velizar T.; Christiansen, Jason; Velleca, Mark; Kingsmore, Stephen F.

    2010-01-01

    Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1β, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-α treatment. Because microarrays can accommodat ~1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys. PMID:11923841

  4. Mitochondrial Protein Profile in Mice with Low or Excessive Selenium Diets

    PubMed Central

    Hu, Lianmei; Wang, Congcong; Zhang, Qin; Yan, Hao; Li, Ying; Pan, Jiaqiang; Tang, Zhaoxin

    2016-01-01

    Dietary selenium putatively prevents oxidative damage, whereas excessive selenium may lead to animal disorder. In this study, we investigated the effects of low and excessive levels of dietary selenium on oxidative stress and mitochondrial proteins in mouse liver. Six to eight week old mice were fed a diet with low, excessive, or moderate (control) levels of selenium (sodium selenite). The selenium concentration and oxidative stress-related parameters in hepatic mitochondria were evaluated. Two-dimensional electrophoresis and mass spectrometry were applied to identify the differentially-expressed proteins associated with dietary selenium. The selenium content of the livers in mice with the low selenium diet was significantly lower than that of the control, while that of mice fed excessive levels was significantly higher. In both groups oxidative stress in hepatic mitochondria was found; accompanied by lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) levels and higher malondialdehyde (MDA) content, compared with the control group. Furthermore, ten proteins in the hepatic mitochondria of the selenium-low or -excessive groups with more than two-fold differences in abundance compared with the control group were identified. The differentially-expressed proteins in hepatic mitochondria may be associated with dietary (low or excessive) selenium-induced oxidative stress. PMID:27428959

  5. Protein signaling networks from single cell fluctuations and information theory profiling.

    PubMed

    Shin, Young Shik; Remacle, F; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R D; Heath, James R

    2011-05-18

    Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

  6. Distinct protein classes in human red cell proteome revealed by similarity of phylogenetic profiles.

    PubMed

    Szczesny, Paweł; Mykowiecka, Agnieszka; Pawłowski, Krzysztof; Grynberg, Marcin

    2013-01-01

    The minimal set of proteins necessary to maintain a vertebrate cell forms an interesting core of cellular machinery. The known proteome of human red blood cell consists of about 1400 proteins. We treated this protein complement of one of the simplest human cells as a model and asked the questions on its function and origins. The proteome was mapped onto phylogenetic profiles, i.e. vectors of species possessing homologues of human proteins. A novel clustering approach was devised, utilising similarity in the phylogenetic spread of homologues as distance measure. The clustering based on phylogenetic profiles yielded several distinct protein classes differing in phylogenetic taxonomic spread, presumed evolutionary history and functional properties. Notably, small clusters of proteins common to vertebrates or Metazoa and other multicellular eukaryotes involve biological functions specific to multicellular organisms, such as apoptosis or cell-cell signaling, respectively. Also, a eukaryote-specific cluster is identified, featuring GTP-ase signalling and ubiquitination. Another cluster, made up of proteins found in most organisms, including bacteria and archaea, involves basic molecular functions such as oxidation-reduction and glycolysis. Approximately one third of erythrocyte proteins do not fall in any of the clusters, reflecting the complexity of protein evolution in comparison to our simple model. Basically, the clustering obtained divides the proteome into old and new parts, the former originating from bacterial ancestors, the latter from inventions within multicellular eukaryotes. Thus, the model human cell proteome appears to be made up of protein sets distinct in their history and biological roles. The current work shows that phylogenetic profiles concept allows protein clustering in a way relevant both to biological function and evolutionary history. PMID:23349899

  7. Protein expression profile changes in human fibroblasts induced by low dose energetic protons

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Clement, Jade Q.; Gridley, Daila S.; Rodhe, Larry H.; Wu, Honglu

    2009-12-01

    Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposures of astronauts in the space radiation environment, relies in part on the mechanistic understanding of radiation induced biological consequences at the molecular level. While some genomic data at the mRNA level are available for cells or animals exposed to radiation, the data at the protein level are still lacking. Here, we studied protein expression profile changes using Panorama antibody microarray chips that contain antibodies to 224 proteins (or their phosphorylated forms) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured until fully confluent and then exposed to 2 cGy of 150 MeV protons at high-dose rate. The proteins were isolated at 2 or 6 h after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loading onto the protein microarray chips. The intensities of the protein spots were analyzed using ScanAlyze software and normalized by the summed fluorescence intensities and the housekeeping proteins. The results showed that low dose protons altered the expression of more than 10% of the proteins listed in the microarray analysis in various protein functional groups. Cell cycle (24%) related proteins were induced by protons and most of them were regulators of G1/S-transition phase. Comparison of the overall protein expression profiles, cell cycle related proteins, cytoskeleton and signal transduction protein groups showed significantly more changes induced by protons compared with other protein functional groups.

  8. Chemically modified diamond-like carbon (DLC) for protein enrichment and profiling by MALDI-MS.

    PubMed

    Najam-ul-Haq, M; Rainer, M; Huck, C W; Ashiq, M N; Bonn, G K

    2012-08-01

    The development of new high throughput methods based on different materials with chemical modifications for protein profiling of complex mixtures leads towards biomarkers; used particularly for early diagnosis of a disease. In this work, diamond-like carbon (DLC) is developed and optimized for serum protein profiling by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). This study is carried out in connection with a material-based approach, termed as material-enhanced laser desorption ionization mass spectrometry. DLC is selected as carrier surface which provides large surface to volume ratio and offers high sensitivity. DLC has a dual role of working as MALDI target while acting as an interface for protein profiling by specifically binding peptides and proteins out of serum samples. Serum constituents are bound through immobilized metal ion affinity chromatography (IMAC) functionality, created through glycidyl methacrylate polymerization under ultraviolet light followed by further derivatization with iminodiacetic acid and copper ion loading. Scanning electron microscopy highlights the morphological characteristics of DLC surface. It could be demonstrated that IMAC functionalized DLC coatings represent a powerful material in trapping biomolecules for their further analysis by MALDI-MS resulting in improved sensitivity, specificity and capacity in comparison to other protein-profiling methods.

  9. New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts

    PubMed Central

    Aloisi, Iris; Parrotta, Luigi; Ruiz, Karina B.; Landi, Claudia; Bini, Luca; Cai, Giampiero; Biondi, Stefania; Del Duca, Stefano

    2016-01-01

    Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the

  10. New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts.

    PubMed

    Aloisi, Iris; Parrotta, Luigi; Ruiz, Karina B; Landi, Claudia; Bini, Luca; Cai, Giampiero; Biondi, Stefania; Del Duca, Stefano

    2016-01-01

    Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the

  11. New Insight into Quinoa Seed Quality under Salinity: Changes in Proteomic and Amino Acid Profiles, Phenolic Content, and Antioxidant Activity of Protein Extracts.

    PubMed

    Aloisi, Iris; Parrotta, Luigi; Ruiz, Karina B; Landi, Claudia; Bini, Luca; Cai, Giampiero; Biondi, Stefania; Del Duca, Stefano

    2016-01-01

    Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the

  12. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    PubMed

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  13. Vectorially oriented membrane protein monolayers: profile structures via x-ray interferometry/holography.

    PubMed Central

    Chupa, J A; McCauley, J P; Strongin, R M; Smith, A B; Blasie, J K; Peticolas, L J; Bean, J C

    1994-01-01

    X-ray interferometry/holography was applied to meridional x-ray diffraction data to determine uniquely the profile structures of a single monolayer of an integral membrane protein and a peripheral membrane protein, each tethered to the surface of a solid inorganic substrate. Bifunctional, organic self-assembled monolayers (SAMs) were utilized to tether the proteins to the surface of Ge/Si multilayer substrates, fabricated by molecular beam epitaxy, to facilitate the interferometric/holographic x-ray structure determination. The peripheral membrane protein yeast cytochrome c was covalently tethered to the surface of a sulfhydryl-terminated 11-siloxyundecanethiol SAM via a disulfide linkage with residue 102. The detergent-solubilized, photosynthetic reaction center integral membrane protein was electrostatically tethered to the surface of an analogous amine-terminated SAM. Optical absorption measurements performed on these two tethered protein monolayer systems were consistent with the x-ray diffraction results indicating the reversible formation of densely packed single monolayers of each fully functional membrane protein on the surface of the respective SAM. The importance of utilizing the organic self-assembled monolayers (as opposed to Langmuir-Blodgett) lies in their ability to tether specifically both soluble peripheral membrane proteins and detergent-solubilized integral membrane proteins. The vectorial orientations of the cytochrome c and the reaction center molecules were readily distinguishable in the profile structure of each monolayer at a spatial resolution of 7 A. Images FIGURE 9 FIGURE 10 PMID:7919004

  14. dRHP-PseRA: detecting remote homology proteins using profile-based pseudo protein sequence and rank aggregation.

    PubMed

    Chen, Junjie; Long, Ren; Wang, Xiao-Long; Liu, Bin; Chou, Kuo-Chen

    2016-01-01

    Protein remote homology detection is an important task in computational proteomics. Some computational methods have been proposed, which detect remote homology proteins based on different features and algorithms. As noted in previous studies, their predictive results are complementary to each other. Therefore, it is intriguing to explore whether these methods can be combined into one package so as to further enhance the performance power and application convenience. In view of this, we introduced a protein representation called profile-based pseudo protein sequence to extract the evolutionary information from the relevant profiles. Based on the concept of pseudo proteins, a new predictor, called "dRHP-PseRA", was developed by combining four state-of-the-art predictors (PSI-BLAST, HHblits, Hmmer, and Coma) via the rank aggregation approach. Cross-validation tests on a SCOP benchmark dataset have demonstrated that the new predictor has remarkably outperformed any of the existing methods for the same purpose on ROC50 scores. Accordingly, it is anticipated that dRHP-PseRA holds very high potential to become a useful high throughput tool for detecting remote homology proteins. For the convenience of most experimental scientists, a web-server for dRHP-PseRA has been established at http://bioinformatics.hitsz.edu.cn/dRHP-PseRA/. PMID:27581095

  15. dRHP-PseRA: detecting remote homology proteins using profile-based pseudo protein sequence and rank aggregation

    PubMed Central

    Chen, Junjie; Long, Ren; Wang, Xiao-long; Liu, Bin; Chou, Kuo-Chen

    2016-01-01

    Protein remote homology detection is an important task in computational proteomics. Some computational methods have been proposed, which detect remote homology proteins based on different features and algorithms. As noted in previous studies, their predictive results are complementary to each other. Therefore, it is intriguing to explore whether these methods can be combined into one package so as to further enhance the performance power and application convenience. In view of this, we introduced a protein representation called profile-based pseudo protein sequence to extract the evolutionary information from the relevant profiles. Based on the concept of pseudo proteins, a new predictor, called “dRHP-PseRA”, was developed by combining four state-of-the-art predictors (PSI-BLAST, HHblits, Hmmer, and Coma) via the rank aggregation approach. Cross-validation tests on a SCOP benchmark dataset have demonstrated that the new predictor has remarkably outperformed any of the existing methods for the same purpose on ROC50 scores. Accordingly, it is anticipated that dRHP-PseRA holds very high potential to become a useful high throughput tool for detecting remote homology proteins. For the convenience of most experimental scientists, a web-server for dRHP-PseRA has been established at http://bioinformatics.hitsz.edu.cn/dRHP-PseRA/. PMID:27581095

  16. Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

    PubMed

    Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

    2016-01-01

    BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

  17. 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.

  18. 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

  19. A SELDI-TOF approach to ecotoxicology: comparative profiling of low molecular weight proteins from a marine diatom exposed to CdSe/ZnS quantum dots.

    PubMed

    Scebba, Francesca; Tognotti, Danika; Presciuttini, Gianluca; Gabellieri, Edi; Cioni, Patrizia; Angeloni, Debora; Basso, Barbara; Morelli, Elisabetta

    2016-01-01

    Quantum dots (QDs), namely semiconductor nanocrystals, due to their particular optical and electronic properties, have growing applications in device technology, biotechnology and biomedical fields. Nevertheless, the possible threat to human health and the environment have attracted increasing attention as the production and applications of QDs increases rapidly while standard evaluation of safety lags. In the present study we performed proteomic analyses, by means of 2D gel electrophoresis and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS). We aimed to identify potential biomarkers of exposure to CdSe/ZnS quantum dots. The marine diatom Phaeodactylum tricornutum exposed to 2.5nM QDs was used as a model system. Both 2DE and SELDI showed the presence of differentially expressed proteins. By Principal Component Analysis (PCA) we were able to show that the differentially expressed proteins can discriminate between exposed and not exposed cells. Furthermore, a protein profile specific for exposed cells was obtained by SELDI analysis. To our knowledge, this is the first example of the application of SELDI technology to the analysis of microorganisms used as biological sentinel model of marine environmental pollution.

  20. Capillary zone electrophoresis

    SciTech Connect

    Jorgenson, J.W.; Lukacs, K.D.

    1983-10-21

    Zone electrophoresis in capillaries is a technique complementary to electrophoresis in supporting media, and each approach has its own particular advantages. Efficient heat transfer from small-diameter capillaries permits use of unusually high voltages, resulting in both high resolution and rapid analysis. Capillaries also seem well suited for automation. Our present electromigration injection technique is relatively straightforward and should be simple to automate. Capillaries are reusable, which is an advantage over gels. On-line electronic detection permits good quantification, further enhancing possibilities for fully automatic operation. The greatest obstacle to further development and utilization of capillaries is the requirement of extremely sensitive detectors, and more types of detectors with higher sensitivity are greatly needed. A better understanding of capillary surface modification will also be important, both for improved capillary surface deactivation and for better control over electroosmotic flow. Capillaries should provide an ideal system in which to explore nonaqueous separation media. The prospects for nonaqueous media in electrophoresis are similar to those in electrochemistry, and capillaries should prove an excellent system in which to begin their study. 18 refs., 8 figs.

  1. 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.

  2. 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.

  3. Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation.

    PubMed

    Moen, Aurora; Lind, Anne-Li; Thulin, Måns; Kamali-Moghaddam, Masood; Røe, Cecilie; Gjerstad, Johannes; Gordh, Torsten

    2016-01-01

    Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ullevål, Norway, during the period 2007-2009. The new multiplex proximity extension assay (PEA) technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR) of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase), epidermal growth factor (EGF; 142% increase), and monocyte chemotactic protein 4 (MCP-4; 70% increase). Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation. PMID:27293953

  4. Inflammatory Serum Protein Profiling of Patients with Lumbar Radicular Pain One Year after Disc Herniation

    PubMed Central

    Moen, Aurora; Lind, Anne-Li; Thulin, Måns; Kamali-Moghaddam, Masood; Røe, Cecilie; Gordh, Torsten

    2016-01-01

    Earlier studies suggest that lumbar radicular pain following disc herniation may be associated with a local or systemic inflammatory process. In the present study, we investigated the serum inflammatory protein profile of such patients. All 45 patients were recruited from Oslo University Hospital, Ullevål, Norway, during the period 2007–2009. The new multiplex proximity extension assay (PEA) technology was used to analyze the levels of 92 proteins. Interestingly, the present data showed that patients with radicular pain 12 months after disc herniation may be different from other patients with regard to many measurable serum cytokines. Given a false discovery rate (FDR) of 0.10 and 0.05, we identified 41 and 13 proteins, respectively, which were significantly upregulated in the patients with severe pain one year after disc herniation. On the top of the list ranked by estimated increase we found C-X-C motif chemokine 5 (CXCM5; 217% increase), epidermal growth factor (EGF; 142% increase), and monocyte chemotactic protein 4 (MCP-4; 70% increase). Moreover, a clear overall difference in the serum cytokine profile between the chronic and the recovered patients was demonstrated. Thus, the present results may be important for future protein serum profiling of lumbar radicular pain patients with regard to prognosis and choice of treatment. We conclude that serum proteins may be measurable molecular markers of persistent pain after disc herniation. PMID:27293953

  5. ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles.

    PubMed

    Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G; Gelly, Jean-Christophe

    2016-01-01

    Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation -with Protein Blocks-, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the 'Hard' category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/. PMID:27319297

  6. ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles

    PubMed Central

    Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G.; Gelly, Jean-Christophe

    2016-01-01

    Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation —with Protein Blocks—, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the ‘Hard’ category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/. PMID:27319297

  7. ORION: a web server for protein fold recognition and structure prediction using evolutionary hybrid profiles.

    PubMed

    Ghouzam, Yassine; Postic, Guillaume; Guerin, Pierre-Edouard; de Brevern, Alexandre G; Gelly, Jean-Christophe

    2016-06-20

    Protein structure prediction based on comparative modeling is the most efficient way to produce structural models when it can be performed. ORION is a dedicated webserver based on a new strategy that performs this task. The identification by ORION of suitable templates is performed using an original profile-profile approach that combines sequence and structure evolution information. Structure evolution information is encoded into profiles using structural features, such as solvent accessibility and local conformation -with Protein Blocks-, which give an accurate description of the local protein structure. ORION has recently been improved, increasing by 5% the quality of its results. The ORION web server accepts a single protein sequence as input and searches homologous protein structures within minutes. Various databases such as PDB, SCOP and HOMSTRAD can be mined to find an appropriate structural template. For the modeling step, a protein 3D structure can be directly obtained from the selected template by MODELLER and displayed with global and local quality model estimation measures. The sequence and the predicted structure of 4 examples from the CAMEO server and a recent CASP11 target from the 'Hard' category (T0818-D1) are shown as pertinent examples. Our web server is accessible at http://www.dsimb.inserm.fr/ORION/.

  8. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  9. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

    PubMed

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  10. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  11. Expression profiling of a complex thaumatin-like protein family in western white pine.

    PubMed

    Liu, Jun-Jun; Zamani, Arezoo; Ekramoddoullah, Abul K M

    2010-02-01

    The protein content in the plant apoplast is believed to change dramatically as a result of host defense response upon infection with various pathogens. In this study, six novel thaumatin-like proteins (TLPs) were identified in western white pine (Pinus monticola) needle apoplast by a proteomic strategy using two-dimensional protein electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sequent cDNA cloning found that ten P. monticola TLP genes (PmTLP-L1 to -L6 and -S1 to -S4) were expressed in various tissues. Phylogenetic analysis demonstrated that these PmTLP genes belong to a large, complex, and highly diverse plant TLP family. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) using gene-specific primer pairs showed that each PmTLP gene exhibited a characteristic pattern of mRNA expression based on their unique organ distribution, seasonal regulation, and response to abiotic and biotic stresses. A time-course analysis at the early stages of infection by white pine blister rust pathogen Cronartium ribicola revealed that a coordinated upregulation of multiple PmTLP genes was involved in P. monticola major gene (Cr2) resistance. The structural and expressional differentiations suggest that the PmTLP family may contribute to host defense as well as other mechanism. PMID:19997927

  12. Different Polar Metabolites and Protein Profiles between High- and Low-Quality Japanese Ginjo Sake

    PubMed Central

    Takahashi, Kei; Kohno, Hiromi

    2016-01-01

    Japanese ginjo sake is a premium refined sake characterized by a pleasant fruity apple-like flavor and a sophisticated taste. Because of technical difficulties inherent in brewing ginjo sake, off-flavors sometimes occur. However, the metabolites responsible for off-flavors as well as those present or absent in higher quality ginjo sake remain uncertain. Here, the relationship between 202 polar chemical compounds in sake identified using capillary electrophoresis coupled with time-of-flight mass spectrometry and its organoleptic properties, such as quality and off-flavor, was examined. First, we found that some off-flavored sakes contained higher total amounts of metabolites than other sake samples. The results also identified that levels of 2-oxoglutaric acid and fumaric acid, metabolites in the tricarboxylic acid cycle, were highly but oppositely correlated with ginjo sake quality. Similarly, pyridoxine and pyridoxamine, co-enzymes for amino transferase, were also highly but oppositely correlated with ginjo sake quality. Additionally, pyruvic acid levels were associated with good quality as well. Compounds involved in the methionine salvage cycle, oxidative glutathione derivatives, and amino acid catabolites were correlated with low quality. Among off-flavors, an inharmonious bitter taste appeared attributable to polyamines. Furthermore, protein analysis displayed that a diversity of protein components and yeast protein (triosephosphate isomerase, TPI) leakage was linked to the overall metabolite intensity in ginjo sake. This research provides insight into the relationship between sake components and organoleptic properties. PMID:26939054

  13. Different Polar Metabolites and Protein Profiles between High- and Low-Quality Japanese Ginjo Sake.

    PubMed

    Takahashi, Kei; Kohno, Hiromi

    2016-01-01

    Japanese ginjo sake is a premium refined sake characterized by a pleasant fruity apple-like flavor and a sophisticated taste. Because of technical difficulties inherent in brewing ginjo sake, off-flavors sometimes occur. However, the metabolites responsible for off-flavors as well as those present or absent in higher quality ginjo sake remain uncertain. Here, the relationship between 202 polar chemical compounds in sake identified using capillary electrophoresis coupled with time-of-flight mass spectrometry and its organoleptic properties, such as quality and off-flavor, was examined. First, we found that some off-flavored sakes contained higher total amounts of metabolites than other sake samples. The results also identified that levels of 2-oxoglutaric acid and fumaric acid, metabolites in the tricarboxylic acid cycle, were highly but oppositely correlated with ginjo sake quality. Similarly, pyridoxine and pyridoxamine, co-enzymes for amino transferase, were also highly but oppositely correlated with ginjo sake quality. Additionally, pyruvic acid levels were associated with good quality as well. Compounds involved in the methionine salvage cycle, oxidative glutathione derivatives, and amino acid catabolites were correlated with low quality. Among off-flavors, an inharmonious bitter taste appeared attributable to polyamines. Furthermore, protein analysis displayed that a diversity of protein components and yeast protein (triosephosphate isomerase, TPI) leakage was linked to the overall metabolite intensity in ginjo sake. This research provides insi