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Sample records for intact protein profiling

  1. Proteomic profiling of intact proteins using WAX-RPLC 2-D separations and FTICR mass spectrometry

    SciTech Connect

    Sharma, Seema; Simpson, David C.; Tolic, Nikola; Jaitly, Navdeep; Mayampurath, Anoop M.; Smith, Richard D.; Pasa-Tolic, Liljiana

    2007-02-01

    We investigated the combination of weak anion exchange (WAX) fractionation and on-line reversed phase liquid chromatography (RPLC) separation using a 12 T FTICR mass spectrometer for the detection of intact proteins from a Shewanella oneidensis MR-1 cell lysate. 715 intact proteins were detected and the combined results from the WAX fractions and the unfractionated cell lysate were aligned using LC-MS features to facilitate protein abundance measurements. Protein identifications and post translational modifications were assigned for ~10% of the detected proteins by comparing intact protein mass measurements to proteins identified in peptide MS/MS analysis of an aliquot of the same fraction. Intact proteins were also detected for S. oneidensis lysates obtained from cells grown on 13C, 15N depleted media under aerobic and sub-oxic conditions. This work aimed at optimizing intact protein detection for profiling proteins at a level that incorporates their modification complement. The strategy can be readily applied for measuring differential protein abundances, and provides a platform for high-throughput selection of biologically relevant targets for further characterization.

  2. (E)-Propyl α-Cyano-4-Hydroxyl Cinnamylate: A High Sensitive and Salt Tolerant Matrix for Intact Protein Profiling by MALDI Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Wang, Sheng; Xiao, Zhaohui; Xiao, Chunsheng; Wang, Huixin; Wang, Bing; Li, Ying; Chen, Xuesi; Guo, Xinhua

    2016-04-01

    Low-abundance samples and salt interference are always of great challenges for the practical protein profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Herein, a series of carboxyl-esterified derivatives of α-cyano-4-hydroxycinnamic acid (CHCA) were synthesized and evaluated as matrices for MALDI-MS analysis of protein. Among them, (E)-propyl α-cyano-4-hydroxyl cinnamylate (CHCA-C3) was found to exhibit excellent assay performance for intact proteins by improving the detection sensitivity 10 folds compared with the traditional matrices [i.e., super2,5-dihydroxybenzoic acid (superDHB), sinapic acid (SA), and CHCA]. In addition, CHCA-C3 was shown to have high tolerance to salts, the ion signal of myoglobin was readily detected even in the presence of urea (8 M), NH4HCO3 (2 M), and KH2PO4 (500 mM), meanwhile sample washability was robust. These achievements were mainly attributed to improved ablation ability and increased hydrophobicity or affinity of CHCA-C3 to proteins in comparison with hydrophilic matrixes, leading to more efficient ionization of analyte. Furthermore, direct analysis of proteins from crude egg white demonstrated that CHCA-C3 was a highly efficient matrix for the analysis of low-abundance proteins in complex biological samples. These outstanding performances indicate the tremendous potential use of CHCA-C3 in protein profiling by MALDI-MS.

  3. Discrimination of different species from the genus Drosophila by intact protein profiling using matrix-assisted laser desorption ionization mass spectrometry

    PubMed Central

    2010-01-01

    Background The use of molecular biology-based methods for species identification and establishing phylogenetic relationships has supplanted traditional methods relying on morphological characteristics. While PCR-based methods are now the commonly accepted gold standards for these types of analysis, relatively high costs, time-consuming assay development or the need for a priori information about species-specific sequences constitute major limitations. In the present study, we explored the possibility to differentiate between 13 different species from the genus Drosophila via a molecular proteomic approach. Results After establishing a simple protein extraction procedure and performing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) with intact proteins and peptides, we could show that most of the species investigated reproducibly yielded mass spectra that were adequate for species classification. Furthermore, a dendrogram generated by cluster analysis of total protein patterns agrees reasonably well with established phylogenetic relationships. Conclusion Considering the intra- and interspecies similarities and differences between spectra obtained for specimens of closely related Drosophila species, we estimate that species typing of insects and possibly other multicellular organisms by intact protein profiling (IPP) can be established successfully for species that diverged from a common ancestor about 3 million years ago. PMID:20374617

  4. Mass Spectrometry of Intact Membrane Protein Complexes

    PubMed Central

    Laganowsky, Arthur; Reading, Eamonn; Hopper, Jonathan T.S.; Robinson, Carol V.

    2014-01-01

    Mass spectrometry of intact soluble protein complexes has emerged as a powerful technique to study the stoichiometry, structure-function and dynamics of protein assemblies. Recent developments have extended this technique to the study of membrane protein complexes where it has already revealed subunit stoichiometries and specific phospholipid interactions. Here, we describe a protocol for mass spectrometry of membrane protein complexes. The protocol begins with preparation of the membrane protein complex enabling not only the direct assessment of stoichiometry, delipidation, and quality of the target complex, but also evaluation of the purification strategy. A detailed list of compatible non-ionic detergents is included, along with a protocol for screening detergents to find an optimal one for mass spectrometry, biochemical and structural studies. This protocol also covers the preparation of lipids for protein-lipid binding studies and includes detailed settings for a Q-ToF mass spectrometer after introduction of complexes from gold-coated nanoflow capillaries. PMID:23471109

  5. Protein methylation reactions in intact pea chloroplasts

    SciTech Connect

    Niemi, K.J. )

    1989-04-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ({sup 3}H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented.

  6. Shining a spotlight on intact proteins

    SciTech Connect

    Pasa-Tolic, Ljiljana; Masselon, Christophe

    2014-05-01

    Cells react to cues from their environment using various mechanisms that include changes in metabolites, gene expression, protein binding partners, protein localization, and protein posttranslational modifications (PTMs), all of which contribute to altered cellular signatures that enable appropriate cellular responses. Given the seemingly infinite number of mechanisms available to affect protein function and modulate biological processes, the question arises as to how cells manage to interpret protein readouts to accomplish the appropriate cell-type specific response to a particular stimulus.

  7. Structural determination of intact proteins using mass spectrometry

    DOEpatents

    Kruppa, Gary; Schoeniger, Joseph S.; Young, Malin M.

    2008-05-06

    The present invention relates to novel methods of determining the sequence and structure of proteins. Specifically, the present invention allows for the analysis of intact proteins within a mass spectrometer. Therefore, preparatory separations need not be performed prior to introducing a protein sample into the mass spectrometer. Also disclosed herein are new instrumental developments for enhancing the signal from the desired modified proteins, methods for producing controlled protein fragments in the mass spectrometer, eliminating complex microseparations, and protein preparatory chemical steps necessary for cross-linking based protein structure determination.Additionally, the preferred method of the present invention involves the determination of protein structures utilizing a top-down analysis of protein structures to search for covalent modifications. In the preferred method, intact proteins are ionized and fragmented within the mass spectrometer.

  8. Intracellular Delivery of Proteins via Fusion Peptides in Intact Plants

    PubMed Central

    Ng, Kiaw Kiaw; Motoda, Yoko; Watanabe, Satoru; Sofiman Othman, Ahmad; Kigawa, Takanori; Kodama, Yutaka; Numata, Keiji

    2016-01-01

    In current plant biotechnology, the introduction of exogenous DNA encoding desired traits is the most common approach used to modify plants. However, general plant transformation methods can cause random integration of exogenous DNA into the plant genome. To avoid these events, alternative methods, such as a direct protein delivery system, are needed to modify the plant. Although there have been reports of the delivery of proteins into cultured plant cells, there are currently no methods for the direct delivery of proteins into intact plants, owing to their hierarchical structures. Here, we demonstrate the efficient fusion-peptide-based delivery of proteins into intact Arabidopsis thaliana. Bovine serum albumin (BSA, 66 kDa) was selected as a model protein to optimize conditions for delivery into the cytosol. The general applicability of our method to large protein cargo was also demonstrated by the delivery of alcohol dehydrogenase (ADH, 150 kDa) into the cytosol. The compatibility of the fusion peptide system with the delivery of proteins to specific cellular organelles was also demonstrated using the fluorescent protein Citrine (27 kDa) conjugated to either a nuclear localization signal (NLS) or a peroxisomal targeting signal (PTS). In conclusion, our designed fusion peptide system can deliver proteins with a wide range of molecular weights (27 to 150 kDa) into the cells of intact A. thaliana without interfering with the organelle-targeting peptide conjugated to the protein. We expect that this efficient protein delivery system will be a powerful tool in plant biotechnology. PMID:27100681

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

    PubMed

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

    2016-01-01

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

  10. Integrating Mass Spectrometry of Intact Protein Complexes into Structural Proteomics

    PubMed Central

    Hyung, Suk-Joon; Ruotolo, Brandon T.

    2013-01-01

    Summary Mass spectrometry analysis of intact protein complexes has emerged as an established technology for assessing the composition and connectivity within dynamic, heterogeneous multiprotein complexes at low concentrations and in the context of mixtures. As this technology continues to move forward, one of the main challenges is to integrate the information content of such intact protein complex measurements with other mass spectrometry approaches in structural biology. Methods such as H/D exchange, oxidative foot-printing, chemical cross-linking, affinity purification, and ion mobility separation add complementary information that allows access to every level of protein structure and organization. Here, we survey the structural information that can be retrieved by such experiments, demonstrate the applicability of integrative mass spectrometry approaches in structural proteomics, and look to the future to explore upcoming innovations in this rapidly-advancing area. PMID:22611037

  11. Selective dansylation of M protein within intact influenza virions

    SciTech Connect

    Robertson, B.H.; Bennett, J.C.; Compans, R.W.

    1982-12-01

    Exposure of purified influenza virions to (/sup 14/C)dansyl chloride resulted in the covalent attachment of the dansyl chromophore to the virion. Gel electrophoresis revealed that the dansyl chromophore was specifically coupled to the internal membrane (M) protein. Purification of the M protein by gel filtration followed by cyanogen bromide cleavage and peptide fractionation revealed that four of six peptide peaks contained dansyl label. Acid hydrolysis of the separated peptide peaks followed by thin-layer chromatography revealed that dansyl label was coupled to lysine residues present in these peptides. The results of these investigations have demonstrated that the M protein molecule is the major viral polypeptide labeled when intact virions are exposed to dansyl chloride.

  12. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

    PubMed Central

    Whitelegge, J. P.; Gundersen, C. B.; Faull, K. F.

    1998-01-01

    Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments. PMID:9655347

  13. THE ENDOCRINE PROFILE OF INTACT FEMALE RATS ON THE DAY OF PROESTRUS FOLLOWING EXPOSURE TO ATRAZINE

    EPA Science Inventory

    The Endocrine Profile of Intact Female Rats on the Day of Proestrus Following Exposure to Atrazine.
    RL Cooper, A Buckalew, SC Laws and TE Stoker
    Endocrinology Branch, RTD, NHEERL, ORD, U.S. EPA, RTP, NC, 27711.

    The chlorotriazine herbicide, atrazine, has been sho...

  14. Quantitative Measurement of Intact Alpha-Synuclein Proteoforms from Post-Mortem Control and Parkinson's Disease Brain Tissue by Intact Protein Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Kellie, John F.; Higgs, Richard E.; Ryder, John W.; Major, Anthony; Beach, Thomas G.; Adler, Charles H.; Merchant, Kalpana; Knierman, Michael D.

    2014-07-01

    A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

  15. Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry.

    PubMed

    Kellie, John F; Higgs, Richard E; Ryder, John W; Major, Anthony; Beach, Thomas G; Adler, Charles H; Merchant, Kalpana; Knierman, Michael D

    2014-01-01

    A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03). PMID:25052239

  16. Quantification of protein isoforms in mesenchymal stem cells by reductive dimethylation of lysines in intact proteins.

    PubMed

    She, Yi-Min; Rosu-Myles, Michael; Walrond, Lisa; Cyr, Terry D

    2012-02-01

    Mass spectrometry (MS)-based quantification of highly homologous proteins in complex samples has proven difficult due to subtle sequence variations and the wide dynamic range of protein isoforms present. Herein, we report the use of reductive dimethylation on intact proteins to quantitatively compare protein isoform expression in the nucleus and cytoplasm of mesenchymal stem cells (MSC) and normal stroma. By coupling fixed-charge MS/MS scanning, high-resolution UPLC FT-MS data-dependent acquisition and MASCOT-based data mining, hydrogen/deuterium-labeled dimethyl-lysine peptides were simultaneously captured allowing the accurate comparison of 123 protein isoforms in parallel LC MS/MS runs. Thirty-four isoforms were identified that had expression levels specific to MSC. Where possible, proteomic analyses were verified by Western blotting and were demonstrated to be divergent from the level of gene transcription detected for certain proteins. Our analysis provides a protein isoform signature specific to MSC and demonstrates the suitability of dimethyl-lysine labeling on intact proteins for quantifying highly homologous proteins on a proteome-wide scale.

  17. Radioiodination of an outer membrane protein in intact Rickettsia prowazekii

    SciTech Connect

    Smith, D.K.; Winkler, H.H.

    1980-08-01

    Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the /sup 125/I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane.

  18. Population Studies of Intact Vitamin D Binding Protein by Affinity Capture ESI-TOF-MS

    PubMed Central

    Borges, Chad R.; Jarvis, Jason W.; Oran, Paul E.; Rogers, Stephen P.; Nelson, Randall W.

    2008-01-01

    Blood plasma proteins with molecular weights greater than approximately 30 kDa are refractory to comprehensive, high-throughput qualitative characterization of microheterogeneity across human populations. Analytical techniques for obtaining high mass resolution for targeted, intact protein characterization and, separately, high sample throughput exist, but efficient means of coupling these assay characteristics remain rather limited. This article discusses the impetus for analyzing intact proteins in a targeted manner across populations and describes the methodology required to couple mass spectrometric immunoassay with electrospray ionization mass spectrometry for the purpose of qualitatively characterizing a prototypical large plasma protein, vitamin D binding protein, across populations. PMID:19137103

  19. Modeling of Recovery Profiles in Mentally Disabled and Intact Patients after Sevoflurane Anesthesia; A Pharmacodynamic Analysis

    PubMed Central

    Shin, Teo Jeon; Noh, Gyu-Jeong; Koo, Yong-Seo

    2014-01-01

    Purpose Mentally disabled patients show different recovery profiles compared to normal patients after general anesthesia. However, the relationship of dose-recovery profiles of mentally disabled patients has never been compared to that of normal patients. Materials and Methods Twenty patients (10 mentally disabled patients and 10 mentally intact patients) scheduled to dental surgery under general anesthesia was recruited. Sevoflurane was administered to maintain anesthesia during dental treatment. At the end of the surgery, sevoflurane was discontinued. End-tidal sevoflurane and recovery of consciousness (ROC) were recorded after sevoflurane discontinuation. The pharmacodynamic relation between the probability of ROC and end-tidal sevoflurane concentration was analyzed using NONMEM software (version VII). Results End-tidal sevoflurane concentration associated with 50% probability of ROC (C50) and γ value were lower in the mentally disabled patients (C50=0.37 vol %, γ=16.5 in mentally intact patients, C50=0.19 vol %, γ=4.58 in mentally disabled patients). Mentality was a significant covariate of C50 for ROC and γ value to pharmacodynamic model. Conclusion A sigmoid Emanx model explains the pharmacodynamic relationship between end-tidal sevoflurane concentration and ROC. Mentally disabled patients may recover slower from anesthesia at lower sevoflurane concentration at ROC an compared to normal patients. PMID:25323901

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

    NASA Astrophysics Data System (ADS)

    Wang, Evelyn H.; Combe, Peter C.; Schug, Kevin A.

    2016-05-01

    Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 μg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

  1. Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry

    NASA Astrophysics Data System (ADS)

    Chi, An; Bai, Dina L.; Geer, Lewis Y.; Shabanowitz, Jeffrey; Hunt, Donald F.

    2007-01-01

    Direct analysis of intact proteins on a chromatographic time scale is demonstrated on a modified linear ion trap mass spectrometer using sequential ion/ion reactions, electron transfer and proton transfer, to dissociate the sample and to convert the resulting peptide fragments to a mixture of singly and doubly charged species. Proteins are converted to gas-phase, multiply charged, positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random fragmentation of amide bonds along the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion/ion reaction with even-electron benzoate anions. m/z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 amino acids at both the N-terminus and the C-terminus of the protein. This information, along with the measured mass of the intact protein, are employed to identify known proteins and to detect the presence of post-translational modifications. In this study, we analyze intact proteins from the Escherchia coli 70S ribosomal protein complex and identify 46 of the 55 known unique components in a single, 90 min, on-line, chromatography experiment. Truncated versions of the above proteins along with several post-translational modifications are also detected.

  2. Near-infrared reflectance spectroscopy for predicting amino acids content in intact processed animal proteins.

    PubMed

    De la Haba, Maria José; Garrido-Varo, Ana; Guerrero-Ginel, José Emilio; Pérez-Marín, Dolores C

    2006-10-01

    Near-infrared calibrations were developed for the instantaneous prediction of amino acids composition of processed animal proteins (PAPs). Two sample presentation modes were compared (ground vs intact) for demonstrating the viability of the analysis in the intact form, avoiding the need for milling. Modified partial least-squares (MPLS) equations for the prediction of amino acids in PAPs were developed using the same set of samples (N = 92 PAPs) analyzed in ground and intact form and in three cups differing in the optical window size. The standard error for cross validation (SECV) and the coefficient of determination (1-VR) values yielded with the calibrations developed using the samples analyzed in the intact form showed similar or even better accuracy than those obtained with finely ground samples. The excellent predictive ability (1-VR > 0.90; CV < 3.0%) obtained for the prediction of amino acids in intact processed animal proteins opens an enormous expectative for the on-line implementation of NIRS technology in the processing and marketing of these important protein feed ingredients, alleviating the costs and time associated with the routine quality controls.

  3. 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.

  4. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation.

    PubMed

    Eitzinger, Nicole; Wagner, Volker; Weisheit, Wolfram; Geimer, Stefan; Boness, David; Kreimer, Georg; Mittag, Maria

    2015-01-01

    Flagellate green algae possess a visual system, the eyespot. In Chlamydomonas reinhardtii it is situated at the edge of the chloroplast and consists of two carotenoid rich lipid globule layers subtended by thylakoid membranes (TM) that are attached to both chloroplast envelope membranes and a specialized area of the plasma membrane (PM). A former analysis of an eyespot fraction identified 203 proteins. To increase the understanding of eyespot related processes, knowledge of the protein composition of the membranes in its close vicinity is desirable. Here, we present a purification procedure that allows isolation of intact eyespots. This gain in intactness goes, however, hand in hand with an increase of contaminants from other organelles. Proteomic analysis identified 742 proteins. Novel candidates include proteins for eyespot development, retina-related proteins, ion pumps, and membrane-associated proteins, calcium sensing proteins as well as kinases, phosphatases and 14-3-3 proteins. Methylation of proteins at Arg or Lys is known as an important posttranslational modification involved in, e.g., signal transduction. Here, we identify several proteins from eyespot fractions that are methylated at Arg and/or Lys. Among them is the eyespot specific SOUL3 protein that influences the size and position of the eyespot and EYE2, a protein important for its development. PMID:26697039

  5. Proteomic Analysis of a Fraction with Intact Eyespots of Chlamydomonas reinhardtii and Assignment of Protein Methylation

    PubMed Central

    Eitzinger, Nicole; Wagner, Volker; Weisheit, Wolfram; Geimer, Stefan; Boness, David; Kreimer, Georg; Mittag, Maria

    2015-01-01

    Flagellate green algae possess a visual system, the eyespot. In Chlamydomonas reinhardtii it is situated at the edge of the chloroplast and consists of two carotenoid rich lipid globule layers subtended by thylakoid membranes (TM) that are attached to both chloroplast envelope membranes and a specialized area of the plasma membrane (PM). A former analysis of an eyespot fraction identified 203 proteins. To increase the understanding of eyespot related processes, knowledge of the protein composition of the membranes in its close vicinity is desirable. Here, we present a purification procedure that allows isolation of intact eyespots. This gain in intactness goes, however, hand in hand with an increase of contaminants from other organelles. Proteomic analysis identified 742 proteins. Novel candidates include proteins for eyespot development, retina-related proteins, ion pumps, and membrane-associated proteins, calcium sensing proteins as well as kinases, phosphatases and 14-3-3 proteins. Methylation of proteins at Arg or Lys is known as an important posttranslational modification involved in, e.g., signal transduction. Here, we identify several proteins from eyespot fractions that are methylated at Arg and/or Lys. Among them is the eyespot specific SOUL3 protein that influences the size and position of the eyespot and EYE2, a protein important for its development. PMID:26697039

  6. MALDI FTICR IMS of Intact Proteins: Using Mass Accuracy to Link Protein Images with Proteomics Data

    NASA Astrophysics Data System (ADS)

    Spraggins, Jeffrey M.; Rizzo, David G.; Moore, Jessica L.; Rose, Kristie L.; Hammer, Neal D.; Skaar, Eric P.; Caprioli, Richard M.

    2015-06-01

    MALDI imaging mass spectrometry is a highly sensitive and selective tool used to visualize biomolecules in tissue. However, identification of detected proteins remains a difficult task. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. Here, we demonstrate the capabilities of MALDI FTICR MS for imaging intact proteins. MALDI FTICR IMS provides an unprecedented combination of mass resolving power (~75,000 at m/z 5000) and accuracy (<5ppm) for proteins up to ~12kDa, enabling identification based on correlation with LC-MS/MS proteomics data. Analysis of rat brain tissue was performed as a proof-of-concept highlighting the capabilities of this approach by imaging and identifying a number of proteins including N-terminally acetylated thymosin β4 ( m/z 4,963.502, 0.6ppm) and ATP synthase subunit ɛ ( m/z 5,636.074, -2.3ppm). MALDI FTICR IMS was also used to differentiate a series of oxidation products of S100A8 ( m/z 10,164.03, -2.1ppm), a subunit of the heterodimer calprotectin, in kidney tissue from mice infected with Staphylococcus aureus. S100A8 - M37O/C42O3 ( m/z 10228.00, -2.6ppm) was found to co-localize with bacterial microcolonies at the center of infectious foci. The ability of MALDI FTICR IMS to distinguish S100A8 modifications is critical to understanding calprotectin's roll in nutritional immunity.

  7. Tolerance of a standard intact protein formula versus a partially hydrolyzed formula in healthy, term infants

    PubMed Central

    Berseth, Carol Lynn; Mitmesser, Susan Hazels; Ziegler, Ekhard E; Marunycz, John D; Vanderhoof, Jon

    2009-01-01

    Background Parents who perceive common infant behaviors as formula intolerance-related often switch formulas without consulting a health professional. Up to one-half of formula-fed infants experience a formula change during the first six months of life. Methods The objective of this study was to assess discontinuance due to study physician-assessed formula intolerance in healthy, term infants. Infants (335) were randomized to receive either a standard intact cow milk protein formula (INTACT) or a partially hydrolyzed cow milk protein formula (PH) in a 60 day non-inferiority trial. Discontinuance due to study physician-assessed formula intolerance was the primary outcome. Secondary outcomes included number of infants who discontinued for any reason, including parent-assessed. Results Formula intolerance between groups (INTACT, 12.3% vs. PH, 13.7%) was similar for infants who completed the study or discontinued due to study physician-assessed formula intolerance. Overall study discontinuance based on parent- vs. study physician-assessed intolerance for all infants (14.4 vs.11.1%) was significantly different (P = 0.001). Conclusion This study demonstrated no difference in infant tolerance of intact vs. partially hydrolyzed cow milk protein formulas for healthy, term infants over a 60-day feeding trial, suggesting nonstandard partially hydrolyzed formulas are not necessary as a first-choice for healthy infants. Parents frequently perceived infant behavior as formula intolerance, paralleling previous reports of unnecessary formula changes. Trial Registration clinicaltrials.gov: NCT00666120 PMID:19545360

  8. Polyvinylpyrrolidone microneedles enable delivery of intact proteins for diagnostic and therapeutic applications.

    PubMed

    Sun, Wenchao; Araci, Zeynep; Inayathullah, Mohammed; Manickam, Sathish; Zhang, Xuexiang; Bruce, Marc A; Marinkovich, M Peter; Lane, Alfred T; Milla, Carlos; Rajadas, Jayakumar; Butte, Manish J

    2013-08-01

    We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy.

  9. 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.

  10. In vitro bioactive properties of intact and enzymatically hydrolysed whey protein: targeting the enteroinsular axis.

    PubMed

    Power-Grant, O; Bruen, C; Brennan, L; Giblin, L; Jakeman, P; FitzGerald, R J

    2015-03-01

    Enzymatically hydrolysed milk proteins have a variety of biofunctional effects some of which may be beneficial in the management of type 2 diabetes mellitus. The purpose of this study was to evaluate the effect of commercially available intact and hydrolysed whey protein ingredients (DH 32, DH 45) on markers of the enteroinsular axis (glucagon like peptide-1 secretion, dipeptidyl peptidase IV inhibition, insulin secretion and antioxidant activity) before and after simulated gastrointestinal digestion (SGID). A whey protein hydrolysate, DH32, significantly enhanced (P < 0.05) insulin secretion from BRIN BD11 β-cells compared to the positive control (16.7 mM glucose and 10 mM Ala). The whey protein hydrolysates inhibited dipeptidyl peptidase IV activity, yielding half maximal inhibitory concentration values (IC50) of 1.5 ± 0.1 and 1.1 ± 0.1 mg mL(-1) for the DH 32 and DH 45, samples respectively, and were significantly more potent than the intact whey (P < 0.05). Enzymatic hydrolysis of whey protein significantly enhanced (P < 0.05) its antioxidant activity compared to intact whey, as measured by the oxygen radical absorbance capacity assay (ORAC). This antioxidant activity was maintained (DH 32, P > 0.05) or enhanced (DH 45, P < 0.05) following SGID. Intact whey stimulated GLP-1 secretion from enteroendocrine cells compared to vehicle control (P < 0.05). This data confirm that whey proteins and peptides can act through multiple targets within the enteroinsular axis and as such may have glucoregulatory potential.

  11. Enhanced Dissociation of Intact Proteins with High Capacity Electron Transfer Dissociation

    NASA Astrophysics Data System (ADS)

    Riley, Nicholas M.; Mullen, Christopher; Weisbrod, Chad R.; Sharma, Seema; Senko, Michael W.; Zabrouskov, Vlad; Westphall, Michael S.; Syka, John E. P.; Coon, Joshua J.

    2016-03-01

    Electron transfer dissociation (ETD) is a valuable tool for protein sequence analysis, especially for the fragmentation of intact proteins. However, low product ion signal-to-noise often requires some degree of signal averaging to achieve high quality MS/MS spectra of intact proteins. Here we describe a new implementation of ETD on the newest generation of quadrupole-Orbitrap-linear ion trap Tribrid, the Orbitrap Fusion Lumos, for improved product ion signal-to-noise via ETD reactions on larger precursor populations. In this new high precursor capacity ETD implementation, precursor cations are accumulated in the center section of the high pressure cell in the dual pressure linear ion trap prior to charge-sign independent trapping, rather than precursor ion sequestration in only the back section as is done for standard ETD. This new scheme increases the charge capacity of the precursor accumulation event, enabling storage of approximately 3-fold more precursor charges. High capacity ETD boosts the number of matching fragments identified in a single MS/MS event, reducing the need for spectral averaging. These improvements in intra-scan dynamic range via reaction of larger precursor populations, which have been previously demonstrated through custom modified hardware, are now available on a commercial platform, offering considerable benefits for intact protein analysis and top down proteomics. In this work, we characterize the advantages of high precursor capacity ETD through studies with myoglobin and carbonic anhydrase.

  12. Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Samperi, Roberto; Laganà, Aldo

    2011-12-01

    Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.

  13. Web and database software for identification of intact proteins using "top down" mass spectrometry.

    PubMed

    Taylor, Gregory K; Kim, Yong-Bin; Forbes, Andrew J; Meng, Fanyu; McCarthy, Ryan; Kelleher, Neil L

    2003-08-15

    For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/). PMID:14632120

  14. Web and database software for identification of intact proteins using "top down" mass spectrometry.

    PubMed

    Taylor, Gregory K; Kim, Yong-Bin; Forbes, Andrew J; Meng, Fanyu; McCarthy, Ryan; Kelleher, Neil L

    2003-08-15

    For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).

  15. Quantification of intact covalently metal labeled proteins using ESI-MS/MS.

    PubMed

    Benda, David; Schwarz, Gunnar; Beck, Sebastian; Linscheid, Michael W

    2014-01-01

    Mass spectrometric methods matured from the successful qualitative characterization of proteins in complex mixtures into methods for quantitative proteomics often based on chemical tags with stable isotope labeling. In the study presented here, we extended the application of lanthanide-ion-based tags from the quantification using inductively coupled plasma-MS into the quantification of labeled intact proteins using electrospray ionization (ESI)-MS and ESI-MS/MS. We applied the metal chelate tag MeCAT-iodoacetamide (IA) (1,4,7,10-tetraazacyclododecane N,N',N″,N″ '-tetra acetic acid with a IA reactive site). Labeled proteins were separated using C3-reversed phase-high-performance liquid chromatography interfaced to ESI-MS. We could prove that even large proteins were completely labeled at all available cysteine residues using MeCAT-IA with only a small excess of reagent. Fragmentation of labeled proteins either using infrared multiphoton dissociation in Fourier transform ion cyclotron resonance-MS or higher-energy collision dissociation with an Orbitrap gave characteristic fragments. We used these fragments to quantify several intact proteins avoiding digestion. To demonstrate the applicability, human serum albumin was quantified in blood serum. The high-performance liquid chromatography/ESI-MS/MS quantification data were validated using inductively coupled plasma-MS. Because the metal within the tag may be any of the lanthanides, multiplexing capabilities are inherent.

  16. Singlet oxygen triplet energy transfer-based imaging technology for mapping protein-protein proximity in intact cells.

    PubMed

    To, Tsz-Leung; Fadul, Michael J; Shu, Xiaokun

    2014-01-01

    Many cellular processes are carried out by large protein complexes that can span several tens of nanometres. Whereas forster resonance energy transfer has a detection range of <10 nm, here we report the theoretical development and experimental demonstration of a new fluorescence-imaging technology with a detection range of up to several tens of nanometres: singlet oxygen triplet energy transfer. We demonstrate that our method confirms the topology of a large protein complex in intact cells, which spans from the endoplasmic reticulum to the outer mitochondrial membrane and the matrix. This new method is thus suited for mapping protein proximity in large protein complexes.

  17. Singlet oxygen Triplet Energy Transfer based imaging technology for mapping protein-protein proximity in intact cells

    PubMed Central

    To, Tsz-Leung; Fadul, Michael J.; Shu, Xiaokun

    2014-01-01

    Many cellular processes are carried out by large protein complexes that can span several tens of nanometers. Whereas Forster resonance energy transfer has a detection range of <10 nm, here we report the theoretical development and experimental demonstration of a new fluorescence imaging technology with a detection range of up to several tens of nanometers: singlet oxygen triplet energy transfer. We demonstrate that our method confirms the topology of a large protein complex in intact cells, which spans from the endoplasmic reticulum to the outer mitochondrial membrane and the matrix. This new method is thus suited for mapping protein proximity in large protein complexes. PMID:24905026

  18. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking

    SciTech Connect

    Bleil, J.D.; Wassarman, P.M. )

    1990-07-01

    During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker (Denny-Jaffee reagent) covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a ZP3-binding protein that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.

  19. Polyvinylpyrrolidone microneedles enable delivery of intact proteins for diagnostic and therapeutic applications

    PubMed Central

    Sun, Wenchao; Araci, Zeynep; Inayathullah, Mohammed; Manickam, Sathish; Zhang, Xuexiang; Bruce, Marc A.; Marinkovich, M. Peter; Lane, Alfred T.; Milla, Carlos; Rajadas, Jayakumar; Butte, Manish J.

    2013-01-01

    We present a method of fabricating microneedles from polyvinylpyrrolidone (PVP) that enables delivery of intact proteins (or peptides) to the dermal layers of the skin. PVP is known to self-assemble into branched hollow fibers in aqueous and alcoholic solutions; we utilized this property to develop dissolvable patches of microneedles. Proteins were dissolved in concentrated PVP solution in both alcohol and water, poured into polydimethylsiloxane templates shaped as microneedles and, upon evaporation of solvent, formed into concentric, fibrous, layered structures. This approach of making PVP microneedles overcomes problems in dosage, uniform delivery and stability of protein formulation as compared to protein-coated metallic microneedles or photopolymerized PVP microneedles. Here we characterize the PVP microneedles and measure the delivery of proteins into skin. We show that our method of fabrication preserves the protein conformation. These microneedles can serve as a broadly useful platform for delivering protein antigens and therapeutic proteins to the skin, for example for allergen skin testing or immunotherapy. PMID:23648574

  20. Accumulation of helper component/proteinase and coat protein of turnip mosaic virus in intact plants.

    PubMed

    Ohshima, K

    1999-02-01

    The helper component/proteinase (HC/Pro) protein of turnip mosaic virus (TuMV) was fused with glutathione S-transferase (GST) and expressed as a fusion protein in Escherichia coli. The quality of antiserum raised against the GST-HC/Pro fusion protein was compared to that of antiserum raised against coat protein (CP) by image analyser. The result showed that these antisera were of similar quality. Then the both antisera were used to follow the time course of accumulation of HC/Pro protein and CP in intact TuMV-infected leaves. CP appeared first at day 3 post inoculation (p.i.) and gradually accumulated in uninoculated upper leaves, whereas HC/Pro protein appeared first at day 4 p.i., accumulated up to day 7 p.i. and then gradually decreased. Potyvirus proteins are encoded by a single translation unit spanning most of the genome and are presumably synthesized in equimolar ratios. Therefore, the reduced accumulation of HC/Pro protein in relation to CP at one month p.i. in infected plants is presumed to be the result of its degradation. PMID:10672341

  1. Simple, Scalable Proteomic Imaging for High-Dimensional Profiling of Intact Systems.

    PubMed

    Murray, Evan; Cho, Jae Hun; Goodwin, Daniel; Ku, Taeyun; Swaney, Justin; Kim, Sung-Yon; Choi, Heejin; Park, Young-Gyun; Park, Jeong-Yoon; Hubbert, Austin; McCue, Margaret; Vassallo, Sara; Bakh, Naveed; Frosch, Matthew P; Wedeen, Van J; Seung, H Sebastian; Chung, Kwanghun

    2015-12-01

    Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels. PMID:26638076

  2. Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

    PubMed

    Kwon, Kwang-Chul; Verma, Dheeraj; Jin, Shuangxia; Singh, Nameirakpam D; Daniell, Henry

    2013-01-01

    Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

  3. Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain.

    PubMed

    Reinhold, A K; Batti, L; Bilbao, D; Buness, A; Rittner, H L; Heppenstall, P A

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG. PMID:25880204

  4. Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain.

    PubMed

    Reinhold, A K; Batti, L; Bilbao, D; Buness, A; Rittner, H L; Heppenstall, P A

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG.

  5. Differential Transcriptional Profiling of Damaged and Intact Adjacent Dorsal Root Ganglia Neurons in Neuropathic Pain

    PubMed Central

    Reinhold, A. K.; Batti, L.; Bilbao, D.; Buness, A.; Rittner, H. L.; Heppenstall, P. A.

    2015-01-01

    Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and “bystanders,” thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG. PMID:25880204

  6. Rapid fingerprinting of milk thermal processing history by intact protein mass spectrometry with nondenaturing chromatography.

    PubMed

    Johnson, Phil; Philo, Mark; Watson, Andrew; Mills, E N Clare

    2011-12-14

    Thermal processing of foods results in proteins undergoing conformational changes, aggregation, and chemical modification notably with sugars via the Maillard reaction. This can impact their functional, nutritional, and allergenic properties. Native size-exclusion chromatography with online electrospray mass spectrometry (SEC-ESI-MS) was used to characterize processing-induced changes in milk proteins in a range of milk products. Milk products could be readily grouped into either pasteurized liquid milks, heavily processed milks, or milk powders by SEC behavior, particularly by aggregation of whey proteins by thermal processing. Maillard modification of all major milk proteins by lactose was observed by MS and was primarily present in milk powders. The method developed is a rapid tool for fingerprinting the processing history of milk and has potential as a quality control method for food ingredient manufacture. The method described here can profile milk protein oligomeric state, aggregation, and Maillard modification in a single shot, rapid analysis. PMID:22007861

  7. Cell-free methods to produce structurally intact mammalian membrane proteins

    PubMed Central

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1–1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  8. Cell-free methods to produce structurally intact mammalian membrane proteins.

    PubMed

    Shinoda, Takehiro; Shinya, Naoko; Ito, Kaori; Ishizuka-Katsura, Yoshiko; Ohsawa, Noboru; Terada, Takaho; Hirata, Kunio; Kawano, Yoshiaki; Yamamoto, Masaki; Tomita, Taisuke; Ishibashi, Yohei; Hirabayashi, Yoshio; Kimura-Someya, Tomomi; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2016-01-01

    The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin. PMID:27465719

  9. Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR

    PubMed Central

    Lee, Sang-Won; Berger, Scott J.; Martinović, Suzana; Paša-Tolić, Ljiljana; Anderson, Gordon A.; Shen, Yufeng; Zhao, Rui; Smith, Richard D.

    2002-01-01

    Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by “mass locking” with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations. PMID:11983894

  10. FT-ICR MS optimization for the analysis of intact proteins

    SciTech Connect

    Tolmachev, Aleksey V.; Robinson, Errol W.; Wu, Si; Pasa-Tolic, Ljiljana; Smith, Richard D.

    2009-10-15

    Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) remains the technique of choice for the analysis of intact proteins from complex biological systems, i.e. top-down proteomics. Recently, we have implemented a compensated open cylindrical ion trapping cell into a 12 T FT-ICR mass spectrometer. This new cell has previously demonstrated improved sensitivity, dynamic range, and mass measurement accuracy for the analysis of relatively small tryptic peptides. These improvements are due to the improved trapping potential of the cell which is significantly closer to the ideal harmonic trapping potential. Here we report the instrument optimization for the analysis of large macro-molecular ions, such as proteins. Also, presented are first principle theoretical considerations to account for different optimum conditions for the analysis of large macro-molecules. The proposed high energy ion loss mechanism is further supported by experimental results of bovine ubiquitin and serum albumin. We find that the analysis of large macro-molecules can be significantly improved by the further reduction of pressure in the ion trapping cell. This will reduce the impact of the high energy ion loss mechanism and enable increased sensitivity and mass measurement accuracy to be realized without compromising resolution. Further, these results appear to be applicable to FTMS in general, and the high energy ion loss mechanism applies to Orbitrap mass analyzers as well.

  11. Intact vinculin protein is required for control of cell shape, cell mechanics, and rac-dependent lamellipodia formation

    NASA Technical Reports Server (NTRS)

    Goldmann, Wolfgang H.; Ingber, Donald E.

    2002-01-01

    Studies were carried out using vinculin-deficient F9 embryonic carcinoma (gamma229) cells to analyze the relationship between structure and function within the focal adhesion protein vinculin, in the context of control of cell shape, cell mechanics, and movement. Atomic force microscopy studies revealed that transfection of the head (aa 1-821) or tail (aa 811-1066) domain of vinculin, alone or together, was unable to fully reverse the decrease in cell stiffness, spreading, and lamellipodia formation caused by vinculin deficiency. In contrast, replacement with intact vinculin completely restored normal cell mechanics and spreading regardless of whether its tyrosine phosphorylation site was deleted. Constitutively active rac also only induced extension of lamellipodia when microinjected into cells that expressed intact vinculin protein. These data indicate that vinculin's ability to physically couple integrins to the cytoskeleton, to mechanically stabilize cell shape, and to support rac-dependent lamellipodia formation all appear to depend on its intact three-dimensional structure.

  12. Purification and properties of the intact P-700 and Fx-containing Photosystem I core protein.

    PubMed

    Parrett, K G; Mehari, T; Warren, P G; Golbeck, J H

    1989-02-28

    The intact Photosystem I core protein, containing the psaA and psaB polypeptides, and electron transfer components P-700 through FX, was isolated from cyanobacterial and higher plant Photosystem I complexes with chaotropic agents followed by sucrose density ultracentrifugation. The concentrations of NaClO4, NaSCN, NaI, NaBr or urea required for the functional removal of the 8.9 kDa, FA/FB polypeptide was shown to be inversely related to the strength of the chaotrope. The Photosystem I core protein, which was purified to homogeniety, contains 4 mol of acid-labile sulfide and has the following properties: (i) the FX-containing core consists of the 82 and 83 kDa reaction center polypeptides but is totally devoid of the low-molecular-mass polypeptides; (ii) methyl viologen and other bipyridilium dyes have the ability to accept electrons directly from FX; (iii) the difference spectrum of FX from 400 to 900 nm is characteristic of an iron-sulfur cluster; (iv) the midpoint potential of FX, determined optically at room temperature, is 60 mV more positive than in the control; (v) there is indication by ESR spectroscopy of low-temperature heterogeneity within FX; and (vi) the heterogeneity is seen by optical spectroscopy as inefficiency in low-temperature electron flow to FX. The constraints imposed by the amount of non-heme iron and labile sulfide in the Photosystem I core protein, the cysteine content of the psaA and psaB polypeptides, and the stoichiometry of high-molecular-mass polypeptides, cause us to re-examine the possibility that FX is a [4Fe-4S] rather than a [2Fe-2S] cluster ligated by homologous cysteine residues on the psaA and psaB heterodimer.

  13. Radial Turgor and Osmotic Pressure Profiles in Intact and Excised Roots of Aster tripolium 1

    PubMed Central

    Zimmermann, Ulrich; Rygol, Joachim; Balling, Angelika; Klöck, Gerd; Metzler, Alexander; Haase, Axel

    1992-01-01

    High-resolution nuclear magnetic resonance images (using very short spin-echo times of 3.8 milliseconds) of cross-sections of excised roots of the halophyte Aster tripolium showed radial cell strands separated by air-filled spaces. Radial insertion of the pressure probe (along the cell strands) into roots of intact plants revealed a marked increase of the turgor pressure from the outermost to the sixth cortical layer (from about 0.1-0.6 megapascals). Corresponding measurements of intracellular osmotic pressure in individual cortical cells (by means of a nanoliter osmometer) showed an osmotic pressure gradient of equal magnitude to the turgor pressure. Neither gradient changed significantly when the plants were grown in, or exposed for 1 hour to, media of high salinity. Differences were recorded in the ability of salts and nonelectrolytes to penetrate the apoplast in the root. The reflection coefficients of the cortical cells were approximately 1 for all the solutes tested. Excision of the root from the stem resulted in a collapse of the turgor and osmotic pressure gradients. After about 15 to 30 minutes, the turgor pressure throughout the cortex attained an intermediate (quasistationary) level of about 0.3 megapascals. This value agreed well with the osmotic value deduced from plasmolysis experiments on excised root segments. These and other data provided conclusions about the driving forces for water and solute transport in the roots and about the function of the air-filled radial spaces in water transport. They also showed that excised roots may be artifactual systems. ImagesFigure 1Figure 2 PMID:16668848

  14. FT-ICR MS optimization for the analysis of intact proteins.

    PubMed

    Tolmachev, Aleksey V; Robinson, Errol W; Wu, Si; Paša-Tolić, Ljiljana; Smith, Richard D

    2009-10-15

    Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) remains the technique of choice for the analysis of intact proteins from complex biological systems, i.e. top-down proteomics. Recently, we have implemented a compensated open cylindrical ion trapping cell into a 12 T FT-ICR mass spectrometer. This new cell has previously demonstrated improved sensitivity, dynamic range, and mass measurement accuracy for the analysis of relatively small tryptic peptides. These improvements are due to the modified trapping potential of the cell which closely approximates the ideal harmonic trapping potential. Here, we report the instrument optimization for the analysis of large macro-molecular ions, such as proteins. Single transient mass spectra of multiply charged bovine ubiquitin ions with sub-ppm mass measurement accuracy, improved signal intensity, and increased dynamic range were obtained using this new cell with increased post-excitation cyclotron radii. The increased cyclotron radii correspond to increased ion kinetic energy and collisions between neutrals and ions with sufficient kinetic energy can exceed a threshold of single collision ion fragmentation. A transition then occurs from relatively long signal lifetimes at low excitation radii to potentially shorter lifetimes, defined by the average ion-neutral collision time. The proposed high energy ion loss mechanism is evaluated and compared with experimental results for bovine ubiquitin and serum albumin. We find that the analysis of large macro-molecules can be significantly improved by the further reduction of pressure in the ion trapping cell. This reduces the high energy ion losses and can enable increased sensitivity and mass measurement accuracy to be realized without compromising resolution. Further, these results appear to be generally applicable to FTMS, and it is expected that the high energy ion loss mechanism also applies to Orbitrap mass analyzers. PMID:20473360

  15. FT-ICR MS optimization for the analysis of intact proteins

    PubMed Central

    Tolmachev, Aleksey V.; Robinson, Errol W.; Wu, Si; Paša-Tolić, Ljiljana; Smith, Richard D.

    2010-01-01

    Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) remains the technique of choice for the analysis of intact proteins from complex biological systems, i.e. top-down proteomics. Recently, we have implemented a compensated open cylindrical ion trapping cell into a 12 T FT-ICR mass spectrometer. This new cell has previously demonstrated improved sensitivity, dynamic range, and mass measurement accuracy for the analysis of relatively small tryptic peptides. These improvements are due to the modified trapping potential of the cell which closely approximates the ideal harmonic trapping potential. Here, we report the instrument optimization for the analysis of large macro-molecular ions, such as proteins. Single transient mass spectra of multiply charged bovine ubiquitin ions with sub-ppm mass measurement accuracy, improved signal intensity, and increased dynamic range were obtained using this new cell with increased post-excitation cyclotron radii. The increased cyclotron radii correspond to increased ion kinetic energy and collisions between neutrals and ions with sufficient kinetic energy can exceed a threshold of single collision ion fragmentation. A transition then occurs from relatively long signal lifetimes at low excitation radii to potentially shorter lifetimes, defined by the average ion-neutral collision time. The proposed high energy ion loss mechanism is evaluated and compared with experimental results for bovine ubiquitin and serum albumin. We find that the analysis of large macro-molecules can be significantly improved by the further reduction of pressure in the ion trapping cell. This reduces the high energy ion losses and can enable increased sensitivity and mass measurement accuracy to be realized without compromising resolution. Further, these results appear to be generally applicable to FTMS, and it is expected that the high energy ion loss mechanism also applies to Orbitrap mass analyzers. PMID:20473360

  16. Metabolic Profiling of Intact Arabidopsis thaliana Leaves during Circadian Cycle Using 1H High Resolution Magic Angle Spinning NMR

    PubMed Central

    van Schadewijk, R.; de Groot, H. J. M.; Alia, A.

    2016-01-01

    Arabidopsis thaliana is the most widely used model organism for research in plant biology. While significant advances in understanding plant growth and development have been made by focusing on the molecular genetics of Arabidopsis, extracting and understanding the functional framework of metabolism is challenging, both from a technical perspective due to losses and modification during extraction of metabolites from the leaves, and from the biological perspective, due to random variation obscuring how well the function is performed. The purpose of this work is to establish the in vivo metabolic profile directly from the Arabidopsis thaliana leaves without metabolite extraction, to reduce the complexity of the results by multivariate analysis, and to unravel the mitigation of cellular complexity by predominant functional periodicity. To achieve this, we use the circadian cycle that strongly influences metabolic and physiological processes and exerts control over the photosynthetic machinery. High resolution-magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to obtain the metabolic profile directly from intact Arabidopsis leaves. Combining one- and two-dimensional 1H HR-MAS NMR allowed the identification of several metabolites including sugars and amino acids in intact leaves. Multivariate analysis on HR-MAS NMR spectra of leaves throughout the circadian cycle revealed modules of primary metabolites with significant and consistent variations of their molecular components at different time points of the circadian cycle. Since robust photosynthetic performance in plants relies on the functional periodicity of the circadian rhythm, our results show that HR-MAS NMR promises to be an important non-invasive method that can be used for metabolomics of the Arabidopsis thaliana mutants with altered physiology and photosynthetic efficiency. PMID:27662620

  17. Complementary LC-MS/MS-Based N-Glycan, N-Glycopeptide, and Intact N-Glycoprotein Profiling Reveals Unconventional Asn71-Glycosylation of Human Neutrophil Cathepsin G

    PubMed Central

    Loke, Ian; Packer, Nicolle H.; Thaysen-Andersen, Morten

    2015-01-01

    Neutrophil cathepsin G (nCG) is a central serine protease in the human innate immune system, but the importance of its N-glycosylation remains largely undescribed. To facilitate such investigations, we here use complementary LC-MS/MS-based N-glycan, N-glycopeptide, and intact glycoprotein profiling to accurately establish the micro- and macro-heterogeneity of nCG from healthy individuals. The fully occupied Asn71 carried unconventional N-glycosylation consisting of truncated chitobiose core (GlcNAcβ: 55.2%; Fucα1,6GlcNAcβ: 22.7%), paucimannosidic N-glycans (Manβ1,4GlcNAcβ1,4GlcNAcβ: 10.6%; Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ: 7.9%; Manα1,6Manβ1,4GlcNAcβ1,4GlcNAcβ: 3.7%, trace level of Manα1,6Manβ1,4GlcNAcβ1,4(Fucα1,6)GlcNAcβ), and trace levels of monoantennary α2,6- and α2,3-sialylated complex N-glycans. High-resolution/mass accuracy LC-MS profiling of intact nCG confirmed the Asn71-glycoprofile and identified two C-terminal truncation variants at Arg243 (57.8%) and Ser244 (42.2%), both displaying oxidation of solvent-accessible Met152. Asn71 appeared proximal (~19 Å) to the active site of nCG, but due to the truncated nature of Asn71-glycans (~5–17 Å) we questioned their direct modulation of the proteolytic activity of the protein. This work highlights the continued requirement of using complementary technologies to accurately profile even relatively simple glycoproteins and illustrates important challenges associated with the analysis of unconventional protein N-glycosylation. Importantly, this study now facilitates investigation of the functional role of nCG Asn71-glycosylation. PMID:26274980

  18. 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

  19. 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.

  20. Intact subepidermal nerve fibers mediate mechanical hypersensitivity via the activation of protein kinase C gamma in spared nerve injury

    PubMed Central

    Ko, Miau-Hwa; Yang, Ming-Ling; Youn, Su-Chung; Tseng, To-Jung

    2016-01-01

    Background Spared nerve injury is an important neuropathic pain model for investigating the role of intact primary afferents in the skin on pain hypersensitivity. However, potential cellular mechanisms remain poorly understood. In phosphoinositide-3 kinase pathway, pyruvate dehydrogenase kinase 1 (PDK1) participates in the regulation of neuronal plasticity for central sensitization. The downstream cascades of PDK1 include: (1) protein kinase C gamma (PKCγ) controls the trafficking and phosphorylation of ionotropic glutamate receptor; (2) protein kinase B (Akt)/the mammalian target of rapamycin (mTOR) signaling is responsible for local protein synthesis. Under these statements, we therefore hypothesized that an increase of PKCγ activation and mTOR-dependent PKCγ synthesis in intact primary afferents after SNI might contribute to pain hypersensitivity. Results The variants of spared nerve injury were performed in Sprague-Dawley rats by transecting any two of the three branches of the sciatic nerve, leaving only one branch intact. Following SNIt (spared tibial branch), mechanical hyperalgesia and mechanical allodynia, but not thermal hyperalgesia, were significantly induced. In the first footpad, normal epidermal innervations were verified by the protein gene product 9.5 (PGP9.5)- and growth-associated protein 43 (GAP43)-immunoreactive (IR) intraepidermal nerve fibers (IENFs) densities. Furthermore, the rapid increases of phospho-PKCγ- and phospho-mTOR-IR subepidermal nerve fibers (SENFs) areas were distinct gathered from the results of PGP9.5-, GAP43-, and neurofilament 200 (NF200)-IR SENFs areas. The efficacy of PKC inhibitor (GF 109203X) or mTOR complex 1 inhibitor (rapamycin) for attenuating mechanical hyperalgesia and mechanical allodynia by intraplantar injection was dose-dependent. Conclusions From results obtained in this study, we strongly recommend that the intact SENFs persistently increase PKCγ activation and mTOR-dependent PKCγ synthesis participate

  1. Separation of peptides and intact proteins by electrostatic repulsion reversed phase liquid chromatography.

    PubMed

    Gritti, Fabrice; Guiochon, Georges

    2014-12-29

    A new brand of BEH-C18 hybrid particles chemically bonded to a leash carrying an amine group permits the implementation of electrostatic repulsive interactions chromatography. Using columns packed with this material, the influence of the concentration of positive charges bonded to the BEH-C18 surface on the overloaded band profiles of a few positively charged peptides and proteins was investigated in the gradient elution mode. Three columns packed with endcapped BEH-C18 particles bonded with three different surface-charge densities (LOW, MEDIUM and HIGH) were used and compared with those provided by a column packed with non-doped, endcapped BEH-C18 particles. The surface concentrations of fixed charges in the LOW, MEDIUM and HIGH columns were estimated at 0.029, 0.050, and 0.064μmol/m(2), for example, about two orders of magnitude smaller than the surface density of bonded C18 chains (2.1μmol/m(2)). Three different mobile phase additives (0.1% v/v of trifluoro-acetic, phosphoric, and formic acid) were used to optimize the purification levels of proteins under different loading conditions. The weak ion-pairing ions (formate and phosphate) generate smaller retention but broader, more fronting band profiles than those eluted with a stronger ion-pairing ion (trifluoroactate). This effect is worse in the presence of fixed charges at the surface of the BEH-C18 particles. This was explained by an enhanced anti-Langmuirian adsorption behavior of the charged proteins in the presence of fixed surface charges. As the protein concentration increases in the bulk, so does the internal ionic strength, the electrostatic repulsive interactions weaken, and retention increases. Band fronting is mostly eliminated by replacing weak ion-pairing acids with TFA with which the adsorption isotherm remains weakly langmuirian. Faster but still complete gradient separation of insulin and myoglobin were achieved with the HIGH column than with the reference neutral column, despite a measurable

  2. 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

  3. 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.

  4. 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.

  5. Protein separation and characterization by np-RP-HPLC followed by intact MALDI-TOF mass spectrometry and peptide mass mapping analyses.

    PubMed

    Dauly, Claire; Perlman, David H; Costello, Catherine E; McComb, Mark E

    2006-07-01

    Because of their complexity, the separation of intact proteins from complex mixtures is an important step to comparative proteomics and the identification and characterization of the proteins by mass spectrometry (MS). In the study reported, we evaluated the use of nonporous-reversed-phase (np-RP)-HPLC for intact protein separation prior to MS analyses. The separation system was characterized and compared to 1D-SDS-PAGE electrophoresis in terms of resolution and sensitivity. We demonstrate that np-RP-HPLC protein separation is highly reproducible and provides intact protein fractions which can be directly analyzed by MALDI-TOF-MS for intact molecular weight determination. An in-well digestion protocol was developed, allowing for rapid protein identification by peptide mass fingerprinting (PMF) and resulted in comparable or improved peptide recovery compared with in-gel digestion. The np-RP sensitivity of detection by UV absorbance at 214 nm for intact proteins was at the low ng level and the sensitivity of peptide analysis by MALDI-TOF-MS was in the 10-50 fmol level. A membrane protein fraction was characterized to demonstrate application of this methodology. Among the identified proteins, multiple forms of vimentin were observed. Overall, we demonstrate that np-RP-HPLC followed by MALDI-TOF-MS allows for rapid, sensitive, and reproducible protein fractionation and very specific protein characterization by integration of PMF analysis with MS intact molecular weight information.

  6. 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

  7. 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

  8. RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming

    PubMed Central

    Bai, Lin; Li, Mengqi; Sun, Junli; Yang, Xiaogan; Lu, Yangqing; Lu, Shengsheng; Lu, Kehuan

    2016-01-01

    The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes. PMID:27070804

  9. RNA-Seq Profiling of Intact and Enucleated Oocyte SCNT Embryos Reveals the Role of Pig Oocyte Nucleus in Somatic Reprogramming.

    PubMed

    Bai, Lin; Li, Mengqi; Sun, Junli; Yang, Xiaogan; Lu, Yangqing; Lu, Shengsheng; Lu, Kehuan

    2016-01-01

    The specific molecular mechanisms involved in somatic reprogramming remain unidentified. Removal of the oocyte genome is one of the primary causes of developmental failure in cloned embryos, whereas intact oocyte shows stronger reprogramming capability than enucleated oocyte. To identify the reason for the low efficiency of cloning and elucidate the mechanisms involved in somatic reprogramming by the oocyte nucleus, we injected pig cumulus cells into 539 intact MII oocytes and 461 enucleated MII oocytes. Following activation, 260 polyploidy embryos developed to the blastocyst stage whereas only 93 traditionally cloned embryos (48.2% vs. 20.2%, P < 0.01) reached blastocyst stage. Blastocysts generated from intact oocytes also had more cells than those generated from enucleated oocytes (60.70 vs. 46.65, P < 0.01). To identify the genes that contribute to this phenomenon, two early embryos in 2-cell and 4-cell stages were collected for single-cell RNA sequencing. The two kinds of embryos were found to have dramatically different transcriptome profiles. Intact oocyte nuclear transfer embryos showed 1,738 transcripts that were up-regulated relative to enucleated cloned embryos at the 2-cell stage and 728 transcripts that were down-regulated (|log2Ratio| ≥ 5). They showed 2,941 transcripts that were up-regulated during the 4-cell stage and 1,682 that were down-regulated (|log2Ratio| ≥ 5). The most significantly enriched gene ontology categories were those involved in the regulation of binding, catalytic activity, and molecular transducer activity. Other genes that were notably up-regulated and expressed in intact oocyte nuclear transfer embryos were metabolic process. This study provides a comprehensive profile of the differences in gene expression between intact oocyte nuclear transfer embryos and traditional nuclear transfer embryos. This work thus paves the way for further research on the mechanisms underlying somatic reprogramming by oocytes. PMID:27070804

  10. Membrane structure in isolated and intact myelins.

    PubMed Central

    Inouye, H; Karthigasan, J; Kirschner, D A

    1989-01-01

    The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions. PMID:2752082

  11. 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.

  12. Relationships between intact parathyroid hormone 24-hour profiles, sleep-wake cycle, and sleep electroencephalographic activity in man.

    PubMed

    Chapotot, F; Gronfier, C; Spiegel, K; Luthringer, R; Brandenberger, G

    1996-10-01

    To determine whether the 24-h intact PTH (iPTH) profile is influenced by the sleep-wake cycle, and whether iPTH pulses show a temporal relationship with internal sleep structure, eight normal young men were studied during 24 h under basal conditions, once with normal nighttime sleep from 2300-0700 h and once after a night of sleep deprivation followed by an 8-h period of daytime sleep from 0700-1500 h. During the 8-h nighttime sleep period, mean iPTH levels were significantly increased by +13% and mean iPTH pulse amplitudes by +31% as compared with the 8-h subsequent waking periods. During the 8 h of total sleep deprivation, mean iPTH levels were not significantly different from the corresponding period in nighttime sleep condition, but mean iPTH pulse amplitudes were significantly lower (P < 0.01). The 8-h daytime sleep period was associated with increased mean iPTH levels and mean iPTH pulse amplitudes (+15% and +57%, respectively, as compared with the corresponding period in nighttime sleep condition). The number of pulses was similar in both experimental series and was not influenced by sleep or by time of day. Analysis of coincidence between iPTH pulses, plasma ionized calcium and plasma phosphate pulses, and slow wave sleep, as well as with rapid eye movement sleep episodes, did not reveal any significant association. Cross-correlation analysis between iPTH, plasma ionized calcium, and plasma phosphate fluctuations during sleep also showed no systematic association. Seven other subjects were studied during a nighttime sleep period in which temporal relationships between iPTH and internal sleep structure were reevaluated using spectral analysis of the sleep electroencephalogram. Cross-correlation analysis between iPTH levels and delta-relative power fluctuations showed nonsignificant results, which confirms the lack of relationship with slow wave sleep. This study demonstrates that the iPTH 24-h profile is influenced by sleep processes with a weak circadian

  13. Profiling intact steroid sulfates and unconjugated steroids in biological fluids by liquid chromatography-tandem mass spectrometry (LC-MS-MS).

    PubMed

    Galuska, Christina E; Hartmann, Michaela F; Sánchez-Guijo, Alberto; Bakhaus, Katharina; Geyer, Joachim; Schuler, Gerhard; Zimmer, Klaus-Peter; Wudy, Stefan A

    2013-07-01

    Within the combined DFG research project "Sulfated Steroids in Reproduction" an analytical method was needed for determining sulfated and unconjugated steroids with highest specificity out of different biological matrices such as aqueous solution, cell lysate and serum. With regard to this analytical challenge, LC-MS-MS presents the technique of choice because it permits (1) analysis of the intact steroid conjugate, (2) allows for simultaneous determination of multiple analytes (profiling, targeted metabolomics approach) and (3) is independent of phenomena such as cross-reactivity. Sample work up consisted of incubation of sample with internal standards (deuterium labeled steroids) followed by solid phase extraction. Only serum samples required a protein precipitation step prior to solid phase extraction. The extract was divided in two parts: six steroid sulfates (E1S, E2S, AS, 16-OH-DHEAS, PREGS, DHEAS) were analyzed by C18aQ-ESI-MS-MS in negative ion mode and eleven unconjugated steroids (E3, 16-OH-DHEA, E1, E2, (4)A, DHEA, T, 17-OH-PREG, Prog, An, PREG) were analyzed by C18-APCI-MS-MS in positive ion mode. For steroid sulfates, we found high sensitivities with LoQ values ranging from 0.08 to 1 ng mL(-1). Unconjugated steroids showed LoQ values between 0.5 and 10 ng mL(-1). Calibration plots showed excellent linearity. Mean intra- and inter-assay CVs were 2.4% for steroid sulfates and 6.4% for unconjugated steroids. Accuracy - determined in a two-level spike experiment - showed mean relative errors of 5.9% for steroid sulfates and 6.1% for unconjugated steroids. In summary, we describe a novel LC-MS-MS procedure capable of profiling six steroid sulfates and eleven unconjugated steroids from various biological matrices.

  14. Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared, Intact Mouse Brains

    PubMed Central

    Schwarz, Martin K.; Scherbarth, Annemarie; Sprengel, Rolf; Engelhardt, Johann; Theer, Patrick; Giese, Guenter

    2015-01-01

    In order to observe and quantify long-range neuronal connections in intact mouse brain by light microscopy, it is first necessary to clear the brain, thus suppressing refractive-index variations. Here we describe a method that clears the brain and preserves the signal from proteinaceous fluorophores using a pH-adjusted non-aqueous index-matching medium. Successful clearing is enabled through the use of either 1-propanol or tert-butanol during dehydration whilst maintaining a basic pH. We show that high-resolution fluorescence imaging of entire, structurally intact juvenile and adult mouse brains is possible at subcellular resolution, even following many months in clearing solution. We also show that axonal long-range projections that are EGFP-labelled by modified Rabies virus can be imaged throughout the brain using a purpose-built light-sheet fluorescence microscope. To demonstrate the viability of the technique, we determined a detailed map of the monosynaptic projections onto a target cell population in the lateral entorhinal cortex. This example demonstrates that our method permits the quantification of whole-brain connectivity patterns at the subcellular level in the uncut brain. PMID:25993380

  15. Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts.

    PubMed

    Buyel, Johannes F; Hubbuch, Jürgen; Fischer, Rainer

    2016-01-01

    Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy. PMID:27584939

  16. Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts.

    PubMed

    Buyel, Johannes F; Hubbuch, Jürgen; Fischer, Rainer

    2016-01-01

    Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 °C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

  17. Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses

    PubMed Central

    Vincent, Delphine; Elkins, Aaron; Condina, Mark R.; Ezernieks, Vilnis; Rochfort, Simone

    2016-01-01

    Cow’s milk is an important source of proteins in human nutrition. On average, cow’s milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600–3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels. PMID:27749892

  18. Autophosphorylation and dephosphorylation of type II cAMP-dependent protein kinase in intact tissue and cells

    SciTech Connect

    Scott, C.W.

    1988-01-01

    Monoclonal antibodies were used to quantitate changes in the extent of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase in intact bovine tracheal smooth muscle and rat hepatocytes. The autophosphorylated and dephosphorylated forms of the regulatory subunit (RII) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by immunoblot analysis. Incubating smooth muscle strips with agents which stimulate cAMP production caused a rapid and reversible dephosphorylation of phospho RII. In contrast, phospho RII levels in rat hepatocytes were unaffected by elevations in cAMP concentration. Insulin was capable of inhibiting cAMP-dependent protein kinase in hepatocytes. Tissue extracts were tested to identify the basis for the lack of RII dephosphorylation in intact hepatocytes. Rat liver extract contained 4 fold less RII and had an 80 fold slower rate of dephosphorylation of endogenous RII compared to bovine smooth muscle extract. The different rates were not observed using purified, /sup 32/P-labelled RII and tissue extracts suggesting the decreased RII dephosphorylation rate in liver was due to a difference in the availability of endogenous RII rather than a difference in measurable phosphatase activity.

  19. A new method for the experimental heating of intact soil profiles for application to climate change experiments

    SciTech Connect

    Hanson, Paul J; Childs, Kenneth W; Wullschleger, Stan D; Riggs, Jeffery S; Thomas, Warren Kyle; Todd Jr, Donald E; Warren, Jeffrey

    2011-01-01

    systems to address uncertainties in process-level responses of microbial, plant, and animal communities in whole, intact ecosystems using this new heating method that capture expected future warming and temperature dynamics throughout the soil profile.

  20. Whole intact rapeseeds or sunflower oil in high-forage or high-concentrate diets affects milk yield, milk composition, and mammary gene expression profile in goats.

    PubMed

    Ollier, S; Leroux, C; de la Foye, A; Bernard, L; Rouel, J; Chilliard, Y

    2009-11-01

    This study aimed to ascertain the response of goat mammary metabolic pathways to concentrate and lipid feeding in relation to milk fatty acid (FA) composition and secretion. Sixteen midlactation multiparous goats received diets differing in forage-to-concentrate ratio [high forage (HF) 64:36, and low forage (LF) 43:57] supplemented or not with lipids [HF with 130 g/d of oil from whole intact rapeseeds (RS) and LF with 130 g/d of sunflower oil (SO)] in a 4 x 4 Latin square design. Milk yield, milk composition, FA profile, and FA secretion were measured, as well as the expression profiles of key genes in mammary metabolism and of 8,382 genes, using a bovine oligonucleotide microarray. After 3 wk of treatment, milk, lactose, and protein yields were lower with HF-RS than with the other diets, whereas treatment had no effect on milk protein content. Milk fat content was higher with the HF-RS and LF-SO diets than with the HF and LF diets, and SO supplementation increased milk fat yield compared with the LF diet. Decreasing the forage-to-concentrate ratio from 64:36 to 43:57 had a limited effect on goat milk FA concentrations and secretions. Supplementing the LF diet with SO changed almost all the FA concentrations, including decreases in medium-chain saturated FA and large increases in trans C18:1 and C18:2 isomers (particularly trans-11 C18:1 and cis-9, trans-11 conjugated linoleic acid), without significant changes in C18:0 and cis-9 C18:1, whereas supplementing the HF diet with RS led to a strong decrease in short- and medium-chain saturated FA and a very strong increase in C18:0 and cis-9 C18:1, without significant changes in trans C18:1 and conjugated linoleic acid. Despite the decreases in milk lactose and protein yields observed with HF-RS, and despite the decrease in milk medium-chain FA and the increase in C18 FA secretion with RS or SO supplementation, none of the dietary treatments had any effect on mammary mRNA expression of the key genes involved in lactose

  1. Whole intact rapeseeds or sunflower oil in high-forage or high-concentrate diets affects milk yield, milk composition, and mammary gene expression profile in goats.

    PubMed

    Ollier, S; Leroux, C; de la Foye, A; Bernard, L; Rouel, J; Chilliard, Y

    2009-11-01

    This study aimed to ascertain the response of goat mammary metabolic pathways to concentrate and lipid feeding in relation to milk fatty acid (FA) composition and secretion. Sixteen midlactation multiparous goats received diets differing in forage-to-concentrate ratio [high forage (HF) 64:36, and low forage (LF) 43:57] supplemented or not with lipids [HF with 130 g/d of oil from whole intact rapeseeds (RS) and LF with 130 g/d of sunflower oil (SO)] in a 4 x 4 Latin square design. Milk yield, milk composition, FA profile, and FA secretion were measured, as well as the expression profiles of key genes in mammary metabolism and of 8,382 genes, using a bovine oligonucleotide microarray. After 3 wk of treatment, milk, lactose, and protein yields were lower with HF-RS than with the other diets, whereas treatment had no effect on milk protein content. Milk fat content was higher with the HF-RS and LF-SO diets than with the HF and LF diets, and SO supplementation increased milk fat yield compared with the LF diet. Decreasing the forage-to-concentrate ratio from 64:36 to 43:57 had a limited effect on goat milk FA concentrations and secretions. Supplementing the LF diet with SO changed almost all the FA concentrations, including decreases in medium-chain saturated FA and large increases in trans C18:1 and C18:2 isomers (particularly trans-11 C18:1 and cis-9, trans-11 conjugated linoleic acid), without significant changes in C18:0 and cis-9 C18:1, whereas supplementing the HF diet with RS led to a strong decrease in short- and medium-chain saturated FA and a very strong increase in C18:0 and cis-9 C18:1, without significant changes in trans C18:1 and conjugated linoleic acid. Despite the decreases in milk lactose and protein yields observed with HF-RS, and despite the decrease in milk medium-chain FA and the increase in C18 FA secretion with RS or SO supplementation, none of the dietary treatments had any effect on mammary mRNA expression of the key genes involved in lactose

  2. Interactions of Intact Unfractionated Heparin with Its Client Proteins Can Be Probed Directly Using Native Electrospray Ionization Mass Spectrometry.

    PubMed

    Zhao, Yunlong; Abzalimov, Rinat R; Kaltashov, Igor A

    2016-02-01

    Heparin and related members of the glycosaminoglycan (GAG) family are highly polyanionic linear saccharides that play important roles in a variety of physiological processes ranging from blood coagulation to embryo- and oncogenesis, tissue regeneration, and immune response regulation. These diverse functions are executed via a variety of mechanisms, including protein sequestration, activation, and facilitation of their interactions with cell-surface receptors, but deciphering the specific molecular mechanisms is frequently impossible due to the extremely high degree of GAG heterogeneity. As a result, the vast majority of studies of heparin (or related GAGs) interactions with its client proteins use synthetically produced heparin mimetics with defined structure or short heparin fragments. In this work we use native electrospray ionization mass spectrometry (ESI MS) in combination with limited charge reduction in the gas phase to obtain meaningful information on noncovalent complexes formed by intact unfractionated heparin and antithrombin-III, interaction which is central to preventing blood clotting. Complexes of different stoichiometries are observed ranging from 1:1 to 1:3 (heparin/protein ratio). In addition to binding stoichiometry, the measurements allow the range of heparin chain lengths to be obtained for each complex and the contribution of each complex to the total ionic signal to be calculated. Incorporation of ion mobility measurements in the experimental workflow allows the total analysis time to be shortened very significantly and the charge state assignment for the charge-reduced species to be verified. The possibility to study interactions of intact unfractionated heparin with a client protein carried out directly by native ESI MS without the need to use relatively homogeneous surrogates demonstrated in this work opens up a host of new exciting opportunities and goes a long way toward ameliorating the persistent but outdated view of the

  3. 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.

  4. Comparative studies of peak intensities and chromatographic separation of proteolytic digests, PTMs, and intact proteins obtained by nanoLC-ESI MS analysis at room and elevated temperatures.

    PubMed

    Moskovets, Eugene V; Ivanov, Alexander R

    2016-06-01

    This work demonstrates that the chromatographic separation performed at highly stabilized elevated temperature results in significant improvements in sensitivity, quantitative accuracy, chromatographic resolution, and run-to-run reproducibility of nanoLC-MS analysis of complex peptides mixtures. A newly developed platform was shown to provide conditions for accurate temperature stabilization and temperature homogeneity when performing nanoLC-ESI MS analysis. We quantitatively assessed and compared the recovery of peptides and small proteins from nanoLC columns at room and elevated temperatures. We found that analyses performed at highly stabilized elevated temperatures led to improved detection sensitivity, reproducibility, and chromatographic resolution in reversed-phase LC separation of unmodified peptides (both hydrophilic and hydrophobic), post-translationally modified peptides (O-phosphorylated), and small intact proteins. The analytical benefits of elevated temperatures for qualitative and quantitative proteomic LC-MS profiling were demonstrated using mixtures of synthetic peptides, tryptic digests of mixtures of model proteins, and digested total lysates of isolated rat kidney mitochondria. The effect of elevated temperature on the ion suppression was also demonstrated. Graphical Abstract A fragment of overlaid LC retention time-m/z planar views demonstrates the improved separation performance in the analysis of a complex peptide mixture at elevated temperature. Retention time-m/z 2D peptide features detected at 60 °C (magenta) were matched and aligned with features detected at room temperature (green).

  5. Reproductive hacking. A male seminal protein acts through intact reproductive pathways in female Drosophila.

    PubMed

    Rubinstein, C Dustin; Wolfner, Mariana F

    2014-01-01

    Seminal proteins are critical for reproductive success in all animals that have been studied. Although seminal proteins have been identified in many taxa, and female reproductive responses to receipt of these proteins have been documented in several, little is understood about the mechanisms by which seminal proteins affect female reproductive physiology. To explore this topic, we investigated how a Drosophila seminal protein, ovulin, increases ovulation rate in mated females. Ovulation is a relatively simple physiological process, with known female regulators: previous studies have shown that ovulation rate is promoted by the neuromodulator octopamine (OA) in D. melanogaster and other insects. We found that ovulin stimulates ovulation by increasing OA signaling in the female. This finding supports a model in which a male seminal protein acts through "hacking" a well-conserved, regulatory system females use to adjust reproductive output, rather than acting downstream of female mechanisms of control or in parallel pathways altogether. We also discuss similarities between 2 forms of intersexual control of behavior through chemical communication: seminal proteins and pheromones. PMID:25483253

  6. Reproductive hacking. A male seminal protein acts through intact reproductive pathways in female Drosophila.

    PubMed

    Rubinstein, C Dustin; Wolfner, Mariana F

    2014-01-01

    Seminal proteins are critical for reproductive success in all animals that have been studied. Although seminal proteins have been identified in many taxa, and female reproductive responses to receipt of these proteins have been documented in several, little is understood about the mechanisms by which seminal proteins affect female reproductive physiology. To explore this topic, we investigated how a Drosophila seminal protein, ovulin, increases ovulation rate in mated females. Ovulation is a relatively simple physiological process, with known female regulators: previous studies have shown that ovulation rate is promoted by the neuromodulator octopamine (OA) in D. melanogaster and other insects. We found that ovulin stimulates ovulation by increasing OA signaling in the female. This finding supports a model in which a male seminal protein acts through "hacking" a well-conserved, regulatory system females use to adjust reproductive output, rather than acting downstream of female mechanisms of control or in parallel pathways altogether. We also discuss similarities between 2 forms of intersexual control of behavior through chemical communication: seminal proteins and pheromones.

  7. High resolution time-of-flight mass analysis of the entire range of intact singly-charged proteins.

    PubMed

    Lee, Jeonghoon; Chen, Huijuan; Liu, Tiancheng; Berkman, Clifford E; Reilly, Peter T A

    2011-12-15

    The proof of principle for high-resolution analysis of intact singly charged proteins of any size is presented. Singly charged protein ions were produced by electrospray ionization followed by surface-induced charge reduction at atmospheric pressure. The inlet and trapping system "stops" the forward momentum of the protein ions over a very broad range to be captured by the digitally produced electric fields of a large radius linear ion trap whereupon they are moved into a smaller radius linear ion trap and collected and concentrated in front of its exit end-cap electrode using digital waveform manipulation. The protein ions are then ejected on demand from the end of the small radius linear quadrupole in a tightly collimated ion beam with an instrumentally defined kinetic energy into the acceleration region of an orthogonal acceleration reflectron time-of-flight mass analyzer where their flight times were measured and detected with a Photonis BiPolar TOF detector. We present results that clearly prove that massive singly charged ions can yield high-resolution mass spectra with very low chemical noise and without loss of sensitivity with increasing mass across the entire spectrum. Analysis of noncovalently bound protein complexes was demonstrated with streptavidin-Cy5 bound with a biotinylated peptide mimic. Our results suggest proteins across the entire range can be directly quantified using our mass analysis technique. We present evidence that solvent molecules noncovalently adduct onto the proteins while yielding consistent flight time distributions. Finally, we provide a look into future that will result from the ability to rapidly measure and quantify protein distributions.

  8. 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

  9. Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei

    PubMed Central

    1996-01-01

    Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery. PMID:8655587

  10. Characterization of metabolic profile of intact non-tumor and tumor breast cells by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy.

    PubMed

    Maria, Roberta M; Altei, Wanessa F; Andricopulo, Adriano D; Becceneri, Amanda B; Cominetti, Márcia R; Venâncio, Tiago; Colnago, Luiz A

    2015-11-01

    (1)H high-resolution magic angle spinning nuclear magnetic resonance ((1)H HR-MAS NMR) spectroscopy was used to analyze the metabolic profile of an intact non-tumor breast cell line (MCF-10A) and intact breast tumor cell lines (MCF-7 and MDA-MB-231). In the spectra of MCF-10A cells, six metabolites were assigned, with glucose and ethanol in higher concentrations. Fifteen metabolites were assigned in MCF-7 and MDA-MB-231 (1)H HR-MAS NMR spectra. They did not show glucose and ethanol, and the major component in both tumor cells was phosphocholine (higher in MDA-MB-231 than in MCF-7), which can be considered as a tumor biomarker of breast cancer malignant transformation. These tumor cells also show acetone signal that was higher in MDA-MB-231 cells than in MCF-7 cells. The high acetone level may be an indication of high demand for energy in MDA-MB-231 to maintain cell proliferation. The higher acetone and phosphocholine levels in MDA-MB-231 cells indicate the higher malignance of the cell line. Therefore, HR-MAS is a rapid reproducible method to study the metabolic profile of intact breast cells, with minimal sample preparation and contamination, which are critical in the analyses of slow-growth cells. PMID:26247715

  11. Characterization of metabolic profile of intact non-tumor and tumor breast cells by high-resolution magic angle spinning nuclear magnetic resonance spectroscopy.

    PubMed

    Maria, Roberta M; Altei, Wanessa F; Andricopulo, Adriano D; Becceneri, Amanda B; Cominetti, Márcia R; Venâncio, Tiago; Colnago, Luiz A

    2015-11-01

    (1)H high-resolution magic angle spinning nuclear magnetic resonance ((1)H HR-MAS NMR) spectroscopy was used to analyze the metabolic profile of an intact non-tumor breast cell line (MCF-10A) and intact breast tumor cell lines (MCF-7 and MDA-MB-231). In the spectra of MCF-10A cells, six metabolites were assigned, with glucose and ethanol in higher concentrations. Fifteen metabolites were assigned in MCF-7 and MDA-MB-231 (1)H HR-MAS NMR spectra. They did not show glucose and ethanol, and the major component in both tumor cells was phosphocholine (higher in MDA-MB-231 than in MCF-7), which can be considered as a tumor biomarker of breast cancer malignant transformation. These tumor cells also show acetone signal that was higher in MDA-MB-231 cells than in MCF-7 cells. The high acetone level may be an indication of high demand for energy in MDA-MB-231 to maintain cell proliferation. The higher acetone and phosphocholine levels in MDA-MB-231 cells indicate the higher malignance of the cell line. Therefore, HR-MAS is a rapid reproducible method to study the metabolic profile of intact breast cells, with minimal sample preparation and contamination, which are critical in the analyses of slow-growth cells.

  12. Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.

    PubMed

    Peckys, Diana B; Alansary, Dalia; Niemeyer, Barbara A; de Jonge, Niels

    2016-08-01

    ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.

  13. Coupling detergent lysis/clean-up methodology with intact protein fractionation for enhanced proteome characterization

    SciTech Connect

    Sharma, Ritin; Dill, Brian; Chourey, Karuna; Shah, Manesh B; Verberkmoes, Nathan C; Hettich, Robert {Bob} L

    2012-01-01

    The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four different detergent clean-up methods (Trichloroacetic acid (TCA) precipitation, Chloroform/Methanol/Water (CMW) extraction, commercial detergent removal spin column method (DRS) and filter-aided sample preparation(FASP)) with respect to varying amounts of protein biomass in the samples, and provide efficiency benchmarks with respect to protein, peptide, and spectral identifications for each method. Our results show that for protein limited samples, FASP outperforms the other three clean-up methods, while at high protein amount all the methods are comparable. This information was used in a dual strategy of comparing molecular weight based fractionated and unfractionated lysates from three increasingly complex samples (Escherichia coli, a five microbial isolate mixture, and a natural microbial community groundwater sample), which were all lysed with SDS and cleaned up using FASP. The two approaches complemented each other by enhancing the number of protein identifications by 8%-25% across the three samples and provided broad pathway coverage.

  14. Effective protein inhibition in intact mouse oocytes through peptide nanoparticle-mediated antibody transfection

    PubMed Central

    Li, Ruichao; Jin, Zhen; Gao, Leilei; Liu, Peng

    2016-01-01

    Female meiosis is a fundamental area of study in reproductive medicine, and the mouse oocyte model of in vitro maturation (IVM) is most widely used to study female meiosis. To investigate the probable role(s) of an unknown protein in female meiosis, the method traditionally used involves microinjecting a specific antibody into mouse oocytes. Recently, in studies on somatic cells, peptide nanoparticle-mediated antibody transfection has become a popular tool because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, untill now no researchers have tried using this technique on mouse oocytes because the zona pellucida surrounding the oocyte membrane (vitelline membrane) is usually thought or proved to be a tough barrier to macromolecules such as antibodies and proteins. Therefore, we attempted to introduce an antibody into mouse oocytes using a peptide nanoparticle. Here we show for the first time that with our optimized method, an antibody can be effectively delivered into mouse oocytes and inhibit its target protein with high specificity. We obtained significant results using small GTPase Arl2 as a test subject protein. We propose peptide nanoparticle-mediated antibody transfection to be a superior alternative to antibody microinjection for preliminary functional studies of unknown proteins in mouse oocytes. PMID:27114861

  15. Comparison of Intact Arabidopsis thaliana Leaf Transcript Profiles during Treatment with Inhibitors of Mitochondrial Electron Transport and TCA Cycle

    PubMed Central

    Yu, Jianping; Ruckle, Michael E.; McIntosh, Lee; Hock, Jeffery J.; Bingham, Scott; White, Samuel J.; George, Rajani M.; Subbaiah, Chalivendra C.; Rhoads, David M.

    2012-01-01

    Plant mitochondria signal to the nucleus leading to altered transcription of nuclear genes by a process called mitochondrial retrograde regulation (MRR). MRR is implicated in metabolic homeostasis and responses to stress conditions. Mitochondrial reactive oxygen species (mtROS) are a MRR signaling component, but whether all MRR requires ROS is not established. Inhibition of the cytochrome respiratory pathway by antimycin A (AA) or the TCA cycle by monofluoroacetate (MFA), each of which initiates MRR, can increase ROS production in some plant cells. We found that for AA and MFA applied to leaves of soil-grown Arabidopsis thaliana plants, ROS production increased with AA, but not with MFA, allowing comparison of transcript profiles under different ROS conditions during MRR. Variation in transcript accumulation over time for eight nuclear encoded mitochondrial protein genes suggested operation of both common and distinct signaling pathways between the two treatments. Consequences of mitochondrial perturbations for the whole transcriptome were examined by microarray analyses. Expression of 1316 and 606 genes was altered by AA and MFA, respectively. A subset of genes was similarly affected by both treatments, including genes encoding photosynthesis-related proteins. MFA treatment resulted in more down-regulation. Functional gene category (MapMan) and cluster analyses showed that genes with expression levels affected by perturbation from AA or MFA inhibition were most similarly affected by biotic stresses such as pathogens. Overall, the data provide further evidence for the presence of mtROS-independent MRR signaling, and support the proposed involvement of MRR and mitochondrial function in plant responses to biotic stress. PMID:23028523

  16. Real-Time Trapping of Intact Singly-Charged Bovine Serum Albumin Proteins with a Big Frequency-Adjusted Quadrupole

    SciTech Connect

    Koizumi, Hideya; Whitten, William B; Reilly, Pete

    2008-01-01

    High-resolution real-time particle mass measurements have not been achievable because the enormous amount of kinetic energy imparted to the particles upon expansion into vacuum competes with and overwhelms the forces applied to the charged particles within the mass spectrometer. It is possible to reduce the kinetic energy of a collimated particulate ion beam through collisions with a buffer gas while radially constraining their motion using a quadrupole guide or trap over a limited mass range. Controlling the pressure drop of the final expansion into a quadrupole trap permits a much broader mass range at the cost of sacrificing collimation. To achieve high-resolution mass analysis of massive particulate ions, an efficient trap with a large tolerance for radial divergence of the injected ions was developed that permits trapping a large range of ions for on-demand injection into an awaiting mass analyzer. The design specifications required that frequency of the trapping potential be adjustable to cover a large mass range and the trap radius be increased to increase the tolerance to divergent ion injection. The large-radius linear quadrupole ion trap was demonstrated by trapping singly-charged bovine serum albumin ions for on-demand injection into a mass analyzer. Additionally, this work demonstrates the ability to measure an electrophoretic mobility cross section (or ion mobility) of singly-charged intact proteins in the low-pressure regime. This work represents a large step toward the goal of high-resolution analysis of intact proteins, RNA, DNA, and viruses.

  17. Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions.

    PubMed

    Liljeroos, Lassi; Huiskonen, Juha T; Ora, Ari; Susi, Petri; Butcher, Sarah J

    2011-11-01

    Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix-matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices.

  18. Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions

    PubMed Central

    Liljeroos, Lassi; Huiskonen, Juha T.; Ora, Ari; Susi, Petri; Butcher, Sarah J.

    2011-01-01

    Measles virus is a highly infectious, enveloped, pleomorphic virus. We combined electron cryotomography with subvolume averaging and immunosorbent electron microscopy to characterize the 3D ultrastructure of the virion. We show that the matrix protein forms helices coating the helical ribonucleocapsid rather than coating the inner leaflet of the membrane, as previously thought. The ribonucleocapsid is folded into tight bundles through matrix–matrix interactions. The implications for virus assembly are that the matrix already tightly interacts with the ribonucleocapsid in the cytoplasm, providing a structural basis for the previously observed regulation of RNA transcription by the matrix protein. Next, the matrix-covered ribonucleocapsids are transported to the plasma membrane, where the matrix interacts with the envelope glycoproteins during budding. These results are relevant to the nucleocapsid organization and budding of other paramyxoviruses, where isolated matrix has been observed to form helices. PMID:22025713

  19. Integration of Ultraviolet Photodissociation with Proton Transfer Reactions and Ion Parking for Analysis of Intact Proteins.

    PubMed

    Holden, Dustin D; McGee, William M; Brodbelt, Jennifer S

    2016-01-01

    We report the implementation of proton transfer reactions (PTR) and ion parking on an Orbitrap mass spectrometer. PTR/ion parking allows charge states of proteins to be focused into a single lower charge state via sequential deprotonation reactions with a proton scavenging reagent, in this case, a nitrogen-containing adduct of fluoranthene. Using PTR and ion parking, we evaluate the charge state dependence of fragmentation of ubiquitin (8.6 kDa), myoglobin (17 kDa), and carbonic anhydrase (29 kDa) upon higher energy collisional dissociation (HCD) or ultraviolet photodissociation (UVPD). UVPD exhibited less charge state dependence, thus yielding more uniform distributions of cleavages along the protein backbone and consequently higher sequence coverage than HCD. HCD resulted in especially prominent cleavages C-terminal to amino acids containing acidic side-chains and N-terminal to proline residues; UVPD did not exhibit preferential cleavage adjacent to acidic residues but did show enhancement next to proline and phenylalanine. PMID:26633754

  20. Microwave-assisted acid and base hydrolysis of intact proteins containing disulfide bonds for protein sequence analysis by mass spectrometry.

    PubMed

    Reiz, Bela; Li, Liang

    2010-09-01

    Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein.

  1. 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

  2. Effect of enzyme-aided cell wall disintegration on protein extractability from intact and dehulled rapeseed (Brassica rapa L. and Brassica napus L.) press cakes.

    PubMed

    Rommi, Katariina; Hakala, Terhi K; Holopainen, Ulla; Nordlund, Emilia; Poutanen, Kaisa; Lantto, Raija

    2014-08-13

    Cell-wall- and pectin-degrading enzyme preparations were used to enhance extractability of proteins from rapeseed press cake. Rapeseed press cakes from cold pressing of intact Brassica rapa and partially dehulled Brassica napus seeds, containing 36-40% protein and 35% carbohydrates, were treated with pectinolytic (Pectinex Ultra SP-L), xylanolytic (Depol 740L), and cellulolytic (Celluclast 1.5L) enzyme preparations. Pectinex caused effective disintegration of embryonic cell walls through hydrolysis of pectic polysaccharides and glucans and increased protein extraction by up to 1.7-fold in comparison to treatment without enzyme addition. Accordingly, 56% and 74% of the total protein in the intact and dehulled press cakes was extracted. Light microscopy of the press cakes suggested the presence of pectins colocalized with proteins inside the embryo cells. Hydrolysis of these intracellular pectins and deconstruction of embryonic cell walls during Pectinex treatment were concluded to relate with enhanced protein release.

  3. 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.

  4. Electrically-assisted delivery of an anionic protein across intact skin: cathodal iontophoresis of biologically active ribonuclease T1.

    PubMed

    Dubey, S; Kalia, Y N

    2011-06-30

    Cathodal iontophoresis of anionic macromolecules has been considered a major challenge owing to (i) the presence of a negative charge on the skin under physiological conditions and (ii) the electroosmotic solvent flow in the (opposite) anode-to-cathode direction. Moreover, electroosmosis, and not electromigration, was considered as the likely electrotransport mechanism for high molecular weight cations. However, it was recently shown that electromigration governed anodal iontophoretic transport of Cytochrome c (12.4 kDa) and Ribonuclease A (RNAse A; 13.6 kDa). Thus, the objective of this study was to investigate the feasibility of iontophoresing a negatively charged protein, the enzyme Ribonuclease T1 (RNAse T1, 11.1 kDa), from the cathode across intact skin. Cumulative permeation and skin deposition of RNAse T1 were investigated as a function of current density (0.15, 0.3 and 0.5 mA/cm(2) applied for 8h) using porcine ear skin and quantified by an enzymatic activity assay. Although RNAse T1 permeation was dependent upon current density (22.41 ± 8.10, 76.41 ± 56.98 and 142.19 ± 62.23μg/cm(2), respectively), no such relationship was observed with respect to skin deposition (9.78 ± 2.39, 7.76 ± 4.34 and 8.70 ± 2.94 μg/cm(2), respectively). MALDI-TOF spectra and the activity assay confirmed that RNAse T1 retained structural integrity and enzymatic function post-iontophoresis. Acetaminophen iontophoresis demonstrated the anode-to-cathode directionality of electroosmotic solvent flow confirming that RNAse T1 electrotransport was due entirely to electromigration. Interestingly, despite its lower net charge and higher molecular weight, electromigration of cationic Ribonuclease A was superior to that of RNAse T1 after iontophoresis at 0.5 mA/cm(2) for 8h. These results provide further evidence that charge to mass ratio and hence electric mobility might not alone be sufficient to predict protein electrotransport across the skin; three dimensional structures and the

  5. Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells.

    PubMed Central

    Häring, H U; White, M F; Machicao, F; Ermel, B; Schleicher, E; Obermaier, B

    1987-01-01

    It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, we labeled intact rat fat cells with [32P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, we found, in addition to the 95-kDa beta-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor beta-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. The insulin effect starts after 30 sec, is maximal at 150 sec, and declines to almost basal values by 5 min. Furthermore, the antiphosphotyrosine antibody precipitated at least five proteins in the soluble fraction of the fat cell. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32P incorporation into a 116-kDa band, a 62-kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. We suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission. Images PMID:3540953

  6. Characterization of Protein N-Glycosylation by Analysis of ZIC-HILIC-Enriched Intact Proteolytic Glycopeptides.

    PubMed

    Pohlentz, Gottfried; Marx, Kristina; Mormann, Michael

    2016-01-01

    Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) solid-phase extraction (SPE) combined with direct-infusion nanoESI mass spectrometry (MS) and tandem MS/MS is a well-suited method for the analysis of protein N-glycosylation. A site-specific characterization of N-glycopeptides is achieved by the combination of proteolytic digestions employing unspecific proteases, glycopeptide enrichment by use of ZIC-HILIC SPE, and subsequent mass spectrometric analysis. The use of thermolysin or a mixture of trypsin and chymotrypsin leads per se to a mass-based separation, that is, small nonglycosylated peptides and almost exclusively glycopeptides at higher m/z values. As a result of their higher hydrophilicity N-glycopeptides comprising short peptide backbones are preferably accumulated by the ZIC-HILIC-based separation procedure. By employing this approach complications associated with low ionization efficiencies of N-glycopeptides resulting from signal suppression in the presence of highly abundant nonglycosylated peptides can be largely reduced. Here, we describe a simple protocol aimed at the enrichment of N-glycopeptides derived from in-solution and in-gel digestions of SDS-PAGE-separated glycoproteins preceding mass spectrometric analysis.

  7. A setup for simultaneous measurement of infrared spectra and light scattering signals: Watching amyloid fibrils grow from intact proteins

    SciTech Connect

    Li, Yang; Maurer, Jürgen; Roth, Andreas; Vogel, Vitali; Winter, Ernst; Mäntele, Werner

    2014-08-15

    A setup for the simultaneous measurement of mid-infrared spectra and static light scattering is described that can be used for the analysis of the formation of nanoscale and microscopic aggregates from smaller molecules to biopolymers. It can be easily integrated into sample chambers of infrared spectrometers or combined with laser beams from tunable infrared lasers. Here, its use for the analysis of the formation of amyloid fibrils from intact proteins is demonstrated. The formation of amyloid fibrils or plaques from proteins is a widespread and pathogenetic relevant process, and a number of diseases are caused and correlated with the deposition of amyloid fibrils in cells and tissues. The molecular mechanisms of these transformations, however, are still unclear. We report here the simultaneous measurement of infrared spectra and static light scattering for the analysis of fibril formation from egg-white lysozyme. The transformation of the native form into non-native forms rich in β-sheet structure is measured by analysis of the amide I spectral region in the infrared spectra, which is sensitive for local structures. At the same time, light scattering signals at forward direction as well as the forward/backward ratio, which are sensitive for the number of scattering centers and their approximate sizes, respectively, are collected for the analysis of fibril growth. Thermodynamic and kinetic parameters as well as mechanistic information are deduced from the combination of the two complementary techniques.

  8. Purification of antigenically intact Ro ribonucleoproteins; biochemical and immunological evidence that the 52-kD protein is not a Ro protein.

    PubMed Central

    Boire, G; Gendron, M; Monast, N; Bastin, B; Ménard, H A

    1995-01-01

    Anti-Ro sera immunoprecipitate Ro ribonucleoproteins (RNPs) from human cell extracts. Ro RNPs are biochemically heterogeneous particles whose functions are unknown and whose exact composition remains controversial. In addition to 60-kD Ro and to La proteins, a 52-kD polypeptide (p52) has been proposed to be a stable component of the Ro RNPs. To confirm the immunological studies supporting this hypothesis, we have biochemically purified Ro RNPs from HeLa cells using non-denaturing conditions. Ro RNPs segregated into three distinct populations, one of which only contained hY5 RNA (RohY5 RNPs). No p52 co-purified with Ro RNPs. Despite the absence of p52, purified Ro RNPs had biochemical and immunological properties identical to those of unfractionated Ro RNPs. Many anti-Ro sera only recognize p52 in immunoblots, and are said to be monospecific anti-p52. Preincubation with purified RohY5 RNPs (free of p52) of all human anti-Ro (including so-called monospecific anti-p52) sera abolished their capacity to immunoprecipitate Ro RNPs from unfractionated HeLa cell extracts. Conversely, preincubation of anti-Ro sera with purified p52 protein specifically inhibited recognition of p52 in immunoblots, but did not interfere with immunoprecipitation of Ro RNPs. Our data demonstrate that anti-p52 antibodies do not target intact Ro RNPs, nor do they target the native 60-kD Ro protein. Contrary to previous reports, p52 protein is not a stable component of antigenically intact Ro RNPs. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7774061

  9. 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

  10. 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

  11. 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.

  12. Gel-aided sample preparation (GASP)--a simplified method for gel-assisted proteomic sample generation from protein extracts and intact cells.

    PubMed

    Fischer, Roman; Kessler, Benedikt M

    2015-04-01

    We describe a "gel-assisted" proteomic sample preparation method for MS analysis. Solubilized protein extracts or intact cells are copolymerized with acrylamide, facilitating denaturation, reduction, quantitative cysteine alkylation, and matrix formation. Gel-aided sample preparation has been optimized to be highly flexible, scalable, and to allow reproducible sample generation from 50 cells to milligrams of protein extracts. This methodology is fast, sensitive, easy-to-use on a wide range of sample types, and accessible to nonspecialists. PMID:25515006

  13. ATR–FTIR spectroscopy reveals involvement of lipids and proteins of intact pea pollen grains to heat stress tolerance

    PubMed Central

    Lahlali, Rachid; Jiang, Yunfei; Kumar, Saroj; Karunakaran, Chithra; Liu, Xia; Borondics, Ferenc; Hallin, Emil; Bueckert, Rosalind

    2014-01-01

    With climate change, pea will be more frequently subjected to heat stress in semi-arid regions like Saskatchewan during flowering. The pollen germination percentage of two pea cultivars was reduced by heat stress (36°C) with an important decrease in cultivar ‘CDC Golden’ compared to ‘CDC Sage.’ Lipids, protein and other pollen coat compositions of whole intact pollen grains of both pea cultivars were investigated using mid infrared (mid-IR) attenuated total reflectance (ATR)–Fourier transform infrared (FTIR) spectroscopy. Curve fitting of ATR absorbance spectra in the protein region enabled estimation and comparison of different protein secondary structures between the two cultivars. CDC Sage had relatively greater amounts of α-helical structures (48.6–43.6%; band at 1654 cm-1) and smaller amounts of β-sheets (41.3–46%) than CDC Golden. The CDC Golden had higher amounts of β-sheets (46.3–51.7%) compared to α-helical structures (35.3–36.2%). Further, heat stress resulted in prominent changes in the symmetrical and asymmetrical CH2 bands from lipid acyl chain, ester carbonyl band, and carbohydrate region. The intensity of asymmetric and symmetric CH2 vibration of heat stressed CDC Golden was reduced considerably in comparison to the control and the decrease was higher compared to CDC Sage. In addition, CDC Golden showed an increase in intensity at the oxidative band of 3015 cm-1. These results reveal that the whole pollen grains of both pea cultivars responded differently to heat stress. The tolerance of CDC Sage to heat stress (expressed as pollen germination percentage) may be due to its protein richness with α-helical structures which would protect against the destructive effects of dehydration due to heat stress. The low pollen germination percentage of CDC Golden after heat stress may be also due to its sensitivity to lipid changes due to heat stress. PMID:25566312

  14. RELATIONSHIP BETWEEN INSULIN-RESISTANCE PROCESSING SPEED AND SPECIFIC EXECUTIVE FUNCTION PROFILES IN NEUROLOGICALLY-INTACT OLDER ADULTS

    PubMed Central

    Frazier, Darvis T.; Bettcher, Brianne M.; Dutt, Shubir; Patel, Nihar; Mungas, Dan; Miller, Joshua; Green, Ralph; Kramer, Joel H.

    2016-01-01

    Objective This study investigated the relationship between insulin-resistance and constituent components of executive function in a sample of neurologically-intact older adult subjects using the homeostasis model assessment (HOMA-IR) and latent factors of working memory, cognitive control and processing speed derived from confirmatory factor analysis. Low-density lipoprotein (LDL), mean arterial pressure (MAP), along with body mass index (BMI) and white matter hypointensity (WMH) were used to control for vascular risk factors, adiposity and cerebrovascular injury. Methods The study included 119 elderly subjects recruited from the University of California, San Francisco Memory and Aging Center. Subjects underwent neuropsychological assessment, fasting blood draw and brain magnetic resonance imaging (MRI). Partial correlations and linear regression models were used to examine the HOMA-IR-executive function relationship. Results Pearson correlation adjusting for age showed a significant relationship between HOMA-IR and working memory (rp=−.18, p=.047), a trend with cognitive control (rp=−.17, p=.068), and no relationship with processing speed (rp=.013, p=.892). Linear regression models adjusting for demographic factors (age, education and gender), LDL, MAP, BMI and WMH indicated that HOMA-IR was negatively associated with cognitive control (r=−.256; p=.026) and working memory (r=−.234; p=.054). Conclusions These results suggest a greater level of peripheral insulin-resistance is associated with decreased cognitive control and working memory. After controlling for demographic factors, vascular risk, adiposity and cerebrovascular injury, HOMA-IR remained significantly associated with cognitive control, with working memory showing a trend. These findings substantiate the insulin-resistance-executive function hypothesis and suggest a complex interaction, demonstrated by the differential impact of insulin-resistance on processing speed and specific aspects of

  15. Novel molecular events associated with altered steroidogenesis induced by exposure to atrazine in the intact and castrate male rat.

    PubMed

    Riffle, Brandy W; Klinefelter, Gary R; Cooper, Ralph L; Winnik, Witold M; Swank, Adam; Jayaraman, Saro; Suarez, Juan; Best, Deborah; Laws, Susan C

    2014-08-01

    Toxicology is increasingly focused on molecular events comprising adverse outcome pathways. Atrazine activates the hypothalamic-pituitary adrenal axis, but relationships to gonadal alterations are unknown. We characterized hormone profiles and adrenal (intact and castrate) and testis (intact) proteomes in rats after 3 days of exposure. The adrenal accounted for most of the serum progesterone and all of the corticosterone increases in intact and castrated males. Serum luteinizing hormone, androstenedione, and testosterone in intact males shared a non-monotonic response suggesting transition from an acute stimulatory to a latent inhibitory response to exposure. Eight adrenal proteins were significantly altered with dose. There were unique proteomic changes between the adrenals of intact and castrated males. Six testis proteins in intact males had non-monotonic responses that significantly correlated with serum testosterone. Different dose-response curves for steroids and proteins in the adrenal and testis reveal novel adverse outcome pathways in intact and castrated male rats.

  16. Comprehensive Profiling of Amino Acid Response Uncovers Unique Methionine-Deprived Response Dependent on Intact Creatine Biosynthesis

    PubMed Central

    Tang, Xiaohu; Keenan, Melissa M.; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J. Will; Freedland, Stephen J.; Murphy, Susan K.; Chi, Jen-Tsan

    2015-01-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  17. Comprehensive profiling of amino acid response uncovers unique methionine-deprived response dependent on intact creatine biosynthesis.

    PubMed

    Tang, Xiaohu; Keenan, Melissa M; Wu, Jianli; Lin, Chih-An; Dubois, Laura; Thompson, J Will; Freedland, Stephen J; Murphy, Susan K; Chi, Jen-Tsan

    2015-04-01

    Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine

  18. Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling

    PubMed Central

    Shang, Jing; Zrazhevskiy, Pavel; Postupna, Nadia; Keene, C. Dirk; Montine, Thomas J.; Gao, Xiaohu

    2015-01-01

    In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, β-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the “Alzheimer’s disease” cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics. PMID:26328896

  19. Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients.

    PubMed

    Bieghs, Liesbeth; Brohus, Malene; Kristensen, Ida B; Abildgaard, Niels; Bøgsted, Martin; Johnsen, Hans E; Conover, Cheryl A; De Bruyne, Elke; Vanderkerken, Karin; Overgaard, Michael T; Nyegaard, Mette

    2016-01-01

    Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration. PMID:27111220

  20. Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients

    PubMed Central

    Bieghs, Liesbeth; Brohus, Malene; Kristensen, Ida B.; Abildgaard, Niels; Bøgsted, Martin; Johnsen, Hans E.; Conover, Cheryl A.; De Bruyne, Elke; Vanderkerken, Karin

    2016-01-01

    Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5–3.8 fold) and decrease in intact IGFBP-3 (0.6–0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration. PMID:27111220

  1. 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.

  2. 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

  3. 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

  4. Phosphorylation by protein kinase C of the 20,000-dalton light chain of myosin in intact and chemically skinned vascular smooth muscle.

    PubMed

    Sutton, T A; Haeberle, J R

    1990-02-15

    In the present study we tested the hypothesis that phosphorylation of the 20,000-dalton light chain subunit of smooth muscle myosin (LC20) by the calcium-activated and phospholipid-dependent protein kinase C regulates contraction of chemically-permeabilized (glycerinated) porcine carotid artery smooth muscle. Purified protein kinase C and oleic acid were used to phosphorylate LC20 in glycerinated muscles in the presence of a CaEGTA/EGTA buffer system (pCa 8) to prevent activation of myosin light chain kinase. Phosphorylation of the light chain to 1.3 mol of PO4/mol of LC20 did not stimulate contraction. Tryptic digests of glycerinated carotid artery LC20 contained two major phosphopeptides which contained phosphoserine but not phosphothreonine. Incubation of glycerinated muscles with calcium (20 microM) and calmodulin (10 microM) resulted in contraction and LC20 phosphorylation to 1.1 mol of PO4/mol of LC20; tryptic digests of LC20 from these muscles contained a single phosphopeptide which could be distinguished by phosphopeptide mapping from the two phosphopeptides derived from muscles phosphorylated with protein kinase C. Further phosphorylation of Ca2+/calmodulin-activated muscles to 2.0 mol of PO4/mol of LC20, by incubation with protein kinase C, had no effect on either the level of isometric force or the lightly-loaded shortening velocity (after-load = 0.1 peak active force); removal of Ca2+ and calmodulin, but not protein kinase C and oleic acid, resulted in normal relaxation in spite of maintained phosphorylation to 1.2 mol of PO4/mol of LC20. Comparison of LC20 phosphopeptide maps from glycerinated muscles incubated with protein kinase C plus Ca2+/calmodulin (2.0 mol of PO4/mol of LC20) to maps from intact muscles stimulated with 10(-6) M phorbol 12,13-dibutyrate (0.05 mol of PO4/mol of LC20) showed that the same three phosphopeptides were present in both the intact and glycerinated muscles. These findings show that phosphorylation of LC20 by protein kinase

  5. 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

  6. PTMSearchPlus: Software Tool for Automated Protein Identification and Post-Translational and Post-Translational Modification Characterization by Integrating Accurate Intact Protein Mass and Bottom-Up Mass Spectrometric Data Searches

    SciTech Connect

    Kertesz, Vilmos; Connelly, Heather M; Erickson, Brian K; Hettich, Robert {Bob} L

    2009-01-01

    PTMSearchPlus is a software tool for the automated integration of accurate intact protein mass (AIPM) and bottom-up (BU) mass spectra searches/data in order to both confidently identify the intact proteins and to characterize their post-translational modifications (PTMs). The development of PTMSearchPlus was motivated by the desire to effectively integrate high-resolution intact protein molecular masses with bottom-up peptide MS/MS data. PTMSearchPlus requires as input both intact protein and proteolytic peptide mass spectra collected from the same protein mixture, a FASTA protein database, and a selection of possible PTMs, the types and ranges of which can be specified. The output of PTMSearchPlus is a list of intact and modified proteins matching the AIPM data concomitant with their respective peptides found by the BU search. This list also contains protein and peptide sequence coverage information, scores, etc. that can be used for further evaluation or refiltering of the results. Corresponding and annotated AIPM and BU mass spectra are also displayed for visual inspection when a listed protein or a peptide is selected. These and other controls ensure that the user can manually evaluate, modify (e.g., remove obvious false positives, low quality spectra etc.), and save the results of the automated search if necessary. Driven by the exponential growth in the number of possible peptide candidates in a BU search when multiple PTMs are probed, the advantages on search speed by limiting the total number of possible PTMs on a peptide in the BU search or by performing an AIPM predicted BU search are also discussed in addition to the integration approach. The features of PTMSearchPlus are demonstrated using both a protein standard mixture and a complex protein mixture from Escherichia coli. Experimental data revealed a unique advantage of coupling AIPM and the BU data sets that is mutually beneficial for both approaches. Namely, AIPM data can confirm that no PTM peptides

  7. PTMSearchPlus: A Software Tool for Automated Protein Identification and Post-Translational Modification Characterization by Integrating Accurate Intact Protein Mass and Bottom-Up Mass Spectrometric Data Searches

    SciTech Connect

    Kertesz, Vilmos; Connelly, Heather M; Erickson, Brian K; Hettich, Robert {Bob} L

    2009-01-01

    PTMSearchPlus is a software tool for the automated integration of accurate intact protein mass (AIPM) and bottom-up (BU) mass spectra searches/data in order to both confidently identify the intact proteins and to characterize their post-translational modifications (PTMs). The development of PTMSearchPlus was motivated by the desire to effectively integrate high resolution intact protein molecular masses with bottom-up peptide MS/MS data. PTMSearchPlus requires as input both intact protein and proteolytic peptide mass spectra collected from the same protein mixture, a FASTA protein database, and a selection of possible PTMs, the types and ranges of which can be specified. The output of PTMSearchPlus is a list of intact and modified proteins matching the AIPM data concomitant with their respective peptides found by the BU search. This list also contains protein and peptide sequence coverage information, scores, etc. that can be used for further evaluation or refiltering of the results. Corresponding and annotated AIPM and BU mass spectra are also displayed for visual inspection when a listed protein or a peptide is selected. These and other controls ensure that the user can manually evaluate, modify (e.g. remove obvious false positives, low quality spectra etc.), and save the results of the automated search if necessary. Driven by the exponential growth in the number of possible peptide candidates in a BU search when multiple PTMs are probed, the advantages on search speed by limiting the total number of possible PTMs on a peptide in the BU search or by performing an AIPM predicted BU search are also discussed in addition to the integration approach. The features of PTMSearchPlus are demonstrated using both a protein standard mixture and a complex protein mixture from Escherichia coli. Experimental data revealed a unique advantage of coupling AIPM and the BU datasets that is mutually beneficial for both approaches. Namely, AIPM data can confirm that no PTM peptides

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Genetics coupled to quantitative intact proteomics links heritable aphid and endosymbiont protein expression to circulative polerovirus transmission.

    PubMed

    Cilia, M; Tamborindeguy, C; Fish, T; Howe, K; Thannhauser, T W; Gray, S

    2011-03-01

    Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid

  15. Intravenous or luminal amino acids are insufficient to maintain pancreatic growth and digestive enzyme expression in the absence of intact dietary protein

    PubMed Central

    Baumler, Megan D.; Koopmann, Matthew C.; Thomas, Diana D. H.; Ney, Denise M.

    2010-01-01

    We previously reported that rats receiving total parenteral nutrition (TPN) undergo significant pancreatic atrophy characterized by reduced total protein and digestive enzyme expression due to a lack of intestinal stimulation by nutrients (Baumler MD, Nelson DW, Ney DM, Groblewski GE. Am J Physiol Gastrointest Liver Physiol 292: G857–G866, 2007). Essentially identical results were recently reported in mice fed protein-free diets (Crozier SJ, D'Alecy LG, Ernst SA, Ginsburg LE, Williams JA. Gastroenterology 137: 1093–1101, 2009), provoking the question of whether reductions in pancreatic protein and digestive enzyme expression could be prevented by providing amino acids orally or by intravenous (IV) infusion while maintaining intestinal stimulation with fat and carbohydrate. Controlled studies were conducted in rats with IV catheters including orally fed/saline infusion or TPN-fed control rats compared with rats fed a protein-free diet, oral amino acid, or IV amino acid feeding, all with oral carbohydrate and fat. Interestingly, neither oral nor IV amino acids were sufficient to prevent the pancreatic atrophy seen for TPN controls or protein-free diets. Oral and IV amino acids partially attenuated the 75–90% reductions in pancreatic amylase and trypsinogen expression; however, values remained 50% lower than orally fed control rats. Lipase expression was more modestly reduced by a lack of dietary protein but did respond to IV amino acids. In comparison, chymotrypsinogen expression was induced nearly twofold in TPN animals but was not altered in other experimental groups compared with oral control animals. In contrast to pancreas, protein-free diets had no detectable effects on jejunal mucosal villus height, total mass, protein, DNA, or sucrase activity. These data underscore that, in the rat, intact dietary protein is essential in maintaining pancreatic growth and digestive enzyme adaptation but has surprisingly little effect on small intestinal mucosa. PMID

  16. Generation and Surface Localization of Intact M Protein in Streptococcus pyogenes Are Dependent on sagA

    PubMed Central

    Biswas, Indranil; Germon, Pierre; McDade, Kathleen; Scott, June R.

    2001-01-01

    The M protein is an important surface-located virulence factor of Streptococcus pyogenes, the group A streptococcus (GAS). Expression of M protein is primarily controlled by Mga, a transcriptional activator protein. A recent report suggested that the sag locus, which includes nine genes necessary and sufficient for production of streptolysin S, another GAS virulence factor, is also needed for transcription of emm, encoding the M protein (Z. Li, D. D. Sledjeski, B. Kreikemeyer, A. Podbielski, and M. D. Boyle, J. Bacteriol. 181:6019–6027, 1999). To investigate this in more detail, we constructed an insertion-deletion mutation in sagA, the first gene in the sag locus, in the M6 strain JRS4. The resulting strain, JRS470, produced no detectable streptolysin S and showed a drastic reduction in cell surface-associated M protein, as measured by cell aggregation and Western blot analysis. However, transcription of the emm gene was unaffected by the sagA mutation. Detailed analysis with monoclonal antibodies and an antipeptide antibody showed that the M protein in the sagA mutant strain was truncated so that it lacks the C-repeat region and the C-terminal domain required for anchoring it to the cell surface. This truncated M protein was largely found, as expected, in the culture supernatant. Lack of surface-located M protein made the sagA mutant strain susceptible to phagocytosis. Thus, although sagA does not affect transcription of the M6 protein gene, it is needed for the surface localization of this important virulence factor. PMID:11598078

  17. 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.

  18. 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.

  19. 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.

  20. 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/.

  1. Ca/sup + +/- and cyclic AMP-induced changes in intact cell phosphorylation of ileal microvillus membrane proteins

    SciTech Connect

    Sharp, G.W.G.; Hannah, C.M.; Cohen, M.; Donowitz, M.

    1986-03-05

    Pieces of rabbit distal ileal mucosa, with the muscularis propria and serosa removed, were incubated for 90 minutes in Krebs-Ringer bicarborate buffer (KRB) with /sup 32/PO/sub 4/ to label the intracellular nucleotide pools. After rinsing, the mucosal pieces were transferred to KRB in the absence and presence of 10 ..mu..M A23187 or 10 mM theophylline. After a further 10 minutes the cells were scraped off and microvillus membranes prepared. The membranes were solubilized, subjected to two dimensional gel electrophoresis and autoradiography, and analyzed by densitometry. A23187 increased the phosphorylation of four microvillus membrane proteins with M/sub r/ of 32, 52, 110 and 116K. Increased phosphorylation of the 52 and 116K proteins has also been detected in microvillus membranes subjected to Ca/sup + +/ and calmodulin in the presence of ..gamma..-/sup 32/P-ATP. Theophylline increased the phosphorylation of the same 32 and 52K proteins and, additionally, of a second 32K peptide. While any of these proteins could be involved in the control of electrolyte transport, it is noteworthy that increased Ca/sup + +/, and increased cyclic AMP levels exert similar effects upon intestinal electrolyte transport. That A23187 and theophylline both increase the phosphorylation of the 32 and 52K proteins increases the possibility that these are involved in ion transport.

  2. Rapid activity-dependent delivery of the neurotrophic protein CPG15 to the axon surface of neurons in intact Xenopus tadpoles.

    PubMed

    Cantallops, Isabel; Cline, Hollis T

    2008-05-01

    CPG15 (aka neuritin) is an activity-induced GPI-anchored axonal protein that promotes dendritic and axonal growth, and accelerates synaptic maturation in vivo. Here we show that CPG15 is distributed inside axons and on the axon surface. CPG15 is trafficked to and from the axonal surface by membrane depolarization. To assess CPG15 trafficking in vivo, we expressed an ecliptic pHluorin (EP)-CPG15 fusion protein in optic tectal explants and in retinal ganglion cells of intact Xenopus tadpoles. Depolarization by KCl increased EP-CPG15 fluorescence on axons. Intraocular kainic acid (KA) injection rapidly increased cell-surface EP-CPG15 in retinotectal axons, but coinjection of TTX and KA did not. Consistent with this, we find that intracellular CPG15 is localized to vesicles and endosomes in presynaptic terminals and colocalizes with synaptic vesicle proteins. The results indicate that the delivery of the neurotrophic protein CPG15 to the axon surface can be regulated on a rapid time scale by activity-dependent mechanisms in vivo. PMID:18383547

  3. Vaccinia Virus Protein Synthesis Has a Low Requirement for the Intact Translation Initiation Factor eIF4F, the Cap-Binding Complex, within Infected Cells

    PubMed Central

    Mulder, Jacqueline; Robertson, Morwenna E. M.; Seamons, Rachael A.; Belsham, Graham J.

    1998-01-01

    The role of the cap-binding complex, eIF4F, in the translation of vaccinia virus mRNAs has been analyzed within infected cells. Plasmid DNAs, which express dicistronic mRNAs containing a picornavirus internal ribosome entry site, produced within vaccinia virus-infected cells both β-glucuronidase and a cell surface-targeted single-chain antibody (sFv). Cells expressing sFv were selected from nonexpressing cells, enabling analysis of protein synthesis specifically within the transfected cells. Coexpression of poliovirus 2A or foot-and-mouth disease virus Lb proteases, which cleaved translation initiation factor eIF4G, greatly inhibited cap-dependent protein (β-glucuronidase) synthesis. Under these conditions, internal ribosome entry site-directed expression of sFv continued and cell selection was maintained. Furthermore, vaccinia virus protein synthesis persisted in the selected cells containing cleaved eIF4G. Thus, late vaccinia virus protein synthesis has a low requirement for the intact cap-binding complex eIF4F. This may be attributed to the short unstructured 5′ noncoding regions of the vaccinia virus mRNAs, possibly aided by the presence of poly(A) at both 5′ and 3′ termini. PMID:9765426

  4. Targeted absolute quantification of intact proteins by reversed phase liquid chromatography-mass spectrometry, charge reduced electrospray, and condensation particle counting.

    PubMed

    Adou, Kouame; Johnston, Murray V; Dykins, John L

    2012-08-21

    A novel approach involving the use of reversed phase liquid chromatography-mass spectrometry (RPLC-MS), charge reduced electrospray (CRES), and condensation particle counting (CPC) for the absolute quantification of intact proteins in liquid solutions is introduced. Under analysis conditions optimized for the quantification of select proteins within their predetermined linear ranges, a set of at least five protein standards with molecular weights (MW) spanning the dynamic ranges of both a quadrupole time-of-flight (QTOF) MS and a suitably selected RPLC column is used to generate a calibration curve of CPC detection efficiency (DE) as a function of the square root of MW. Next, the sample of interest is analyzed, and from the MS-generated MW data, the DE of each target protein is determined from the calibration curve. On the basis of MW, DE, and number concentration (molecules/unit volume), absolute quantification is achieved for each protein of interest. Application of this approach to the absolute quantification of cytochrome C (as target compound) in a commercial protein mixture is demonstrated with a deviation of 8%, a coefficient of variation (CV) of 5%, and a quantification limit of 432 fmol. For nontarget components of the mixture (ribonuclease A, holotransferrin, and apomyoglobin), the percent deviation from the stated concentrations and the CV varied from 0.20 to 23 and from 4.1 to 18, respectively. Performance of the method was further assessed by analyzing a laboratory quality control mixture comprising 0.33 μM of cytochrome C. The calculated value was 0.34 (CV: 5.1%). Universal in essence, the new technique holds strong promise for the absolute quantification of select proteins in liquid samples under conditions of good peak resolution and stable baseline.

  5. Removal of Available Decorin Core-Protein from Powdered Bovine Hide by Treatments used to Process Intact Hides into Leather

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a modification of a previously developed sandwich Elisa procedure to measure decorin core-protein (DCP), we determined the available decorin content of a sample of raw powdered bovine hide before and after treatment with the reagents used in the early steps of the process for converting a hide...

  6. Proteins in vacuo: A molecular dynamics study of the unfolding behavior of highly charged disulfide-bond-intact lysozyme subjected to a temperature pulse

    NASA Astrophysics Data System (ADS)

    Reimann, C. T.; Velázquez, I.; Bittner, M.; Tapia, O.

    1999-12-01

    Molecular dynamics simulations were used to interpret a variety of experimental data on highly charged disulfide-bond-intact lysozyme in vacuo. The simulation approach involved submitting a model of the protein [Reimann, Velázquez, and Tapia, J. Phys. Chem. B 102, 9344 (1998)] in a given charge state to a 3-ns-long heat pulse (usually at 500 K) followed by cooling or relaxation for 1 ns back to room temperature (293 K). This treatment yielded a charge threshold around Q0=8+ for obtaining significant unfolding, as indicated by an enhancement in collision cross section and conformer length. The collision cross sections and lengths theoretically obtained, along with the threshold charge state for initiating unfolding, were compatible with experimental results on lysozyme in vacuo. The unfolded, highly elongated conformations obtained for Q>=9+ displayed a significant level of non-native β-sheet content which appeared to be additionally stabilized by charge self-solvation.

  7. Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns.

    PubMed

    Tvardovskiy, Andrey; Wrzesinski, Krzysztof; Sidoli, Simone; Fey, Stephen J; Rogowska-Wrzesinska, Adelina; Jensen, Ole N

    2015-12-01

    Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.

  8. 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.

  9. 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.

  10. An essential virulence protein of Agrobacterium tumefaciens, VirB4, requires an intact mononucleotide binding domain to function in transfer of T-DNA.

    PubMed

    Fullner, K J; Stephens, K M; Nester, E W

    1994-12-15

    The 11 gene products of the Agrobacterium tumefaciens virB operon, together with the VirD4 protein, are proposed to form a membrane complex which mediates the transfer of T-DNA to plant cells. This study examined one putative component of that complex, VirB4. A deletion of the virB4 gene on the Ti plasmid pTiA6NC was constructed by replacing the virB4 gene with the kanamycin resistance-conferring nptII gene. The virB4 gene was found to be necessary for virulence on plants and for the transfer of IncQ plasmids to recipient cells of A. tumefaciens. Genetic complementation of the deletion strain by the virB4 gene under control of the virB promoter confirmed that the deletion was nonpolar on downstream virB genes. Genetic complementation was also achieved with the virB4 gene placed under control of the lac promoter, even though synthesis of the VirB4 protein from this promoter is far below wild-type levels. Having shown a role for the VirB4 protein in DNA transfer, lysine-439, found within the conserved mononucleotide binding domain of VirB4, was changed to a glutamic acid, methionine, or arginine by oligonucleotide-directed mutagenesis. virB4 genes bearing these mutations were unable to complement the virB4 deletion for either virulence or for IncQ transfer, showing that an intact mononucleotide binding site is necessary for the function of VirB4 in DNA transfer. The necessity of the VirB4 protein with an intact mononucleotide binding site for extracellular complementation of virE2 mutants was also shown. In merodiploid studies, lysine-439 mutations present in trans decreased IncQ plasmid transfer frequencies, suggesting that VirB4 functions within a complex to facilitate DNA transfer. PMID:7830718

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. Characterization of intact protein conjugates and biopharmaceuticals using ion-exchange chromatography with online detection by native electrospray ionization mass spectrometry and top-down tandem mass spectrometry.

    PubMed

    Muneeruddin, Khaja; Nazzaro, Mark; Kaltashov, Igor A

    2015-10-01

    Characterization of biopharmaceutical products is a challenging task, which needs to be carried out at several different levels (including both primary structure and conformation). An additional difficulty frequently arises due to the structural heterogeneity inherent to many protein-based therapeutics (e.g., extensive glycosylation or "designer" modifications such as chemical conjugation) or introduced postproduction as a result of stress (e.g., oxidation and deamidation). A combination of ion-exchange chromatography (IXC) with online detection by native electrospray ionization mass spectrometry (ESI MS) allows characterization of complex and heterogeneous therapeutic proteins and protein conjugates to be accomplished at a variety of levels without compromising their conformational integrity. The IXC/ESI MS measurements allow protein conjugates to be profiled by analyzing conjugation stoichiometry and the presence of multiple positional isomers, as well as to establish the effect of chemical modifications on the conformational integrity of each species. While mass profiling alone is not sufficient for identification of nonenzymatic post-translational modifications (PTMs) that result in a very small mass change of the eluting species (e.g., deamidation), this task can be completed using online top-down structural analysis, as demonstrated using stressed interferon-β as an example. The wealth of information that can be provided by IXC/native ESI MS and tandem mass spectrometry (MS/MS) on protein-based therapeutics will undoubtedly make it a very valuable addition to the experimental toolbox of biopharmaceutical analysis.

  16. [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

  17. Selective and nonselective cleavages in positive and negative CID of the fragments generated from in-source decay of intact proteins in MALDI-MS.

    PubMed

    Takayama, Mitsuo; Sekiya, Sadanori; Iimuro, Ryunosuke; Iwamoto, Shinichi; Tanaka, Koichi

    2014-01-01

    Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z'-ions originating from cleavage at the N-Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z'-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a "mobile proton" are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

  18. Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

    NASA Astrophysics Data System (ADS)

    Takayama, Mitsuo; Sekiya, Sadanori; Iimuro, Ryunosuke; Iwamoto, Shinichi; Tanaka, Koichi

    2014-01-01

    Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z'-ions originating from cleavage at the N-Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z'-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a "mobile proton" are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

  19. Ribonucleic acid-protein cross-linking within the intact Escherichia coli ribosome, utilizing ethylene glycol bis[3-(2-ketobutyraldehyde) ether], a reversible, bifunctional reagent: identification of 30S proteins.

    PubMed

    Brewer, L A; Noller, H F

    1983-08-30

    To obtain detailed topographical information concerning the spatial arrangement of the multitude of ribosomal proteins with respect to specific sequences in the three RNA chains of intact ribosomes, a reagent capable of covalently and reversibly joining RNA to protein has been synthesized [Brewer, L.A., Goelz, S., & Noller, H. F. (1983) Biochemistry (preceding paper in this issue)]. This compound, ethylene glycol bis[3-(2-ketobutyraldehyde) ether] which we term "bikethoxal", possesses two reactive ends similar to kethoxal. Accordingly, it reacts selectively with guanine in single-stranded regions of nucleic acid and with arginine in protein. The cross-linking is reversible in that the arginine- and guanine-bikethoxal linkage can be disrupted by treatment with mild base, allowing identification of the linked RNA and protein components by standard techniques. Further, since the sites of kethoxal modification within the RNA sequences of intact subunits are known, the task of identifying the components of individual ribonucleoprotein complexes should be considerably simplified. About 15% of the ribosomal protein was covalently cross-linked to 16S RNA by bikethoxal under our standard reaction conditions, as monitored by comigration of 35S-labeled protein with RNA on Sepharose 4B in urea. Cross-linked 30S proteins were subsequently removed from 16S RNA by treatment with T1 ribonuclease and/or mild base cleavage of the reagent and were identified by two-dimensional polyacrylamide gel electrophoresis. The major 30S proteins found in cross-linked complexes are S4, S5, S6, S7, S8, S9 (S11), S16, and S18. The minor ones are S2, S3, S12, S13, S14, S15, and S17.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. Novel sheathless CE-MS interface as an original and powerful infusion platform for nanoESI study: from intact proteins to high molecular mass noncovalent complexes.

    PubMed

    Gahoual, Rabah; Busnel, Jean-Marc; Wolff, Philippe; François, Yannis Nicolas; Leize-Wagner, Emmanuelle

    2014-02-01

    Development of nano-electrospray (nanoESI) sources allowed to increase significantly the sensitivity which is often lacking when studying biological noncovalent assemblies. However, the flow rate used to infuse the sample into the mass spectrometer cannot be precisely controlled with nanoESI and the robustness of the system could represent an issue. In this study, we have used a sheathless capillary electrophoresis-mass spectrometry (CESI) prototype as a nanoESI infusion device. The hydrodynamic mobilization of the capillary content was characterized and the ability of the system to generate a stable electrospray under controlled flow rate conditions ranging from 4 up to 900 nL/min was demonstrated. The effect of the infusing flow rate on the detection of an intact model protein analyzed under native conditions was investigated. Results demonstrated a significant increase in sensitivity of 46-fold and a signal-to-noise ratio improvement of nearly 5-fold when using an infusing flow rate from 456.9 down to 13.7 nL/min. The CESI prototype was further used to detect successfully the β ring homodimer in its native conformation. Obtained results were compared with those achieved with conventional ESI. Intensity signals were increased by a factor of 5, while sample consumption decreased 80 times. β ring complexed with the P14 peptide was also studied. Finally, the CESI interface was used to observe the quaternary structure of native hemocyanins from Carcinus maenas crabs; this high molecular complex coexisting under various degrees of complexation and resulting in masses ranging from 445 kDa to 1.34 MDa. PMID:23881366

  10. 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

  11. Construction of hormonally responsive intact cell hybrids by cell fusion: transfer of. beta. -adrenergic receptor and nucleotide regulatory protein(s) in normal and desensitized cells

    SciTech Connect

    Schulster, D.; Salmon, D.M.

    1985-01-01

    Fusion of normal, untreated human erythrocytes with desensitized turkey erythrocytes increases isoproterenol stimulation of cyclic (/sup 3/H)AMP accumulation over basal rates. Moreover, pretreatment of the human erythrocytes with cholera toxin before they are fused with desensitized turkey erthythrocytes leads to a large stimulation with isoproterenol. This is even greater and far more rapid than the response obtained if turkey erythrocytes are treated directly with cholera toxin. It is concluded that the stimulation in the fused system is due to the transfer of an ADP-ribosylated subunit of nucleotide regulatory protein.

  12. Radial Turgor and Osmotic Pressure Profiles in Intact and Excised Roots of Aster tripolium: Pressure Probe Measurements and Nuclear Magnetic Resonance-Imaging Analysis.

    PubMed

    Zimmermann, U; Rygol, J; Balling, A; Klöck, G; Metzler, A; Haase, A

    1992-05-01

    High-resolution nuclear magnetic resonance images (using very short spin-echo times of 3.8 milliseconds) of cross-sections of excised roots of the halophyte Aster tripolium showed radial cell strands separated by air-filled spaces. Radial insertion of the pressure probe (along the cell strands) into roots of intact plants revealed a marked increase of the turgor pressure from the outermost to the sixth cortical layer (from about 0.1-0.6 megapascals). Corresponding measurements of intracellular osmotic pressure in individual cortical cells (by means of a nanoliter osmometer) showed an osmotic pressure gradient of equal magnitude to the turgor pressure. Neither gradient changed significantly when the plants were grown in, or exposed for 1 hour to, media of high salinity. Differences were recorded in the ability of salts and nonelectrolytes to penetrate the apoplast in the root. The reflection coefficients of the cortical cells were approximately 1 for all the solutes tested. Excision of the root from the stem resulted in a collapse of the turgor and osmotic pressure gradients. After about 15 to 30 minutes, the turgor pressure throughout the cortex attained an intermediate (quasistationary) level of about 0.3 megapascals. This value agreed well with the osmotic value deduced from plasmolysis experiments on excised root segments. These and other data provided conclusions about the driving forces for water and solute transport in the roots and about the function of the air-filled radial spaces in water transport. They also showed that excised roots may be artifactual systems.

  13. 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

  14. 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.

  15. Single-Cell Metabolite Profiling of Stalk and Glandular Cells of Intact Trichomes with Internal Electrode Capillary Pressure Probe Electrospray Ionization Mass Spectrometry.

    PubMed

    Nakashima, Taiken; Wada, Hiroshi; Morita, Satoshi; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nonami, Hiroshi

    2016-03-15

    In this report, we developed the pressure probe electrospray ionization-mass spectrometry with internal electrode capillary (IEC-PPESI-MS) which enables high spatial-resolution cell sampling, precise postsampling manipulation, and high detection sensitivity. Using this technique, a comparative in situ single-cell metabolite profiling of stalk and glandular cells, the two adjacent cell types comprising a trichome unit in tomato plants (Solanum lycopersicum L.), were performed to clarify the extent of metabolic differentiation between two cell types as well as among different types of trichomes. Owing to high sensitivity of the system, less than a picoliter cell sap from a single stalk cell sufficiently yielded a number of peaks of amino acids, organic acids, carbohydrates, and flavonoids. The minimal cell sap removal from a stalk cell without severe disturbance of trichome structure enabled sequential analysis of adjacent glandular cell on the same trichome, which showed the presence of striking differences in metabolite compositions between two adjacent cell types. Comparison among different types of trichome also revealed significant variations in metabolite profiles, particularly in flavonoids and acyl sugars compositions. Some metabolites were found only in specific cell types or particular trichome types. Although extensive metabolomics analysis of glandular cells of tomato trichomes has been previously documented, this is the first report describing cell-to-cell variations in metabolite compositions of stalk and glandular cells as well as in different trichome types. Further application of this technique may provide new insights into distinct metabolism in plant cells displaying variations in shape, size, function and physicochemical properties. PMID:26845634

  16. Single-Cell Metabolite Profiling of Stalk and Glandular Cells of Intact Trichomes with Internal Electrode Capillary Pressure Probe Electrospray Ionization Mass Spectrometry.

    PubMed

    Nakashima, Taiken; Wada, Hiroshi; Morita, Satoshi; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nonami, Hiroshi

    2016-03-15

    In this report, we developed the pressure probe electrospray ionization-mass spectrometry with internal electrode capillary (IEC-PPESI-MS) which enables high spatial-resolution cell sampling, precise postsampling manipulation, and high detection sensitivity. Using this technique, a comparative in situ single-cell metabolite profiling of stalk and glandular cells, the two adjacent cell types comprising a trichome unit in tomato plants (Solanum lycopersicum L.), were performed to clarify the extent of metabolic differentiation between two cell types as well as among different types of trichomes. Owing to high sensitivity of the system, less than a picoliter cell sap from a single stalk cell sufficiently yielded a number of peaks of amino acids, organic acids, carbohydrates, and flavonoids. The minimal cell sap removal from a stalk cell without severe disturbance of trichome structure enabled sequential analysis of adjacent glandular cell on the same trichome, which showed the presence of striking differences in metabolite compositions between two adjacent cell types. Comparison among different types of trichome also revealed significant variations in metabolite profiles, particularly in flavonoids and acyl sugars compositions. Some metabolites were found only in specific cell types or particular trichome types. Although extensive metabolomics analysis of glandular cells of tomato trichomes has been previously documented, this is the first report describing cell-to-cell variations in metabolite compositions of stalk and glandular cells as well as in different trichome types. Further application of this technique may provide new insights into distinct metabolism in plant cells displaying variations in shape, size, function and physicochemical properties.

  17. 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

  18. 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.

  19. 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

  20. 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/.

  1. Rapid Identification and Subtyping of Helicobacter cinaedi Strains by Intact-Cell Mass Spectrometry Profiling with the Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

    PubMed Central

    Taniguchi, Takako; Sekiya, Ayumi; Higa, Mariko; Saeki, Yuji; Umeki, Kazumi; Okayama, Akihiko; Hayashi, Tetsuya

    2014-01-01

    Helicobacter cinaedi infection is recognized as an increasingly important emerging disease in humans. Although H. cinaedi-like strains have been isolated from a variety of animals, it is difficult to identify particular isolates due to their unusual phenotypic profiles and the limited number of biochemical tests for detecting helicobacters. Moreover, analyses of the 16S rRNA gene sequences are also limited due to the high levels of similarity among closely related helicobacters. This study was conducted to evaluate intact-cell mass spectrometry (ICMS) profiling using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) as a tool for the identification of H. cinaedi. A total of 68 strains of H. cinaedi isolated from humans, dogs, a cat, and hamsters were examined in addition to other Helicobacter species. The major ICMS profiles of H. cinaedi were identical and differed from those of Helicobacter bilis, which show >98% sequence similarity at the 16S rRNA sequence level. A phyloproteomic analysis of the H. cinaedi strains examined in this work revealed that human isolates formed a single cluster that was distinct from that of the animal isolates, with the exception of two strains from dogs. These phyloproteomic results agreed with those of the phylogenetic analysis based on the nucleotide sequences of the hsp60 gene. Because they formed a distinct cluster in both analyses, our data suggest that animal strains may not be a major source of infection in humans. In conclusion, the ICMS profiles obtained using a MALDI-TOF MS approach may be useful for the identification and subtyping of H. cinaedi. PMID:24153128

  2. Noncovalent association of protein and capsular polysaccharide on bacteria-sized latex beads as a model for polysaccharide-specific humoral immunity to intact gram-positive extracellular bacteria.

    PubMed

    Colino, Jesus; Duke, Leah; Snapper, Clifford M

    2013-09-15

    Intact Streptococcus pneumoniae expressing type 14 capsular polysaccharide (PPS14) and type III S. agalactiae containing a PPS14 core capsule identical to PPS14 exhibit noncovalent associations of PPS14 and bacterial protein, in contrast to soluble covalent conjugates of these respective Ags. Both bacteria and conjugates induce murine PPS14-specific IgG responses dependent on CD4⁺ T cells. Further, secondary immunization with conjugate and S. agalactiae, although not S. pneumoniae, results in a boosted response. However, in contrast to conjugate, PPS14-specific IgG responses to bacteria lack affinity maturation use the 44.1-idiotype and are dependent on marginal zone B cells. To better understand the mechanism underlying this dichotomy, we developed a minimal model of intact bacteria in which PPS14 and pneumococcal surface protein A (PspA) were stably attached to 1 μm (bacteria-sized) latex beads, but not directly linked to each other, in contrast to PPS14-PspA conjugate. Beads coated simultaneously with PPS14+[PspA], similar to conjugate, induced in mice boosted PPS14-specific IgG secondary responses, dependent on T cells and ICOS-dependent costimulation, and in which priming could be achieved with PspA alone. In contrast to conjugate, but similar to intact bacteria, the primary PPS14-specific IgG response to beads coated simultaneously with PPS14+[PspA] peaked rapidly, with the secondary response highly enriched for the 44.1-idiotype and lacking affinity maturation. These results demonstrate that noncovalent association in a particle, of polysaccharide and protein, recapitulates essential immunologic characteristics of intact bacteria that are distinct from soluble covalent conjugates of these respective Ags.

  3. 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.

  4. Corticosteroid-binding globulin reactive centre loop antibodies recognise only the intact natured protein: elastase cleaved and uncleaved CBG may coexist in circulation.

    PubMed

    Lewis, John G; Elder, Peter A

    2011-11-01

    Corticosteroid-binding globulin (CBG) is the principal carrier of cortisol in circulation and is a non-inhibitory member of the serpin family of serine proteinase inhibitors. It possesses an exposed elastase specific site which, when cleaved, allows a conformational change promoting the delivery of cortisol to sites of inflammation. Previously there was no ability to independently distinguish between the uncleaved, stressed, conformer of CBG and total CBG in circulation. Here we raised and characterized monoclonal antibodies generated against a synthetic peptide spanning the elastase cleavage site within the exposed reactive centre loop (RCL) and measured changes in CBG by ELISA following treatment with human neutrophil elastase. The antibodies recognized the synthetic peptide as well as intact CBG and the epitope (STGVTLNL) spanned the elastase cleavage site. Treatment of plasma with elastase resulted in a complete loss of CBG levels determined using these RCL antibodies whereas CBG levels measured with an unrelated CBG monoclonal antibody were unaffected. We also compared plasma levels of CBG measured by RCL antibodies and an unrelated CBG antibody and showed discordance in some samples. This study shows for the first time the ability to measure the intact, stressed conformer of CBG. We report discordance with total CBG in some samples implying the presence of cleaved CBG in circulation. This is an important finding as it has implications for free cortisol which hitherto have been determined from total cortisol and total CBG levels. This antibody could be used for determining the time course of intact CBG in various relevant patient cohorts and for structure/function studies on the biology of human CBG.

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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).

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. (Photosynthesis in intact plants)

    SciTech Connect

    Not Available

    1990-01-01

    Progress in the two years since the last renewal application has been excellent. We have made substantial contributions on both main fronts of the projects, and are particularly happy with the progress of our research on intact plants. The approach of basing our field work on a sound foundation of laboratory studies has enabled is to use methods which provide unambiguous assays of well characterized reactions. We have also made excellent progress in several laboratory studies which will have direct applications in future field work, and have introduced to the laboratory a range of molecular genetics techniques which will allow us to explore new options in the attempt to understand function at the level of molecular structure.

  19. 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.

  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. 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

  2. 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

  3. 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.

  4. 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).

  5. Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery.

    PubMed

    Pirmoradian, Mohammad; Astorga-Wells, Juan; Zubarev, Roman A

    2015-12-01

    Prefractionation of proteins is often employed to improve analysis specificity in proteomics. Prefractionation based on the isoelectric point (pI) is particularly attractive because pI is a well-defined parameter and it is orthogonal to hydrophobicity on which reversed-phase chromatography is based. However, direct capillary electrophoresis of blood proteins is challenging due to its high content of salts and charged small molecules. Here, we couple an online desalinator device to our multijunction capillary isoelectric focusing (MJ-CIEF) instrument and perform direct isoelectric separation of human blood plasma. In a proof-of-principle experiment, pooled samples of patients with progressive mild cognitive impairment and corresponding healthy controls were investigated. Injection of 3 μL of plasma containing over 100 μg of proteins into the desalinator was followed by pI fractionation with MJ-CIEF in less than 1 h. Shotgun proteomics of 12 collected fractions from each of the 5 replicates of pooled samples resulted in the identification and accurate quantification (median CV between the replicates is <4%) of nearly 365 protein groups from 4030 unique peptides (with <1% FDR for both peptides and proteins). The obtained results include several proteins previously reported as AD markers. The isoelectric point of each quantified protein was calculated using a set of 7 synthetic peptides spiked into the samples. Several proteins with a significant pI shift between their isoforms in the patient and control samples were identified. The presented method is straightforward, robust, and scalable; therefore, it can be used in both biological and clinical applications.

  6. RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells

    PubMed Central

    Roussis, Ioannis M.; Guille, Matthew; Myers, Fiona A.; Scarlett, Garry P.

    2016-01-01

    Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation–Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos. PMID:26824753

  7. RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

    PubMed

    Roussis, Ioannis M; Guille, Matthew; Myers, Fiona A; Scarlett, Garry P

    2016-01-01

    Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation-Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos. PMID:26824753

  8. Effects of activation of protein kinase C (PKC) on the hormonal stimulation and inhibition of cAMP formation in intact human platelets

    SciTech Connect

    Williams, K.A.; Haslam, R.J.

    1986-05-01

    Washed platelets, labelled by preincubation with (/sup 3/H)adenine and (/sup 32/P)P/sub i/, were studied in the presence of indomethacin, phosphocreatine and creatine phosphokinase to block thromboxane A/sub 2/ formation and inhibitory effects of released ADP. Addition of phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-glycerol (diC/sub 8/) decreased the initial rate of accumulation of (/sup 3/H)cAMP observed with PGE/sub 1/ and 3-isobutyl 1- methylxanthine. Maximal decreases of 31% (1 ..mu..M PMA) and 42% (100 ..mu..M diC/sub 8/) were obtained. Also, the inhibition of (/sup 3/H)cAMP formation by epinephrine (5 ..mu..M) was decreased from 68% to 16% and 31% by 1..mu..M PMA and 100 ..mu..M diC/sub 8/, respectively. The effects of increasing concentrations of PMA and diC/sub 8/ on the stimulation of (/sup 3/H)cAMp formation by PGE/sub 1/ and on the inhibitory action of epinephrine correlated with increases in /sup 32/P incorporation into the major substrate of PKC (P47) and into two other polypeptides (P41 and P20). These results suggested that activation of PKC might explain the failure of some aggregating agents (e.g. PAF and vasopressin) to inhibit adenylate cyclase in intact platelets, although they are inhibitory with isolated membranes. However, comparison of the effects of PMA and these aggregating agents on the phosphorylation of platelet polypeptides indicated that activation of PKC by aggregating agents is inadequate to block their inhibitory effects on adenylate cyclase, when PGE/sub 1/ is present.

  9. 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.

  10. The physics of intact capture

    NASA Technical Reports Server (NTRS)

    Tsou, Peter; Griffiths, D. J.; Albee, A. L.

    1994-01-01

    The ability to capture projectiles intact at hypervelocities in underdense media open a new area of study in physics. Underdense material behaves markedly different than solid, liquid, or gas upon hypervelocity impact. This new phenomenon enables applications in science that would either not be possible or would be very costly by other means. This phenomenon has been fully demonstrated in the laboratory and validated in space. Even more interesting is the fact that this hypervelocity intact capture was accomplished passively. A better understanding of the physics of intact capture will lead to improvements in intact capture. A collection of physical observations of this phenomenon is presented here.

  11. 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

  12. 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.

  13. Homogeneous human complex-type oligosaccharides in correctly folded intact glycoproteins: evaluation of oligosaccharide influence on protein folding, stability, and conformational properties.

    PubMed

    Kajihara, Yasuhiro; Tanabe, Yasutaka; Sasaoka, Shun; Okamoto, Ryo

    2012-05-01

    The N-glycosylation of proteins is generated at the consensus sequence NXS/T (where X is any amino acid except proline) by the biosynthetic process, and occurs in the endoplasmic reticulum and Golgi apparatus. In order to investigate the influence of human complex-type oligosaccharides on counterpart protein conformation, crambin and ovomucoide, which consist of 46 and 56 amino acid residues, respectively, were selected for synthesis of model glycoproteins. These small glycoproteins were intentionally designed to be glycosylated at the α-helix (crambin: 8 position), β-sheet (crambin: 2 position) and loop position between the antiparallel β-sheets (ovomucoide: 28 position), and were synthesized by using a peptide-segment coupling strategy. After preparation of these glycosylated polypeptide chains, protein folding experiments were performed under redox conditions by using cysteine-cystine. Although the small glycoproteins bearing intentional glycosylation at the α-helix and β-sheet exhibited a suitable folding process, glycosylation at the loop position between the antiparallel β-strands caused multiple products. The conformational differences in the isolated homogeneous glycoproteins compared with non-glycosylated counterparts were evaluated by circular dichroism (CD) and NMR spectroscopy. These analyses suggested that this intentional N-glycosylation did not result in large conformational changes in the purified protein structures, including the case of glycosylation at the loop position between the antiparallel β-strands. In addition to these experiments, the conformational properties of three glycoproteins were evaluated by CD spectroscopy under different temperatures. The oligosaccharides on the protein surface fluctuated considerably; this was dependent on the increase in the solution temperature and was thought to disrupt the protein tertiary structure. Based on the measurement of the CD spectra, however, the glycoproteins bearing three disulfide

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. Specificity of interaction between carcinogenic polynuclear aromatic hydrocarbons and nuclear proteins: widespread occurrence of a restricted pattern of histone-binding in intact cells

    SciTech Connect

    MacLeod, M.C.; Pelling, J.C.; Slaga, T.J.; Nikbakht-Noghrei, P.A.; Mansfield, B.K.; Selkirk, J.K.

    1982-01-01

    Metabolic activation of benzo(a)pyrene (B(a)P) produces a number of potentially reactive metabolites. The endproducts of one metabolic pathway, 7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydro-B(a)P (BPDE) are responsible for essentially all DNA adduct formation in animal cells treated with B(a)P, and a particular stereoisomer, designated (+)-anti-BPDE is thought to be the ultimate carcinogenic derivative of B(a)P. In hamster embryo cell nuclei treated with (+)-anti-BPDE, two of the histones of the nucleosomal core, H3 and H2A, are covalently modified, while the remaining core histones, H4 and H2B, are essentially unmodified. All four purified core histones, however, serve as targets. 7,12-dimethylbenz(a)anthracene and 3-methylcholanthrene show the same pattern of histone binding in hamster embryo cells. Treatment of mouse embryo cells with (/sup 3/H)-BPDE results in covalent binding of the hydrocarbon to histones H3 and H2A among the many cellular targets, while histones H2B and H4 are not bound. Similar binding patterns are seen in mouse embryo cells, a permanent murine, fibroblastic cell line, and a human mammary epithelial cell line, T47D, treated with (/sup 3/H)B(a)P. Again, the histones are unevenly labeled, displaying the H3 and H2A pattern. Histone-binding in the human cells may also be mediated by BPDE. Similar BPDE binding patterns were observed in other murine and human cell lines and in primary cultures of murine epidermal epithelial cells. The restriction of histone H2B and H4 binding appears to be general when intact cultured cells are studied. This specificity was not observed in a mixed reconstituted system in which rat liver microsomes were used to activate B(a)P. This finding reinforces reservations concerning the use of microsomal systems to probe the interactions of carcinogens with macromolecules and the relationships of adduct formation with the processes of carcinogenesis. (ERB)

  2. 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.

  3. Isoprenoid, lipid, and protein contents in intact plastids isolated from mesocarp cells of traditional and high-pigment tomato cultivars at different ripening stages.

    PubMed

    Lenucci, Marcello S; Serrone, Lucia; De Caroli, Monica; Fraser, Paul D; Bramley, Peter M; Piro, Gabriella; Dalessandro, Giuseppe

    2012-02-22

    This study reports quali-quantitative analyses on isoprenoids, phospholipids, neutral lipids, phytosterols, and proteins in purified plastids isolated from fresh fruits of traditional (Donald and Incas) and high-pigment (Kalvert and HLY-18) tomato cultivars at four ripening stages. In all of the investigated cultivars, lycopene, β-catotene, lutein, and total carotenoids varied significantly during ripening. Chromoplasts of red-ripe tomato fruits of high-pigment cultivars accumulated twice as much as lycopene (307.6 and 319.2 μg/mg of plastid proteins in Kalvert and HLY-18, respectively) than ordinary cultivars (178.6 and 151.7 μg/mg of plastid proteins in Donald and Incas, respectively); differences in chlorophyll and α-tocopherol contents were also evidenced. Phospholipids and phytosterols increased during ripening, whereas triglycerides showed a general decrease. Regardless of the stage of ripening, palmitic acid was the major fatty acid in all cultivars (ranging from 35 to 52% of the total fatty acids), followed by stearic, oleic, linoleic, linolenic, and myristic acids, but their relative percentage was affected by ripening. Most of the bands detected on the SDS-PAGEs of plastid proteins were constantly present during chloroplast-to-chromoplast conversion, some others disappeared, and only one, with a molecular weight of ~41.6 kDa, was found to increase in intensity.

  4. 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

  5. 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

  6. Space research with intact organisms

    NASA Technical Reports Server (NTRS)

    Phillips, Robert W.; Haddy, Francis J.

    1992-01-01

    Effects of space exposure on intact organisms are briefly reviewed, and examples of future experiments that might provide new information on the role of gravity in the evolution of life are suggested. It is noted that long term experiments with intact plant and animals for studying gravitational thresholds will provide important new insights.

  7. 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.

  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 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. β-Apo-10'-carotenoids Modulate Placental Microsomal Triglyceride Transfer Protein Expression and Function to Optimize Transport of Intact β-Carotene to the Embryo.

    PubMed

    Costabile, Brianna K; Kim, Youn-Kyung; Iqbal, Jahangir; Zuccaro, Michael V; Wassef, Lesley; Narayanasamy, Sureshbabu; Curley, Robert W; Harrison, Earl H; Hussain, M Mahmood; Quadro, Loredana

    2016-08-26

    β-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, β-carotene 15,15'-oxygenase (BCO1) converts β-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. β-Carotene can also be cleaved by β-carotene 9',10'-oxygenase (BCO2) to form β-apo-10'-carotenal, a precursor of retinoic acid and a transcriptional regulator per se The mammalian embryo obtains β-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that β-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that β-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that β-apo-10'-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of β-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that β-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of β-carotene for proper embryogenesis. PMID:27402843

  15. Monoclonal antibody to a conserved epitope on proteins encoded by Babesia bigemina and present on the surface of intact infected erythrocytes.

    PubMed

    Shompole, S; Perryman, L E; Rurangirwa, F R; McElwain, T F; Jasmer, D P; Musoke, A J; Wells, C W; McGuire, T C

    1995-09-01

    To define Babesia bigemina-specific antigens on the surface of infected erythrocytes, monoclonal antibodies (MAbs) were identified by live-cell immunofluorescence. As determined by live-cell immunofluorescence, two MAbs made to the Mexico strain reacted with the Mexico strain and three Kenya strains, while three MAbs made to the Kenya-Ngong strain reacted with the Kenya strains but not the Mexico strain. Binding of MAb 44.18 (made to the Mexico strain) to a strain-common epitope was confirmed by immunoelectron microscopy and by surface-specific immunoprecipitation of [35S]methionine-labeled proteins (200, 28, and 16 kDa in size), which also demonstrated that the MAb recognized an epitope on proteins encoded by B. bigemina. In immunoblots, the MAb bound to predominant antigens with sizes of 200 and 220 kDa in erythrocyte lysates infected with strains from Puerto Rico, St. Croix, Texcoco (Mexico), Kenya, and Mexico. Major antigens with sizes of 200 and 220 kDa were isolated from a MAb 44.18 affinity matrix. Calf serum antibodies to these isolated antigens bound to erythrocytes infected with either the Mexico or Kenya strains as determined by live-cell immunofluorescence, allowing the conclusion that at least one conserved surface epitope was recognized. Calf serum antibodies identified major labeled proteins with sizes of 200 and 72 kDa by surface-specific immunoprecipitation, and infected erythrocytes sensitized with these antibodies were phagocytized by cultured bovine peripheral blood monocytes. These results provide a rationale for evaluating antigens identified by MAb 44.18 individually and as components of subunit vaccines.

  16. 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.

  17. 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

  18. 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

  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. 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

  3. 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

  4. 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

  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. 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.

  10. 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

  11. Stimulation of host centriolar antigen in TC7 cells by simian virus 40: requirement for RNA and protein syntheses and an intact simian virus 40 small-t gene function.

    PubMed

    Shyamala, M; Atcheson, C L; Kasamatsu, H

    1982-08-01

    Simian virus 40 (SV 40) stimulated a host cell antigen in the centriolar region after infection of African green monkey kidney (AGMK) cells. The addition of puromycin and actinomycin D to cells infected with SV40 within 5 h after infection inhibited the stimulation of the host cell antigen, indicating that de novo protein and RNA syntheses that occurred within the first 5 h after infection were essential for the stimulation. Early viable deletion mutants of SV40 with deletions mapping between 0.54 and 0.59 map units on the SV40 genome, dl2000, dl2001, dl2003, dl2004, dl2005, dl2006, and dl2007, did not stimulate the centriolar antigen above the level of uninfected cells. This indicated that an intact, functional small-t protein was essential for the SV40-mediated stimulation of the host cell antigen. Our studies, using cells infected with nondefective adenovirus-SV40 hybrid viruses that lack the small-t gene region of SV40 (Ad2+ND1, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5), revealed that the lack of small-t gene function of SV40 could be complemented by a gene function of the adenovirus-SV40 hybrid viruses for the centriolar antigen stimulation. Thus, adenovirus 2 has a gene(s) that is analogous to the small-t gene of SV40 for the stimulation of the host cell antigen in AGMK cells.

  12. 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

  13. 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

  14. 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.

  15. DNA Binding Mode Transitions of Escherichia coli HUαβ: Evidence for Formation of a Bent DNA – Protein Complex on Intact, Linear Duplex DNA

    PubMed Central

    Koh, Junseock; Saecker, Ruth M.; Record, M. Thomas

    2008-01-01

    Escherichia coli HUαβ, a major nucleoid associated protein (NAP), organizes the DNA chromosome and facilitates numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA lengths (8, 15, 34, 38 and 160 base pairs) we establish that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA mole ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 base pairs), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 (± 0.4) × 106 M−1, and a binding enthalpy of +7.7 (± 0.6) kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω ~ 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 (± 0.2) × 105 M−1 for the 10 bp mode, 3.5 (± 1.4) × 104 M−1 for the 6 bp mode) and binding enthalpies (4.2 (± 0.3) kcal/mol for the 10 bp mode, −1.6 (±0.3) kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. FRET data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement, AFM), fluorescence, and electrophoretic gel mobility shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. PMID:18657548

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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/.

  1. 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

  2. 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

  3. 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

  4. Protein profile in vascular wall of atherosclerotic mice analyzed ex vivo using FT-IR spectroscopy

    NASA Astrophysics Data System (ADS)

    Wrobel, Tomasz P.; Majzner, Katarzyna; Baranska, Malgorzata

    2012-10-01

    The structure of proteins in a tissue can undergo changes on account of disease state such as diabetes or atherosclerosis. In this work the protein profile in atherosclerotic tissue is monitored by FT-IR imaging coupled with Hierarchical Cluster Analysis (HCA). Additionally, a model for prediction of secondary structure of proteins content based on amide I and II range is used to show the distribution of analyzed proteins. A new protein class emerged in atherosclerotic tissue in the region of the plaque and additionally the plaque was found to be strongly mixed with smooth muscle cell. The calculated secondary structure contents of proteins in atherosclerotic tissue in comparison to healthy tissue showed an increase of structures related to beta-sheet (E and T) and a decrease of helical (H) and unassigned arrangements.

  5. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  6. Effects of agricultural production systems and their components on protein profiles of potato tubers.

    PubMed

    Lehesranta, Satu J; Koistinen, Kaisa M; Massat, Nathalie; Davies, Howard V; Shepherd, Louise V T; McNicol, James W; Cakmak, Ismail; Cooper, Julia; Lück, Lorna; Kärenlampi, Sirpa O; Leifert, Carlo

    2007-02-01

    A range of studies have compared the level of nutritionally relevant compounds in crops from organic and nonorganic farming systems, but there is very limited information on the effect of farming systems and their key components on the protein composition of plants. We addressed this gap by quantifying the effects of different farming systems and key components of such systems on the protein profiles of potato tubers. Tuber samples were produced in the Nafferton factorial systems study, a group of long-term, replicated factorial field experiments designed to identify and quantify the effect of fertility management methods, crop protection practices and rotational designs used in organic, low input and conventional production systems. Protein profiles were determined by 2-DE and subsequent protein identification by HPLC-ESI-MS/MS. Principal component analysis of 2-DE data showed that only fertility management practices (organic matter vs. mineral fertiliser based) had a significant effect on protein composition. Quantitative differences were detected in 160 of the 1100 tuber proteins separated by 2-DE. Proteins identified by MS are involved in protein synthesis and turnover, carbon and energy metabolism and defence responses, suggesting that organic fertilisation leads to an increased stress response in potato tubers.

  7. Alteration of protein profile in rat liver of animals exposed to subacute diazinon: a proteomic approach.

    PubMed

    Lari, Parisa; Rashedinia, Marzieh; Abnous, Khalil; Hosseinzadeh, Hossein

    2014-05-01

    Diazinon, an organophosphorus insecticide, is employed to control pests in agriculture. Diazinon may contaminate the environment during the manufacturing process or agricultural application. Previous studies have revealed that diazinon may induce alteration in the protein profile of the liver. Here, a proteomics approach was used to investigate the effects on the protein profile in the liver of rats of subacute oral exposures at 15 mg/kg of diazinon. Liver proteins were separated using 2D-PAGE, and stained by MS-compatible silver staining and/or the fluorescent SYPRO® Ruby protein gel stain. Gels were scanned and analyzed using the Image Master software. Differentially displayed protein species were identified using MALDI-TOF/TOF and MASCOT software. Significantly altered protein species were identified to be involved in apoptosis, cell metabolism, transport, and antioxidant systems. Exposure to diazinon decreased levels of some species of catalase, peroxiredoxin-6, 3-ketoacyl-CoA thiolase, and glucose regulated protein78, whereas the level of protein disulfide-isomerase A3 increased. Our results suggested that diazinon may induce hepatotoxicity through oxidative stress, apoptosis, and metabolic disorders in rat liver.

  8. TMBETADISC-RBF: Discrimination of beta-barrel membrane proteins using RBF networks and PSSM profiles.

    PubMed

    Ou, Yu-Yen; Gromiha, M Michael; Chen, Shu-An; Suwa, Makiko

    2008-06-01

    Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. We have developed a method based on radial basis function networks and position specific scoring matrix (PSSM) profiles generated by PSI-BLAST and non-redundant protein database. Our approach with PSSM profiles has correctly predicted the OMPs with a cross-validated accuracy of 96.4% in a set of 1251 proteins, which contain 206 OMPs, 667 globular proteins and 378 alpha-helical inner membrane proteins. Furthermore, we applied our method on a dataset containing 114 OMPs, 187 TMH proteins and 195 globular proteins obtained with less than 20% sequence identity and obtained the cross-validated accuracy of 95%. This accuracy of discriminating OMPs is higher than other methods in the literature and our method could be used as an effective tool for dissecting OMPs from genomic sequences. We have developed a prediction server, TMBETADISC-RBF, which is available at http://rbf.bioinfo.tw/~sachen/OMP.html.

  9. Accounting for Observed Small Angle X-ray Scattering Profile in the Protein-Protein Docking Server ClusPro

    PubMed Central

    Xia, Bing; Mamonov, Artem; Leysen, Seppe; Allen, Karen N; Strelkov, Sergei V.; Paschalidis, Ioannis Ch.; Vajda, Sandor; Kozakov, Dima

    2015-01-01

    The protein-protein docking server ClusPro is used by thousands of laboratories, and models built by the server have been reported in over 300 publications. Although the structures generated by the docking include near-native ones for many proteins, selecting the best model is difficult due to the uncertainty in scoring. Small Angle X-ray Scattering (SAXS) is an experimental technique for obtaining low resolution structural information in solution. While not sufficient on its own to uniquely predict complex structures, accounting for SAXS data improves the ranking of models and facilitates the identification of the most accurate structure. Although SAXS profiles are currently available only for a small number of complexes, due to its simplicity the method is becoming increasingly popular. Since combining SAXS experiments will provide a viable strategy for fairly high-throughput determination of protein complex structures, the option of using SAXS restraints is added to the ClusPro server. PMID:26095982

  10. Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing.

    PubMed

    2015-01-01

    The author's email has been corrected in the publication of Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to: j.zhou@uws.edu.au from: jzho7551@mail.usyd.edu.au. PMID:26167960

  11. Effects of Fe deficiency on the protein profile of Brassica napus phloem sap

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2-DE (IEF-SDS PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Two hundred sixty-three spots were consistently detected...

  12. PLASMA PROTEIN PROFILING AS A HIGH THROUGHPUT TOOL FOR CHEMICAL SCREENING USING A SMALL FISH MODEL

    EPA Science Inventory

    Hudson, R. Tod, Michael J. Hemmer, Kimberly A. Salinas, Sherry S. Wilkinson, James Watts, James T. Winstead, Peggy S. Harris, Amy Kirkpatrick and Calvin C. Walker. In press. Plasma Protein Profiling as a High Throughput Tool for Chemical Screening Using a Small Fish Model (Abstra...

  13. Genetic differences in the serum proteome of horses, donkeys and mules are detectable by protein profiling.

    PubMed

    Henze, Andrea; Aumer, Franziska; Grabner, Arthur; Raila, Jens; Schweigert, Florian J

    2011-10-01

    Although horses and donkeys belong to the same genus, their genetic characteristics probably result in specific proteomes and post-translational modifications (PTM) of proteins. Since PTM can alter protein properties, specific PTM may contribute to species-specific characteristics. Therefore, the aim of the present study was to analyse differences in serum protein profiles of horses and donkeys as well as mules, which combine the genetic backgrounds of both species. Additionally, changes in PTM of the protein transthyretin (TTR) were analysed. Serum protein profiles of each species (five animals per species) were determined using strong anion exchanger ProteinChips® (Bio-Rad, Munich, Germany) in combination with surface-enhanced laser desorption ionisation-time of flight MS. The PTM of TTR were analysed subsequently by immunoprecipitation in combination with matrix-assisted laser desorption ionisation-time of flight MS. Protein profiling revealed species-specific differences in the proteome, with some protein peaks present in all three species as well as protein peaks that were unique for donkeys and mules, horses and mules or for horses alone. The molecular weight of TTR of horses and donkeys differed by 30 Da, and both species revealed several modified forms of TTR besides the native form. The mass spectra of mules represented a merging of TTR spectra of horses and donkeys. In summary, the present study indicated that there are substantial differences in the proteome of horses and donkeys. Additionally, the results probably indicate that the proteome of mules reveal a higher similarity to donkeys than to horses.

  14. Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization

    PubMed Central

    Peffers, M.J.; McDermott, B.; Clegg, P.D.; Riggs, C.M.

    2015-01-01

    Summary Objective The aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins. Design SF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. In addition immunohistochemistry, western blotting and mRNA expression analysis was undertaken on selected joint tissues. Results The number of protein identifications was increased by 33% in the ProteoMiner™ treated SF compared to undepleted SF. A total of 764 proteins (462 with≥2 significant peptides) were identified in SF. A subset of 10 proteins were identified which were differentially expressed in OA SF. S100-A10, a calcium binding protein was upregulated in OA and validated with western blotting and immunohistochemistry. Several new OA specific peptide fragments (neopeptides) were identified. Conclusion The protein equalisation method compressed the dynamic range of the synovial proteins identifying the most comprehensive SF proteome to date. A number of proteins were identified for the first time in SF which may be involved in the pathogenesis of OA. We identified a distinct set of proteins and neopeptides that may act as potential biomarkers to distinguish between normal and OA joints. PMID:25819577

  15. Major urinary protein (MUP) profiles show dynamic changes rather than individual ‘barcode’ signatures

    PubMed Central

    Thoß, M.; Luzynski, K.C.; Ante, M.; Miller, I.; Penn, D.J.

    2016-01-01

    House mice (Mus musculus) produce a variable number of major urinary proteins (MUPs), and studies suggest that each individual produces a unique MUP profile that provides a distinctive odor signature controlling individual and kin recognition. This ‘barcode hypothesis’ requires that MUP urinary profiles show high individual variability within populations and also high individual consistency over time, but tests of these assumptions are lacking. We analyzed urinary MUP profiles of 66 wild-caught house mice from eight populations using isoelectric focusing. We found that MUP profiles of wild male house mice are not individually unique, and though they were highly variable, closer inspection revealed that the variation strongly depended on MUP band type. The prominent (‘major) bands were surprisingly homogenous (and hence most MUPs are not polymorphic), but we also found inconspicuous (‘minor’) bands that were highly variable and therefore potential candidates for individual fingerprints. We also examined changes in urinary MUP profiles of 58 males over time (from 6 to 24 weeks of age), and found that individual MUP profiles and MUP concentration were surprisingly dynamic, and showed significant changes after puberty and during adulthood. Contrary to what we expected, however, the minor bands were the most variable over time, thus no good candidates for individual fingerprints. Although MUP profiles do not provide individual fingerprints, we found that MUP profiles were more similar among siblings than non-kin despite considerable fluctuation. Our findings show that MUP profiles are not highly stable over time, they do not show strong individual clustering, and thus challenge the barcode hypothesis. Within-individual dynamics of MUP profiles indicate a different function of MUPs in individual recognition than previously assumed and advocate an alternative hypothesis (‘dynamic changes’ hypothesis). PMID:26973837

  16. Protein profiles associated with context fear conditioning and their modulation by memantine.

    PubMed

    Ahmed, Md Mahiuddin; Dhanasekaran, A Ranjitha; Block, Aaron; Tong, Suhong; Costa, Alberto C S; Gardiner, Katheleen J

    2014-04-01

    Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimer's Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimer's Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein

  17. Generating a detailed protein profile of Fasciola hepatica during the chronic stage of infection in cattle.

    PubMed

    Haçarız, Orçun; Baykal, Ahmet Tarık; Akgün, Mete; Kavak, Pınar; Sağıroğlu, Mahmut Şamil; Sayers, Gearóid Patrick

    2014-06-01

    Fasciola hepatica is a trematode helminth causing a damaging disease, fasciolosis, in ruminants and humans. Comprehensive proteomic studies broaden our knowledge of the parasite's protein profile, and provide new insights into the development of more effective strategies to deal with fasciolosis. The objective of this study was to generate a comprehensive profile of F. hepatica proteins expressed during the chronic stage of infection in cattle by building on previous efforts in this area. The approach included an improved sample preparation procedure for surface and internal layers of the parasite, the application of nano-UPLC-ESI-qTOF-MS (nano-ultra-performance LC and ESI quadrupole TOF MS) integrated with different acquisition methods and in silico database search against various protein databases and a transcript database including a new assembly of publically available EST. Of a total of 776 identified proteins, 206 and 332 were specific to the surface and internal layers of the parasite, respectively. Furthermore, 238 proteins were common to both layers, with comparative differences of 172 proteins detected. Specific proteins not previously identified in F. hepatica, but shown to be immunomodulatory or potential drug targets for other parasites, are discussed.

  18. Generating a detailed protein profile of Fasciola hepatica during the chronic stage of infection in cattle.

    PubMed

    Haçarız, Orçun; Baykal, Ahmet Tarık; Akgün, Mete; Kavak, Pınar; Sağıroğlu, Mahmut Şamil; Sayers, Gearóid Patrick

    2014-06-01

    Fasciola hepatica is a trematode helminth causing a damaging disease, fasciolosis, in ruminants and humans. Comprehensive proteomic studies broaden our knowledge of the parasite's protein profile, and provide new insights into the development of more effective strategies to deal with fasciolosis. The objective of this study was to generate a comprehensive profile of F. hepatica proteins expressed during the chronic stage of infection in cattle by building on previous efforts in this area. The approach included an improved sample preparation procedure for surface and internal layers of the parasite, the application of nano-UPLC-ESI-qTOF-MS (nano-ultra-performance LC and ESI quadrupole TOF MS) integrated with different acquisition methods and in silico database search against various protein databases and a transcript database including a new assembly of publically available EST. Of a total of 776 identified proteins, 206 and 332 were specific to the surface and internal layers of the parasite, respectively. Furthermore, 238 proteins were common to both layers, with comparative differences of 172 proteins detected. Specific proteins not previously identified in F. hepatica, but shown to be immunomodulatory or potential drug targets for other parasites, are discussed. PMID:24733753

  19. Intact capture of hypervelocity particles

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    1986-01-01

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  20. Intact capture of hypervelocity particles

    NASA Astrophysics Data System (ADS)

    Tsou, P.; Brownlee, D. E.; Albee, A. L.

    Knowledge of the phase, structure, and crystallography of cosmic particles, as well as their elemental and isotopic compositions, would be very valuable information toward understanding the nature of our solar system. This information can be obtained from the intact capture of large mineral grains of cosmic particles from hypervelocity impacts. Hypervelocity experiments of intact capture in underdense media have indicated realistic potential in this endeaver. The recovery of the thermal blankets and louvers from the Solar Max spacecraft have independently verified this potential in the unintended capture of cosmic materials from hypervelocity impacts. Passive underdense media will permit relatively simple and inexpensive missions to capture cosmic particles intact, either by going to a planetary body or by waiting for the particles to come to the Shuttle or the Space Station. Experiments to explore the potential of using various underdense media for an intact comet sample capture up to 6.7 km/s were performed at NASA Ames Research Center Vertical Gun Range. Explorative hypervelocity experiments up to 7.9 km/s were also made at the Ernst Mach Institute. These experiments have proven that capturing intact particles at hypervelocity impacts is definitely possible. Further research is being conducted to achieve higher capture ratios at even higher hypervelocities for even smaller projectiles.

  1. Measuring mitochondrial function in intact cardiac myocytes

    PubMed Central

    Dedkova, Elena N.; Blatter, Lothar A.

    2011-01-01

    Mitochondria are involved in cellular functions that go beyond the traditional role of these organelles as the power plants of the cell. Mitochondria have been implicated in several human diseases, including cardiac dysfunction, and play a role in the aging process. Many aspects of our knowledge of mitochondria stem from studies performed on the isolated organelle. Their relative inaccessibility imposes experimental difficulties to study mitochondria in their natural environment – the cytosol of intact cells – and has hampered a comprehensive understanding of the plethora of mitochondrial functions. Here we review currently available methods to study mitochondrial function in intact cardiomyocytes. These methods primarily use different flavors of fluorescent dyes and genetically encoded fluorescent proteins in conjunction with high-resolution imaging techniques. We review methods to study mitochondrial morphology, mitochondrial membrane potential, Ca2+ and Na+ signaling, mitochondrial pH regulation, redox state and ROS production, NO signaling, oxygen consumption, ATP generation and the activity of the mitochondrial permeability transition pore. Where appropriate we complement this review on intact myocytes with seminal studies that were performed on isolated mitochondria, permeabilized cells, and in whole hearts. PMID:21964191

  2. Intact capture of cosmic dust

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1991-01-01

    The focus of this development effort is to capture dust particles at hypervelocities intact and unmelted in order to preserve volatile organics. At the same time, the capture process must minimize any organic elemental or compound contamination to prevent any compromise of exobiological analyses. Inorganic silicate aerogel has been developed as a successful capture medium to satisfy both requirements of intact capture and minimal organic contamination. Up to 6 km/s, silicate projectiles from a few microns up to 100 microns have been captured intact without any melting and with minimal loss of mass. Carbon in silicate aerogel can be reduced to less than 1 part in 1000 and hydrogen 3 parts in 1000 when baked in air. Under controlled inert gas environments, additional hydrocarbon reduction can be achieved.

  3. How the diffusivity profile reduces the arbitrariness of protein folding free energies

    NASA Astrophysics Data System (ADS)

    Hinczewski, M.; von Hansen, Y.; Dzubiella, J.; Netz, R. R.

    2010-06-01

    The concept of a protein diffusing in its free-energy folding landscape has been fruitful for both theory and experiment. Yet the choice of the reaction coordinate (RC) introduces an undesirable degree of arbitrariness into the problem. We analyze extensive simulation data of an α-helix in explicit water solvent as it stochastically folds and unfolds. The free-energy profiles for different RCs exhibit significant variations, some having an activation barrier, while others not. We show that this variation has little effect on the predicted folding kinetics if the diffusivity profiles are properly taken into account. This kinetic quasi-universality is rationalized by an RC rescaling, which, due to the reparameterization invariance of the Fokker-Planck equation, allows the combination of free-energy and diffusivity effects into a single function, the rescaled free-energy profile. This rescaled free energy indeed shows less variation among different RCs than the bare free energy and diffusivity profiles separately do, if we properly distinguish between RCs that contain knowledge of the native state and those that are purely geometric in nature. Our method for extracting diffusivity profiles is easily applied to experimental single molecule time series data and might help to reconcile conflicts that arise when comparing results from different experimental probes for the same protein.

  4. UFO: a web server for ultra-fast functional profiling of whole genome protein sequences

    PubMed Central

    Meinicke, Peter

    2009-01-01

    Background Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Description Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. Conclusion For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address. PMID:19725959

  5. Evolution of liquid holdup profile in a standing protein stabilized foam.

    PubMed

    Wang, Zebin; Narsimhan, Ganesan

    2004-12-01

    Evolution of liquid holdup profile in a standing foam formed by whipping and stabilized by sodium caseinate in the presence of xanthan gum when subjected to 16 and 29g centrifugal force fields was measured using magnetic resonance imaging for different pH, ionic strength, protein and xanthan gum concentrations. Drainage resulted in the formation of a separate liquid layer at the bottom at longer times. Foam drainage was slowest at pH 7, lower ionic strength, higher protein and gum concentrations. Foam was found to be most stable at pH 5.1 near the isoelectric point of protein, lower ionic strength and higher protein and xanthan gum concentrations. A predicted equilibrium liquid holdup profile based on a previous model (G. Narsimhan, J. Food Eng. 14 (1991) 139) agreed well with experimental values at sufficiently long times. A proposed model for velocity of drainage of a power law fluid in a Plateau border for two different simplified geometries was incorporated in a previously developed model for foam drainage (G. Narsimhan, J. Food Eng. 14 (1991) 139) to predict the evolution of liquid holdup profiles. The model predictions for simplified circular geometry of Plateau border compared well with the experimental data of liquid holdup profiles at small times. At longer times, however, the predicted liquid holdup profile was larger than the observed, this discrepancy being due to coarsening of bubble size and decrease in foam height not accounted for in the model. A Newtonian model for foam drainage under predicted drainage rates did not agree with the experimental data. PMID:15476794

  6. Quantitative proteomic analysis of intact plastids.

    PubMed

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described. PMID:24136541

  7. Quantitative proteomic analysis of intact plastids.

    PubMed

    Shiraya, Takeshi; Kaneko, Kentaro; Mitsui, Toshiaki

    2014-01-01

    Plastids are specialized cell organelles in plant cells that are differentiated into various forms including chloroplasts, chromoplasts, and amyloplasts, and fulfill important functions in maintaining the overall cell metabolism and sensing environmental factors such as sunlight. It is therefore important to grasp the mechanisms of differentiation and functional changes of plastids in order to enhance the understanding of vegetality. In this chapter, details of a method for the extraction of intact plastids that makes analysis possible while maintaining the plastid functions are provided; in addition, a quantitative shotgun method for analyzing the composition and changes in the content of proteins in plastids as a result of environmental impacts is described.

  8. Effect of high hydrostatic pressure on the profile of proteins extracted from Betula pendula pollens

    NASA Astrophysics Data System (ADS)

    Murat Altuner, Ergin; Çeter, Talip; Alpas, Hami

    2014-10-01

    High hydrostatic pressure (HHP) has high success potential in pollen protein extraction, but its effect on pollen protein profiles has not been studied yet. The aim of this study is to put forward whether HHP processing causes a change in the protein profiles extracted from pollens or not. In this study, proteins extracted from Betula pendula pollens were studied at 100, 200 and 300 MPa at room temperature for 5 min. In addition, the efficiency of three different extraction solvents, namely phosphate buffer saline (PBS) buffer pH 7.5, trichloroacetic acid-acetone and Tris-HCl buffer pH 8.8, was also observed, and the results were compared with the conventional pollen protein extraction procedure. As a result, it is concluded that 200 MPa for 5 min has extracted similar amounts of protein compared with the conventional extraction method which lasted for 24 h, which lasted for 24 h. On the other hand, the application time for 200 MPa for 5 min is extremely shorter when it is compared to the conventional extraction method.

  9. EvoDesign: De novo protein design based on structural and evolutionary profiles.

    PubMed

    Mitra, Pralay; Shultis, David; Zhang, Yang

    2013-07-01

    Protein design aims to identify new protein sequences of desirable structure and biological function. Most current de novo protein design methods rely on physics-based force fields to search for low free-energy states following Anfinsen's thermodynamic hypothesis. A major obstacle of such approaches is the inaccuracy of the force field design, which cannot accurately describe the atomic interactions or distinguish correct folds. We developed a new web server, EvoDesign, to design optimal protein sequences of given scaffolds along with multiple sequence and structure-based features to assess the foldability and goodness of the designs. EvoDesign uses an evolution-profile-based Monte Carlo search with the profiles constructed from homologous structure families in the Protein Data Bank. A set of local structure features, including secondary structure, torsion angle and solvation, are predicted by single-sequence neural-network training and used to smooth the sequence motif and accommodate the physicochemical packing. The EvoDesign algorithm has been extensively tested in large-scale protein design experiments, which demonstrate enhanced foldability and structural stability of designed sequences compared with the physics-based designing methods. The EvoDesign server is freely available at http://zhanglab.ccmb.med.umich.edu/EvoDesign.

  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. 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.

  12. Biochemical composition and protein profile of alpaca (Vicugna pacos) oviductal fluid.

    PubMed

    Apichela, S A; Argañaraz, M E; Zampini, R; Vencato, J; Miceli, D C; Stelletta, C

    2015-03-01

    Knowledge and assessment of the constituents of the oviductal fluid (OF) in camelids is necessary for a correct formulation of specific culture media for the development of reproductive biotechnology. This study is the first describing the biochemical composition and SDS-PAGE protein profile of alpaca oviductal fluid in non-pregnant animals and animals that have completed the first month and second month of gestation. Samples were also classified into oviducts that were ipsilateral or contralateral to the ovary with corpus luteum. No differences were found between both oviducts, whereas pregnant and non-pregnant females displayed significant differences in the biochemical composition and protein profile of the oviductal fluid. Relative albumin content was higher in non-pregnant females. Relative creatinine content in OF from females that have completed the second month of gestation was lower than non-pregnant females and females that have completed the first month of gestation. Ion Na(+) concentration was higher in OF from non-pregnant females when compared with pregnant ones. The protein profile of non-pregnant females showed five protein bands of 70, 42, 25, 24 and 19kDa that were significantly more intense compared with pregnant animals. Bands were identified as moesin, actin cytoplasmic 2, hydroxypyruvate isomerase, ferritin light chain and peroxiredoxin-6 with MALDI/MS. Our results encourage more thorough future studies, in order to unravel the complex reproductive processes of the South American camelid oviduct.

  13. Divergent Evolution Within Protein Superfolds Inferred from Profile-based Phylogenetics

    PubMed Central

    Theobald, Douglas L.; Wuttke, Deborah S.

    2006-01-01

    Many dissimilar protein sequences fold into similar structures. A central and persistent challenge facing protein structural analysis is the discrimination between homology and convergence for structurally similar domains that lack significant sequence similarity. Classic examples are the OB-fold and SH3 domains, both small, modular β-barrel protein superfolds. The similarities among these domains have variously been attributed to common descent or to convergent evolution. Using a sequence profile-based phylogenetic technique, we analyzed all structurally characterized OB-fold, SH3, and PDZ domains with less than 40% mutual sequence identity. An all-against-all, profile-versus-profile analysis of these domains revealed many previously undetectable significant interrelationships. The matrices of scores were used to infer phylogenies based on our derivation of the relationships between sequence similarity E-values and evolutionary distances. The resulting clades of domains correlate remarkably well with biological function, as opposed to structural similarity, indicating that the functionally distinct sub-families within these superfolds are homologous. This method extends phylogenetics into the challenging “twilight zone” of sequence similarity, providing the first objective resolution of deep evolutionary relationships among distant protein families. PMID:16266719

  14. Combining Phylogenetic Profiling-Based and Machine Learning-Based Techniques to Predict Functional Related Proteins

    PubMed Central

    Lin, Tzu-Wen; Wu, Jian-Wei; Chang, Darby Tien-Hao

    2013-01-01

    Annotating protein functions and linking proteins with similar functions are important in systems biology. The rapid growth rate of newly sequenced genomes calls for the development of computational methods to help experimental techniques. Phylogenetic profiling (PP) is a method that exploits the evolutionary co-occurrence pattern to identify functional related proteins. However, PP-based methods delivered satisfactory performance only on prokaryotes but not on eukaryotes. This study proposed a two-stage framework to predict protein functional linkages, which successfully enhances a PP-based method with machine learning. The experimental results show that the proposed two-stage framework achieved the best overall performance in comparison with three PP-based methods. PMID:24069454

  15. Anti-heat shock protein autoantibody profiling in breast cancer using customized protein microarray.

    PubMed

    Shi, Liu; Gehin, Thomas; Chevolot, Yann; Souteyrand, Eliane; Mangé, Alain; Solassol, Jérôme; Laurenceau, Emmanuelle

    2016-02-01

    Heat shock proteins (HSPs) are over-expressed in a wide range of human cancers. It results in the stimulation of the immune system and consequently in elevated concentration of anti-HSP autoantibodies. Elevated anti-HSP autoantibodies were found in breast cancer patients, and they are associated with tumor metastasis. Therefore, screening these autoantibodies could be of diagnostic and prognostic values. Protein microarrays have already demonstrated their great potential as a diagnostic tool. However, protein diversity requires optimization of the microarray fabrication to achieve high sensitivity and specificity. In this study, seven HSPs were immobilized on six different surface chemistries. After evaluation and optimization with purified antibodies of the six surface chemistries, two surfaces were selected to detect anti-HSP autoantibodies in breast cancer sera. Multiplex detection of anti-HSP autoantibodies allowed discrimination of breast cancer patients (50) from healthy controls (26) with a sensitivity of 86% and a specificity of 100%. PMID:26715250

  16. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells

    PubMed Central

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  17. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells.

    PubMed

    Xie, Yajun; Wang, Jinlong; Zhang, Yuanya; Liu, Xiaofei; Wang, Xiaorong; Liu, Kehui; Huang, Xiahe; Wang, Yingchun

    2016-01-01

    Protein tyrosine phosphorylation is an important mechanism that regulates cytoskeleton reorganization and cell spreading of migratory cells. A number of cytoskeletal proteins are known to be tyrosine phosphorylated (pY) in different cellular processes. However, the profile of pY proteins during different stages of cell spreading has not been available. Using immunoafffinity enrichment of pY proteins coupled with label free quantitative proteomics, we quantitatively identified 447 pY proteins in the migratory ECV-304 cells at the early spreading (adhesion) and the active spreading stages. We found that pY levels of the majority of the quantified proteins were significantly increased in the active spreading stage compared with the early spreading stage, suggesting that active cell spreading is concomitant with extra tyrosine phosphorylation. The major categories of proteins impacted by tyrosine phosphorylation are involved in cytoskeleton and focal adhesion regulation, protein translation and degradation. Our findings, for the first time, dissect the cell spreading-specific pY signals from the adhesion induced pY signals, and provide a valuable resource for the future mechanistic research regarding the regulation of cell spreading. PMID:27554326

  18. Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays.

    PubMed

    Sill, Martin; Schröder, Christoph; Shen, Ying; Marzoq, Aseel; Komel, Radovan; Hoheisel, Jörg D; Nienhüser, Henrik; Schmidt, Thomas; Kastelic, Damjana

    2016-01-01

    In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL-10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins' capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches. PMID:27600085

  19. Application of activity-based protein profiling to study enzyme function in adipocytes.

    PubMed

    Galmozzi, Andrea; Dominguez, Eduardo; Cravatt, Benjamin F; Saez, Enrique

    2014-01-01

    Activity-based protein profiling (ABPP) is a chemical proteomics approach that utilizes small-molecule probes to determine the functional state of enzymes directly in native systems. ABPP probes selectively label active enzymes, but not their inactive forms, facilitating the characterization of changes in enzyme activity that occur without alterations in protein levels. ABPP can be a tool superior to conventional gene expression and proteomic profiling methods to discover new enzymes active in adipocytes and to detect differences in the activity of characterized enzymes that may be associated with disorders of adipose tissue function. ABPP probes have been developed that react selectively with most members of specific enzyme classes. Here, using as an example the serine hydrolase family that includes many enzymes with critical roles in adipocyte physiology, we describe methods to apply ABPP analysis to the study of adipocyte enzymatic pathways.

  20. Proteomic profile of carbonylated proteins in rat liver: discovering possible mechanisms for tetracycline-induced steatosis.

    PubMed

    Deng, Zhenglu; Yan, Siyu; Hu, Hui; Duan, Zhigui; Yin, Lanxuan; Liao, Shenke; Sun, Yubai; Yin, Dazhong; Li, Guolin

    2015-01-01

    To investigate biochemical mechanisms for the tetracycline-induced steatosis in rats, targeted proteins of oxidative modification were profiled. The results showed that tetracycline induced lipid accumulation, oxidative stress, and cell viability decline in HepG2 cells only under the circumstances of palmitic acid overload. Tetracycline administration in rats led to significant decrement in blood lipids, while resulted in more than four times increment in intrahepatic triacylglycerol and typical microvesicular steatosis in the livers. The triacylglycerol levels were positively correlated with oxidative stress. Proteomic profiles of carbonylated proteins revealed 26 targeted proteins susceptible to oxidative modification and most of them located in mitochondria. Among them, the long-chain specific acyl-CoA dehydrogenase was one of the key enzymes regulating fatty acid β-oxidation. Oxidative modification of the enzyme in the tetracycline group depressed its enzymatic activity. In conclusion, the increased influx of lipid into the livers is the first hit of tetracycline-induced microvesicular steatosis. Oxidative stress is an essential part of the second hit, which may arise from the lipid overload and attack a series of functional proteins, aggravating the development of steatosis. The 26 targeted proteins revealed here provide a potential direct link between oxidative stress and tetracycline-induced steatosis.

  1. 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.

  2. Prediction of mitochondrial protein function by comparative physiology and phylogenetic profiling.

    PubMed

    Cheng, Yiming; Perocchi, Fabiana

    2015-01-01

    According to the endosymbiotic theory, mitochondria originate from a free-living alpha-proteobacteria that established an intracellular symbiosis with the ancestor of present-day eukaryotic cells. During the bacterium-to-organelle transformation, the proto-mitochondrial proteome has undergone a massive turnover, whereby less than 20 % of modern mitochondrial proteomes can be traced back to the bacterial ancestor. Moreover, mitochondrial proteomes from several eukaryotic organisms, for example, yeast and human, show a rather modest overlap, reflecting differences in mitochondrial physiology. Those differences may result from the combination of differential gain and loss of genes and retargeting processes among lineages. Therefore, an evolutionary signature, also called "phylogenetic profile", could be generated for every mitochondrial protein. Here, we present two evolutionary biology approaches to study mitochondrial physiology: the first strategy, which we refer to as "comparative physiology," allows the de novo identification of mitochondrial proteins involved in a physiological function; the second, known as "phylogenetic profiling," allows to predict protein functions and functional interactions by comparing phylogenetic profiles of uncharacterized and known components.

  3. Protein corona change the drug release profile of nanocarriers: the "overlooked" factor at the nanobio interface.

    PubMed

    Behzadi, Shahed; Serpooshan, Vahid; Sakhtianchi, Ramin; Müller, Beate; Landfester, Katharina; Crespy, Daniel; Mahmoudi, Morteza

    2014-11-01

    The emergence of nanocarrier systems in drug delivery applications has ushered in rapid development of new classes of therapeutic agents which can provide an essential breakthrough in the fight against refractory diseases. However, successful clinical application of nano-drug delivery devices has been limited mainly due to the lack of control on sustained release of therapeutics from the carriers. A wide range of sophisticated approaches employs the formation of crosslinkable, non-crosslinkable, stimuli-responsive polymer nanocarriers in order to enhance their delivery efficiency. Despite the extensive research conducted on the development of various nanocarriers, the effect of the biological milieu on the drug release profile of these constructs is not yet fully investigated. In particular, the formation of a protein corona on the surface of nanocarriers, when they interact with living organisms in vivo is largely decisive for their biological function. Using a number of synthetized (i.e., superparamagnetic iron oxide nanoparticles and polymeric nanocapsules) and commercialized nanocarriers (i.e., Abraxane®, albumin-bound paclitaxel drug), this study demonstrates that the protein corona can shield the nanocarriers and, consequently, alters the release profile of the drugs from the nanocarriers. More specifically, the protein corona could significantly reduce the burst effect of either protein conjugated nanocarriers or carriers with surface loaded drug (i.e., SPIONs). However, the corona shell only slightly changed the release profile of polymeric nanocapsules. Therefore, the intermediary, buffer effect of the protein shells on the surface of nanoscale carriers plays a crucial role in their successful high-yield applications in vivo.

  4. 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.

  5. Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays

    PubMed Central

    Sill, Martin; Schröder, Christoph; Shen, Ying; Marzoq, Aseel; Komel, Radovan; Hoheisel, Jörg D.; Nienhüser, Henrik; Schmidt, Thomas; Kastelic, Damjana

    2016-01-01

    In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL‐10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins’ capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches. PMID:27600085

  6. Differential protein expression profiles of cyst fluid from papillary thyroid carcinoma and benign thyroid lesions.

    PubMed

    Dinets, Andrii; Pernemalm, Maria; Kjellin, Hanna; Sviatoha, Vitalijs; Sofiadis, Anastasios; Juhlin, C Christofer; Zedenius, Jan; Larsson, Catharina; Lehtiö, Janne; Höög, Anders

    2015-01-01

    Cystic papillary thyroid carcinoma (cPTC) is a subgroup of PTC presenting a diagnostic challenge at fine needle aspiration biopsy (FNAB). To further investigate this entity we aimed to characterize protein profiles of cyst fluids from cPTC and benign thyroid cystic lesions. In total, 20 cPTCs and 56 benign thyroid cystic lesions were studied. Profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on cyst fluids from a subset of cases after depletion, and selected proteins were further analyzed by Western blot (WB), immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). A total of 1,581 proteins were detected in cyst fluids, of which 841 were quantified in all samples using LC-MS/MS. Proteins with different expression levels between cPTCs and benign lesions were identified by univariate analysis (41 proteins) and multivariate analysis (59 proteins in an orthogonal partial least squares model). WB analyses of cyst fluid and IHC on corresponding tissue samples confirmed a significant up-regulation of cytokeratin 19 (CK-19/CYFRA 21-1) and S100A13 in cPTC vs. benign lesions. These findings were further confirmed by ELISA in an extended material of non-depleted cyst fluids from cPTCs (n = 17) and benign lesions (n = 55) (p<0.05). Applying a cut-off at >55 ng/ml for CK-19 resulted in 82% specificity and sensitivity. For S100A13 a cut-off at >230 pg/ml revealed a 94% sensitivity, but only 35% specificity. This is the first comprehensive catalogue of the protein content in fluid from thyroid cysts. The up-regulations of CK-19 and S100A13 suggest their possible use in FNAB based preoperative diagnostics of cystic thyroid lesions. PMID:25978681

  7. Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab

    PubMed Central

    Mukaihara, Kenta; Suehara, Yoshiyuki; Kohsaka, Shinji; Akaike, Keisuke; Tanabe, Yu; Kubota, Daisuke; Ishii, Midori; Fujimura, Tsutomu; Kazuno, Saiko; Okubo, Taketo; Takagi, Tatsuya; Yao, Takashi; Kaneko, Kazuo; Saito, Tsuyoshi

    2016-01-01

    Giant cell tumors of bone (GCTB) are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the protein expression

  8. PhyloGene server for identification and visualization of co-evolving proteins using normalized phylogenetic profiles

    PubMed Central

    Sadreyev, Ilyas R.; Ji, Fei; Cohen, Emiliano; Ruvkun, Gary; Tabach, Yuval

    2015-01-01

    Proteins that function in the same pathways, protein complexes or the same environmental conditions can show similar patterns of sequence conservation across phylogenetic clades. In species that no longer require a specific protein complex or pathway, these proteins, as a group, tend to be lost or diverge. Analysis of the similarity in patterns of sequence conservation across a large set of eukaryotes can predict functional associations between different proteins, identify new pathway members and reveal the function of previously uncharacterized proteins. We used normalized phylogenetic profiling to predict protein function and identify new pathway members and disease genes. The phylogenetic profiles of tens of thousands conserved proteins in the human, mouse, Caenorhabditis elegans and Drosophila genomes can be queried on our new web server, PhyloGene. PhyloGene provides intuitive and user-friendly platform to query the patterns of conservation across 86 animal, fungal, plant and protist genomes. A protein query can be submitted either by selecting the name from whole-genome protein sets of the intensively studied species or by entering a protein sequence. The graphic output shows the profile of sequence conservation for the query and the most similar phylogenetic profiles for the proteins in the genome of choice. The user can also download this output in numerical form. PMID:25958392

  9. PhyloGene server for identification and visualization of co-evolving proteins using normalized phylogenetic profiles.

    PubMed

    Sadreyev, Ilyas R; Ji, Fei; Cohen, Emiliano; Ruvkun, Gary; Tabach, Yuval

    2015-07-01

    Proteins that function in the same pathways, protein complexes or the same environmental conditions can show similar patterns of sequence conservation across phylogenetic clades. In species that no longer require a specific protein complex or pathway, these proteins, as a group, tend to be lost or diverge. Analysis of the similarity in patterns of sequence conservation across a large set of eukaryotes can predict functional associations between different proteins, identify new pathway members and reveal the function of previously uncharacterized proteins. We used normalized phylogenetic profiling to predict protein function and identify new pathway members and disease genes. The phylogenetic profiles of tens of thousands conserved proteins in the human, mouse, Caenorhabditis elegans and Drosophila genomes can be queried on our new web server, PhyloGene. PhyloGene provides intuitive and user-friendly platform to query the patterns of conservation across 86 animal, fungal, plant and protist genomes. A protein query can be submitted either by selecting the name from whole-genome protein sets of the intensively studied species or by entering a protein sequence. The graphic output shows the profile of sequence conservation for the query and the most similar phylogenetic profiles for the proteins in the genome of choice. The user can also download this output in numerical form.

  10. Profiling of the cell surface proteome.

    PubMed

    Jang, Jun Ho; Hanash, Samir

    2003-10-01

    The in depth-mining of the proteome necessitates the comprehensive analysis of proteins in individual subcellular compartments to uncover interesting patterns of protein expression that include assessment of protein location, trafficking and of post-translational modifications that are location specific. One of the compartments of substantial interest from a diagnostic and therapeutic point of view is the plasma membrane which contains intrinsic membrane proteins and other proteins expressed on the cell surface. Technologies are currently available for the comprehensive profiling of the cell surface proteome that rely on protein tagging of intact cells. Studies are emerging that point to unexpected patterns of expression of specific proteins on the cell surface, with a common occurrence of proteins previously considered to occur predominantly in other compartments, notably the endoplasmic reticulum. The profiling of the cell surface and plasma membrane proteomes will likely provide novel insights and uncover disease related alterations. PMID:14625857

  11. Protein profiles of bovine placenta derived from somatic cell nuclear transfer.

    PubMed

    Kim, Hong Rye; Kang, Jae Ku; Yoon, Jong Taek; Seong, Hwan Hoo; Jung, Jin Kwan; Lee, Hong Mie; Sik Park, Chang; Jin, Dong Il

    2005-11-01

    Practical application of animal cloning by somatic cell nuclear transfer (SCNT) has been hampered by an extremely low success rate. To address whether placental dysfunction in SCNT causes fetal loss during pregnancy, we have used a global proteomics approach using 2-DE and MS to analyze the differential protein patterns of three placentae from the afterbirth of cases of postnatal death, derived from SCNT of Korean Native cattle, and three normal placentae obtained from the afterbirth of fetuses derived from artificial insemination. Proteins within a pI range of 4.0-7.0 and 6.0-9.0 were analyzed separately by 2-DE in triplicate. A total of approximately 2000 spots were detected in placental 2-DE gels stained with CBB. In the comparison of normal and SCNT samples, 60 spots were identified as differentially expressed proteins, of which 33 spots were up-regulated proteins in SCNT placentae, while 27 spots were down-regulated proteins. Most of the proteins identified in this analysis appeared to be related with protein repair or protection, cytoskeleton, signal transduction, immune system, metabolism, extracellular matrix and remodeling, transcription regulation, cell structure or differentiation and ion transport. One of up-regulated proteins in SCNT was TIMP-2 protein known to be related to extracellular matrix and remodeling during pregnancy. Western blot analysis showed an increased level of TIMP-2 in SCNT placenta compared to normal. Our results revealed composite profiles of key proteins involved in abnormal placenta derived from SCNT, and suggested expression abnormality of these genes in SCNT placenta, resulting in fetal losses following SCNT.

  12. 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.

  13. 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.

  14. 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

  15. Monitoring Intact Viruses Using Aptamers

    PubMed Central

    Kumar, Penmetcha K. R.

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  16. Monitoring Intact Viruses Using Aptamers.

    PubMed

    Kumar, Penmetcha K R

    2016-01-01

    Viral diagnosis and surveillance are necessary steps in containing the spread of viral diseases, and they help in the deployment of appropriate therapeutic interventions. In the past, the commonly employed viral detection methods were either cell-culture or molecule-level assays. Most of these assays are laborious and expensive, require special facilities, and provide a slow diagnosis. To circumvent these limitations, biosensor-based approaches are becoming attractive, especially after the successful commercialization of glucose and other biosensors. In the present article, I have reviewed the current progress using the biosensor approach for detecting intact viruses. At the time of writing this review, three types of bioreceptor surfaces (antibody-, glycan-, and aptamer-based) have been explored on different sensing platforms for detecting intact viruses. Among these bioreceptors, aptamer-based sensors have been increasingly explored for detecting intact viruses using surface plasmon resonance (SPR) and other platforms. Special emphasis is placed on the aptamer-based SPR platform in the present review. PMID:27527230

  17. Understanding the blood-brain barrier using gene and protein expression profiling technologies.

    PubMed

    Pottiez, Gwënaël; Flahaut, Christophe; Cecchelli, Roméo; Karamanos, Yannis

    2009-12-11

    The blood-brain barrier (BBB) contributes to the brain homeostasis by regulating the passage of endogenous and exogenous compounds. This function is in part due to well-known proteins such as tight junction proteins, plasma membrane transporters and metabolic barrier proteins. Over the last decade, genomics and proteomics have emerged as supplementary tools for BBB research. The development of genomic and proteomic technologies has provided several means to extend the BBB knowledge and to investigate additional routes for the bypass of this barrier. These profiling technologies have been used on BBB models to decipher the physiological characteristics and, under stress conditions, to understand the molecular mechanisms of brain diseases. In this review, we will report and discuss the genomic and proteomic studies recently carried out to enhance the understanding of BBB features.

  18. Profiling lethal factor interacting proteins from human stomach using T7 phage display screening.

    PubMed

    Cardona-Correa, Albin; Rios-Velazquez, Carlos

    2016-05-01

    The anthrax lethal factor (LF) is a zinc dependent metalloproteinase that cleaves the majority of mitogen-activated protein kinase kinases and a member of NOD-like receptor proteins, inducing cell apoptosis. Despite efforts to fully understand the Bacillus anthracis toxin components, the gastrointestinal (GI) anthrax mechanisms have not been fully elucidated. Previous studies demonstrated gastric ulceration, and a substantial bacterial growth rate in Peyer's patches. However, the complete molecular pathways of the disease that results in tissue damage by LF proteolytic activity remains unclear. In the present study, to identify the profile of the proteins potentially involved in GI anthrax, protein‑protein interactions were investigated using human stomach T7 phage display (T7PD) cDNA libraries. T7PD is a high throughput technique that allows the expression of cloned DNA sequences as peptides on the phage surface, enabling the selection and identification of protein ligands. A wild type and mutant LF (E687A) were used to differentiate interaction sites. A total of 124 clones were identified from 194 interacting‑phages, at both the DNA and protein level, by in silico analysis. Databases revealed that the selected candidates were proteins from different families including lipase, peptidase‑A1 and cation transport families, among others. Furthermore, individual T7PD candidates were tested against LF in order to detect their specificity to the target molecule, resulting in 10 LF‑interacting peptides. With a minimum concentration of LF for interaction at 1 µg/ml, the T7PD isolated pepsin A3 pre‑protein (PAP) demonstrated affinity to both types of LF. In addition, PAP was isolated in various lengths for the same protein, exhibiting common regions following PRALINE alignment. These findings will help elucidate and improve the understanding of the molecular pathogenesis of GI anthrax, and aid in the development of potential therapeutic agents. PMID

  19. Protein array–based profiling of CSF identifies RBPJ as an autoantigen in multiple sclerosis

    PubMed Central

    Querol, Luis; Clark, Pamela L.; Bailey, Mary A.; Cotsapas, Chris; Cross, Anne H.; Hafler, David A.; Kleinstein, Steven H.; Lee, Jae-Yun; Yaari, Gur

    2013-01-01

    Objective: To profile the reactivity of CSF-derived immunoglobulin from patients with multiple sclerosis (MS) against a large panel of antigens, to identify disease-specific reactivities. Methods: CSF from subjects with MS with elevated immunoglobulin G and CSF from control subjects presenting with other inflammatory neurologic disease were screened against a protein array consisting of 9,393 proteins. Reactivity to a candidate protein identified using these arrays was confirmed with ELISA and immunocytochemistry. Results: Autoantibodies against one protein on the array, recombination signal binding protein for immunoglobulin kappa J region (RBPJ), discriminated between patients with MS and controls (p = 0.0052). Using a large validation cohort, we found a higher prevalence of autoantibodies against RBPJ in the CSF of patients with MS (12.5%) compared with the CSF of patients with other neurologic diseases (1.6%; p = 0.02) by ELISA. This difference in reactivity was restricted to the CSF as serum reactivity against RBPJ did not differ between patients and controls. The presence of CSF autoantibodies against RBPJ was further confirmed by immunocytochemistry. Conclusions: These data indicate that RBPJ, a ubiquitous protein of the Notch signaling pathway that plays an important role in Epstein-Barr virus infection, is a novel MS autoantigen candidate that is recognized by CSF-derived immunoglobulin G in a subset of patients with MS. PMID:23921886

  20. High light induced changes in organization, protein profile and function of photosynthetic machinery in Chlamydomonas reinhardtii.

    PubMed

    Nama, Srilatha; Madireddi, Sai Kiran; Devadasu, Elsin Raju; Subramanyam, Rajagopal

    2015-11-01

    The green alga Chlamydomonas (C.) reinhardtii is used as a model organism to understand the efficiency of photosynthesis along with the organization and protein profile of photosynthetic apparatus under various intensities of high light exposure for 1h. Chlorophyll (Chl) a fluorescence induction, OJIPSMT transient was decreased with increase in light intensity indicating the reduction in photochemical efficiency. Further, circular dichroism studies of isolated thylakoids from high light exposed cells showed considerable change in the pigment-pigment interactions and pigment-proteins interactions. Furthermore, the organization of supercomplexes from thylakoids is studied, in which, one of the hetero-trimer of light harvesting complex (LHC) II is affected significantly in comparison to other complexes of LHC's monomers. Also, other supercomplexes, photosystem (PS)II reaction center dimer and PSI complexes are reduced. Additionally, immunoblot analysis of thylakoid proteins revealed that PSII core proteins D1 and D2 were significantly decreased during high light treatment. Similarly, the PSI core proteins PsaC, PsaD and PsaG were drastically changed. Further, the LHC antenna proteins of PSI and PSII were differentially affected. From our results it is clear that LHCs are damaged significantly, consequently the excitation energy is not efficiently transferred to the reaction center. Thus, the photochemical energy transfer from PSII to PSI is reduced. The inference of the study deciphers the structural and functional changes driven by light may therefore provide plants/alga to regulate the light harvesting capacity in excess light conditions.

  1. 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

  2. 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).

  3. Language and Williams syndrome: how intact is "intact"?

    PubMed

    Karmiloff-Smith, A; Grant, J; Berthoud, I; Davies, M; Howlin, P; Udwin, O

    1997-04-01

    It has been claimed that Williams syndrome (WS), a rare neurodevelopmental disorder, is characterized by serious cognitive deficits alongside intact language. The syndrome is often used as a prime example of the modularity of an innate faculty for morphosyntactic rules. We challenge this claim and hypothesize that morphosyntax, although surprisingly good given WS level of mental retardation, is by no means intact. We make an initial test of this hypothesis through an analysis of the receptive language of a group of English-speaking WS individuals on a standardized morphosyntactic test. We then present an experimental study of expressive language that examines grammatical gender assignment in French-speaking WS patients. Despite a Verbal Mental Age selected to be higher than the chronological age of the young control group, these people with WS continue even in adulthood to show clear-cut deficits in their production of an aspect of morphosyntax that normal children acquire effortlessly very early. The results of the 2 studies, one focusing on receptive language and the other on expressive language, challenge the notion that comprehension and use of morphosyntactic rules in WS individuals are intact. The Within-domain dissociations regarding the use of grammatical gender assignment across several sentence clements and their difficulties in understanding embedded sentences-two quintessentially linguistic skills-suggest that we must rethink the notion of spared, modular, language capacities in Williams syndrome. We conclude that WS language follows a different path to normal acquisition and may turn out to be more like second language learning. PMID:9180000

  4. Influence of peptides-phenolics interaction on the antioxidant profile of protein hydrolysates from Brassica napus.

    PubMed

    Hernández-Jabalera, Anaid; Cortés-Giraldo, Isabel; Dávila-Ortíz, Gloria; Vioque, Javier; Alaiz, Manuel; Girón-Calle, Julio; Megías, Cristina; Jiménez-Martínez, Cristian

    2015-07-01

    The role of the peptides-phenolic compounds (PC) interaction on the antioxidant capacity profile (ACP) of protein hydrolysates from rapeseed (Brassica napus) was studied in 36 hydrolysates obtained from a PC-rich and PC-reduced protein substrate. The latent profile analysis (LPA), with data of seven in vitro methods and one assay for cellular antioxidant activity (CAA), allowed identifying five distinctive groups of hydrolysates, each one with distinctive ACP. The interaction of peptides with naturally present PC diminished in vitro antioxidant activity in comparison with their PC-reduced counterparts. However, CAA increased when peptides-PC interaction occurred. The profile with the highest average CAA (62.41 ± 1.48%), shown by hydrolysates obtained by using alcalase, shared typical values of Cu(2+)-catalysed β-carotene oxidation (62.41 ± 0.43%), β-carotene bleaching inhibition (91.75 ± 0.22%) and Cu(2+)-chelating activity (74.53 ± 0.58%). The possibilities for a sample to exhibit ACP with higher CAA increased with each unit of positively charged amino acids, according to multinomial logistic regression analysis.

  5. Serum pattern profiling for analyzing different types of stress by protein chip technology: a preliminary study.

    PubMed

    Liu, Hui; Hou, Diandong; Wu, Da; Yin, Hong; Wu, Xiaoyi

    2010-01-01

    ProteinChip is a widely accepted tool for exploring serum pattern profile to evaluate the risk of somatic diseases from different stressors. In this study, by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-ToF), the serum proteome from mice under restraint and thermal stresses were profiled in detail and compared with the control group. Around 150 characteristic peaks were detected in all three groups, with m/z ranging from 1500 to 50,000, with most peaks being within the 2000 m/z to 20,000 m/z range. Compared with the control group, three significant protein peaks with m/z values of 2780, 3303 and 3450 appeared specifically in the restrained stress group and four other peaks with m/z values of 7500, 7811, 29,950 and 38,565 in the thermal stress group. Unexpectedly, no universal positive stress peaks were detected. These preliminary results clearly suggested that there might not be a common mechanism shared by various psychophysiological disorders under different stressors. By fast serum proteomics profiling, SELDI-ToF may be a convenient tool for evaluating the risk of stress-induced illness.

  6. 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

  7. Reliability of human serum protein profiles generated with C8 magnetic beads assisted MALDI-TOF mass spectrometry.

    PubMed

    de Noo, Mirre E; Tollenaar, Rob A E M; Ozalp, Aliye; Kuppen, Peter J K; Bladergroen, Marco R; Eilers, Paul H C; Deelder, André M

    2005-11-15

    Protein profiling with mass spectrometry is a promising approach for classification and identification of biomarkers; however, there is debate about measurement quality and reliability. Here, we present a pipeline for preprocessing, statistical data analysis and presentation. Serum samples of 16 healthy individuals are used to generate protein profiles with high-resolution MALDI-TOF after isolation of peptides with C8 magnetic beads. Analysis of variance was performed after binning, baseline correction and normalization of the mean spectra. Relative variations in the spectra are expressed as coefficient of variation, which depending on the respective preanalytical variation parameter investigated, was found to range between 0.15 and 0.67 in this study. With this novel method, the reproducibility of our protein profiling procedure could be quantified. We showed that circadian rhythm and the number of freeze-thaw cycles had relatively limited influence on serum protein profiles, whereas the period between collection and serum centrifugation had a more pronounced effect.

  8. Effects of Fe deficiency on the protein profile of Brassica napus phloem sap.

    PubMed

    Gutierrez-Carbonell, Elain; Lattanzio, Giuseppe; Albacete, Alfonso; Rios, Juan José; Kehr, Julia; Abadía, Anunciación; Grusak, Michael A; Abadía, Javier; López-Millán, Ana Flor

    2015-11-01

    The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2DE (IEF-SDS-PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Phloem sap purity was assessed by measuring sugar concentrations. Two hundred sixty-three spots were consistently detected and 15.6% (41) of them showed significant changes in relative abundance (22 decreasing and 19 increasing) as a result of Fe deficiency. Among them, 85% (35 spots), were unambiguously identified. Functional categories containing the largest number of protein species showing changes as a consequence of Fe deficiency were signaling and regulation (32%), and stress and redox homeostasis (17%). The Phloem sap showed a higher oxidative stress and significant changes in the hormonal profile as a result of Fe deficiency. Results indicate that Fe deficiency elicits major changes in signaling pathways involving Ca and hormones, which are generally associated with flowering and developmental processes, causes an alteration in ROS homeostasis processes, and induces decreases in the abundances of proteins involved in sieve element repair, suggesting that Fe-deficient plants may have an impaired capacity to heal sieve elements upon injury.

  9. Integrated quantitative analysis of nitrogen stress response in Chlamydomonas reinhardtii using metabolite and protein profiling.

    PubMed

    Wase, Nishikant; Black, Paul N; Stanley, Bruce A; DiRusso, Concetta C

    2014-03-01

    Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.

  10. Variance decomposition of protein profiles from antibody arrays using a longitudinal twin model

    PubMed Central

    2011-01-01

    Background The advent of affinity-based proteomics technologies for global protein profiling provides the prospect of finding new molecular biomarkers for common, multifactorial disorders. The molecular phenotypes obtained from studies on such platforms are driven by multiple sources, including genetic, environmental, and experimental components. In characterizing the contribution of different sources of variation to the measured phenotypes, the aim is to facilitate the design and interpretation of future biomedical studies employing exploratory and multiplexed technologies. Thus, biometrical genetic modelling of twin or other family data can be used to decompose the variation underlying a phenotype into biological and experimental components. Results Using antibody suspension bead arrays and antibodies from the Human Protein Atlas, we study unfractionated serum from a longitudinal study on 154 twins. In this study, we provide a detailed description of how the variation in a molecular phenotype in terms of protein profile can be decomposed into familial i.e. genetic and common environmental; individual environmental, short-term biological and experimental components. The results show that across 69 antibodies analyzed in the study, the median proportion of the total variation explained by familial sources is 12% (IQR 1-22%), and the median proportion of the total variation attributable to experimental sources is 63% (IQR 53-72%). Conclusion The variability analysis of antibody arrays highlights the importance to consider variability components and their relative contributions when designing and evaluating studies for biomarker discoveries with exploratory, high-throughput and multiplexed methods. PMID:22093360

  11. Protein profiling reveals consequences of lifestyle choices on predicted biological aging

    PubMed Central

    Enroth, Stefan; Enroth, Sofia Bosdotter; Johansson, Åsa; Gyllensten, Ulf

    2015-01-01

    Ageing is linked to a number of changes in how the body and its organs function. On a molecular level, ageing is associated with a reduction of telomere length, changes in metabolic and gene-transcription profiles and an altered DNA-methylation pattern. Lifestyle factors such as smoking or stress can impact some of these molecular processes and thereby affect the ageing of an individual. Here we demonstrate by analysis of 77 plasma proteins in 976 individuals, that the abundance of circulating proteins accurately predicts chronological age, as well as anthropometrical measurements such as weight, height and hip circumference. The plasma protein profile can also be used to identify lifestyle factors that accelerate and decelerate ageing. We found smoking, high BMI and consumption of sugar-sweetened beverages to increase the predicted chronological age by 2–6 years, while consumption of fatty fish, drinking moderate amounts of coffee and exercising reduced the predicted age by approximately the same amount. This method can be applied to dried blood spots and may thus be useful in forensic medicine to provide basic anthropometrical measures for an individual based on a biological evidence sample. PMID:26619799

  12. Simultaneous measurement of multiple radiation-induced protein expression profiles using the Luminex(TM) system

    NASA Technical Reports Server (NTRS)

    Desai, N.; Wu, H.; George, K.; Gonda, S. R.; Cucinotta, F. A.; Cucniotta, F. A. (Principal Investigator)

    2004-01-01

    Space flight results in the exposure of astronauts to a mixed field of radiation composed of energetic particles of varying energies, and biological indicators of space radiation exposure provides a better understanding of the associated long-term health risks. Current methods of biodosimetry have employed the use of cytogenetic analysis for biodosimetry, and more recently the advent of technological progression has led to advanced research in the use of genomic and proteomic expression profiling to simultaneously assess biomarkers of radiation exposure. We describe here the technical advantages of the Luminex(TM) 100 system relative to traditional methods and its potential as a tool to simultaneously profile multiple proteins induced by ionizing radiation. The development of such a bioassay would provide more relevant post-translational dynamics of stress response and will impart important implications in the advancement of space and other radiation contact monitoring. c2004 COSPAR. Published by Elsevier Ltd. All rights reserved.

  13. 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.

  14. 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

  15. 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.

  16. Splicing profile based protein categorization between human and mouse genomes by use of the DDBJ Web services.

    PubMed

    Västermark, Ake; Shigemoto, Yasumasa; Abe, Takashi; Sugawara, Hideaki

    2004-01-01

    In one scenario of gene evolution, exon shuffling plays a fundamental role in increasing gene diversity. This paper is an appraisal of the biological relevance of categorising proteins by their splicing profiles (exon-intron structures). The central question is whether protein function is more correlated with splicing profiles than sequence similarity, or not. To approach this question, a splicing profile similarity (SPS) index, which measures relative exon length discrepancy, was devised. Arbitrary human proteins were compared, in terms of SPS and amino acid sequence similarity, to their 1) mouse orthologues and 2) human paralogues, which epitomise functional equivalence and non-equivalence, respectively, to methodically elucidate the global relationship between a) biological function, b) splicing profile similarity, and c) sequence similarity. Protein function is more correlated with splicing profile similarity than sequence similarity as demonstrated by the fact that human-mouse orthologues (HMOs) display significantly higher splicing profile similarity than do human-human paralogues (HHPs), despite the mutual sequence similarity between these two categories. This finding indicates that splicing profile-based protein categorisation is biologically meaningful.

  17. Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

    PubMed

    Bal, Gürkan; Futschik, Matthias E; Hartl, Daniela; Ringel, Frauke; Kamhieh-Milz, Julian; Sterzer, Viktor; Hoheisel, Jörg D; Alhamdani, Mohamed S S; Salama, Abdulgabar

    2016-02-01

    The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration.

  18. Profiling and relationship of water-soluble sugar and protein compositions in soybean seeds.

    PubMed

    Yu, Xiaomin; Yuan, Fengjie; Fu, Xujun; Zhu, Danhua

    2016-04-01

    Sugar and protein are important quality traits in soybean seeds for making soy-based food products. However, the investigations on both compositions and their relationship have rarely been reported. In this study, a total of 35 soybean germplasms collected from Zhejiang province of China, were evaluated for both water-soluble sugar and protein. The total water-soluble sugar (TWSS) content of the germplasms studied ranged from 84.70 to 140.91 mg/g and the water-soluble protein (WSP) content varied from 26.5% to 36.0%. The WSP content showed positive correlations with the TWSS and sucrose contents but negative correlations with the fructose and glucose contents. The clustering showed the 35 germplasms could be divided into four groups with specific contents of sugar and protein. The combination of water-soluble sugar and protein profiles provides useful information for future breeding and genetic research. This investigation will facilitate future work for seed quality improvement.

  19. Single-Cell Protein Profiling of Wastewater Enterobacterial Communities Predicts Disinfection Efficiency

    PubMed Central

    Ponniah, Gomathinayagam; Chen, Han; Michielutti, Ronda; Salonen, Nancy; Blum, Paul

    2003-01-01

    The efficiency of enterobacterial disinfection is dependent largely on enterobacterial community physiology. However, the relationship between enterobacterial community physiology and wastewater processing is unclear. The purpose of this study was to investigate this relationship. The influence of wastewater treatment processes on enterobacterial community physiology was examined at the single-cell level by using culture-independent methods. Intracellular concentrations of two conserved proteins, the growth-related protein Fis and the stationary-phase protein Dps, were analyzed by epifluoresence microscopy of uncultivated cells by using enterobacterial group-specific polyclonal fluorochrome-coupled antibodies. Enterobacterial single-cell community protein profiles were distinct for different types of biological treatment. The differences were not apparent when bulk methods of protein analysis were used. Trickling filter wastewater yielded Fis-enriched communities compared to the communities in submerged aeration basin wastewater. Community differences in Fis and Dps contents were used to predict disinfection efficiency. Disinfection of community samples by heat exposure combined with cultivation in selective media confirmed that enterobacterial communities exhibited significant differences in sensitivity to disinfection. These findings provide strategies that can be used to increase treatment plant performance, reduce the enterobacterial content in municipal wastewater, and minimize the release of disinfection by-products into receiving water. PMID:12839804

  20. Expression Profiles of Vpx/Vpr Proteins Are Co-related with the Primate Lentiviral Lineage

    PubMed Central

    Sakai, Yosuke; Miyake, Ariko; Doi, Naoya; Sasada, Hikari; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    Viruses of human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency virus (SIV) lineages carry a unique accessory protein called Vpx. Vpx is essential or critical for viral replication in natural target cells such as macrophages and T lymphocytes. We have previously shown that a poly-proline motif (PPM) located at the C-terminal region of Vpx is required for its efficient expression in two strains of HIV-2 and SIVmac, and that the Vpx expression levels of the two clones are significantly different. Notably, the PPM sequence is conserved and confined to Vpx and Vpr proteins derived from certain lineages of HIV-2/SIVs. In this study, Vpx/Vpr proteins from diverse primate lentiviral lineages were experimentally and phylogenetically analyzed to obtain the general expression picture in cells. While both the level and PPM-dependency of Vpx/Vpr expression in transfected cells varied among viral strains, each viral group, based on Vpx/Vpr amino acid sequences, was found to exhibit a characteristic expression profile. Moreover, phylogenetic tree analyses on Gag and Vpx/Vpr proteins gave essentially the same results. Taken together, our study described here suggests that each primate lentiviral lineage may have developed a unique expression pattern of Vpx/Vpr proteins for adaptation to its hostile cellular and species environments in the process of viral evolution. PMID:27536295

  1. Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality.

    PubMed

    Wu, Nicholas C; Olson, C Anders; Du, Yushen; Le, Shuai; Tran, Kevin; Remenyi, Roland; Gong, Danyang; Al-Mawsawi, Laith Q; Qi, Hangfei; Wu, Ting-Ting; Sun, Ren

    2015-07-01

    Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.

  2. Profiling and relationship of water-soluble sugar and protein compositions in soybean seeds.

    PubMed

    Yu, Xiaomin; Yuan, Fengjie; Fu, Xujun; Zhu, Danhua

    2016-04-01

    Sugar and protein are important quality traits in soybean seeds for making soy-based food products. However, the investigations on both compositions and their relationship have rarely been reported. In this study, a total of 35 soybean germplasms collected from Zhejiang province of China, were evaluated for both water-soluble sugar and protein. The total water-soluble sugar (TWSS) content of the germplasms studied ranged from 84.70 to 140.91 mg/g and the water-soluble protein (WSP) content varied from 26.5% to 36.0%. The WSP content showed positive correlations with the TWSS and sucrose contents but negative correlations with the fructose and glucose contents. The clustering showed the 35 germplasms could be divided into four groups with specific contents of sugar and protein. The combination of water-soluble sugar and protein profiles provides useful information for future breeding and genetic research. This investigation will facilitate future work for seed quality improvement. PMID:26593554

  3. Identification of novel biomarkers in chronic immune thrombocytopenia (ITP) by microarray-based serum protein profiling.

    PubMed

    Bal, Gürkan; Futschik, Matthias E; Hartl, Daniela; Ringel, Frauke; Kamhieh-Milz, Julian; Sterzer, Viktor; Hoheisel, Jörg D; Alhamdani, Mohamed S S; Salama, Abdulgabar

    2016-02-01

    The pathological mechanisms underlying the development of immune thrombocytopenia (ITP) are unclear and its diagnosis remains a process of exclusion. Currently, there are no known specific biomarkers for ITP to support differential diagnosis and treatment decisions. Profiling of serum proteins may be valuable for identifying such biomarkers. Sera from 46 patients with primary chronic ITP and 34 healthy blood donors were analysed using a microarray of 755 antibodies. We identified 161 differentially expressed proteins. In addition to oncoproteins and tumour-suppressor proteins, including apoptosis regulator BCL2, breast cancer type 1 susceptibility protein (BRCA1), Fanconi anaemia complementation group C (FANCC) and vascular endothelial growth factor A (VEGFA), we detected six anti-nuclear autoantibodies in a subset of ITP patients: anti-PCNA, anti-SmD, anti-Ro/SSA60, anti-Ro/SSA52, anti-La/SSB and anti-RNPC antibodies. This finding may provide a rational explanation for the association of ITP with malignancies and other autoimmune diseases. While RUNX1mRNA expression in the peripheral blood mononuclear cells (PBMC) of patients was significantly downregulated, an accumulation of RUNX1 protein was observed in the platelets of ITP patients. This may indicate dysregulation of RUNX1 expression in PBMC and megakaryocytes and may lead to an imbalanced immune response and impaired thrombopoiesis. In conclusion, we provide novel insights into the pathogenic mechanisms of ITP that warrant further exploration. PMID:26628061

  4. Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

    SciTech Connect

    Islam, Kabirul; Chen, Yuling; Wu, Hong; Bothwell, Ian R.; Blum, Gil J.; Zeng, Hong; Dong, Aiping; Zheng, Weihong; Min, Jinrong; Deng, Haiteng; Luo, Minkui

    2013-11-18

    Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. With two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.

  5. Intact human holo-transferrin interaction with oxaliplatin.

    PubMed

    Zhao, Yuan-Yuan; Mandal, Rupasri; Li, Xing-Fang

    2005-01-01

    We report the interaction of intact human holo-transferrin (holo-Tf) with oxaliplatin (an anticancer drug), and the characterization of a complex composed of (1:1) intact holo-Tf and the parent oxaliplatin molecule using nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS). The molecular weight of this complex was determined to be 80,077 Da, which was an increase of 397 mass units compared to the protein alone (79,680 Da), suggesting that a parent drug molecule was bound to the intact protein. We further examined the interaction between the intact protein and oxaliplatin using size-exclusion high-performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICPMS). The protein complex and free oxaliplatin were separated by HPLC and quantitatively determined by simultaneous monitoring of both 195Pt and 56Fe using ICPMS. The HPLC/ICPMS detected both Pt and Fe signals at retention time of 2.6 min, identifying the protein-drug complex. The Fe signal at 2.6 min did not change with the increase in incubation time of the reaction mixture containing holo-Tf and oxaliplatin, while the Pt signal at the same retention time increased over time, further demonstrating that the formation of this complex does not affect the protein-bound Fe. The binding constant of the (1:1) intact human holo-Tf-oxaliplatin complex was determined to be 7.7x10(5) M-1. Both nanoESI-MS and HPLC/ICPMS results support that the holo-Tf and parent oxaliplatin molecules form complexes through non-covalent binding, suggesting that holo-Tf may be a useful carrier for oxaliplatin delivery.

  6. Using SEC-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling

    PubMed Central

    Simpson, David C.; Ahn, Seonghee; Pasa-Tolic, Ljiljana; Bogdanov, Bogdan; Mottaz, Heather M.; Vilkov, Andrey N.; Anderson, Gordon A.; Lipton, Mary S.; Smith, Richard D.

    2007-01-01

    Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated capability for broad proteome coverage and good throughput. However, due to incomplete sequence coverage, this approach is not ideally suited to the study of modified proteins. The modification complement of a protein can best be elucidated by analyzing the intact protein. Two-dimensional gel electrophoresis, typically coupled with the analysis of peptides that result from in-gel digestion, is the most frequently applied protein separation technique in MS-based proteomics. As an alternative, numerous column-based liquid phase techniques, which are generally more amenable to automation, are being investigated. In this work, the combination of size exclusion chromatography (SEC) fractionation with reversed-phase liquid chromatography (RPLC)-Fourier-transform ion cyclotron resonance (FTICR)-mass spectrometry (MS) is compared with the combination of RPLC fractionation with capillary isoelectric focusing (CIEF)-FTICR-MS for the analysis of the Shewanella oneidensis proteome. SEC-RPLC-FTICR-MS allowed the detection of 297 proteins, as opposed to 166 using RPLC-CIEF-FTICR-MS, indicating that approaches based on LC-MS provide better coverage. However, there were significant differences in the sets of proteins detected and both approaches provide a basis for accurately quantifying changes in protein and modified protein abundances. PMID:16732621

  7. Comparative analysis of click chemistry mediated activity-based protein profiling in cell lysates.

    PubMed

    Yang, Yinliang; Yang, Xiaomeng; Verhelst, Steven H L

    2013-01-01

    Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I)-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions. PMID:24126377

  8. Phosphorylation of intact erythrocytes in human muscular dystrophy

    SciTech Connect

    Johnson, R.M.; Nigro, M.

    1986-04-01

    The uptake of exogenous /sup 32/Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-(/sup 32/P)ATP, and suggests a possible reinterpretation of those experiments.

  9. 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

  10. Protein kinase target discovery from genome-wide messenger RNA expression profiling.

    PubMed

    Ma'ayan, Avi; He, John C

    2010-01-01

    Genome-wide messenger RNA profiling provides a snapshot of the global state of the cell under different experimental conditions such as diseased versus normal cellular states. However, because measurements are in the form of quantitative changes in messenger RNA levels, such experimental data does not provide direct understanding of the regulatory molecular mechanisms responsible for the observed changes. Identifying potential cell signaling regulatory mechanisms responsible for changes in gene expression under different experimental conditions or in different tissues has been the focus of many computational systems biology studies. Most popular approaches include promoter analysis, gene ontology, or pathway enrichment analysis, as well as reverse engineering of networks from messenger RNA expression data. Here we present a rational approach for identifying and ranking protein kinases that are likely responsible for observed changes in gene expression. By combining promoter analysis; data from various chromatin immunoprecipitation studies such as chromatin immunoprecipitation sequencing, chromatin immunoprecipitation coupled with paired-end ditag, and chromatin immunoprecipitation-on-chip; protein-protein interactions; and kinase-protein phosphorylation reactions collected from the literature, we can identify and rank candidate protein kinases for knock-down, or other types of functional validations, based on genome-wide changes in gene expression. We describe how protein kinase candidate identification and ranking can be made robust by cross-validation with phosphoproteomics data as well as through a literature-based text-mining approach. In conclusion, data integration can produce robust candidate rankings for understanding cell regulation through identification of protein kinases responsible for gene expression changes, and thus rapidly advancing drug target discovery and unraveling drug mechanisms of action.

  11. Proteomic profiling reveals a severely perturbed protein expression pattern in aged skeletal muscle.

    PubMed

    O'Connell, Kathleen; Gannon, Joan; Doran, Philip; Ohlendieck, Kay

    2007-08-01

    Extended longevity is often accompanied by frailty and increased susceptibility to a variety of crippling disorders. One of the most striking features of human aging is sarcopenia, which is defined as the age-related decline in skeletal muscle mass and strength. Although various metabolic and functional defects in aging muscle fibres have been described over the last decade, it is not known whether a pathophysiological hierarchy exists within degenerative pathways leading to muscle wasting. Hence, in order to identify novel biomarkers of age-dependent skeletal muscle degeneration, we have here applied mass spectrometry-based proteomics for studying global muscle protein expression patterns. As a model system of sarcopenia, we have employed crude extracts from senescent rat gastrocnemius muscle, as compared to young adult tissue preparations. Using the highly sensitive protein dye Deep Purple for the analysis of the 2-D separated muscle proteome and peptide mass fingerprinting for the identification of individual protein spots, a differential expression pattern was observed for contractile proteins, metabolic factors, regulatory components and heat shock elements. A drastic increase was shown for alpha B-crystallin, myosin light chain MLC-1, phosphoglycerate kinase, adenylate kinase, triosephosphate isomerase, albumin, aconitase and nucleoside-diphosphate kinase in aged fibres. In contrast, the expression of pyruvate kinase, aldolase, creatine kinase, transferrin, alpha-tropomyosin and myosin light chain MLC-3 was decreased in old skeletal muscle. Comparative 2-D immunoblotting of selected candidate proteins has confirmed the effect of aging on the skeletal muscle proteome. These findings demonstrate a severely perturbed protein expression pattern in aged skeletal muscle, which reflects the underlying molecular alterations causing a drastic decline of muscle strength in the senescent organism. In the long-term, the systematic deduction of abnormal protein expression

  12. 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

  13. Force profiles of protein pulling with or without cytoskeletal links studied by AFM

    SciTech Connect

    Afrin, Rehana; Ikai, Atsushi . E-mail: aikai@bio.titech.ac.jp

    2006-09-15

    To test the capability of the atomic force microscope for distinguishing membrane proteins with/without cytoskeletal associations, we studied the pull-out mechanics of lipid tethers from the red blood cell (RBC). When wheat germ agglutinin, a glycophorin A (GLA) specific lectin, was used to pull out tethers from RBC, characteristic force curves for tether elongation having a long plateau force were observed but without force peaks which are usually attributed to the forced unbinding of membrane components from the cytoskeleton. The result was in agreement with the reports that GLA is substantially free of cytoskeletal interactions. On the contrary, when the Band 3 specific lectin, concanavalin A, was used, the force peaks were indeed observed together with a plateau supporting its reported cytoskeletal association. Based on these observations, we postulate that the state of cytoskeletal association of particular membrane proteins can be identified from the force profiles of their pull-out mechanics.

  14. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle.

    PubMed

    Uezumi, Akiyoshi; Nakatani, Masashi; Ikemoto-Uezumi, Madoka; Yamamoto, Naoki; Morita, Mitsuhiro; Yamaguchi, Asami; Yamada, Harumoto; Kasai, Takehiro; Masuda, Satoru; Narita, Asako; Miyagoe-Suzuki, Yuko; Takeda, Shin'ichi; Fukada, So-Ichiro; Nishino, Ichizo; Tsuchida, Kunihiro

    2016-08-01

    Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases. PMID:27509136

  15. Design and synthesis of an activity-based protein profiling probe derived from cinnamic hydroxamic acid.

    PubMed

    Ai, Teng; Qiu, Li; Xie, Jiashu; Geraghty, Robert J; Chen, Liqiang

    2016-02-15

    In our continued effort to discover new anti-hepatitis C virus (HCV) agents, we validated the anti-replicon activity of compound 1, a potent and selective anti-HCV hydroxamic acid recently reported by us. Generally favorable physicochemical and in vitro absorption, distribution, metabolism, and excretion (ADME) properties exhibited by 1 made it an ideal parent compound from which activity-based protein profiling (ABPP) probe 3 was designed and synthesized. Evaluation of probe 3 revealed that it possessed necessary anti-HCV activity and selectivity. Therefore, we have successfully obtained compound 3 as a suitable ABPP probe to identify potential molecular targets of compound 1. Probe 3 and its improved analogs are expected to join a growing list of ABPP probes that have made important contributions to not only the studies of biochemical and cellular functions but also discovery of selective inhibitors of protein targets.

  16. Protein Profiling of Human Breast Tumor Cells Identifies Novel Biomarkers Associated with Molecular Subtypes*S⃞

    PubMed Central

    Gonçalves, Anthony; Charafe-Jauffret, Emmanuelle; Bertucci, François; Audebert, Stéphane; Toiron, Yves; Esterni, Benjamin; Monville, Florence; Tarpin, Carole; Jacquemier, Jocelyne; Houvenaeghel, Gilles; Chabannon, Christian; Extra, Jean-Marc; Viens, Patrice; Borg, Jean-Paul; Birnbaum, Daniel

    2008-01-01

    Molecular subtypes of breast cancer with relevant biological and clinical features have been defined recently, notably ERBB2-overexpressing, basal-like, and luminal-like subtypes. To investigate the ability of mass spectrometry-based proteomics technologies to analyze the molecular complexity of human breast cancer, we performed a SELDI-TOF MS-based protein profiling of human breast cell lines (BCLs). Triton-soluble proteins from 27 BCLs were incubated with ProteinChip arrays and subjected to SELDI analysis. Unsupervised global hierarchical clustering spontaneously discriminated two groups of BCLs corresponding to “luminal-like” cell lines and to “basal-like” cell lines, respectively. These groups of BCLs were also different in terms of estrogen receptor status as well as expression of epidermal growth factor receptor and other basal markers. Supervised analysis revealed various protein biomarkers with differential expression in basal-like versus luminal-like cell lines. We identified two of them as a carboxyl terminus-truncated form of ubiquitin and S100A9. In a small series of frozen human breast tumors, we confirmed that carboxyl terminus-truncated ubiquitin is observed in primary breast samples, and our results suggest its higher expression in luminal-like tumors. S100A9 up-regulation was found as part of the transcriptionally defined basal-like cluster in DNA microarrays analysis of human tumors. S100A9 association with basal subtypes as well as its poor prognosis value was demonstrated on a series of 547 tumor samples from early breast cancer deposited in a tissue microarray. Our study shows the potential of integrated genomics and proteomics profiling to improve molecular knowledge of complex tumor phenotypes and identify biomarkers with valuable diagnostic or prognostic values. PMID:18426791

  17. Gene expression profiles on predicting protein interaction network and exploring of new treatments for lung cancer.

    PubMed

    Yang, Zehui; Zheng, Rui; Gao, Yuan; Zhang, Qiang

    2014-12-01

    In the present study, we aimed to explore disease-associated genes and their functions in lung cancer. We downloaded the gene expression profile GSE4115 from Gene Expression Omnibus (GEO) database. Total 97 lung cancer and 90 adjacent non-tumor lung tissue (normal) samples were applied to identify the differentially expressed genes (DEGs) by paired t test and variance analysis in spectral angle mapper (SAM) package in R. Gene Ontology (GO) functional enrichment analysis of DEGs were performed with Database for Annotation Visualization and Integrated Discovery, followed by construction of protein-protein interaction (PPI) network from Human Protein Reference Database (HPRD). Finally, network modules were analyzed by the MCODE algorithm to detect protein complexes in the PPI network. Total 3,102 genes were identified as DEGs at FDR < 0.05, including 1,146 down-regulated and 1,956 up-regulated DEGs. GO functional enrichment analysis revealed that up-regulated DEGs mainly participated in cell cycle and intracellular related functions, and down-regulated DEGs might influence cell functions. There were 39,240 pairs of PPIs in human obtained from HPRD databases, 3,102 DEGs were mapped to this PPI network, in which 2,429 pairs of PPIs and 1,342 genes were identified. With MCODE algorithm, 48 modules were selected, including five corresponding modules and three modules with differences in gene expressing profiles. In addition, three DGEs, FXR2, ARFGAP1 and ELAVL1 were discovered as potential lung cancer related genes. The discovery of featured genes which were probably related to lung cancer, has a great significance on studying mechanism, distinguishing normal and cancer tissues, and exploring new treatments for lung cancer. PMID:25205123

  18. Do cultural conditions induce differential protein expression: Profiling of extracellular proteome of Aspergillus terreus CM20.

    PubMed

    M, Saritha; Singh, Surender; Tiwari, Rameshwar; Goel, Renu; Nain, Lata

    2016-11-01

    The present study reports the diversity in extracellular proteins expressed by the filamentous fungus, Aspergillus terreus CM20 with respect to differential hydrolytic enzyme production profiles in submerged fermentation (SmF) and solid-state fermentation (SSF) conditions, and analysis of the extracellular proteome. The SSF method was superior in terms of increase in enzyme activities resulting in 1.5-3 fold enhancement as compared to SmF, which was explained by the difference in growth pattern of the fungus under the two culture conditions. As revealed by zymography, multiple isoforms of endo-β-glucanase, β-glucosidase and xylanase were expressed in SSF, but not in SmF. Extracellular proteome profiling of A. terreus CM20 under SSF condition using liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) identified 63 proteins. Functional classification revealed the hydrolytic system to be composed of glycoside hydrolases (56%), proteases (16%), oxidases and dehydrogenases (6%), decarboxylases (3%), esterases (3%) and other proteins (16%). Twenty families of glycoside hydrolases (GH) (1, 3, 5, 7, 10, 11, 12, 15, 16, 28, 30, 32, 35, 43, 54, 62, 67, 72, 74 and 125), and one family each of auxiliary activities (AA7) and carbohydrate esterase (CE1) were detected, unveiling the vast diversity of synergistically acting biomass-cleaving enzymes expressed by the fungus. Saccharification of alkali-pretreated paddy straw with A. terreus CM20 proteins released high amounts of glucose (439.63±1.50mg/gds), xylose (121.04±1.25mg/gds) and arabinose (56.13±0.56mg/gds), thereby confirming the potential of the enzyme cocktail in bringing about considerable conversion of lignocellulosic polysaccharides to sugar monomers. PMID:27664725

  19. Insulin Receptor Substrate Adaptor Proteins Mediate Prognostic Gene Expression Profiles in Breast Cancer

    PubMed Central

    Becker, Marc A.; Ibrahim, Yasir H.; Oh, Annabell S.; Fagan, Dedra H.; Byron, Sara A.; Sarver, Aaron L.; Lee, Adrian V.; Shaw, Leslie M.; Fan, Cheng; Perou, Charles M.; Yee, Douglas

    2016-01-01

    Therapies targeting the type I insulin-like growth factor receptor (IGF-1R) have not been developed with predictive biomarkers to identify tumors with receptor activation. We have previously shown that the insulin receptor substrate (IRS) adaptor proteins are necessary for linking IGF1R to downstream signaling pathways and the malignant phenotype in breast cancer cells. The purpose of this study was to identify gene expression profiles downstream of IGF1R and its two adaptor proteins. IRS-null breast cancer cells (T47D-YA) were engineered to express IRS-1 or IRS-2 alone and their ability to mediate IGF ligand-induced proliferation, motility, and gene expression determined. Global gene expression signatures reflecting IRS adaptor specific and primary vs. secondary ligand response were derived (Early IRS-1, Late IRS-1, Early IRS-2 and Late IRS-2) and functional pathway analysis examined. IRS isoforms mediated distinct gene expression profiles, functional pathways, and breast cancer subtype association. For example, IRS-1/2-induced TGFb2 expression and blockade of TGFb2 abrogated IGF-induced cell migration. In addition, the prognostic value of IRS proteins was significant in the luminal B breast tumor subtype. Univariate and multivariate analyses confirmed that IRS adaptor signatures correlated with poor outcome as measured by recurrence-free and overall survival. Thus, IRS adaptor protein expression is required for IGF ligand responses in breast cancer cells. IRS-specific gene signatures represent accurate surrogates of IGF activity and could predict response to anti-IGF therapy in breast cancer. PMID:26991655

  20. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella

    PubMed Central

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I-Ping; Camargo, Eddie; Tzen, Jason T. C.; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-01-01

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates. PMID:26481560

  1. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella

    NASA Astrophysics Data System (ADS)

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I.-Ping; Camargo, Eddie; Tzen, Jason T. C.; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-10-01

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

  2. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella.

    PubMed

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I-Ping; Camargo, Eddie; Tzen, Jason T C; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-10-20

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

  3. Construction and analysis of a profile library characterizing groups of structurally known proteins.

    PubMed Central

    Ogiwara, A.; Uchiyama, I.; Takagi, T.; Kanehisa, M.

    1996-01-01

    A new sequence motif library StrProf was constructed characterizing the groups of related proteins in the PDB three-dimensional structure database. For a representative member of each protein family, which was identified by cross-referencing the PDB with the PIR superfamily classification, a group of related sequences was collected by the BLAST search against the nonredundant protein sequence database. For every group, the motifs were identified automatically according to the criteria of conservation and uniqueness of pentapeptide patterns and with a dual dynamic programming algorithm. In the StrProf library, motifs are represented by profile matrices rather than consensus patterns to allow more flexible search capabilities. Another dynamic programming algorithm was then developed to search this motif library. When the computationally derived StrProf was compared with PROSITE, which is a manually derived motif library in the best consensus pattern representation, the numbers of identified patterns were comparable. StrProf missed about one third of the PROSITE motifs, but there were also new motifs lacking in PROSITE. The new library was incorporated in SMART (Sequence Motif Analysis and Retrieval Tool), a computer tool designed to help search and annotate biologically important sites in an unknown protein sequence. The client program is available free of charge through the Internet. PMID:8897599

  4. Cancer cell profiling by barcoding allows multiplexed protein analysis in fine needle aspirates

    PubMed Central

    Ullal, Adeeti V.; Peterson, Vanessa; Agasti, Sarit S.; Tuang, Suan; Juric, Dejan; Castro, Cesar M.; Weissleder, Ralph

    2014-01-01

    Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. Here, we introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine needle aspirates (FNA), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing where barcodes can be photo-cleaved and digitally detected without any amplification steps. Following extensive benchmarking in cell lines, this method showed high reproducibility and achieved single cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials. PMID:24431113

  5. Characterization and differentiation of diverse transgenic and nontransgenic soybean varieties from CE protein profiles.

    PubMed

    García-Ruiz, Carmen; García, Maria Concepción; Cifuentes, Alejandro; Marina, Maria Luisa

    2007-07-01

    Nowadays, soybeans are commercialized in a wide variety of colors and tones. Moreover, some pigmented seeds are being commercialized as soybeans while, on other occasions, these seeds are labeled as mung beans, azuki beans or soybean frijoles generating confusion on their identity. In this work, CE has been applied for the first time for the characterization and differentiation of different pigmented beans commercialized as soybeans. Other seeds commercialized as azuki, mung green soybeans or soybean frijoles were also analyzed. Borate buffer (at pH 8.5) containing 20% v/v ACN was used as the separation media and solution containing ACN/water (75:25 v/v) with 0.3% v/v acetic acid was used to solubilize the proteins from the samples. A 50 cm bare fused-silica capillary was employed for obtaining adequate separations in about 12 min. The CE protein pattern observed for yellow soybeans was different from that corresponding to green and red soybeans. The seeds commercialized as black soybean presented electropherograms identical or similar to those yielded by the yellow seeds with the exception of the sample labeled as black soybeans frijoles that presented a totally different pattern. In addition, CE protein profiles obtained for azuki and mung green soybeans were very similar to those corresponding to red soybeans and green soybeans, respectively. Finally, the CE method was also applied to differentiate transgenic and nontransgenic soybean varieties. Discriminant analysis, using several protein peak areas as variable, was used to successfully classify these samples.

  6. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella.

    PubMed

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I-Ping; Camargo, Eddie; Tzen, Jason T C; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2015-01-01

    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates. PMID:26481560

  7. Biochemical and protein profile of alpaca (Vicugna pacos) uterine horn fluid during early pregnancy.

    PubMed

    Argañaraz, M E; Apichela, S A; Zampini, R; Vencato, J; Stelletta, C

    2015-02-01

    South American camelids show high embryo loss rate, during the first 60 days of pregnancy. One of the factors which may be related to this situation is that over 98% of the embryos implant in the left uterine horn (LUH) even though both ovaries contribute similarly to ovulation. There is scarce information about the uterine environment of female camelids at any physiological state that could explain the capability of the LUH to attract the embryo and maintain pregnancy. We describe, for the first time, the biochemical and protein profile of uterine fluid (UF), addressing the right and LUH environment in non-pregnant and pregnant alpacas. Different substrates, electrolytes and metabolites were assayed in both uterine horn fluids. Small changes were observed in glucose and total protein levels, which were more noticeable during pregnancy. In addition, 10 specific proteins were found in the left horn fluid in 5-week-pregnant alpacas, and two protein bands were identified in non-pregnant alpaca right horn fluid. These results would provide basic information for identification of possible markers for pregnancy diagnosis, reproductive diseases and hormone-treated animals evaluation and hence contributing to improve the pregnancy rate.

  8. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis

    PubMed Central

    Negm, Ola H.; Hamed, Mohamed R.; Dilnot, Elizabeth M.; Shone, Clifford C.; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E.; Edwards, Laura J.; Tighe, Patrick J.; Wilcox, Mark H.

    2015-01-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated <10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. PMID:26178385

  9. 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

  10. Genome-wide promoter binding profiling of protein phosphatase-1 and its major nuclear targeting subunits.

    PubMed

    Verheyen, Toon; Görnemann, Janina; Verbinnen, Iris; Boens, Shannah; Beullens, Monique; Van Eynde, Aleyde; Bollen, Mathieu

    2015-07-13

    Protein phosphatase-1 (PP1) is a key regulator of transcription and is targeted to promoter regions via associated proteins. However, the chromatin binding sites of PP1 have never been studied in a systematic and genome-wide manner. Methylation-based DamID profiling in HeLa cells has enabled us to map hundreds of promoter binding sites of PP1 and three of its major nuclear interactors, i.e. RepoMan, NIPP1 and PNUTS. Our data reveal that the α, β and γ isoforms of PP1 largely bind to distinct subsets of promoters and can also be differentiated by their promoter binding pattern. PP1β emerged as the major promoter-associated isoform and shows an overlapping binding profile with PNUTS at dozens of active promoters. Surprisingly, most promoter binding sites of PP1 are not shared with RepoMan, NIPP1 or PNUTS, hinting at the existence of additional, largely unidentified chromatin-targeting subunits. We also found that PP1 is not required for the global chromatin targeting of RepoMan, NIPP1 and PNUTS, but alters the promoter binding specificity of NIPP1. Our data disclose an unexpected specificity and complexity in the promoter binding of PP1 isoforms and their chromatin-targeting subunits. PMID:25990731

  11. Protein profiling and histone deacetylation activities in somaclonal variants of oil palm (Elaeis guineensis Jacq.).

    PubMed

    Yaacob, Jamilah Syafawati; Loh, Hwei-San; Mat Taha, Rosna

    2013-01-01

    Mantled fruits as a result of somaclonal variation are often observed from the oil palm plantlets regenerated via tissue culture. The mantling of fruits with finger-like and thick outer coating phenotypes significantly reduces the seed size and oil content, posing a threat to oil palm planters, and may jeopardize the economic growth of countries that depend particularly on oil palm plantation. The molecular aspects of the occurrence of somaclonal variations are yet to be known, possibly due to gene repression such as DNA methylation, histone methylation and histone deacetylation. Histone deacetylases (HDACs), involved in eukaryotic gene regulation by catalyzing the acetyl groups are removal from lysine residues on histone, hence transcriptionally repress gene expression. This paper described the total protein polymorphism profiles of somaclonal variants of oil palm and the effects of histone deacetylation on this phenomenon. Parallel to the different phenotypes, the protein polymorphism profiles of the mantled samples (leaves, fruits, and florets) and the phenotypically normal samples were proven to be different. Higher HDAC activity was found in mantled leaf samples than in the phenotypically normal leaf samples, leading to a preliminary conclusion that histone deacetylation suppressed gene expression and contributed to the development of somaclonal variants. PMID:23844406

  12. Abscisic acid and late embryogenesis abundant protein profile changes in winter wheat under progressive drought stress.

    PubMed

    Vaseva, I I; Grigorova, B S; Simova-Stoilova, L P; Demirevska, K N; Feller, U

    2010-09-01

    Three varieties (cv. Pobeda, Katya and Sadovo) of winter wheat (Triticum aestivum), differing in their agronomic characteristics, were analysed during progressive soil water stress and recovery at early vegetation stages. Changes in abscisic acid content, SDS-PAGE and immunoblot profiles of proteins that remained soluble upon heating were monitored. Initially higher ABA content in control Pobeda and Katya corresponded to earlier expression of the studied late embryogenesis abundant (LEA) proteins. A combination of higher ABA content, early immunodetection of dehydrins, and a significant increase of WZY2 transcript levels were observed in drought-stressed leaves of the tolerant variety Katya. One-step RT-PCR analyses of some acidic dehydrin genes (WCOR410b, TADHN) documented their relatively constant high expression levels in leaves under drought stress during early vegetative development. Neutral WZY2 dehydrin, TaLEA2 and TaLEA3 transcripts accumulated gradually with increasing water deficit. Delayed expression of TaLEA2 and TaLEA3 genes was found in the least drought-tolerant wheat, Sadovo. The expression profile of WZY2 revealed two distinct and separate bands, suggesting alternative splicing, which altered as water stress increased.

  13. Adjuvant-induced Human Monocyte Secretome Profiles Reveal Adjuvant- and Age-specific Protein Signatures.

    PubMed

    Oh, Djin-Ye; Dowling, David J; Ahmed, Saima; Choi, Hyungwon; Brightman, Spencer; Bergelson, Ilana; Berger, Sebastian T; Sauld, John F; Pettengill, Matthew; Kho, Alvin T; Pollack, Henry J; Steen, Hanno; Levy, Ofer

    2016-06-01

    Adjuvants boost vaccine responses, enhancing protective immunity against infections that are most common among the very young. Many adjuvants activate innate immunity, some via Toll-Like Receptors (TLRs), whose activities varies with age. Accordingly, characterization of age-specific adjuvant-induced immune responses may inform rational adjuvant design targeting vulnerable populations. In this study, we employed proteomics to characterize the adjuvant-induced changes of secretomes from human newborn and adult monocytes in response to Alum, the most commonly used adjuvant in licensed vaccines; Monophosphoryl Lipid A (MPLA), a TLR4-activating adjuvant component of a licensed Human Papilloma Virus vaccine; and R848 an imidazoquinoline TLR7/8 agonist that is a candidate adjuvant for early life vaccines. Monocytes were incubated in vitro for 24 h with vehicle, Alum, MPLA, or R848 and supernatants collected for proteomic analysis employing liquid chromatography-mass spectrometry (LC-MS) (data available via ProteomeXchange, ID PXD003534). 1894 non-redundant proteins were identified, of which ∼30 - 40% were common to all treatment conditions and ∼5% were treatment-specific. Adjuvant-stimulated secretome profiles, as identified by cluster analyses of over-represented proteins, varied with age and adjuvant type. Adjuvants, especially Alum, activated multiple innate immune pathways as assessed by functional enrichment analyses. Release of lactoferrin, pentraxin 3, and matrix metalloproteinase-9 was confirmed in newborn and adult whole blood and blood monocytes stimulated with adjuvants alone or adjuvanted licensed vaccines with distinct clinical reactogenicity profiles. MPLA-induced adult monocyte secretome profiles correlated in silico with transcriptome profiles induced in adults immunized with the MPLA-adjuvanted RTS,S malaria vaccine (Mosquirix™). Overall, adjuvants such as Alum, MPLA and R848 give rise to distinct and age-specific monocyte secretome profiles

  14. Adjuvant-induced Human Monocyte Secretome Profiles Reveal Adjuvant- and Age-specific Protein Signatures*

    PubMed Central

    Oh, Djin-Ye; Dowling, David J.; Ahmed, Saima; Choi, Hyungwon; Brightman, Spencer; Bergelson, Ilana; Berger, Sebastian T.; Sauld, John F.; Pettengill, Matthew; Kho, Alvin T.; Pollack, Henry J.; Steen, Hanno; Levy, Ofer

    2016-01-01

    Adjuvants boost vaccine responses, enhancing protective immunity against infections that are most common among the very young. Many adjuvants activate innate immunity, some via Toll-Like Receptors (TLRs), whose activities varies with age. Accordingly, characterization of age-specific adjuvant-induced immune responses may inform rational adjuvant design targeting vulnerable populations. In this study, we employed proteomics to characterize the adjuvant-induced changes of secretomes from human newborn and adult monocytes in response to Alum, the most commonly used adjuvant in licensed vaccines; Monophosphoryl Lipid A (MPLA), a TLR4-activating adjuvant component of a licensed Human Papilloma Virus vaccine; and R848 an imidazoquinoline TLR7/8 agonist that is a candidate adjuvant for early life vaccines. Monocytes were incubated in vitro for 24 h with vehicle, Alum, MPLA, or R848 and supernatants collected for proteomic analysis employing liquid chromatography-mass spectrometry (LC-MS) (data available via ProteomeXchange, ID PXD003534). 1894 non-redundant proteins were identified, of which ∼30 - 40% were common to all treatment conditions and ∼5% were treatment-specific. Adjuvant-stimulated secretome profiles, as identified by cluster analyses of over-represented proteins, varied with age and adjuvant type. Adjuvants, especially Alum, activated multiple innate immune pathways as assessed by functional enrichment analyses. Release of lactoferrin, pentraxin 3, and matrix metalloproteinase-9 was confirmed in newborn and adult whole blood and blood monocytes stimulated with adjuvants alone or adjuvanted licensed vaccines with distinct clinical reactogenicity profiles. MPLA-induced adult monocyte secretome profiles correlated in silico with transcriptome profiles induced in adults immunized with the MPLA-adjuvanted RTS,S malaria vaccine (Mosquirix™). Overall, adjuvants such as Alum, MPLA and R848 give rise to distinct and age-specific monocyte secretome profiles

  15. Methionine sulfoxide profiling of milk proteins to assess the influence of lipids on protein oxidation in milk.

    PubMed

    Wüst, Johannes; Pischetsrieder, Monika

    2016-06-15

    Thermal treatment of milk and milk products leads to protein oxidation, mainly the formation of methionine sulfoxide. Reactive oxygen species, responsible for the oxidation, can be generated by Maillard reaction, autoxidation of sugars, or lipid peroxidation. The present study investigated the influence of milk fat on methionine oxidation in milk. For this purpose, quantitative methionine sulfoxide profiling of all ten methionine residues of β-lactoglobulin, α-lactalbumin, and αs1-casein was carried out by ultrahigh-performance liquid chromatography-electrospray ionization tandem mass spectrometry with scheduled multiple reaction monitoring (UHPLC-ESI-MS/MS-sMRM). Analysis of defatted and regular raw milk samples after heating for up to 8 min at 120 °C and analysis of ultrahigh-temperature milk samples with 0.1%, 1.5%, and 3.5% fat revealed that methionine oxidation of the five residues of the whey proteins and of residues M 123, M 135, and M 196 of αs1-casein was not affected or even suppressed in the presence of milk fat. Only the oxidation of residues M 54 and M 60 of αs1-casein was promoted by lipids. In evaporated milk samples, formation of methionine sulfoxide was hardly influenced by the fat content of the samples. Thus, it can be concluded that lipid oxidation products are not the major cause of methionine oxidation in milk.

  16. 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

  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.

  18. Profiling Lipid–protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Proteome Microarrays*

    PubMed Central

    Lu, Kuan-Yi; Tao, Sheng-Ce; Yang, Tzu-Ching; Ho, Yu-Hsuan; Lee, Chia-Hsien; Lin, Chen-Ching; Juan, Hsueh-Fen; Huang, Hsuan-Cheng; Yang, Chin-Yu; Chen, Ming-Shuo; Lin, Yu-Yi; Lu, Jin-Ying; Zhu, Heng; Chen, Chien-Sheng

    2012-01-01

    Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 μm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid–protein

  19. New procyanidin B3-human salivary protein complexes by mass spectrometry. Effect of salivary protein profile, tannin concentration, and time stability.

    PubMed

    Perez-Gregorio, Maria Rosa; Mateus, Nuno; De Freitas, Victor

    2014-10-15

    Several factors could influence the tannin-protein interaction such as the human salivary protein profile, the tannin tested, and the tannin/protein ratio. The goal of this study aims to study the effect of different salivas (A, B, and C) and different tannin concentrations (0.5 and 1 mg/mL) on the interaction process as well as the complex's stability over time. This study is focused on the identification of new procyanidin B3-human salivary protein complexes. Thus, 48 major B3-human salivary protein aggregates were identified regardless of the saliva and tannin concentration tested. A higher number of aggregates was found at lower tannin concentration. Moreover, the number of protein moieties involved in the aggregation process was higher when the tannin concentration was also higher. The selectivity of the different groups of proteins to bind tannin was also confirmed. It was also verified that the B3-human salivary protein complexes formed evolved over time.

  20. Post-translational modification profiling - A novel tool for mapping the protein modification landscape in cancer.

    PubMed

    Eisenberg-Lerner, Avital; Ciechanover, Aaron; Merbl, Yifat

    2016-08-01

    The ubiquitin system plays an important role in essentially every cellular process, regulating numerous pathways ranging from development, transcription, DNA damage response, cell cycle, and signal transduction. Its best studied role involves removal of faulty proteins or those that are not necessary anymore. Aberrations in the ubiquitin system have been implicated in various pathologies including cancer, where specific mutations in E3 ligases such as Mdm2, pVHL, and BRCA1 have been linked to disease progression, prognosis, and resistance to drugs. Yet, there are hundreds of E3 ligases in the human genome and our knowledge of their target proteins and their dynamic regulation in the cellular environment is largely limited. In addition, fundamental questions related to recognition and specificity in ubiquitin conjugation remain unanswered. It is thus of major importance to characterize the ubiquitin landscape under various cellular conditions, and study how the regulatory network is altered in health and disease. To do so, analytical tools that allow identification of ubiquitin substrates, the conjugation and removal of ubiquitin, and the nature of specific ubiquitin linkages that are formed are needed. In this mini-review, we discuss common proteomic methodologies applied to studying the ubiquitome, and specifically focus on our recently developed post-translational modification (PTM) profiling approach. PTM profiling is a functional assay, amenable to biochemical manipulation, which allows the detection of protein modifications in a high-throughput manner. We discuss in detail the advantages and limitations of this system, focusing primarily on examples for analyzing the ubiquitin system in cancer. Uncovering the intricate signaling dynamics governed by and regulating ubiquitin modifications should clearly evolve into a new paradigm in understanding the molecular basis of malignant transformation and the development of novel therapeutic modalities. PMID:27229346

  1. 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

  2. Effect of freezing rate and dendritic ice formation on concentration profiles of proteins frozen in cylindrical vessels.

    PubMed

    Rodrigues, Miguel A; Miller, Maria A; Glass, Matt A; Singh, Satish K; Johnston, Keith P

    2011-04-01

    The process of freezing protein solutions can perturb the conformation of the protein and potentially lead to aggregate formation during long-term storage in the frozen state. Radial macroscopic freeze concentration and temperature profiles for bovine serum albumin (BSA) solutions in small cylindrical stainless steel vessels were determined for various freezing rates. The measured concentrations of both BSA and immunoglobulin G2, as well as trehalose in sampled ice sections, increased by up to twofold to threefold toward the bottom and radial center for slow freezing rates produced in stagnant air freezers. The concentration and temperature profiles result in density gradients that transport solutes by convective flow. For faster external cooling by either forced convection of air or a liquid coolant, the increased freezing rate raised the ice front velocity resulting in enhanced dendritic ice growth. The ice trapped the solutes more effectively before they were removed from the ice front by diffusion and convection, resulting in more uniform solute concentration profiles. The dynamic temperature profiles from multiple radial thermocouples were consistent with the independently measured freeze concentration profiles. The ability to control the protein concentration profile in the frozen state offers the potential to improve stability of protein in long-term frozen storage.

  3. Microwave irradiation induced changes in protein molecular structures of barley grains: relationship to changes in protein chemical profile, protein subfractions, and digestion in dairy cows.

    PubMed

    Yan, Xiaogang; Khan, Nazir A; Zhang, Fangyu; Yang, Ling; Yu, Peiqiang

    2014-07-16

    The objectives of this study were to evaluate microwave irradiation (MIR) induced changes in crude protein (CP) subfraction profiles, ruminal CP degradation characteristics and intestinal digestibility of rumen undegraded protein (RUP), and protein molecular structures in barley (Hordeum vulgare) grains. Samples from hulled (n = 1) and hulless cultivars (n = 2) of barley, harvested from four replicate plots in two consecutive years, were evaluated. The samples were either kept as raw or irradiated in a microwave for 3 min (MIR3) or 5 min (MIR5). Compared to raw grains, MIR5 decreased the contents of rapidly degradable CP subfraction (from 45.22 to 6.36% CP) and the ruminal degradation rate (from 8.16 to 3.53%/h) of potentially degradable subfraction. As a consequence, the effective ruminal degradability of CP decreased (from 55.70 to 34.08% CP) and RUP supply (from 43.31 to 65.92% CP) to the postruminal tract increased. The MIR decreased the spectral intensities of amide 1, amide II, α-helix, and β-sheet and increased their ratios. The changes in protein spectral intensities were strongly correlated with the changes in CP subfractions and digestive kinetics. These results show that MIR for a short period (5 min) with a lower energy input can improve the nutritive value and utilization of CP in barely grains.

  4. An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples

    PubMed Central

    Nie, Shuai; Henley, W. Hampton; Miller, Scott E.; Zhang, Huaibin; Mayer, Kathryn M.; Dennis, Patty J.; Oblath, Emily A.; Alarie, Jean Pierre; Wu, Yue; Oppenheim, Frank G.; Little, Frédéric F.; Uluer, Ahmet Z.; Wang, Peidong; Ramsey, J. Michael

    2014-01-01

    During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 μL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5-h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the device’s potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines non-invasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics. PMID:24448498

  5. In silico comparative analysis and expression profile of antioxidant proteins in plants.

    PubMed

    Sheoran, S; Pandey, B; Sharma, P; Narwal, S; Singh, R; Sharma, I; Chatrath, R

    2013-02-27

    The antioxidant system in plants is a very important defensive mechanism to overcome stress conditions. We examined the expression profile of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) using a bioinformatics approach. We explored secondary structure prediction and made detailed studies of signature pattern of antioxidant proteins in four plant species (Triticum aestivum, Arabidopsis thaliana, Oryza sativa, and Brassica juncea). Fingerprinting analysis was done with ScanProsite, which includes a large collection of biologically meaningful signatures. Multiple sequence alignment of antioxidant proteins of the different plant species revealed a conserved secondary structure region, indicating homology at the sequence and structural levels. The secondary structure prediction showed that these proteins have maximum tendency for α helical structure. The sequence level similarities were also analyzed with a phylogenetic tree using neighbor-joining method. In the antioxidant enzymes SOD, CAT and APX, three major families of signature were predominant and common; these were PKC_PHOSPHO_SITE, CK2_PHOSPHO_SITE and N-myristoylation site, which are functionally related to various plant signaling pathways. This study provides new strategies for screening of biomodulators involved in plant stress metabolism that will be useful for designing degenerate primers or probes specific for antioxidant. These enzymes could be the first line of defence in the cellular antioxidant defence pathway, activated due to exposure to abiotic stresses.

  6. MEASUREMENT OF DRUG-PROTEIN DISSOCIATION RATES BY HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PEAK PROFILING

    PubMed Central

    Schiel, John E.; Ohnmacht, Corey M.; Hage, David S.

    2012-01-01

    The rate at which a drug or other small solute interacts with a protein is important in understanding the biological and pharmacokinetic behavior of these agents. One approach that has been developed for examining these rates involves the use of high-performance affinity chromatography (HPAC) and estimates of band-broadening through peak profiling. Previous work with this method has been based on a comparison of the statistical moments for a retained analyte versus non-retained species at a single, high flow rate to obtain information on stationary phase mass transfer. In this study an alternative approach was created that allows a broad range of flow rates to be used for examining solute-protein dissociation rates. Chromatographic theory was employed to derive equations that could be used with this approach on a single column, as well as with multiple columns to evaluate and correct for the impact of stagnant mobile phase mass transfer. The interaction of L-tryptophan with human serum albumin was used as a model system to test this method. A dissociation rate constant of 2.7 (± 0.2) s−1 was obtained by this approach at pH 7.4 and 37°C, which was in good agreement with previous values determined by other methods. The techniques described in this report can be applied to other biomolecular systems and should be valuable for the determination of drug-protein dissociation rates. PMID:19422253

  7. In silico comparative analysis and expression profile of antioxidant proteins in plants.

    PubMed

    Sheoran, S; Pandey, B; Sharma, P; Narwal, S; Singh, R; Sharma, I; Chatrath, R

    2013-01-01

    The antioxidant system in plants is a very important defensive mechanism to overcome stress conditions. We examined the expression profile of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) using a bioinformatics approach. We explored secondary structure prediction and made detailed studies of signature pattern of antioxidant proteins in four plant species (Triticum aestivum, Arabidopsis thaliana, Oryza sativa, and Brassica juncea). Fingerprinting analysis was done with ScanProsite, which includes a large collection of biologically meaningful signatures. Multiple sequence alignment of antioxidant proteins of the different plant species revealed a conserved secondary structure region, indicating homology at the sequence and structural levels. The secondary structure prediction showed that these proteins have maximum tendency for α helical structure. The sequence level similarities were also analyzed with a phylogenetic tree using neighbor-joining method. In the antioxidant enzymes SOD, CAT and APX, three major families of signature were predominant and common; these were PKC_PHOSPHO_SITE, CK2_PHOSPHO_SITE and N-myristoylation site, which are functionally related to various plant signaling pathways. This study provides new strategies for screening of biomodulators involved in plant stress metabolism that will be useful for designing degenerate primers or probes specific for antioxidant. These enzymes could be the first line of defence in the cellular antioxidant defence pathway, activated due to exposure to abiotic stresses. PMID:23512671

  8. An integrated workflow for characterizing intact phosphoproteins from complex mixtures

    PubMed Central

    Wu, Si; Yang, Feng; Zhao, Rui; Tolić, Nikola; Robinson, Errol W.; Camp, David; Smith, Richard D.; Paša-Tolić, Ljiljana

    2014-01-01

    The phosphorylation of any site on a given protein can affect its activity, degradation rate, ability to dock with other proteins or bind divalent cations, and/or its localization. These effects can operate within the same protein; in fact, multisite phosphorylation is a key mechanism for achieving signal integration in cells. Hence, knowing the overall phosphorylation signature of a protein is essential for understanding the "state" of a cell. However, current technologies to monitor the phosphorylation status of proteins are inefficient at determining the relative stoichiometries of phosphorylation at multiple sites. Here we report a new capability for comprehensive liquid chromatography mass spectrometry (LC/MS) analysis of intact phosphoproteins. The technology platform built upon integrated bottom-up and top-down approach that is facilitated by intact protein reversed-phase (RP)LC concurrently coupled with Fourier transform ion cyclotron resonance (FTICR) MS and fraction collection. As the use of conventional RPLC systems for phosphopeptide identification has proven challenging due to the formation of metal ion complexes at various metal surfaces during LC/MS and ESI-MS analysis, we have developed a “metal-free” RPLC-ESI-MS platform for phosphoprotein characterization. This platform demonstrated a significant sensitivity enhancement for phosphorylated casein proteins enriched from a standard protein mixture and revealed the presence of over 20 casein isoforms arising from genetic variants with varying numbers of phosphorylation sites. The integrated workflow was also applied to an enriched yeast phosphoproteome to evaluate the feasibility of this strategy for characterizing complex biological systems, and revealed ~16% of the detected yeast proteins to have multiple phosphorylation isoforms. Intact protein LC/MS platform for characterization of combinatorial posttranslational modifications (PTMs), with special emphasis on multisite phosphorylation, holds

  9. Presence of membranous vesicles in cat seminal plasma: ultrastructural characteristics, protein profile and enzymatic activity.

    PubMed

    Polisca, A; Troisi, A; Minelli, A; Bellezza, I; Fontbonne, A; Zelli, R

    2015-02-01

    This study sought to verify the presence of membranous vesicles in cat seminal plasma by means of transmission electron microscopy and to identify protein profile and some of the enzymatic activities associated with these particles. The transmission electron microscopy observations showed the existence of different sized vesicular membranous structures of more or less spherical shape. These vesicles were surrounded by single-, double- or multiple-layered laminar membranes. The vesicle diameters ranged from 16.3 to 387.4 nm, with a mean of 116.5 ± 70.7 nm. Enzyme activity determinations showed the presence of dipeptilpeptidase IV, aminopeptidase, alkaline and acid phosphatase. To our knowledge, this is the first report that identifies and characterizes the membranous vesicles in cat seminal plasma. However, further studies are necessary to identify the exact site of production of these membranous vesicles in the cat male genital tract and to determine their specific roles in the reproductive events of this species.

  10. Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

    SciTech Connect

    Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; Nandhikonda, Premchendar; Smith, Richard D.; Wright, Aaron T.

    2015-09-25

    Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosic bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.

  11. Ribosome profiling reveals pervasive translation outside of annotated protein-coding genes.

    PubMed

    Ingolia, Nicholas T; Brar, Gloria A; Stern-Ginossar, Noam; Harris, Michael S; Talhouarne, Gaëlle J S; Jackson, Sarah E; Wills, Mark R; Weissman, Jonathan S

    2014-09-11

    Ribosome profiling suggests that ribosomes occupy many regions of the transcriptome thought to be noncoding, including 5' UTRs and long noncoding RNAs (lncRNAs). Apparent ribosome footprints outside of protein-coding regions raise the possibility of artifacts unrelated to translation, particularly when they occupy multiple, overlapping open reading frames (ORFs). Here, we show hallmarks of translation in these footprints: copurification with the large ribosomal subunit, response to drugs targeting elongation, trinucleotide periodicity, and initiation at early AUGs. We develop a metric for distinguishing between 80S footprints and nonribosomal sources using footprint size distributions, which validates the vast majority of footprints outside of coding regions. We present evidence for polypeptide production beyond annotated genes, including the induction of immune responses following human cytomegalovirus (HCMV) infection. Translation is pervasive on cytosolic transcripts outside of conserved reading frames, and direct detection of this expanded universe of translated products enables efforts at understanding how cells manage and exploit its consequences. PMID:25159147

  12. Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

    NASA Astrophysics Data System (ADS)

    Bhargava, Maneesh

    Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

  13. Unbiased Quantitative Models of Protein Translation Derived from Ribosome Profiling Data

    PubMed Central

    Gritsenko, Alexey A.; Hulsman, Marc; Reinders, Marcel J. T.; de Ridder, Dick

    2015-01-01

    Translation of RNA to protein is a core process for any living organism. While for some steps of this process the effect on protein production is understood, a holistic understanding of translation still remains elusive. In silico modelling is a promising approach for elucidating the process of protein synthesis. Although a number of computational models of the process have been proposed, their application is limited by the assumptions they make. Ribosome profiling (RP), a relatively new sequencing-based technique capable of recording snapshots of the locations of actively translating ribosomes, is a promising source of information for deriving unbiased data-driven translation models. However, quantitative analysis of RP data is challenging due to high measurement variance and the inability to discriminate between the number of ribosomes measured on a gene and their speed of translation. We propose a solution in the form of a novel multi-scale interpretation of RP data that allows for deriving models with translation dynamics extracted from the snapshots. We demonstrate the usefulness of this approach by simultaneously determining for the first time per-codon translation elongation and per-gene translation initiation rates of Saccharomyces cerevisiae from RP data for two versions of the Totally Asymmetric Exclusion Process (TASEP) model of translation. We do this in an unbiased fashion, by fitting the models using only RP data with a novel optimization scheme based on Monte Carlo simulation to keep the problem tractable. The fitted models match the data significantly better than existing models and their predictions show better agreement with several independent protein abundance datasets than existing models. Results additionally indicate that the tRNA pool adaptation hypothesis is incomplete, with evidence suggesting that tRNA post-transcriptional modifications and codon context may play a role in determining codon elongation rates. PMID:26275099

  14. DNA Methylation Profile and Expression of Surfactant Protein A2 gene in Lung Cancer

    PubMed Central

    Grageda, Melissa; Silveyra, Patricia; Thomas, Neal J.; DiAngelo, Susan L.; Floros, Joanna

    2014-01-01

    Knowledge of the methylation profile of genes allow for the identification of biomarkers that may guide diagnosis and effective treatment of disease. Human surfactant protein A (SP-A) plays an important role in lung homeostasis and immunity, and is encoded by two genes (SFTPA1 and SFTPA2). The goal of this study was to identify differentially methylated CpG sites in the promoter region of the SFTPA2 gene in lung cancer tissue, and to determine the correlation between the promoter’s methylation profile and gene expression. For this, we collected 28 pairs of cancerous human lung tissue and adjacent non-cancerous (NC) lung tissue: 17 adenocarcinoma (AC), 9 squamous cell carcinoma (SCC), and 2 AC with SCC features, and we evaluated DNA methylation of the SFTPA2 promoter region by bisulfite conversion. Our results identified a higher methylation ratio in one CpG site of the SFTPA2 gene in cancerous tissue vs. NC tissue (0.36 vs. 0.11, p=0.001). When assessing AC samples, we also found cancerous tissues associated with a higher methylation ratio (0.43 vs. 0.10, p=0.02). In the SCC group, although cancerous tissue showed a higher methylation ratio (0.22 vs. 0.11), this difference was not statistically significant (p=0.35). Expression of SFTPA2 mRNA and total SP-A protein was significantly lower in cancer tissue when compared to adjacent NC tissue (p<0.001), and correlated with the hypermethylated status of a SFTPA2 CpG site in AC samples. The findings of this pilot study may hold promise for future use of SFTPA2 as a biomarker for the diagnosis of lung cancer. PMID:25514367

  15. Deconvolution of complex differential scanning calorimetry profiles for protein transitions under kinetic control.

    PubMed

    Toledo-Núñez, Citlali; Vera-Robles, L Iraís; Arroyo-Maya, Izlia J; Hernández-Arana, Andrés

    2016-09-15

    A frequent outcome in differential scanning calorimetry (DSC) experiments carried out with large proteins is the irreversibility of the observed endothermic effects. In these cases, DSC profiles are analyzed according to methods developed for temperature-induced denaturation transitions occurring under kinetic control. In the one-step irreversible model (native → denatured) the characteristics of the observed single-peaked endotherm depend on the denaturation enthalpy and the temperature dependence of the reaction rate constant, k. Several procedures have been devised to obtain the parameters that determine the variation of k with temperature. Here, we have elaborated on one of these procedures in order to analyze more complex DSC profiles. Synthetic data for a heat capacity curve were generated according to a model with two sequential reactions; the temperature dependence of each of the two rate constants involved was determined, according to the Eyring's equation, by two fixed parameters. It was then shown that our deconvolution procedure, by making use of heat capacity data alone, permits to extract the parameter values that were initially used. Finally, experimental DSC traces showing two and three maxima were analyzed and reproduced with relative success according to two- and four-step sequential models. PMID:27402175

  16. Stochastic theory of protein synthesis and polysome: ribosome profile on a single mRNA transcript.

    PubMed

    Sharma, Ajeet K; Chowdhury, Debashish

    2011-11-21

    The process of polymerizing a protein by a ribosome, using a messenger RNA (mRNA) as the corresponding template, is called translation. Ribosome may be regarded as a molecular motor for which the mRNA template serves also as the track. Often several ribosomes may translate the same (mRNA) simultaneously. The ribosomes bound simultaneously to a single mRNA transcript are the members of a polyribosome (or, simply, polysome). Experimentally measured polysome profile gives the distribution of polysome sizes. Recently a breakthrough in determining the instantaneous positions of the ribosomes on a given mRNA track has been achieved and the technique is called ribosome profiling (Ingolia et al., 2009; Guo et al., 2010). Motivated by the success of these techniques, we have studied the spatio-temporal organization of ribosomes by extending a theoretical model that we have reported elsewhere (Sharma and Chowdhury, 2011). This extended version of our model incorporates not only (i) mechano-chemical cycle of individual ribomes, and (ii) their steric interactions, but also (iii) the effects of (a) kinetic proofreading, (b) translational infidelity, (c) ribosome recycling, and (d) sequence inhomogeneities. The theoretical framework developed here will serve in guiding further experiments and in analyzing the data to gain deep insight into various kinetic processes involved in translation.

  17. Deconvolution of complex differential scanning calorimetry profiles for protein transitions under kinetic control.

    PubMed

    Toledo-Núñez, Citlali; Vera-Robles, L Iraís; Arroyo-Maya, Izlia J; Hernández-Arana, Andrés

    2016-09-15

    A frequent outcome in differential scanning calorimetry (DSC) experiments carried out with large proteins is the irreversibility of the observed endothermic effects. In these cases, DSC profiles are analyzed according to methods developed for temperature-induced denaturation transitions occurring under kinetic control. In the one-step irreversible model (native → denatured) the characteristics of the observed single-peaked endotherm depend on the denaturation enthalpy and the temperature dependence of the reaction rate constant, k. Several procedures have been devised to obtain the parameters that determine the variation of k with temperature. Here, we have elaborated on one of these procedures in order to analyze more complex DSC profiles. Synthetic data for a heat capacity curve were generated according to a model with two sequential reactions; the temperature dependence of each of the two rate constants involved was determined, according to the Eyring's equation, by two fixed parameters. It was then shown that our deconvolution procedure, by making use of heat capacity data alone, permits to extract the parameter values that were initially used. Finally, experimental DSC traces showing two and three maxima were analyzed and reproduced with relative success according to two- and four-step sequential models.

  18. GADIS: Algorithm for designing sequences to achieve target secondary structure profiles of intrinsically disordered proteins.

    PubMed

    Harmon, Tyler S; Crabtree, Michael D; Shammas, Sarah L; Posey, Ammon E; Clarke, Jane; Pappu, Rohit V

    2016-09-01

    Many intrinsically disordered proteins (IDPs) participate in coupled folding and binding reactions and form alpha helical structures in their bound complexes. Alanine, glycine, or proline scanning mutagenesis approaches are often used to dissect the contributions of intrinsic helicities to coupled folding and binding. These experiments can yield confounding results because the mutagenesis strategy changes the amino acid compositions of IDPs. Therefore, an important next step in mutagenesis-based approaches to mechanistic studies of coupled folding and binding is the design of sequences that satisfy three major constraints. These are (i) achieving a target intrinsic alpha helicity profile; (ii) fixing the positions of residues corresponding to the binding interface; and (iii) maintaining the native amino acid composition. Here, we report the development of a G: enetic A: lgorithm for D: esign of I: ntrinsic secondary S: tructure (GADIS) for designing sequences that satisfy the specified constraints. We describe the algorithm and present results to demonstrate the applicability of GADIS by designing sequence variants of the intrinsically disordered PUMA system that undergoes coupled folding and binding to Mcl-1. Our sequence designs span a range of intrinsic helicity profiles. The predicted variations in sequence-encoded mean helicities are tested against experimental measurements. PMID:27503953

  19. Differential expression profiling of serum proteins and metabolites for biomarker discovery

    NASA Astrophysics Data System (ADS)

    Roy, Sushmita Mimi; Anderle, Markus; Lin, Hua; Becker, Christopher H.

    2004-11-01

    A liquid chromatography-mass spectrometry (LC-MS) proteomics and metabolomics platform is presented for quantitative differential expression analysis. Proteome profiles obtained from 1.5 [mu]L of human serum show ~5000 de-isotoped and quantifiable molecular ions. Approximately 1500 metabolites are observed from 100 [mu]L of serum. Quantification is based on reproducible sample preparation and linear signal intensity as a function of concentration. The platform is validated using human serum, but is generally applicable to all biological fluids and tissues. The median coefficient of variation (CV) for ~5000 proteomic and ~1500 metabolomic molecular ions is approximately 25%. For the case of C-reactive protein, results agree with quantification by immunoassay. The independent contributions of two sources of variance, namely sample preparation and LC-MS analysis, are respectively quantified as 20.4 and 15.1% for the proteome, and 19.5 and 13.5% for the metabolome, for median CV values. Furthermore, biological diversity for ~20 healthy individuals is estimated by measuring the variance of ~6500 proteomic and metabolomic molecular ions in sera for each sample; the median CV is 22.3% for the proteome and 16.7% for the metabolome. Finally, quantitative differential expression profiling is applied to a clinical study comparing healthy individuals and rheumatoid arthritis (RA) patients.

  20. A novel method for protein secondary structure prediction using dual-layer SVM and profiles.

    PubMed

    Guo, Jian; Chen, Hu; Sun, Zhirong; Lin, Yuanlie

    2004-03-01

    A high-performance method was developed for protein secondary structure prediction based on the dual-layer support vector machine (SVM) and position-specific scoring matrices (PSSMs). SVM is a new machine learning technology that has been successfully applied in solving problems in the field of bioinformatics. The SVM's performance is usually better than that of traditional machine learning approaches. The performance was further improved by combining PSSM profiles with the SVM analysis. The PSSMs were generated from PSI-BLAST profiles, which contain important evolution information. The final prediction results were generated from the second SVM layer output. On the CB513 data set, the three-state overall per-residue accuracy, Q3, reached 75.2%, while segment overlap (SOV) accuracy increased to 80.0%. On the CB396 data set, the Q3 of our method reached 74.0% and the SOV reached 78.1%. A web server utilizing the method has been constructed and is available at http://www.bioinfo.tsinghua.edu.cn/pmsvm.

  1. Data mining of protein-binding profiling data identifies structural modifications that distinguish selective and promiscuous compounds.

    PubMed

    Yongye, Austin B; Medina-Franco, José L

    2012-09-24

    Activity profiling of compound collections across multiple targets is increasingly being used in probe and drug discovery. Herein, we discuss an approach to systematically analyzing the structure-activity relationships of a large screening profile data with emphasis on identifying structural changes that have a significant impact on the number of proteins to which a compound binds. As a case study, we analyzed a recently released public data set of more than 15 000 compounds screened across 100 sequence-unrelated proteins. The screened compounds have different origins and include natural products, synthetic molecules from academic groups, and commercial compounds. Similar synthetic structures from academic groups showed, overall, greater promiscuity differences than do natural products and commercial compounds. The method implemented in this work readily identified structural changes that differentiated highly specific from promiscuous compounds. This approach is general and can be applied to analyze any other large-scale protein-binding profile data.

  2. Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

    PubMed

    Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

    2014-01-01

    Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability.

  3. Dissecting the Influence of Protein Flexibility on the Location and Thermodynamic Profile of Explicit Water Molecules in Protein-Ligand Binding.

    PubMed

    Yang, Ying; Lill, Markus A

    2016-09-13

    Explicit water molecules in the binding site of proteins play a crucial role for protein-ligand association. Recent advances in computer-aided drug discovery methodology allow for an accurate prediction of the localized position and thermodynamic profile of water molecules (i.e., hydration sites) in the binding site. The underlying calculations are based on MD simulations of explicit water molecules in a restrained protein structure. However, the ligand-binding process is typically associated with protein conformational change that influences the position and thermodynamic properties of the hydration site. In this manuscript, we present the developments of two methods to incorporate the influence of protein conformational change on hydration sites either by following the conformational transition step-by-step (method I) or to match the hydration sites of the two transition end states using local coordinate systems (method II). Using these methods, we highlight the difference in the estimated protein desolvation free energy with and without inclusion of protein flexibility. To the best of our knowledge, this is the first study that explicitly studies the influence of protein conformational change on the position and thermodynamic profiles of water molecules and provides methodology to incorporate protein flexibility into the estimation of the desolvation free energy. PMID:27494046

  4. Dissecting the Influence of Protein Flexibility on the Location and Thermodynamic Profile of Explicit Water Molecules in Protein-Ligand Binding.

    PubMed

    Yang, Ying; Lill, Markus A

    2016-09-13

    Explicit water molecules in the binding site of proteins play a crucial role for protein-ligand association. Recent advances in computer-aided drug discovery methodology allow for an accurate prediction of the localized position and thermodynamic profile of water molecules (i.e., hydration sites) in the binding site. The underlying calculations are based on MD simulations of explicit water molecules in a restrained protein structure. However, the ligand-binding process is typically associated with protein conformational change that influences the position and thermodynamic properties of the hydration site. In this manuscript, we present the developments of two methods to incorporate the influence of protein conformational change on hydration sites either by following the conformational transition step-by-step (method I) or to match the hydration sites of the two transition end states using local coordinate systems (method II). Using these methods, we highlight the difference in the estimated protein desolvation free energy with and without inclusion of protein flexibility. To the best of our knowledge, this is the first study that explicitly studies the influence of protein conformational change on the position and thermodynamic profiles of water molecules and provides methodology to incorporate protein flexibility into the estimation of the desolvation free energy.

  5. Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

    PubMed Central

    2012-01-01

    Background Haemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. Results The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. Conclusions The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression. PMID:22703293

  6. Identification of Plasmodium vivax Proteins with Potential Role in Invasion Using Sequence Redundancy Reduction and Profile Hidden Markov Models

    PubMed Central

    Restrepo-Montoya, Daniel; Becerra, David; Carvajal-Patiño, Juan G.; Mongui, Alvaro; Niño, Luis F.; Patarroyo, Manuel E.; Patarroyo, Manuel A.

    2011-01-01

    Background This study describes a bioinformatics approach designed to identify Plasmodium vivax proteins potentially involved in reticulocyte invasion. Specifically, different protein training sets were built and tuned based on different biological parameters, such as experimental evidence of secretion and/or involvement in invasion-related processes. A profile-based sequence method supported by hidden Markov models (HMMs) was then used to build classifiers to search for biologically-related proteins. The transcriptional profile of the P. vivax intra-erythrocyte developmental cycle was then screened using these classifiers. Results A bioinformatics methodology for identifying potentially secreted P. vivax proteins was designed using sequence redundancy reduction and probabilistic profiles. This methodology led to identifying a set of 45 proteins that are potentially secreted during the P. vivax intra-erythrocyte development cycle and could be involved in cell invasion. Thirteen of the 45 proteins have already been described as vaccine candidates; there is experimental evidence of protein expression for 7 of the 32 remaining ones, while no previous studies of expression, function or immunology have been carried out for the additional 25. Conclusions The results support the idea that probabilistic techniques like profile HMMs improve similarity searches. Also, different adjustments such as sequence redundancy reduction using Pisces or Cd-Hit allowed data clustering based on rational reproducible measurements. This kind of approach for selecting proteins with specific functions is highly important for supporting large-scale analyses that could aid in the identification of genes encoding potential new target antigens for vaccine development and drug design. The present study has led to targeting 32 proteins for further testing regarding their ability to induce protective immune responses against P. vivax malaria. PMID:21984903

  7. The prostate cancer immunome: In silico functional analysis of antigenic proteins from microarray profiling with IgG.

    PubMed

    Luna-Coronell, Johana A; Vierlinger, Klemens; Gamperl, Magdalena; Hofbauer, Johann; Berger, Ingrid; Weinhäusel, Andreas

    2016-04-01

    The study of the immunome of prostate cancer (PCa) and characterization of autoantibody signature from differentially reactive antigens can uncover disease stage proteins, reveal enriched networks and even expose aberrant cellular mechanisms during the disease process. By conducting plasma IgG profiling on protein microarrays presenting 5449 unique human proteins expressed in 15 417 E. coli human cDNA expression clones, we elucidated 471 (21 higher reactive in PCa) differentially reactive antigens in 50 PCa versus 49 patients with benign prostate hyperplasia (BPH) at initial diagnosis. Functional analyzes show that the immune-profile of PCa compared to BPH control samples is significantly enriched in features targeting Cellular assembly, Cell death and pathways involved in Cell cycle, translation, and assembly of proteins as EIF2 signaling, PCa related genes as AXIN1 and TP53, and ribosomal proteins (e.g. RPS10). An overlap of 61 (out of 471) DIRAGs with the published 1545 antigens from the SEREX database has been found, however those were higher reactive in BPH. Clinical relevance is shown when antibody-reactivities against eight proteins were significantly (p < 0.001) correlated with Gleason-score. Herewith we provide a biological and pathophysiological characterization of the immunological layer of cancerous (PCa) versus benign (BPH) disease, derived from antibody profiling on protein microarrays. PMID:27089054

  8. Apoptotic protein profile in Leishmania donovani after treatment with hexaazatrinaphthylenes derivatives.

    PubMed

    López-Arencibia, Atteneri; Martín-Navarro, Carmen M; Sifaoui, Ines; Reyes-Batlle, María; Wagner, Carolina; Lorenzo-Morales, Jacob; Piñero, José E

    2016-07-01

    Two hexaazatrinaphthylene derivatives, DGV-B and DGV-C previously known to induce an apoptotic-like process in Leishmania donovani parasites were used in this study. For this purpose, two different human protein commercial arrays were used to determine the proteomic profile of the treated parasites compared to non-treated ones. One of the commercial arrays is able to detect the relative expression of 35 human apoptosis-related proteins and the other one is able to identify 9 different human kinases. The obtained results showed that the two tested molecules were able to activate a programmed cell death process by different pathways in the promastigote stage of the parasite. The present study reports the potential application of two commercialised human apoptotic arrays to evaluate the action mechanism of active compounds at least against Leishmania donovani. The obtained data would be useful to establish the putative activated apoptosis pathways in the treated parasites and to further support the use of hexaazatrinaphthylene derivatives for the treatment of leishmaniasis in the near future. Nevertheless, further molecular studies should be developed in order to design and evaluate specific apoptotic arrays for Leishmania genus.

  9. Genome-wide identification and gene expression profiling of ubiquitin ligases for endoplasmic reticulum protein degradation

    PubMed Central

    Kaneko, Masayuki; Iwase, Ikuko; Yamasaki, Yuki; Takai, Tomoko; Wu, Yan; Kanemoto, Soshi; Matsuhisa, Koji; Asada, Rie; Okuma, Yasunobu; Watanabe, Takeshi; Imaizumi, Kazunori; Nomura, Yausyuki

    2016-01-01

    Endoplasmic reticulum (ER)-associated degradation (ERAD) is a mechanism by which unfolded proteins that accumulate in the ER are transported to the cytosol for ubiquitin–proteasome-mediated degradation. Ubiquitin ligases (E3s) are a group of enzymes responsible for substrate selectivity and ubiquitin chain formation. The purpose of this study was to identify novel E3s involved in ERAD. Thirty-seven candidate genes were selected by searches for proteins with RING-finger motifs and transmembrane regions, which are the major features of ERAD E3s. We performed gene expression profiling for the identified E3s in human and mouse tissues. Several genes were specifically or selectively expressed in both tissues; the expression of four genes (RNFT1, RNF185, CGRRF1 and RNF19B) was significantly upregulated by ER stress. To determine the involvement of the ER stress-responsive genes in ERAD, we investigated their ER localisation, in vitro autoubiquitination activity and ER stress resistance. All were partially localised to the ER, whereas CGRRF1 did not possess E3 activity. RNFT1 and RNF185, but not CGRRF1 and RNF19B, exhibited significant resistance to ER stressor in an E3 activity-dependent manner. Thus, these genes are possible candidates for ERAD E3s. PMID:27485036

  10. Proteomics profiling reveals novel proteins and functions of the plant stigma exudate

    PubMed Central

    Rejón, Juan David; Delalande, François; Castro, Antonio Jesús

    2013-01-01

    Proteomic analysis of the stigmatic exudate of Lilium longiflorum and Olea europaea led to the identification of 51 and 57 proteins, respectively, most of which are described for the first time in this secreted fluid. These results indicate that the stigmatic exudate is an extracellular environment metabolically active, participating in at least 80 different biological processes and 97 molecular functions. The stigma exudate showed a markedly catabolic profile and appeared to possess the enzyme machinery necessary to degrade large polysaccharides and lipids secreted by papillae to smaller units, allowing their incorporation into the pollen tube during pollination. It may also regulate pollen-tube growth in the pistil through the selective degradation of tube-wall components. Furthermore, some secreted proteins were involved in pollen-tube adhesion and orientation, as well as in programmed cell death of the papillae cells in response to either compatible pollination or incompatible pollen rejection. Finally, the results also revealed a putative cross-talk between genetic programmes regulating stress/defence and pollination responses in the stigma. PMID:24151302

  11. Effect of Protein, Polysaccharide, and Oxygen Concentration Profiles on Biofilm Cohesiveness▿

    PubMed Central

    Ahimou, Francois; Semmens, Michael J.; Haugstad, Greg; Novak, Paige J.

    2007-01-01

    It is important to control biofilm cohesiveness to optimize process performance. In this study, a membrane-aerated biofilm reactor inoculated with activated sludge was used to grow mixed-culture biofilms of different ages and thicknesses. The cohesions, or cohesive energy levels per unit volume of biofilm, based on a reproducible method using atomic force microscopy (F. Ahimou, M. J. Semmens, P. J. Novak, and G. Haugstad, Appl. Environ. Microbiol. 73:2897-2904, 2007), were determined at different locations within the depths of the biofilms. In addition, the protein and polysaccharide concentrations within the biofilm depths, as well as the dissolved oxygen (DO) concentration profiles within the biofilms, were measured. It was found that biofilm cohesion increased with depth but not with age. Level of biofilm cohesive energy per unit volume was strongly correlated with biofilm polysaccharide concentration, which increased with depth in the membrane-aerated biofilm. In a 12-day-old biofilm, DO also increased with depth and may therefore be linked to polysaccharide production. In contrast, protein concentration was relatively constant within the biofilm and did not appear to influence cohesion. PMID:17337565

  12. Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423.

    PubMed

    Liu, Beibei; Su, Shengzhong; Wu, Ying; Li, Ying; Shan, Xiaohui; Li, Shipeng; Liu, Hongkui; Dong, Haixiao; Ding, Meiqi; Han, Junyou; Yuan, Yaping

    2015-07-01

    Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize.

  13. Profiling mitochondrial proteins in radiation-induced genome-unstable cell lines with persistent oxidative stress by mass spectrometry

    SciTech Connect

    Miller, John H.; Jin, Shuangshuang; Morgan, William F.; Yang, Austin; Wan, Yunhu; Aypar, Umut; Peters, Jonathan S.; Springer, David L.

    2008-06-01

    Radiation-induced genome instability (RIGI) is a response to radiation exposure in which the progeny of surviving cells exhibit increased frequency of chromosomal changes many generations after the initial insult. Persistently elevated oxidative stress accompanying RIGI and the ability of free-radical scavengers, given before irradiation, to reduce the incidence of instability suggest that radiation induced alterations to mitochondrial function likely play a role in RIGI. To further elucidate this mechanism, we performed high-throughput quantitative mass spectrometry on samples enriched in mitochondrial proteins from three chromosomally-unstable GM10115 Chinese-hamster-ovary cell lines and their stable parental cell line. Out of several hundred identified proteins, sufficient data were collected on 74 mitochondrial proteins to test for statistically significant differences in their abundance between unstable and stable cell lines. Each of the unstable cell lines showed a distinct profile of statistically-significant differential abundant mitochondrial proteins. The LS-12 cell line was characterized by 8 downregulated proteins, whereas the CS-9 cell line exhibited 5 distinct up-regulated proteins. The unstable 115 cell line had two down-regulated proteins, one of which was also downregulated in LS-12, and one up-regulated protein relative to stable parental cells. The mitochondrial protein profiles for LS-12 and C-9 provide further evidence that mitochondrial dysfunction is involved in the genome instability of these cell lines.

  14. EvoDB: a database of evolutionary rate profiles, associated protein domains and phylogenetic trees for PFAM-A.

    PubMed

    Ndhlovu, Andrew; Durand, Pierre M; Hazelhurst, Scott

    2015-01-01

    The evolutionary rate at codon sites across protein-coding nucleotide sequences represents a valuable tier of information for aligning sequences, inferring homology and constructing phylogenetic profiles. However, a comprehensive resource for cataloguing the evolutionary rate at codon sites and their corresponding nucleotide and protein domain sequence alignments has not been developed. To address this gap in knowledge, EvoDB (an Evolutionary rates DataBase) was compiled. Nucleotide sequences and their corresponding protein domain data including the associated seed alignments from the PFAM-A (protein family) database were used to estimate evolutionary rate (ω = dN/dS) profiles at codon sites for each entry. EvoDB contains 98.83% of the gapped nucleotide sequence alignments and 97.1% of the evolutionary rate profiles for the corresponding information in PFAM-A. As the identification of codon sites under positive selection and their position in a sequence profile is usually the most sought after information for molecular evolutionary biologists, evolutionary rate profiles were determined under the M2a model using the CODEML algorithm in the PAML (Phylogenetic Analysis by Maximum Likelihood) suite of software. Validation of nucleotide sequences against amino acid data was implemented to ensure high data quality. EvoDB is a catalogue of the evolutionary rate profiles and provides the corresponding phylogenetic trees, PFAM-A alignments and annotated accession identifier data. In addition, the database can be explored and queried using known evolutionary rate profiles to identify domains under similar evolutionary constraints and pressures. EvoDB is a resource for evolutionary, phylogenetic studies and presents a tier of information untapped by current databases. PMID:26140928

  15. EvoDB: a database of evolutionary rate profiles, associated protein domains and phylogenetic trees for PFAM-A

    PubMed Central

    Ndhlovu, Andrew; Durand, Pierre M.; Hazelhurst, Scott

    2015-01-01

    The evolutionary rate at codon sites across protein-coding nucleotide sequences represents a valuable tier of information for aligning sequences, inferring homology and constructing phylogenetic profiles. However, a comprehensive resource for cataloguing the evolutionary rate at codon sites and their corresponding nucleotide and protein domain sequence alignments has not been developed. To address this gap in knowledge, EvoDB (an Evolutionary rates DataBase) was compiled. Nucleotide sequences and their corresponding protein domain data including the associated seed alignments from the PFAM-A (protein family) database were used to estimate evolutionary rate (ω = dN/dS) profiles at codon sites for each entry. EvoDB contains 98.83% of the gapped nucleotide sequence alignments and 97.1% of the evolutionary rate profiles for the corresponding information in PFAM-A. As the identification of codon sites under positive selection and their position in a sequence profile is usually the most sought after information for molecular evolutionary biologists, evolutionary rate profiles were determined under the M2a model using the CODEML algorithm in the PAML (Phylogenetic Analysis by Maximum Likelihood) suite of software. Validation of nucleotide sequences against amino acid data was implemented to ensure high data quality. EvoDB is a catalogue of the evolutionary rate profiles and provides the corresponding phylogenetic trees, PFAM-A alignments and annotated accession identifier data. In addition, the database can be explored and queried using known evolutionary rate profiles to identify domains under similar evolutionary constraints and pressures. EvoDB is a resource for evolutionary, phylogenetic studies and presents a tier of information untapped by current databases. Database URL: http://www.bioinf.wits.ac.za/software/fire/evodb PMID:26140928

  16. Exploratory investigations of hypervelocity intact capture spectroscopy.

    PubMed

    Tsou, P; Griffiths, D J

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  17. Exploratory investigations of hypervelocity intact capture spectroscopy

    NASA Technical Reports Server (NTRS)

    Tsou, P.; Griffiths, D. J.

    1993-01-01

    The ability to capture hypervelocity projectiles intact opens a new technique available for hypervelocity research. A determination of the reactions taking place between the projectile and the capture medium during the process of intact capture is extremely important to an understanding of the intact capture phenomenon, to improving the capture technique, and to developing a theory describing the phenomenon. The intact capture of hypervelocity projectiles by underdense media generates spectra, characteristic of the material species of projectile and capture medium involved. Initial exploratory results into real-time characterization of hypervelocity intact capture techniques by spectroscopy include ultra-violet and visible spectra obtained by use of reflecting gratings, transmitting gratings, and prisms, and recorded by photographic and electronic means. Spectrometry proved to be a valuable real-time diagnostic tool for hypervelocity intact capture events, offering understanding of the interactions of the projectile and the capture medium during the initial period and providing information not obtainable by other characterizations. Preliminary results and analyses of spectra produced by the intact capture of hypervelocity aluminum spheres in polyethylene (PE), polystyrene (PS), and polyurethane (PU) foams are presented. Included are tentative emission species identifications, as well as gray body temperatures produced in the intact capture process.

  18. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability....

  19. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  20. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  1. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  2. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  3. 46 CFR 174.185 - Intact stability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Intact stability. 174.185 Section 174.185 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Offshore Supply Vessels § 174.185 Intact stability....

  4. 46 CFR 174.015 - Intact stability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Intact stability. 174.015 Section 174.015 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SUBDIVISION AND STABILITY SPECIAL RULES PERTAINING TO SPECIFIC VESSEL TYPES Special Rules Pertaining to Deck Cargo Barges § 174.015 Intact stability. (a)...

  5. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

    PubMed Central

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  6. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    PubMed

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  7. A novel immuno-competitive capture mass spectrometry strategy for protein-protein interaction profiling reveals that LATS kinases regulate HCV replication through NS5A phosphorylation.

    PubMed

    Meistermann, Hélène; Gao, Junjun; Golling, Sabrina; Lamerz, Jens; Le Pogam, Sophie; Tzouros, Manuel; Sankabathula, Sailaja; Gruenbaum, Lore; Nájera, Isabel; Langen, Hanno; Klumpp, Klaus; Augustin, Angélique

    2014-11-01

    Mapping protein-protein interactions is essential to fully characterize the biological function of a protein and improve our understanding of diseases. Affinity purification coupled to mass spectrometry (AP-MS) using selective antibodies against a target protein has been commonly applied to study protein complexes. However, one major limitation is a lack of specificity as a substantial part of the proposed binders is due to nonspecific interactions. Here, we describe an innovative immuno-competitive capture mass spectrometry (ICC-MS) method to allow systematic investigation of protein-protein interactions. ICC-MS markedly increases the specificity of classical immunoprecipitation (IP) by introducing a competition step between free and capturing antibody prior to IP. Instead of comparing only one experimental sample with a control, the methodology generates a 12-concentration antibody competition profile. Label-free quantitation followed by a robust statistical analysis of the data is then used to extract the cellular interactome of a protein of interest and to filter out background proteins. We applied this new approach to specifically map the interactome of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) in a cellular HCV replication system and uncovered eight new NS5A-interacting protein candidates along with two previously validated binding partners. Follow-up biological validation experiments revealed that large tumor suppressor homolog 1 and 2 (LATS1 and LATS2, respectively), two closely related human protein kinases, are novel host kinases responsible for NS5A phosphorylation at a highly conserved position required for optimal HCV genome replication. These results are the first illustration of the value of ICC-MS for the analysis of endogenous protein complexes to identify biologically relevant protein-protein interactions with high specificity.

  8. Nano-LC-ESI MS/MS analysis of proteins in dried sea dragon Solenognathus hardwickii and bioinformatic analysis of its protein expression profiling.

    PubMed

    Zhang, Dong-Mei; Feng, Li-Xing; Li, Lu; Liu, Miao; Jiang, Bao-Hong; Yang, Min; Li, Guo-Qiang; Wu, Wan-Ying; Guo, De-An; Liu, Xuan

    2016-09-01

    The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry (nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and pI values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology (GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING (Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins.

  9. Nano-LC-ESI MS/MS analysis of proteins in dried sea dragon Solenognathus hardwickii and bioinformatic analysis of its protein expression profiling.

    PubMed

    Zhang, Dong-Mei; Feng, Li-Xing; Li, Lu; Liu, Miao; Jiang, Bao-Hong; Yang, Min; Li, Guo-Qiang; Wu, Wan-Ying; Guo, De-An; Liu, Xuan

    2016-09-01

    The sea dragon Solenognathus hardwickii has long been used as a traditional Chinese medicine for the treatment of various diseases, such as male impotency. To gain a comprehensive insight into the protein components of the sea dragon, shotgun proteomic analysis of its protein expression profiling was conducted in the present study. Proteins were extracted from dried sea dragon using a trichloroacetic acid/acetone precipitation method and then separated by SDS-PAGE. The protein bands were cut from the gel and digested by trypsin to generate peptide mixture. The peptide fragments were then analyzed using nano liquid chromatography tandem mass spectrometry (nano-LC-ESI MS/MS). 810 proteins and 1 577 peptides were identified in the dried sea dragon. The identified proteins exhibited molecular weight values ranging from 1 900 to 3 516 900 Da and pI values from 3.8 to 12.18. Bioinformatic analysis was conducted using the DAVID Bioinformatics Resources 6.7 Gene Ontology (GO) analysis tool to explore possible functions of the identified proteins. Ascribed functions of the proteins mainly included intracellular non-membrane-bound organelle, non-membrane-bounded organelle, cytoskeleton, structural molecule activity, calcium ion binding and etc. Furthermore, possible signal networks of the identified proteins were predicted using STRING (Search Tool for the Retrieval of Interacting Genes) database. Ribosomal protein synthesis was found to play an important role in the signal network. The results of this study, to best of our knowledge, were the first to provide a reference proteome profile for the sea dragon, and would aid in the understanding of the expression and functions of the identified proteins. PMID:27667517

  10. Expression Profile of Six RNA-Binding Proteins in Pulmonary Sarcoidosis

    PubMed Central

    Novosadova, Eva; Hagemann-Jensen, Michael; Kullberg, Susanna; Kolek, Vitezslav; Grunewald, Johan; Petrek, Martin

    2016-01-01

    Background Sarcoidosis is characterised by up-regulation of cytokines and chemokine ligands/receptors and proteolytic enzymes. This pro-inflammatory profile is regulated post-transcriptionally by RNA-binding proteins (RBPs). We investigated in vivo expression of six RBPs (AUF1, HuR, NCL, TIA, TIAR, PCBP2) and two inhibitors of proteolytic enzymes (RECK, PTEN) in pulmonary sarcoidosis and compared it to the expression in four control groups of healthy individuals and patients with other respiratory diseases: chronic obstructive pulmonary disease (COPD), asthma and idiopathic interstitial pneumonias (IIPs). Methods RT-PCR was used to quantify the mRNAs in bronchoalveolar (BA) cells obtained from 50 sarcoidosis patients, 23 healthy controls, 30 COPD, 19 asthmatic and 19 IIPs patients. Flow cytometry was used to assess intracellular protein expression of AUF1 and HuR in peripheral blood T lymphocytes (PBTLs) obtained from 9 sarcoidosis patients and 6 healthy controls. Results Taking the stringent conditions for multiple comparisons into consideration, we consistently observed in the primary analysis including all patients regardless of smoking status as well as in the subsequent sub-analysis limited for never smokers that the BA mRNA expression of AUF1 (p<0.001), TIA (p<0.001), NCL (p<0.01) and RECK (p<0.05) was decreased in sarcoidosis compared to healthy controls. TIA mRNA was also decreased in sarcoidosis compared to both obstructive pulmonary diseases (COPD and asthma; p<0.001) but not compared to IIPs. There were several positive correlations between RECK mRNA and RBP mRNAs in BA cells. Also sarcoidosis CD3+, CD4+ and CD8+ PBTLs displayed lower mean fluorescence intensity of AUF1 (p≤0.02) and HuR (p≤0.03) proteins than control healthy PBTLs. Conclusion mRNA expressions of three RBPs (AUF1, TIA and NCL) and their potential target mRNA encoding RECK in BA cells and additionally protein expression of AUF1 and HuR in PBTLs were down-regulated in our sarcoidosis

  11. DEVELOPMENT OF PROTEIN PROFILE TECHNOLOGY TO EVALUATE ECOLOGICAL EFFECTS OF ENVIRONMENTAL CHEMICALS USING A SMALL FISH MODEL

    EPA Science Inventory

    Hemmer, Michael J., Robert T. Hudson and Calvin C. Walker. In press. Development of Protein Profile Technology to Evaluate Ecological Effects of Environmental Chemicals Using a Small Fish Model (Abstract). To be presented at the EPA Science Forum: Healthy Communities and Ecosyste...

  12. ASSESSMENT OF THE SWINE PROTEIN-ANNOTATED OLIGONUCLEOTIDE MICROARRAY AND UTILITY OF THE ARRAYS FOR EQTL AND TRANSCRIPTIONAL PROFILING STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions com...

  13. In-gel activity-based protein profiling of a clickable covalent ERK1/2 inhibitor.

    PubMed

    Lebraud, Honorine; Wright, David J; East, Charlotte E; Holding, Finn P; O'Reilly, Marc; Heightman, Tom D

    2016-08-16

    In-gel activity-based protein profiling (ABPP) offers rapid assessment of the proteome-wide selectivity and target engagement of a chemical tool. Here we demonstrate the use of the inverse electron demand Diels Alder (IEDDA) click reaction for in-gel ABPP by evaluating the selectivity profile and target engagement of a covalent ERK1/2 probe tagged with a trans-cyclooctene group. The chemical probe was shown to bind covalently to Cys166 of ERK2 using protein MS and X-ray crystallography, and displayed submicromolar GI50s in A375 and HCT116 cells. In both cell lines, the probe demonstrated target engagement and a good selectivity profile at low concentrations, which was lost at higher concentrations. The IEDDA cycloaddition enabled fast and quantitative fluorescent tagging for readout with a high background-to-noise ratio and thereby provides a promising alternative to the commonly used copper catalysed alkyne-azide cycloaddition. PMID:27385078

  14. N- and O-linked glycosylation site profiling of the human basic salivary proline-rich protein 3M.

    PubMed

    Manconi, Barbara; Cabras, Tiziana; Sanna, Monica; Piras, Valentina; Liori, Barbara; Pisano, Elisabetta; Iavarone, Federica; Vincenzoni, Federica; Cordaro, Massimo; Faa, Gavino; Castagnola, Massimo; Messana, Irene

    2016-05-01

    In the present study, we show that the heterogeneous mixture of glycoforms of the basic salivary proline-rich protein 3M, encoded by PRB3-M locus, is a major component of the acidic soluble fraction of human whole saliva in the first years of life. Reversed-phase high-performance liquid chromatography with high-resolution electrospray ionization mass spectrometry analysis of the intact proteoforms before and after N-deglycosylation with Peptide-N-Glycosidase F and tandem mass spectrometry sequencing of peptides obtained after Endoproteinase GluC digestion allowed the structural characterization of the peptide backbone and identification of N- and O-glycosylation sites. The heterogeneous mixture of the proteoforms derives from the combination of 8 different neutral and sialylated glycans O-linked to Threonine 50, and 33 different glycans N-linked to Asparagine residues at positions 66, 87, 108, 129, 150, 171, 192, and 213. PMID:26991339

  15. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

    PubMed Central

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris

    2016-01-01

    Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2−) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, and de novo protein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. PMID:26826234

  16. Predicting protein function with hierarchical phylogenetic profiles: the Gene3D Phylo-Tuner method applied to eukaryotic genomes.

    PubMed

    Ranea, Juan A G; Yeats, Corin; Grant, Alastair; Orengo, Christine A

    2007-11-01

    "Phylogenetic profiling" is based on the hypothesis that during evolution functionally or physically interacting genes are likely to be inherited or eliminated in a codependent manner. Creating presence-absence profiles of orthologous genes is now a common and powerful way of identifying functionally associated genes. In this approach, correctly determining orthology, as a means of identifying functional equivalence between two genes, is a critical and nontrivial step and largely explains why previous work in this area has mainly focused on using presence-absence profiles in prokaryotic species. Here, we demonstrate that eukaryotic genomes have a high proportion of multigene families whose phylogenetic profile distributions are poor in presence-absence information content. This feature makes them prone to orthology mis-assignment and unsuited to standard profile-based prediction methods. Using CATH structural domain assignments from the Gene3D database for 13 complete eukaryotic genomes, we have developed a novel modification of the phylogenetic profiling method that uses genome copy number of each domain superfamily to predict functional relationships. In our approach, superfamilies are subclustered at ten levels of sequence identity-from 30% to 100%-and phylogenetic profiles built at each level. All the profiles are compared using normalised Euclidean distances to identify those with correlated changes in their domain copy number. We demonstrate that two protein families will "auto-tune" with strong co-evolutionary signals when their profiles are compared at the similarity levels that capture their functional relationship. Our method finds functional relationships that are not detectable by the conventional presence-absence profile comparisons, and it does not require a priori any fixed criteria to define orthologous genes. PMID:18052542

  17. Proteomic and transcriptomic profiling of Staphylococcus aureus surface LPXTG-proteins: correlation with agr genotypes and adherence phenotypes.

    PubMed

    Ythier, Mathilde; Resch, Grégory; Waridel, Patrice; Panchaud, Alexandre; Gfeller, Aurélie; Majcherczyk, Paul; Quadroni, Manfredo; Moreillon, Philippe

    2012-11-01

    Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence

  18. Proteomic and Transcriptomic Profiling of Staphylococcus aureus Surface LPXTG-proteins: Correlation with agr Genotypes and Adherence Phenotypes*

    PubMed Central

    Ythier, Mathilde; Resch, Grégory; Waridel, Patrice; Panchaud, Alexandre; Gfeller, Aurélie; Majcherczyk, Paul; Quadroni, Manfredo; Moreillon, Philippe

    2012-01-01

    Staphylococcus aureus infections involve numerous adhesins and toxins, which expression depends on complex regulatory networks. Adhesins include a family of surface proteins covalently attached to the peptidoglycan via a conserved LPXTG motif. Here we determined the protein and mRNA expression of LPXTG-proteins of S. aureus Newman in time-course experiments, and their relation to fibrinogen adherence in vitro. Experiments were performed with mutants in the global accessory-gene regulator (agr), surface protein A (Spa), and fibrinogen-binding protein A (ClfA), as well as during growth in iron-rich or iron-poor media. Surface proteins were recovered by trypsin-shaving of live bacteria. Released peptides were analyzed by liquid chromatography coupled to tandem mass-spectrometry. To unambiguously identify peptides unique to LPXTG-proteins, the analytical conditions were refined using a reference library of S. aureus LPXTG-proteins heterogeneously expressed in surrogate Lactococcus lactis. Transcriptomes were determined by microarrays. Sixteen of the 18 LPXTG-proteins present in S. aureus Newman were detected by proteomics. Nine LPXTG-proteins showed a bell-shape agr-like expression that was abrogated in agr-negative mutants including Spa, fibronectin-binding protein A (FnBPA), ClfA, iron-binding IsdA, and IsdB, immunomodulator SasH, functionally uncharacterized SasD, biofilm-related SasG and methicillin resistance-related FmtB. However, only Spa and SasH modified their proteomic and mRNA profiles in parallel in the parent and its agr- mutant, whereas all other LPXTG-proteins modified their proteomic profiles independently of their mRNA. Moreover, ClfA became highly transcribed and active in fibrinogen-adherence tests during late growth (24 h), whereas it remained poorly detected by proteomics. On the other hand, iron-regulated IsdA-B-C increased their protein expression by >10-times in iron-poor conditions. Thus, proteomic, transcriptomic, and adherence

  19. Contribution of antibody-based protein profiling to the human Chromosome-centric Proteome Project (C-HPP).

    PubMed

    Fagerberg, Linn; Oksvold, Per; Skogs, Marie; Algenäs, Cajsa; Lundberg, Emma; Pontén, Fredrik; Sivertsson, Asa; Odeberg, Jacob; Klevebring, Daniel; Kampf, Caroline; Asplund, Anna; Sjöstedt, Evelina; Al-Khalili Szigyarto, Cristina; Edqvist, Per-Henrik; Olsson, Ingmarie; Rydberg, Urban; Hudson, Paul; Ottosson Takanen, Jenny; Berling, Holger; Björling, Lisa; Tegel, Hanna; Rockberg, Johan; Nilsson, Peter; Navani, Sanjay; Jirström, Karin; Mulder, Jan; Schwenk, Jochen M; Zwahlen, Martin; Hober, Sophia; Forsberg, Mattias; von Feilitzen, Kalle; Uhlén, Mathias

    2013-06-01

    A gene-centric Human Proteome Project has been proposed to characterize the human protein-coding genes in a chromosome-centered manner to understand human biology and disease. Here, we report on the protein evidence for all genes predicted from the genome sequence based on manual annotation from literature (UniProt), antibody-based profiling in cells, tissues and organs and analysis of the transcript profiles using next generation sequencing in human cell lines of different origins. We estimate that there is good evidence for protein existence for 69% (n = 13985) of the human protein-coding genes, while 23% have only evidence on the RNA level and 7% still lack experimental evidence. Analysis of the expression patterns shows few tissue-specific proteins and approximately half of the genes expressed in all the analyzed cells. The status for each gene with regards to protein evidence is visualized in a chromosome-centric manner as part of a new version of the Human Protein Atlas ( www.proteinatlas.org ).

  20. 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

  1. Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

    NASA Astrophysics Data System (ADS)

    Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

    2009-09-01

    Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

  2. Comprehensive protein tyrosine phosphatase mRNA profiling identifies new regulators in the progression of glioma.

    PubMed

    Bourgonje, Annika M; Verrijp, Kiek; Schepens, Jan T G; Navis, Anna C; Piepers, Jolanda A F; Palmen, Chantal B C; van den Eijnden, Monique; Hooft van Huijsduijnen, Rob; Wesseling, Pieter; Leenders, William P J; Hendriks, Wiljan J A J

    2016-01-01

    The infiltrative behavior of diffuse gliomas severely reduces therapeutic potential of surgical resection and radiotherapy, and urges for the identification of new drug-targets affecting glioma growth and migration. To address the potential role of protein tyrosine phosphatases (PTPs), we performed mRNA expression profiling for 91 of the 109 known human PTP genes on a series of clinical diffuse glioma samples of different grades and compared our findings with in silico knowledge from REMBRANDT and TCGA databases. Overall PTP family expression levels appeared independent of characteristic genetic aberrations associated with lower grade or high grade gliomas. Notably, seven PTP genes (DUSP26, MTMR4, PTEN, PTPRM, PTPRN2, PTPRT and PTPRZ1) were differentially expressed between grade II-III gliomas and (grade IV) glioblastomas. For DUSP26, PTEN, PTPRM and PTPRT, lower expression levels correlated with poor prognosis, and overexpression of DUSP26 or PTPRT in E98 glioblastoma cells reduced tumorigenicity. Our study represents the first in-depth analysis of PTP family expression in diffuse glioma subtypes and warrants further investigations into PTP-dependent signaling events as new entry points for improved therapy. PMID:27586084

  3. Profiling protein thiol oxidation in tumor cells using sulfenic acid-specific antibodies.

    PubMed

    Seo, Young Ho; Carroll, Kate S

    2009-09-22

    Hydrogen peroxide (H2O2) functions as a second messenger that can activate cell proliferation through chemoselective oxidation of cysteine residues in signaling proteins. The connection between H2O2 signaling, thiol oxidation, and activation of growth pathways has emerged as fertile ground for the development of strategies for cancer treatment. Central to achieving this goal is the development of tools and assays that facilitate characterization of the molecular events associated with tumorigenesis and evaluation of patient response to therapy. Here we report on the development of an immunochemical method for detecting sulfenic acid, the initial oxidation product that results when a thiolate reacts with H2O2. For this approach, the sulfenic acid is derivatized with a chemical tag to generate a unique epitope for recognition. The elicited antibody is exquisitely specific, context-independent, and capable of visualizing sulfenic acid formation in cells. Applying this approach to several systems, including cancer cell lines, shows it can be used to monitor differences in thiol redox status and reveals a diverse pattern of sulfenic acid modifications across different subtypes of breast tumors. These studies demonstrate a general strategy for producing antibodies against a specific oxidation state of cysteine and show the utility of these reagents for profiling thiol oxidation associated with pathological conditions such as breast cancer.

  4. Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling

    DOE PAGES

    Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; Nandhikonda, Premchendar; Smith, Richard D.; Wright, Aaron T.

    2015-09-25

    Here, the development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosicmore » bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.« less

  5. Proteomic profile of carbonylated proteins in rat liver: exercise attenuated oxidative stress may be involved in fatty liver improvement.

    PubMed

    Hu, Xiaofei; Duan, Zhigui; Hu, Hui; Li, Guolin; Yan, Siyu; Wu, Jinfeng; Wang, Jun; Yin, Dazhong; Xie, Qingji

    2013-05-01

    To screen target proteins of oxidative stress which mediate the effects of exercise on preventing nonalcoholic fatty liver disease (NAFLD), the methods for selecting carbonylated proteins were modified, and carbonylated proteins were profiled. The results showed that treadmill training reduced oxidative stress and the levels of intrahepatic triglyceride (IHTG). The changes in IHTG showed a significant positive correlation with oxidative stress as indicated by malondialdehyde level. Further results from proteomics illustrated that 17 functional proteins were susceptible to oxidative modification, and exercise protected three proteins from carbonylation. The latter three proteins may serve as both direct target proteins of oxidative stress and mediators contributing to the beneficial effects of exercise. In particular, a long-chain specific acyl-CoA dehydrogenase (ACADL) which was a key enzyme in lipid metabolism was not carbonylated and with higher activities in exercise group. These findings indicate that this modified technique is practical and powerful in selecting carbonylated proteins. Long-term treadmill training is effective in ameliorating oxidative stress and preventing the accumulation of IHTG. Among the 17 target proteins of oxidative modification, three proteins contribute to the beneficial effects of exercise. Preventing ACADL from carbonylation may be involved in the physiological mechanism of exercise-induced NAFLD improvement.

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

    PubMed

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

    2015-11-02

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

  7. Comparison of protein expression profile changes in human fibroblasts induced by low doses of gamma rays and energetic protons

    NASA Astrophysics Data System (ADS)

    Zhang, Ye; Clement, Jade; Gridley, Diala; Rohde, Larry; Wu, Honglu

    Extrapolation of known radiation risks to the risks from low dose and low dose-rate exposures of human population, especially prolonged exposure 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 more than 200 proteins (or modified proteins) involved in cell signaling that included mostly apoptosis, cytoskeleton, cell cycle and signal transduction. Normal human fibroblasts were cultured till fully confluent and then exposed to 2 cGy of gamma rays at either low (1 cGy/hr) or high (0.2 Gy/min) dose-rate, or to 2 cGy of 150 MeV protons at high dose-rate. The proteins were isolated at 2 and 6 hours after exposure and labeled with Cy3 for the irradiated cells and with Cy5 for the control samples before loaded 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. Comparison of the overall protein expression profiles in gamma-irradiated cells showed significantly higher inductions at the high dose-rate than at the low dose-rate. The protein profile in cells after the proton exposure showed a much earlier induction pattern in comparison to both the high and low dose-rate gamma exposures. The same expression patterns were also found in individual cell signaling cascades. At 6 hours post irradiation, high dose-rate gamma rays induced cellular protein level changes (ratio to control ˜2) mostly in apoptosis, cell cycle and cytoskeleton, while low dose-rate gamma rays induced similar changes with smaller fold-change values. In comparison, protons induced

  8. Quantitative Analysis of Human Salivary Gland-Derived Intact Proteome Using Top-Down Mass Spectrometry

    SciTech Connect

    Wu, Si; Brown, Joseph N.; Tolic, Nikola; Meng, Da; Liu, Xiaowen; Zhang, Haizhen; Zhao, Rui; Moore, Ronald J.; Pevzner, Pavel A.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2014-05-31

    There are several notable challenges inherent to fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, post-translational modifications, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based liquid chromatography-mass spectrometry (LC-MS) approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of post-translational modifications. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin (B2M). In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein (PIP) and O-glycosylated acidic protein rich protein (aPRP). These characterized proteoforms constitute an intact saliva proteoform database, which was used for quantitative comparison of intact salivary proteoforms among six healthy individuals. Human parotid (PS) and submandibular/sublingual gland (SMSL) secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FTICR mass spectrometer. Significantly different protein and PTM patterns were resolved with high reproducibility between PS and SMSL glands. The results from this study provide further insight into the potential mechanisms of PTM pathways in oral glandular secretion, expanding our knowledge of this complex yet easily accessible fluid. Intact protein LC-MS approach presented herein can potentially be applied for rapid and accurate identification of biomarkers from only a few microliters of human glandular saliva.

  9. Peripheral nerve injury activates convergent nociceptive input to dorsal horn neurons from neighboring intact nerve.

    PubMed

    Terayama, Ryuji; Yamamoto, Yuya; Kishimoto, Noriko; Maruhama, Kotaro; Mizutani, Masahide; Iida, Seiji; Sugimoto, Tomosada

    2015-04-01

    Previous studies demonstrated that peripheral nerve injury induced excessive nociceptive response of spinal cord dorsal horn neurons and such change has been proposed to reflect the development of neuropathic pain state. The aim of this study was to examine the spinal dorsal horn for convergence of nociceptive input to second-order neurons deafferented by peripheral nerve injury. Double immunofluorescence labeling for c-Fos and phosphorylated extracellular signal-regulated kinase (p-ERK) was performed to detect convergent synaptic input to spinal dorsal horn neurons after the saphenous nerve injury. c-Fos expression and the phosphorylation of ERK were induced by noxious heat stimulation of the hindpaw and by electrical stimulation of the injured or uninjured saphenous nerve, respectively. Within the central te