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Sample records for intact protein profiling

  1. (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.

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

    PubMed

    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. Graphical Abstract ᅟ.

  3. 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.

  4. 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.

  5. 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.

  6. Calcium Activation Profile In Electrically Stimulated Intact Rat Heart Cells

    NASA Astrophysics Data System (ADS)

    Geerts, Hugo; Nuydens, Rony; Ver Donck, Luc; Nuyens, Roger; De Brabander, Marc; Borgers, Marcel

    1988-06-01

    Recent advances in fluorescent probe technology and image processing equipment have made available the measurement of calcium in living systems on a real-time basis. We present the use of the calcium indicator Fura-2 in intact normally stimulated rat heart cells for the spatial and dynamic measurement of the calcium excitation profile. After electric stimulation (1 Hz), the activation proceeds from the center of the myocyte toward the periphery. Within two frame times (80 ms), the whole cell is activated. The activation is slightly faster in the center of the cell than in the periphery. The mean recovery time is 200-400 ms. There is no difference along the cell's long axis. The effect of a beta-agonist and of a calcium antagonist is described.

  7. Posttranslational protein modification by polyamines in intact and regenerating nerves.

    PubMed

    Chakraborty, G; Leach, T; Zanakis, M F; Sturman, J A; Ingoglia, N A

    1987-03-01

    A 150,000-g supernatant from axoplasm of the giant axon of the stellate nerve of the squid and from rat sciatic and goldfish optic nerves was found to be able to incorporate covalently [3H]putrescine and [3H]spermidine into an exogenous protein (N,N'-dimethylcasein). Incorporation of radioactivity was inhibited by CuSO4, a specific inhibitor of transglutaminases, the enzymes mediating these reactions in other tissues. Analysis of pH and temperature range and enzyme kinetics displayed characteristics predicted for transglutaminase-mediated reactions. Transglutaminase activity increased during regeneration of both vertebrate nerves, but greater activity was found in segments of nerve containing no intact axons than in either intact segments or in segments containing regenerating axons. Polyacrylamide gel electrophoresis of endogenous modified proteins (in the absence of N,N'-dimethylcasein) showed labeling of 18-, 46- and 200-kilodalton proteins by both [3H]putrescine and [3H]spermidine. Analysis of the protein-bound radioactivity from intact and regenerating rat sciatic nerves demonstrated it to be predominantly in the form of the parent radioactive polyamine. These experiments demonstrate the covalent modification of proteins by polyamines at low levels in squid axoplasm and at relatively higher levels in rat sciatic and goldfish optic nerves. In the latter two cases, the activity of these modification reactions may be due in part to the modification of axonal proteins, but the majority of the activity occurs in nonneuronal cells of the nerve.

  8. Analytical strategies for the global quantification of intact proteins.

    PubMed

    Collier, Timothy S; Muddiman, David Charles

    2012-09-01

    The quantification of intact proteins is a relatively recent development in proteomics. In eukaryotic organisms, proteins are present as multiple isoforms as the result of variations in genetic code, alternative splicing, post-translational modification and other processing events. Understanding the identities and biological functions of these isoforms and how their concentrations vary across different states is the central goal of proteomics. To date, the bulk of proteomics research utilizes a "bottom-up" approach, digesting proteins into their more manageable constitutive peptides, but sacrificing information about the specific isoform and combinations of post-translational modifications present on the protein. Very specific strategies for protein quantification such as the enzyme-linked immunosorbent assay and Western blot are commonplace in laboratories and clinics, but impractical for the study of global biological changes. Herein, we describe strategies for the quantification of intact proteins, their distinct advantages, and challenges to their employment. Techniques contained in this review include the more traditional and widely employed methodology of differential gel electrophoresis and more recently developed mass spectrometry-based techniques including metabolic labeling, chemical labeling, and label-free methodologies.

  9. Toponomics: studying protein-protein interactions and protein networks in intact tissue.

    PubMed

    Pierre, Sandra; Scholich, Klaus

    2010-04-01

    The function of a protein is determined on several levels including the genome, transcriptome, proteome, and the recently introduced toponome. The toponome describes the topology of all proteins, protein complexes and protein networks which constitute and influence the microenvironment of a given protein. It has long been known that cellular function or dysfunction of proteins strongly depends on their microenvironment and even small changes in protein arrangements can dramatically alter their activity/function. Thus, deciphering the topology of the multi-dimensional networks which control normal and disease-related pathways will give a better understanding of the mechanisms underlying disease development. While various powerful proteomic tools allow simultaneous quantification of proteins, only a limited number of techniques are available to visualize protein networks in intact cells and tissues. This review discusses a novel approach to map and decipher functional molecular networks of proteins in intact cells or tissues. Multi-epitope-ligand-cartography (MELC) is an imaging technology that identifies and quantifies protein networks at the subcellular level of morphologically-intact specimens. This immunohistochemistry-based method allows serial visualization and biomathematical analysis of up to 100 cellular components using fluorescence-labelled tags. The resulting toponome maps, simultaneously ranging from the subcellular to the supracellular scale, have the potential to provide the basis for a mathematical description of the dynamic topology of protein networks, and will complement current proteomic data to enhance the understanding of physiological and pathophysiological cell functions.

  10. Intact-Protein Analysis System for Discovery of Serum-Based Disease Biomarkers

    PubMed Central

    Wang, Hong; Hanash, Samir

    2015-01-01

    Profiling of serum and plasma proteins has substantial relevance to the discovery of circulating disease biomarkers. However, the extreme complexity and vast dynamic range of protein abundance in serum and plasma present a formidable challenge for protein analysis. Thus, integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomic analysis of serum or plasma. In this chapter, we describe an orthogonal multidimensional intact-protein analysis system (IPAS) (Wang et al., Mol Cell Proteomics 4:618–625, 2005) coupled with protein tagging (Faca et al., J Proteome Res 5:2009–2018, 2006) to profile the serum and plasma proteomes quantitatively, which we have applied in our biomarker discovery studies (Katayama et al., Genome Med 1:47, 2009; Faca et al., PLoS Med 5:e123, 2008; Zhang et al. Genome Biol 9:R93, 2008). PMID:21468941

  11. Intact-protein analysis system for discovery of serum-based disease biomarkers.

    PubMed

    Wang, Hong; Hanash, Samir

    2011-01-01

    Profiling of serum and plasma proteins has substantial relevance to the discovery of circulating disease biomarkers. However, the extreme complexity and vast dynamic range of protein abundance in serum and plasma present a formidable challenge for protein analysis. Thus, integration of multiple technologies is required to achieve high-resolution and high-sensitivity proteomic analysis of serum or plasma. In this chapter, we describe an orthogonal multidimensional intact-protein analysis system (IPAS) (Wang et al., Mol Cell Proteomics 4:618-625, 2005) coupled with protein tagging (Faca et al., J Proteome Res 5:2009-2018, 2006) to profile the serum and plasma proteomes quantitatively, which we have applied in our biomarker discovery studies (Katayama et al., Genome Med 1:47, 2009; Faca et al., PLoS Med 5:e123, 2008; Zhang et al. Genome Biol 9:R93, 2008).

  12. The phosphorylation of coated membrane proteins in intact neurons

    PubMed Central

    1986-01-01

    To complement studies that have demonstrated the prominent phosphorylation of a 50-kD coated vesicle polypeptide in vitro, we have evaluated the phosphorylation of coated membrane proteins in intact cells. A co-assembly assay has been devised in which extracts of cultured rat sympathetic neurons labeled with [32P]-Pi were combined with unlabeled carrier bovine brain coat proteins and reassembled coat structures were isolated by gradient centrifugation. Two groups of phosphorylated polypeptides, of 100-110 kD (pp100-110) and 155 kD (pp155) apparent molecular mass, were incorporated into reassembled coats. The neuronal pp100-110 are structurally and functionally related to the 100-110-kD component of the bovine brain assembly protein (AP), a protein complex that also contains 50-kD and 16.5-kD components and is characterized by its ability to promote the reassembly of clathrin coat structures under physiological conditions of pH and ionic strength (Zaremba, S. and J. H. Keen, 1983, J. Cell Biol., 97:1337-1348). The neuronal pp155 detected in reassembled coat structures was readily observable in total extracts of [32P]-Pi-labeled neurons dissolved in SDS-containing buffer. A bovine brain counterpart to the neuronal pp155 was also observed when brain coated vesicles were subjected to two- dimensional gel electrophoresis. Phosphoserine was the predominant phosphoaminoacid found in both the pp100 and pp155. A structural and functional counterpart to the 50-kD brain assembly polypeptide (AP50) was also identified in these neurons. Although the brain AP50 is prominently phosphorylated by an endogenous protein kinase in isolated coated vesicle preparations, the neuronal AP50 was not detectably phosphorylated in intact cells as assessed by two-dimensional non- equilibrium pH gradient gel electrophoresis of labeled cells dissolved directly in SDS-containing buffers. These results demonstrate that the bovine brain assembly polypeptides of 50 kD and 100-110 kD that we have

  13. Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

    PubMed Central

    Rose, Christopher M.; Russell, Jason D.; Ledvina, Aaron R.; McAlister, Graeme C.; Westphall, Michael S.; Griep-Raming, Jens; Schwartz, Jae C.; Coon, Joshua J.; Syka, John E.P.

    2013-01-01

    We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section RF ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately two-fold higher quadrupole field frequency of this cell relative to that of the A-QLT, enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation. PMID:23609185

  14. Multipurpose dissociation cell for enhanced ETD of intact protein species.

    PubMed

    Rose, Christopher M; Russell, Jason D; Ledvina, Aaron R; McAlister, Graeme C; Westphall, Michael S; Griep-Raming, Jens; Schwartz, Jae C; Coon, Joshua J; Syka, John E P

    2013-06-01

    We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell's longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

  15. 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...

  16. 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).

  17. Applying the INTACT method to purify endosperm nuclei and to generate parental-specific epigenome profiles.

    PubMed

    Moreno-Romero, Jordi; Santos-González, Juan; Hennig, Lars; Köhler, Claudia

    2017-02-01

    The early endosperm tissue of dicot species is very difficult to isolate by manual dissection. This protocol details how to apply the INTACT (isolation of nuclei tagged in specific cell types) system for isolating early endosperm nuclei of Arabidopsis at high purity and how to generate parental-specific epigenome profiles. As a Protocol Extension, this article describes an adaptation of an existing Nature Protocol that details the use of the INTACT method for purification of root nuclei. We address how to obtain the INTACT lines, generate the starting material and purify the nuclei. We describe a method that allows purity assessment, which has not been previously addressed. The purified nuclei can be used for ChIP and DNA bisulfite treatment followed by next-generation sequencing (seq) to study histone modifications and DNA methylation profiles, respectively. By using two different Arabidopsis accessions as parents that differ by a large number of single-nucleotide polymorphisms (SNPs), we were able to distinguish the parental origin of epigenetic modifications. Our protocol describes the only working method to our knowledge for generating parental-specific epigenome profiles of the early Arabidopsis endosperm. The complete protocol, from silique collection to finished libraries, can be completed in 2 d for bisulfite-seq (BS-seq) and 3 to 4 d for ChIP-seq experiments.This protocol is an extension to: Nat. Protoc. 6, 56-68 (2011); doi:10.1038/nprot.2010.175; published online 16 December 2010.

  18. A wide range of protein isoforms in serum and plasma uncovered by a quantitative intact protein analysis system.

    PubMed

    Misek, David E; Kuick, Rork; Wang, Hong; Galchev, Vladimir; Deng, Bin; Zhao, Rong; Tra, John; Pisano, Michael R; Amunugama, Ravi; Allen, David; Walker, Angela K; Strahler, John R; Andrews, Philip; Omenn, Gilbert S; Hanash, Samir M

    2005-08-01

    We have implemented an orthogonal 3-D intact protein analysis system (IPAS) to quantitatively profile protein differences between human serum and plasma. Reference specimens consisting of pooled Caucasian-American serum, citrate-anticoagulated plasma, and EDTA-anticoagulated plasma were each depleted of six highly abundant proteins, concentrated, and labeled with a different Cy dye (Cy5, Cy3, or Cy2). A mixture consisting of each of the labeled samples was subjected to three dimensions of separation based on charge, hydrophobicity, and molecular mass. Differences in the abundance of proteins between each of the three samples were determined. More than 5000 bands were found to have greater than two-fold difference in intensity between any pair of labeled specimens by quantitative imaging. As expected, some of the differences in band intensities between serum and plasma were attributable to proteins related to coagulation. Interestingly, many proteins were identified in multiple fractions, each exhibiting different pI, hydrophobicity, or molecular mass. This is likely reflective of the expression of different protein isoforms or specific protein cleavage products, as illustrated by complement component 3 precursor and clusterin. IPAS provides a high resolution, high sensitivity, and quantitative approach for the analysis of serum and plasma proteins, and allows assessment of PTMs as a potential source of biomarkers.

  19. 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

  20. Fluorescent labeling of tetracysteine-tagged proteins in intact cells

    PubMed Central

    Hoffmann, Carsten; Gaietta, Guido; Zürn, Alexander; Adams, Stephen R; Terrillon, Sonia; Ellisman, Mark H; Tsien, Roger Y; Lohse, Martin J

    2011-01-01

    In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed. PMID:20885379

  1. 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.

  2. Capillary electrophoresis of intact basic proteins using noncovalently triple-layer coated capillaries.

    PubMed

    Haselberg, Rob; de Jong, Gerhardus J; Somsen, Govert W

    2009-07-01

    The usefulness of a noncovalent, positively charged capillary coating for the efficient analysis of intact basic proteins with CE was studied. Capillaries were coated by subsequent flushing with solutions of 10% w/v Polybrene (PB), 3% w/v dextran sulfate (DS), and again 10% w/v PB. Coating characterization studies showed that stable coatings could be produced which exhibited a pH-independent and highly reproducible EOF. The PB-DS-PB coating was evaluated with Tris phosphate BGEs of various pH using the four basic model proteins: alpha-chymotrypsinogen A, ribonuclease A, cytochrome c, and lysozyme. Typical migration time RSDs for the proteins were less than 0.85%, and apparent plate numbers were above 125,000 using a capillary length of 40 cm. The high separation efficiency allowed detection of several minor impurities in the model proteins. Using a BGE of medium pH, the CE system with triple-layer coating appeared to be useful for the repeatable profiling of recombinant humanized mouse monoclonal immunoglobulin G(1) showing a characteristic pattern of glycoforms. The CE system was also applied to the characterization of two llama antibodies, which were produced in Saccharomyces cerevisiae, revealing the presence of a side product in one of the antibodies. The high migration time stability allowed the reliable determination of antibody-antigen binding by monitoring migration time shifts. Finally, the feasibility of using the PB-DS-PB coated capillaries for CE with mass spectrometric detection was shown by the characterization of the impure llama antibody sample.

  3. Detection of intact megaDalton protein assemblies of vanillyl-alcohol oxidase by mass spectrometry.

    PubMed Central

    van Berkel, W. J.; van den Heuvel, R. H.; Versluis, C.; Heck, A. J.

    2000-01-01

    Well-resolved ion signals of intact large protein assemblies, with molecular masses extending above one million Dalton, have been detected and mass analyzed using electrospray ionization mass spectrometry, with an uncertainty in mass of <0.2%. The mass spectral data seem to reflect known solution-phase behavior of the studied protein assembly and have therefore been directly used to probe the protein assembly topology and stability as a function of ionic strength and pH. PMID:10752605

  4. 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.

  5. Optimization of a MALDI TOF-TOF mass spectrometer for intact protein analysis.

    PubMed

    Liu, Zhaoyang; Schey, Kevin L

    2005-04-01

    A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins.

  6. 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

  7. 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.

  8. MALDI FTICR IMS of intact proteins: Using mass accuracy to link protein images with proteomics data

    PubMed Central

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

    2015-01-01

    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 identifications 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 5,000) and accuracy (<5 ppm) for proteins up to ∼12 kDa 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, 0.6 ppm) and ATP Synthase subunit ε (m/z 5,636.074, −2.3 ppm). MALDI FTICR IMS was also used to differentiate a series of oxidation products of S100A8 (m/z 10,164.03, −2.1 ppm), a subunit of the heterodimer calprotectin, in kidney tissue from mice infected with Staphylococcus aureus. S100A8 – M37O/C42O3 (m/z 10228.00, −2.6 ppm) was found to co-localize with bactierial 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. PMID:25904064

  9. 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.

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

    PubMed Central

    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.

    2015-01-01

    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 three 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. PMID:26589699

  11. 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

    2011-12-09

    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.

  12. Detection and Quantitation of Succinimide in Intact Protein via Hydrazine Trapping and Chemical Derivatization

    PubMed Central

    KLAENE, JOSHUA J.; NI, WENQIN; ALFARO, JOSHUA F.; ZHOU, ZHAOHUI SUNNY

    2014-01-01

    Formation of aspartyl succinimide (Asu) is a common post-translational modification (PTM) of protein pharmaceuticals under acidic conditions. We present a method to detect and quantitate succinimide in intact protein via hydrazine trapping and chemical derivatization. Succinimide, which is labile under typical analytical conditions, is first trapped with hydrazine to form stable hydrazide and can be directly analyzed by mass spectrometry. The resulting aspartyl hydrazide can be selectively derivatized by various tags, such as fluorescent rhodamine sulfonyl chloride that absorbs strongly in the visible region (570 nm). Our tagging strategy allows the labeled protein to be analyzed by orthogonal methods, including HPLC-UV, LC-MS, and SDS-PAGE coupled with fluorescence imaging. A unique advantage of our method is that variants containing succinimide, after derivatization, can be readily resolved via either affinity enrichment or chromatographic separation. This allows further investigation of individual factors in a complex protein mixture that affect succinimide formation. Some additional advantages imparted by fluorescence labeling include, the facile detection of the intact protein without proteolytic digestion to peptides; and high sensitivity, e.g. without optimization 0.41% succinimide was readily detected. As such, our method should be useful for rapid screening, optimization of formulation conditions and related processes relevant to protein pharmaceuticals. PMID:25043726

  13. On the utility of predictive chromatography to complement mass spectrometry based intact protein identification.

    PubMed

    Pridatchenko, Marina L; Perlova, Tatyana Yu; Ben Hamidane, Hisham; Goloborodko, Anton A; Tarasova, Irina A; Gorshkov, Alexander V; Evreinov, Victor V; Tsybin, Yury O; Gorshkov, Mikhail V

    2012-03-01

    The amino acid sequence determines the individual protein three-dimensional structure and its functioning in an organism. Therefore, "reading" a protein sequence and determining its changes due to mutations or post-translational modifications is one of the objectives of proteomic experiments. The commonly utilized approach is gradient high-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry. While serving as a way to simplify the protein mixture, the liquid chromatography may be an additional analytical tool providing complementary information about the protein structure. Previous attempts to develop "predictive" HPLC for large biomacromolecules were limited by empirically derived equations based purely on the adsorption mechanisms of the retention and applicable to relatively small polypeptide molecules. A mechanism of the large biomacromolecule retention in reversed-phase gradient HPLC was described recently in thermodynamics terms by the analytical model of liquid chromatography at critical conditions (BioLCCC). In this work, we applied the BioLCCC model to predict retention of the intact proteins as well as their large proteolytic peptides separated under different HPLC conditions. The specific aim of these proof-of-principle studies was to demonstrate the feasibility of using "predictive" HPLC as a complementary tool to support the analysis of identified intact proteins in top-down, middle-down, and/or targeted selected reaction monitoring (SRM)-based proteomic experiments.

  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. 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.

  16. A photoactivable phospholipid analogue that specifically labels membrane cytoskeletal proteins of intact erythrocytes

    SciTech Connect

    Pradhan, D.; Williamson, P.; Schlegel, R.A. )

    1989-08-22

    A radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho({sup 14}C)ethanolamine(({sup 14}C)AzPE), was synthesized. Upon incubation with erythrocytes in the dark, about 90% of ({sup 14}C)AzPE spontaneously incorporated into the cells; of this fraction, about 90% associated with the membrane, all of it noncovalently. Upon photoactivation, 3-4% of the membrane-associated probe was incorporated into protein. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as extraction of labeled membranes with alkali or detergent, showed that the probe preferentially labeled cytoskeletal proteins. ({sup 14}C)AzPE appears to be a useful tool for the study of lipid-protein interactions at the cytoplasmic face of the plasma membrane of intact cells.

  17. Fragmentation of multiply-charged intact protein ions using MALDI TOF-TOF mass spectrometry.

    PubMed

    Liu, Zhaoyang; Schey, Kevin L

    2008-02-01

    Top down proteomics in a TOF-TOF instrument was further explored by examining the fragmentation of multiply charged precursors ions generated by matrix-assisted laser desorption ionization. Evaluation of sample preparation conditions allowed selection of solvent/matrix conditions and sample deposition methods to produce sufficiently abundant doubly and triply charged precursor ions for subsequent CID experiments. As previously reported, preferential cleavage was observed at sites C-terminal to acidic residues and N-terminal to proline residues for all ions examined. An increase in nonpreferential fragmentation as well as additional low mass product ions was observed in the spectra from multiply charged precursor ions providing increased sequence coverage. This enhanced fragmentation from multiply charged precursor ions became increasingly important with increasing protein molecular weight and facilitates protein identification using database searching algorithms. The useable mass range for MALDI TOF-TOF analysis of intact proteins has been expanded to 18.2 kDa using this approach.

  18. 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

  19. Development and validation of an intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses.

    PubMed

    Cromlish, W A; Payette, P; Kennedy, B P

    1999-11-15

    We have developed an intact cell assay to be used in the direct quantitation of protein tyrosine phosphatase (PTP) activity. Utilizing the baculovirus expression system, the assay readily allows for a direct activity readout for PTPs such as PTP1B or CD45. Infected Sf9 cells expressing either full-length PTP1B, full-length CD45, CD45 catalytic domain, or hCOX-1 (mock-infected) are harvested 29 hr post-infection, at which time cells are viable and the expressed proteins are processed, as well as localized to their predicted subcellular compartments. Assays are carried out in a 96-well format, with cells expressing the PTP of interest. Cells are preincubated with or without inhibitor and challenged with substrate, and the phosphatase activity is determined spectrophotometrically by monitoring the conversion of p-nitrophenyl phosphate to p-nitrophenol at OD405. Documented PTP inhibitors have been used to validate this assay system. This study demonstrates that a direct readout of PTP activity in intact cells can be achieved, thus providing a useful cell-based screen for determining selective inhibitors of PTPs.

  20. The intact Kunitz domain protects the amyloid precursor protein from being processed by matriptase-2.

    PubMed

    Beckmann, Anna-Madeleine; Glebov, Konstantin; Walter, Jochen; Merkel, Olaf; Mangold, Martin; Schmidt, Frederike; Becker-Pauly, Christoph; Gütschow, Michael; Stirnberg, Marit

    2016-08-01

    Proteolytic processing of the amyloid precursor protein (APP) leads to amyloid-β (Aβ) peptides. So far, the mechanism of APP processing is insufficiently characterized at the molecular level. Whereas the knowledge of Aβ generation by several proteases has been expanded, the contribution of the Kunitz-type protease inhibitor domain (KPI) present in two major APP isoforms to the complex proteolytic processing of APP is poorly understood. In this study, we have identified KPI-containing APP as a very potent, slow-binding inhibitor for the membrane-bound proteolytic regulator of iron homeostasis matriptase-2 by forming stable complexes with its target protease in HEK cells. Inhibition and complex formation depend on the intact KPI domain. By inhibiting matriptase-2, KPI-containing APP is protected from matriptase-2-mediated proteolysis within the Aβ region, thus preventing the generation of N-terminally truncated Aβ.

  1. Imaging individual proteins and nanodomains on intact cell membranes with a probe-based optical antenna.

    PubMed

    van Zanten, Thomas S; Lopez-Bosque, Maria J; Garcia-Parajo, Maria F

    2010-01-01

    Optical antennas that confine and enhance electromagnetic fields in a nanometric region hold great potential for nanobioimaging and biosensing. Probe-based monopole optical antennas are fabricated to enhance fields localized to <30 nm near the antenna apex in aqueous conditions. These probes are used under appropriate excitation antenna conditions to image individual antibodies with an unprecedented resolution of 26 +/- 4 nm and virtually no surrounding background. On intact cell membranes in physiological conditions, the obtained resolution is 30 +/- 6 nm. Importantly, the method allows individual proteins to be distinguished from nanodomains and the degree of clustering to be quantified by directly measuring physical size and intensity of individual fluorescent spots. Improved antenna geometries should lead to true live cell imaging below 10-nm resolution with position accuracy in the subnanometric range.

  2. Differential isotope dansylation labeling combined with liquid chromatography mass spectrometry for quantification of intact and N-terminal truncated proteins.

    PubMed

    Tang, Yanan; Li, Liang

    2013-08-20

    The N-terminal amino acids of proteins are important structure units for maintaining the biological function, localization, and interaction networks of proteins. Under different biological conditions, one or several N-terminal amino acids could be cleaved from an intact protein due to processes, such as proteolysis, resulting in the change of protein properties. Thus, the ability to quantify the N-terminal truncated forms of proteins is of great importance, particularly in the area of development and production of protein-based drugs where the relative quantity of the intact protein and its truncated form needs to be monitored. In this work, we describe a rapid method for absolute quantification of protein mixtures containing intact and N-terminal truncated proteins. This method is based on dansylation labeling of the N-terminal amino acids of proteins, followed by microwave-assisted acid hydrolysis of the proteins into amino acids. It is shown that dansyl labeled amino acids are stable in acidic conditions and can be quantified by liquid chromatography mass spectrometry (LC-MS) with the use of isotope analog standards.

  3. 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

  4. Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants

    PubMed Central

    Tsunoda, Satoshi; Avezov, Edward; Zyryanova, Alisa; Konno, Tasuku; Mendes-Silva, Leonardo; Pinho Melo, Eduardo; Harding, Heather P; Ron, David

    2014-01-01

    Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER. DOI: http://dx.doi.org/10.7554/eLife.03421.001 PMID:25073928

  5. 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

  6. Effects of hydrolysed casein, intact casein and intact whey protein on energy expenditure and appetite regulation: a randomised, controlled, cross-over study.

    PubMed

    Bendtsen, Line Q; Lorenzen, Janne K; Gomes, Sisse; Liaset, Bjørn; Holst, Jens J; Ritz, Christian; Reitelseder, Søren; Sjödin, Anders; Astrup, Arne

    2014-10-28

    Casein and whey differ in amino acid composition and in the rate of absorption; however, the absorption rate of casein can be increased to mimic that of whey by exogenous hydrolysis. The objective of the present study was to compare the effects of hydrolysed casein (HC), intact casein (IC) and intact whey (IW) on energy expenditure (EE) and appetite regulation, and thereby to investigate the influence of amino acid composition and the rate of absorption. In the present randomised cross-over study, twenty-four overweight and moderately obese young men and women consumed three isoenergetic dietary treatments that varied in protein source. The study was conducted in a respiration chamber, where EE, substrate oxidation and subjective appetite were measured over 24 h at three independent visits. Moreover, blood and urine samples were collected from the participants. The results showed no differences in 24 h and postprandial EE or appetite regulation. However, lipid oxidation, estimated from the respiratory quotient (RQ), was found to be higher after consumption of IW than after consumption of HC during daytime (P= 0·014) as well as during the time after the breakfast meal (P= 0·008) when the food was provided. Likewise, NEFA concentrations were found to be higher after consumption of IW than after consumption of HC and IC (P< 0·01). However, there was no overall difference in the concentration of insulin or glucagon-like peptide 1. In conclusion, dietary treatments when served as high-protein mixed meals induced similar effects on EE and appetite regulation, except for lipid oxidation, where RQ values suggest that it is higher after consumption of IW than after consumption of HC.

  7. 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.

  8. 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

  9. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues.

    PubMed

    Lee, Je Hyuk; Daugharthy, Evan R; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C; Terry, Richard; Turczyk, Brian M; Yang, Joyce L; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M

    2015-03-01

    RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

  10. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

    PubMed Central

    Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Terry, Richard; Turczyk, Brian M.; Yang, Joyce L.; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M.

    2014-01-01

    RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. PMID:25675209

  11. Autopilot: an online data acquisition control system for the enhanced high-throughput characterization of intact proteins.

    PubMed

    Durbin, Kenneth R; Fellers, Ryan T; Ntai, Ioanna; Kelleher, Neil L; Compton, Philip D

    2014-02-04

    The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context.

  12. Autopilot: An Online Data Acquisition Control System for the Enhanced High-Throughput Characterization of Intact Proteins

    PubMed Central

    2015-01-01

    The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context. PMID:24400813

  13. Recombinant chymosin used for exact and complete removal of a prochymosin derived fusion tag releasing intact native target protein

    PubMed Central

    Justesen, Sune F L; Lamberth, Kasper; Nielsen, Lise-Lotte B; Schafer-Nielsen, Claus; Buus, Søren

    2009-01-01

    Fusion tags add desirable properties to recombinant proteins, but they are not necessarily acceptable in the final products. Ideally, fusion tags should be removed releasing the intact native protein with no trace of the tag. Unique endoproteinases with the ability to cleave outside their own recognition sequence can potentially cleave at the boundary of any native protein. Chymosin was recently shown to cleave a pro-chymosin derived fusion tag releasing native target proteins. In our hands, however, not all proteins are chymosin-resistant under the acidic cleavage conditions (pH 4.5) used in this system. Here, we have modified the pro-chymosin fusion tag and demonstrated that chymosin can remove this tag at more neutral pH (pH 6.2); conditions, that are less prone to compromise the integrity of target proteins. Chymosin was successfully used to produce intact native target protein both at the level of small and large-scale preparations. Using short peptide substrates, we further examined the influence of P1′ amino acid (the N-terminus of the native target protein) and found that chymosin accepts many different, although not all, amino acids. We conclude that chymosin has several appealing characteristics for the exact removal of fusion tags. It is readily available in highly purified recombinant versions approved by the FDA for preparation of food for human consumption. We suggest that one should consider extending the use of chymosin to the preparation of pharmaceutical proteins. PMID:19388053

  14. 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

  15. Improved separation of fluorogenic derivatized intact proteins with high resolution and efficiency using a reversed-phase liquid chromatographic system.

    PubMed

    Ichibangase, Tomoko; Nakata, Katsunori; Imai, Kazuhiro

    2014-06-01

    Although the efficient separation of intact protein mixtures is extremely difficult, reversed-phase chromatography is an important technique for performing quantitative, accurate and reproducible proteomics analyses. Here, we show that, despite the operating constraints of conventional high-performance liquid chromatography, such as column temperature, operating pressure and separation time, comprehensive separation of fluorogenic derivatized intact proteins could be achieved with high resolution and separation efficiency. First, amylin was chosen as a model peptide and used to estimate the separation efficiency with respect to column temperature and flow rate, as indicated by peak capacity. Then, an extract of human primary hepatocytes was used to model complex component mixtures and the separation conditions were optimized. The effects of mobile-phase pH, the separation time and the column length were also investigated. Consequently, more than 890 peaks could be separated efficiently in the extract, which is 1.5-fold greater than when using conventional conditions. Finally, it was demonstrated that both longer separation time and column length contributed greatly to the effective separation of the protein mixture. These results are expected to provide insights into the separation of intact proteins.

  16. 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

  17. 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.

  18. 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.

  19. Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes.

    PubMed

    Singhal, S S; Singhal, J; Cheng, J; Pikuła, S; Sharma, R; Zimniak, P; Awasthi, Y C; Awasthi, S

    2001-01-01

    We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.

  20. Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment

    PubMed Central

    Konijnenberg, Albert; Bannwarth, Ludovic; Yilmaz, Duygu; Koçer, Armağan; Venien-Bryan, Catherine; Sobott, Frank

    2015-01-01

    Here we study the intact stoichiometry and top-down fragmentation behavior of three integral membrane proteins which were natively reconstituted into detergent micelles: the mechano-sensitive ion channel of large conductance (MscL), the Kirbac potassium channel and the p7 viroporin from the hepatitis C virus. By releasing the proteins under nondenaturing conditions inside the mass spectrometer, we obtained their oligomeric sizes. Increasing the ion activation (collision energy) causes unfolding and subsequent ejection of a highly charged monomer from the membrane protein complexes. Further increase of the ion activation then causes collision-induced dissociation (CID) of the ejected monomers, with fragments observed which were predominantly found to stem from membrane-embedded regions. These experiments show how in a single experiment, we can probe the relation between higher-order structure and protein sequence, by combining the native MS data with fragmentation obtained from top-down MS. PMID:25970171

  1. Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application.

    PubMed

    He, Huining; Ye, Junxiao; Wang, Yinsong; Liu, Quan; Chung, Hee Sun; Kwon, Young Min; Shin, Meong Cheol; Lee, Kyuri; Yang, Victor C

    2014-02-28

    Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab-low molecular weight protamine (LMWP). l-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different l-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed.

  2. Inducible DNA-protein interactions of the murine kappa immunoglobulin enhancer in intact cells: comparison with in vitro interactions.

    PubMed Central

    Hromas, R; Pauli, U; Marcuzzi, A; Lafrenz, D; Nick, H; Stein, J; Stein, G; Van Ness, B

    1988-01-01

    The large intron of the kappa immunoglobulin gene contains a cis-acting enhancer element, which is important in the tissue-specific expression of the gene. We have confirmed the binding activity of a sequence-specific factor present in lymphoid extracts derived from cell lines expressing, or induced to express, the kappa gene. We have extended these studies to show the binding activity is present in normal activated splenic B cells as well as lambda producing cells, and have demonstrated by DNAse footprint analysis full protection of a sequence containing the 11 bp homology to the SV-40 core enhancer. We have compared these in vitro binding studies with an analysis of protein-DNA interactions in intact murine cell lines using genomic sequencing techniques. We demonstrate significant alterations in DMS reactivity of DNA in the murine 70Z/3 cell line after it is induced to kappa expression. These alterations occur at guanine residues which are part of the the 11 bp core sequence, and are identical to those observed in cells constitutively expressing kappa. This provides direct evidence for the induced binding of the tissue specific factor to intact chromatin. In intact chromatin we also observed significant alteration in the reactivity of a guanine, 3' of the core sequence, which is part of a potential secondary DNA structure, and protection of four residues that are part of a region homologous to the heavy chain enhancer. Images PMID:2830597

  3. Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy

    PubMed Central

    2016-01-01

    The function of bioenergetic membranes is strongly influenced by the spatial arrangement of their constituent membrane proteins. Atomic force microscopy (AFM) can be used to probe protein organization at high resolution, allowing individual proteins to be identified. However, previous AFM studies of biological membranes have typically required that curved membranes are ruptured and flattened during sample preparation, with the possibility of disruption of the native protein arrangement or loss of proteins. Imaging native, curved membranes requires minimal tip–sample interaction in both lateral and vertical directions. Here, long-range tip–sample interactions are reduced by optimizing the imaging buffer. Tapping mode AFM with high-resonance-frequency small and soft cantilevers, in combination with a high-speed AFM, reduces the forces due to feedback error and enables application of an average imaging force of tens of piconewtons. Using this approach, we have imaged the membrane organization of intact vesicular bacterial photosynthetic “organelles”, chromatophores. Despite the highly curved nature of the chromatophore membrane and lack of direct support, the resolution was sufficient to identify the photosystem complexes and quantify their arrangement in the native state. Successive imaging showed the proteins remain surprisingly static, with minimal rotation or translation over several-minute time scales. High-order assemblies of RC-LH1-PufX complexes are observed, and intact ATPases are successfully imaged. The methods developed here are likely to be applicable to a broad range of protein-rich vesicles or curved membrane systems, which are an almost ubiquitous feature of native organelles. PMID:28114766

  4. 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.

  5. 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

  6. Evolution of organelle-associated protein profiling.

    PubMed

    Yan, Wei; Aebersold, Ruedi; Raines, Elaine W

    2009-02-15

    Identification of the protein constituents of cell organelles forms the basis for studies to define the roles of specific proteins in organelle structure and functions. Over the past decade, the use of mass spectrometry-based proteomics has dissected various organelles and allowed the association of many novel proteins with particular organelles. This review chronicles the evolution of organelle proteomics technology, and discusses how many limitations, such as organelle heterogeneity and purity, can be avoided with recently developed quantitative profiling approaches. Although many challenges remain, quantitative profiling of organelles holds the promise to begin to address the complex and dynamic shuttling of proteins among organelles that will be critical for application of this advanced technology to disease-based changes in organelle function.

  7. [Optimization of two-dimensional high performance liquid chromatographic columns for highly efficient separation of intact proteins].

    PubMed

    Hong, Guangfeng; Gao, Mingxia; Yan, Guoquan; Guan, Xia; Tao, Qian; Zhang, Xiangmin

    2010-02-01

    In order to optimize two-dimensional liquid chromatographic (2D-LC) columns for highly efficient separation of proteins, several liquid chromatographic columns were investigated and evaluated. Weak anion-exchange (WAX) column was chosen as the first dimension because of its extensive protein separation power. By comparison of different WAX chromatographic columns for human liver protein separation, TSKgel DEAE-5PW column was selected as the first dimension of a 2D-LC system. For the second dimension, ten typical reversed-phase (RP) LC columns (250 mm x 4.6 mm, 5 microm, 30 nm) were investigated and evaluated. Their silica based RP stationary phases were butyl (C4), octyl (C8) or octadecyl (C18). To evaluate the retention behavior and non-specific protein adsorption ability of these ten columns, four neutral compounds (uracil, nitrobenzene, naphthalene and fluorene) and three standard proteins (cytochrome C, myoglobin and albumin from chicken egg white) were adopted and separated by RPLC. Meantime, WAX fractions were used to investigate the separation ability of different alkyl-bonded silica stationary phase columns for complex protein samples. By comparison of column separation efficiency, adsorption of intact proteins and sample analysis, Jupiter 300 C4 column was finally employed for its excellent separation ability. Optimization of WAX and RPLC columns offers reliable foundation for the construction of 2D-LC protein separation systems.

  8. 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.

  9. 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

  10. 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

  11. Surfactant-free purification of membrane proteins with intact native membrane environment.

    PubMed

    Jamshad, Mohammed; Lin, Yu-Pin; Knowles, Timothy J; Parslow, Rosemary A; Harris, Craig; Wheatley, Mark; Poyner, David R; Bill, Roslyn M; Thomas, Owen R T; Overduin, Michael; Dafforn, Tim R

    2011-06-01

    In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10 nm disc-like structure ideally suited to purification and further biochemical study.

  12. Protein Aggregation Profile of the Bacterial Cytosol

    PubMed Central

    de Groot, Natalia S.; Ventura, Salvador

    2010-01-01

    Background Protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity. Methodology/Principal Findings Here, we use the AGGRESCAN algorithm to approximate the aggregation profile of an experimental cytosolic Escherichia coli proteome. The analysis indicates that the aggregation propensity of bacterial proteins is associated with their length, conformation, location, function, and abundance. The data are consistent with the predictions of other algorithms on different theoretical proteomes. Conclusions/Significance Overall, the study suggests that the avoidance of protein aggregation in functional environments acts as a strong evolutionary constraint on polypeptide sequences in both prokaryotic and eukaryotic organisms. PMID:20195530

  13. Defining intact protein primary structures from saliva: A step toward the human proteome project

    PubMed Central

    Halgand, F.; Zabrouskov, V.; Bassilian, S.; Souda, P.; Loo, J.A.; Faull, K.F.; Wong, D.T.; Whitelegge, J.P.

    2012-01-01

    Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was best satisfied by including a mutation and a covalent modification in the C-terminal part, and the assignment of a sequence of a previously unreported protein of mass 10433 Da. The final characterization of Peptide P-J was achieved and the discovery of a truncated form of this peptide was reported. The first sequence assignment was done at low resolution using a hybrid quadrupole time of flight instrument to quickly identify and characterize proteins and data acquisition was switched to FTICR for proteins that required additional sequence coverage and certainty of assignment. High-resolution and high mass accuracy mass spectrometry on a FTICR-MS instrument combined with ECD provided the most informative datasets, with the more frequent presence of ‘unique’ ions that unambiguously define the primary structure. A mixture of predictable and unusual post-translational modifications in the protein sequence precluded the use of shotgun-annotated databases at this stage, requiring manual iterations of sequence refinement in many cases. This led us to propose guidelines for an iterative processing workflow of MS and MSMS datasets that allow researchers to completely assign the identity and the structure of a protein. PMID:22509742

  14. 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.

  15. 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.

  16. Defining intact protein primary structures from saliva: a step toward the human proteome project.

    PubMed

    Halgand, F; Zabrouskov, V; Bassilian, S; Souda, P; Loo, J A; Faull, K F; Wong, D T; Whitelegge, J P

    2012-05-15

    Top-down mass spectrometry has been used to investigate structural diversity within some abundant salivary protein families. In this study, we report the identification of two isoforms of protein II-2 which differed in mass by less than 1 Da, the determination of a sequence for protein IB8a that was best satisfied by including a mutation and a covalent modification in the C-terminal part, and the assignment of a sequence of a previously unreported protein of mass 10433 Da. The final characterization of Peptide P-J was achieved, and the discovery of a truncated form of this peptide was reported. The first sequence assignment was done at low resolution using a hybrid quadrupole time-of-flight instrument to quickly identify and characterize proteins, and data acquisition was switched to Fourier-transform ion cyclotron resonance (FTICR) for proteins that required additional sequence coverage and certainty of assignment. High-resolution and high mass accuracy mass spectrometry on a FTICR-mass spectrometry (MS) instrument combined with electron-capture dissociation (ECD) provided the most informative data sets, with the more frequent presence of "unique" ions that unambiguously define the primary structure. A mixture of predictable and unusual post-translational modifications in the protein sequence precluded the use of shotgun-annotated databases at this stage, requiring manual iterations of sequence refinement in many cases. This led us to propose guidelines for an iterative processing workflow of MS and MSMS data sets that allow researchers to completely assign the identity and the structure of a protein.

  17. Cotton leaf curl Burewala virus with intact or mutant transcriptional activator proteins: complexity of cotton leaf curl disease.

    PubMed

    Kumar, Jitendra; Gunapati, Samatha; Alok, Anshu; Lalit, Adarsh; Gadre, Rekha; Sharma, Naresh C; Roy, Joy K; Singh, Sudhir P

    2015-05-01

    Cotton leaf curl disease (CLCuD) is a serious disease of cotton on the Indian subcontinent. In the present study, three cotton leaf curl viruses, cotton leaf curl Burewala virus (CLCuBuV), cotton leaf curl Kokhran virus (CLCuKoV) and cotton leaf curl Multan virus (CLCuMV), and their associated satellites, cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellite (CLCuMA), were detected. CLCuBuV with either intact (CLCuBuV-1) or mutant (CLCuBuV-2) transcriptional activator protein (TrAP) were detected in different plants. Agroinoculation with CLCuBuV-1 or CLCuBuV-2 together with CLCuMB and CLCuMA, resulted in typical leaf curling and stunting of tobacco plants. Inoculation with CLCuKoV or an isolate of CLCuMV (CLCuMV-2), together with CLCuMB and CLCuMA, induced severe leaf curling, while the other isolate of CLCuMV (CLCuMV-1), which was recombinant in origin, showed mild leaf curling in tobacco. To investigate the effect of intact or mutant TrAP and also the recombination events, CLCuBuV-1, CLCuBuV-2, CLCuMV-1 or CLCuMV-2 together with the satellites (CLCuMA and CLCuMB) were transferred to cotton via whitefly-mediated transmission. Cotton plants containing CLCuBuV-1, CLCuBuV-2 or CLCuMV-2 together with satellites showed curling and stunting, whereas the plants having CLCuMV-1 and the satellites showed only mild and indistinguishable symptoms. CLCuBuV-1 (intact TrAP) showed severe symptoms in comparison to CLCuBuV-2 (mutant TrAP). The present study reveals that two types of CLCuBuV, one with an intact TrAP and the other with a mutant TrAP, exist in natural infection of cotton in India. Additionally, CLCuMuV-1, which has a recombinant origin, induces mild symptoms in comparison to the other CLCuMV isolates.

  18. 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.

  19. NMR-Profiles of Protein Solutions

    PubMed Central

    Pedrini, Bill; Serrano, Pedro; Mohanty, Biswaranjan; Geralt, Michael; Wüthrich, Kurt

    2014-01-01

    NMR-Profiles are quantitative one-dimensional presentations of two-dimensional [15N,1H]-correlation spectra used to monitor the quality of protein solutions prior to and during NMR structure determinations and functional studies. In our current use in structural genomics projects, a NMR-Profile is recorded at the outset of a structure determination, using a uniformly 15N-labeled micro-scale sample of the protein. We thus assess the extent to which polypeptide backbone resonance assignments can be achieved with given NMR techniques, for example, conventional triple resonance experiments or APSY-NMR. With the availability of sequence-specific polypeptide backbone resonance assignments in the course of the structure determination, an “Assigned NMR-Profile” is generated, which visualizes the variation of the 15N–1H correlation cross peak intensities along the sequence and thus maps the sequence locations of polypeptide segments for which the NMR line shapes are affected by conformational exchange or other processes. The Assigned NMR-Profile provides a guiding reference during later stages of the structure determination, and is of special interest for monitoring the protein during functional studies, where dynamic features may be modulated during physiological functions. PMID:23839514

  20. Gas-phase IR spectra of intact [alpha]-helical coiled coil protein complexes

    NASA Astrophysics Data System (ADS)

    Pagel, Kevin; Kupser, Peter; Bierau, Frauke; Polfer, Nicolas C.; Steill, Jeffrey D.; Oomens, Jos; Meijer, Gerard; Koksch, Beate; von Helden, Gert

    2009-06-01

    Electrospray ionization (ESI) is the softest ionization method that is currently available and it is widely accepted, that ESI generated ions of proteins and protein assemblies at certain conditions retain characteristic aspects of their solution-state conformation. ESI mass spectrometry (MS) therefore evolved as a useful tool to obtain information on composition, stoichiometry, and dynamics of non-covalently associated protein complexes. While tertiary structure information of proteins can be obtained from ion mobility spectrometry (IMS), only a few techniques yield direct information on the secondary structure of gas-phase peptides and proteins. We present here the mid-IR spectroscopic secondary structural analysis of three de novo designed [alpha]-helical coiled coil model peptides and their non-covalently associated complexes in the gas-phase. The conformational stability of such coiled coil peptides in solution is primarily driven by aggregation. Isolated monomers usually remain unfolded. Two of the investigated peptides were designed to assemble into stable [alpha]-helical complexes in acidic solution, while the third one remains monomeric and unfolded at these conditions. Monomer ions of all three peptides show comparable photodissociation IR spectra and therefore suggest an unfolded conformation in the gas phase. In contrast, considerable CO stretch (amide-I) and N-H bend (amide-II) band shifts have been observed for the dimers which is consistent with an elevated H-bond content. These findings provide evidence that at least a fraction of the condensed phase [alpha]-helical structure is retained in the gas-phase coiled coil complexes.

  1. Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells.

    PubMed

    Beazely, Michael A; Alan, Jamie K; Watts, Val J

    2005-01-01

    Adenylyl cyclase type 6 (AC6) activity is inhibited by protein kinase C (PKC) in vitro; however, in intact cells, PKC activation does not inhibit the activity of transiently expressed AC6. To investigate the effects of PKC activation on AC6 activity in intact cells, we constructed human embryonic kidney (HEK) 293 cells that stably express wild-type AC6 (AC6-WT) or an AC6 mutant lacking a PKC and cyclic AMP-dependent protein kinase (PKA) phosphorylation site, Ser674 (AC6-S674A). In contrast to in vitro observations, we observed a PKC-mediated enhancement of forskolin- and isoproterenol-stimulated cyclic AMP accumulation in HEK-AC6 cells. Phorbol 12-myristate 13-acetate also potentiated cyclic AMP accumulation in cells expressing endogenous AC6, including Chinese hamster ovary cells and differentiated Cath.a differentiated cells. In HEK-AC6-S674A cells, the potentiation of AC6 stimulation was significantly greater than in cells expressing AC6-WT. The positive effect of PKC activation on AC6 activity seemed to involve Raf1 kinase because the Raf1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) inhibited the PKC potentiation of AC6 activity. Furthermore, the forskolin-stimulated activity of a recombinant AC6 in which the putative Raf1 regulatory sites have been eliminated was not potentiated by activation of PKC. The ability of Raf1 to regulate AC6 may involve a direct interaction because AC6 and a constitutively active Raf1 construct were coimmunoprecipitated. In addition, we report that epidermal growth factor receptor activation also enhances AC6 signaling in a Raf1-dependent manner. These data suggest that Raf1 potentiates drug-stimulated cyclic AMP accumulation in cells expressing AC6 after activation of multiple signaling pathways.

  2. Fast quantification of recombinant protein inclusion bodies within intact cells by FT-IR spectroscopy.

    PubMed

    Gross-Selbeck, Sven; Margreiter, Gerd; Obinger, Christian; Bayer, Karl

    2007-01-01

    The accomplishment of the quantification of the recombinant protein content of whole bacterial cells by FT-IR spectroscopy by application of chemometrics is shown. Recombinant Escherichia coli cells expressing an inclusion body forming fusion protein were dried on a 96-well silicon plate for the analysis in a high-throughput FT-IR spectrometer. Acquired spectra of additionally conventionally quantified samples were used to establish a multivariate calibration. The obtained method was tested by predicting inclusion body contents of samples not used for the multivariate model. Results from FT-IR spectra coincided well with the data of universalized electrophoresis analysis. Hence FT-IR spectroscopy could prove as a fast and simple alternative to conventional quantification methods.

  3. Activation of Ras in vitro and in intact fibroblasts by the Vav guanine nucleotide exchange protein.

    PubMed Central

    Gulbins, E; Coggeshall, K M; Langlet, C; Baier, G; Bonnefoy-Berard, N; Burn, P; Wittinghofer, A; Katzav, S; Altman, A

    1994-01-01

    We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role of Vav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56lck displayed GEF activity against Ras but not against recombinant RacI, RacII, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a approximately 10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras. GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras. GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation. Images PMID:8289830

  4. Enhancing recombinant protein quality and yield by protein stability profiling.

    PubMed

    Mezzasalma, Tara M; Kranz, James K; Chan, Winnie; Struble, Geoffrey T; Schalk-Hihi, Céline; Deckman, Ingrid C; Springer, Barry A; Todd, Matthew J

    2007-04-01

    The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.

  5. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    PubMed

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    PubMed Central

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

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

    PubMed

    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.

  13. Phylogenetic profiles of all membrane transport proteins

    PubMed Central

    Weiner, January; Kooij, Taco W.A.

    2016-01-01

    In order to combat the on-going malaria epidemic, discovery of new drug targets remains vital. Proteins that are essential to survival and specific to malaria parasites are key candidates. To survive within host cells, the parasites need to acquire nutrients and dispose of waste products across multiple membranes. Additionally, like all eukaryotes, they must redistribute ions and organic molecules between their various internal membrane bound compartments. Membrane transport proteins mediate all of these processes and are considered important mediators of drug resistance as well as drug targets in their own right. Recently, using advanced experimental genetic approaches and streamlined life cycle profiling, we generated a large collection of Plasmodium berghei gene deletion mutants and assigned essential gene functions, highlighting potential targets for prophylactic, therapeutic, and transmission-blocking anti-malarial drugs. Here, we present a comprehensive orthology assignment of all Plasmodium falciparum putative membrane transport proteins and provide a detailed overview of the associated essential gene functions obtained through experimental genetics studies in human and murine model parasites. Furthermore, we discuss the phylogeny of selected potential drug targets identified in our functional screen. We extensively discuss the results in the context of the functional assignments obtained using gene targeting available to date. PMID:28357319

  14. Mass-tag enhanced immuno-laser desorption/ionization mass spectrometry for sensitive detection of intact protein antigens.

    PubMed

    Lorey, Martina; Adler, Belinda; Yan, Hong; Soliymani, Rabah; Ekström, Simon; Yli-Kauhaluoma, Jari; Laurell, Thomas; Baumann, Marc

    2015-05-19

    A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 μL plasma sample, well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We propose the potential use of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection.

  15. Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

    PubMed Central

    Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

    2013-01-01

    Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

  16. Relationship between Insulin-Resistance Processing Speed and Specific Executive Function Profiles in Neurologically Intact Older Adults.

    PubMed

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

    2015-09-01

    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. 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. 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). 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 executive function.

  17. 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

  18. Comparison of immune responses induced by rat RT-1 antigens presented as inserts into liposomes, as protein micelles and as intact cells.

    PubMed

    Hedlund, G; Jansson, B; Sjögren, H O

    1984-09-01

    Partially purified rat transplantation antigens (RT-1) were inserted into liposomes composed of various types of lipids and used for immunization. The immune responses induced by the liposomes were compared with responses induced by RT-1 as protein micelles, alone or emulsified in Freund's incomplete adjuvant, or intact cells. Liposomes gave generally a higher humoral response than protein micelles. Each type of RT-1 immunization gave a particular pattern of specific Ig (sub)class responses. Freund's incomplete adjuvant was not only lacking in potentiating effect on low protein dose immunization but had a significant inhibitory effect. Besides intact cells only distearoyl-phosphatidylcholine liposomes had the potential to induce a cell-mediated cytotoxic response.

  19. Cotton leaf curl disease in resistant cotton is associated with a single begomovirus that lacks an intact transcriptional activator protein.

    PubMed

    Amrao, Luqman; Amin, Imran; Shahid, M Shafiq; Briddon, Rob W; Mansoor, Shahid

    2010-09-01

    Cotton leaf curl disease (CLCuD) is the major limitation to cotton production across Pakistan and northwestern India. The disease first appeared in epidemic form in the 1980s and was shown to be caused by monopartite begomoviruses (seven distinct species have thus far been shown to be involved), frequently as multiple infections. Additionally, the viruses are associated with a specific satellite, the CLCuD betasatellite Cotton leaf curl Multan betasatellite (CLCuMB), which is responsible for the distinctive disease symptoms, and a satellite-like molecule (termed an alphasatellite), the function of which is unclear. During the late 1990s, cotton varieties with conventional resistance were introduced, alleviating losses to cotton production. However, during 2001 a resistance breaking strain of CLCuD (known as the "Burewala" strain) appeared which spread across most cotton producing areas of Pakistan. We have conducted an analysis of the Burewala strain and show that, contrary to the earlier (Multan) strain, it consists of a single begomovirus. The virus is associated with a recombinant betasatellite, derived from the Multan strain, but we were unable to detect the presence of an alphasatellite. Sequence comparisons show the virus to be a new recombinant species, consisting of sequences derived from two of the viruses associated with the first epidemic, for which we propose the name Cotton leaf curl Burewala virus (CLCuBuV). Surprisingly the virus lacks an intact C2 gene, encoding the transcriptional activator protein, which is invariably present in begomoviruses. The possible mechanisms for the selection of a "defective" begomovirus are discussed.

  20. 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

  1. 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

  2. Hormone-induced protein phosphorylation. I. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid

    PubMed Central

    1982-01-01

    We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid lobules were preincubated with 32Pi for 45 min at 37 degrees C, washed, and then incubated at 37 degrees C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic lobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37 degrees C; (b) an analogous phosphorylated polypeptide was observed in isoproterenol- stimulated parotid lobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic lobules and propranolol to isoproterenol-stimulated parotid lobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose- dependent fashion with secretagogue action in both the exocrine pancreas and parotid. PMID:6296160

  3. Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor.

    PubMed

    Otsuki, Noriyuki; Sekizuka, Tsuyoshi; Seki, Fumio; Sakai, Kouji; Kubota, Toru; Nakatsu, Yuichiro; Chen, Surui; Fukuhara, Hideo; Maenaka, Katsumi; Yamaguchi, Ryoji; Kuroda, Makoto; Takeda, Makoto

    2013-01-20

    Recent outbreaks in monkeys have proven that canine distemper virus (CDV) causes diseases in a wide range of mammals. CDV uses SLAM and nectin4 as receptors to replicate in susceptible animals. Here, we show that human nectin4, but not human SLAM, is fully functional as a CDV receptor. The CDV Ac96I strain hardly replicated in nectin4-expressing human epithelial NCI-H358 cells, but readily adapted to grow in them. Unsurprisingly, no amino acid change in the H protein was required for the adaptation. The original Ac96I strain possessed a truncated C protein, and a subpopulation possessing the intact C protein was selected after growth in NCI-H358 cells. Other CDV strains possessing the intact C protein showed significantly higher growth abilities in NCI-H358 cells than the Ac96I strain with the truncated C protein. These findings suggest that the C protein is functional in human epithelial cells and critical for CDV replication in them.

  4. Two variables dominating the retention of intact proteins under gradient elution with simultaneous ultrafast high-resolution separation by hydrophobic interaction chromatography.

    PubMed

    Geng, Xindu; Jia, Xiaodan; Liu, Peng; Wang, Fei; Yang, Xiaoming

    2015-10-07

    The retention of intact proteins under gradient elution in hydrophobic interaction chromatography (HIC) was found to be governed by two variables, the steady region (SR) and the migration region (MR). In the SR, the proteins are immobilized by the strong interactions with the stationary phase such that the retention time is independent of the column length. In the MR, the proteins also interact with the stationary phase, but they move normally, thus the retention time depends on their partition coefficients and the column length. The SR can be used as an operation space (OP) for high-throughput protein analysis by 1D-LC using short columns at high flow rates to maintain a high resolution. The OP can also be employed for all assisted operations in online 2D-LC. Based on the steady region/migration region optimization strategy developed in this study, five successive complete separations of seven intact proteins were performed in a HIC cake in less than 5 min, and a crude extract of ribonuclease A from bovine pancreas was purified using online 2D-LC to 95.8% purity with 93.2% mass recovery in 45 min. This approach can be used to expedite the purification of drug-target proteins and should therefore be of interest to the pharmaceutical industry.

  5. MALDI tissue profiling of integral membrane proteins from ocular tissues.

    PubMed

    Thibault, Danielle B; Gillam, Christopher J; Grey, Angus C; Han, Jun; Schey, Kevin L

    2008-06-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this article, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed age related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods.

  6. Fast, robust and high-resolution glycosylation profiling of intact monoclonal IgG antibodies using nanoLC-chip-QTOF.

    PubMed

    Jacobs, Joannes F M; Wevers, Ron A; Lefeber, Dirk J; van Scherpenzeel, Monique

    2016-10-01

    Optimal glycosylation of immunoglobulins is essential in the generation of therapeutic biologicals with respect to efficacy, pharmacokinetics and immunogenic properties. This challenge in the field of biopharmaceuticals requires technologies for fast, robust and quantitative analysis of glycosylation. Current analyses of monoclonal antibody glycosylation are proteolysis-based mass spectrometry methods, which provide detailed structural information, but suffer a number of drawbacks such as lengthy sample preparation with the possibility to introduce artifacts. Here, we describe a fast, robust and high-resolution nanoLC-chip-QTOF method for quantitative analysis of intact monoclonal IgG glycosylation profiling. The method is able to detect hypoglycosylation, i.e. the lack of whole glycans, which is an important advantage over the well-established methods for free N-glycan or glycopeptide analysis. Moreover, the method is highly amenable to automation and because no digestion steps are involved, it provides direct relative quantitative information of both glycans on each IgG attachment site. We demonstrate that the ease and robustness make this technique ideally suited for quality control of the production process of mAb biopharmaceuticals, and provides new opportunities to study the clinical impact of mAb-glycosylation in patients with monoclonal gammopathies.

  7. Quantitative analysis of human salivary gland-derived intact proteome using top-down mass spectrometry.

    PubMed

    Wu, Si; Brown, Joseph N; Tolić, Nikola; Meng, Da; Liu, Xiaowen; Zhang, Haizhen; Zhao, Rui; Moore, Ronald J; Pevzner, Pavel; Smith, Richard D; Paša-Tolić, Ljiljana

    2014-05-01

    There are several notable challenges inherent for fully characterizing the entirety of the human saliva proteome using bottom-up approaches, including polymorphic isoforms, PTMs, unique splice variants, deletions, and truncations. To address these challenges, we have developed a top-down based LC-MS/MS approach, which cataloged 20 major human salivary proteins with a total of 83 proteoforms, containing a broad range of PTMs. Among these proteins, several previously reported disease biomarker proteins were identified at the intact protein level, such as beta-2 microglobulin. In addition, intact glycosylated proteoforms of several saliva proteins were also characterized, including intact N-glycosylated protein prolactin inducible protein and O-glycosylated acidic protein rich protein. 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 and submandibular/sublingual gland secretion samples (2 μg of protein each) from six healthy individuals were compared using RPLC coupled with the 12T FT-ICR mass spectrometer. Significantly different proteoform profiles were resolved with high reproducibility between parotid secretion and submandibular/sublingual 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.

  8. Liquid Extraction Surface Analysis Mass Spectrometry Coupled with Field Asymmetric Waveform Ion Mobility Spectrometry for Analysis of Intact Proteins from Biological Substrates.

    PubMed

    Sarsby, Joscelyn; Griffiths, Rian L; Race, Alan M; Bunch, Josephine; Randall, Elizabeth C; Creese, Andrew J; Cooper, Helen J

    2015-07-07

    Previously we have shown that liquid extraction surface analysis (LESA) mass spectrometry is suitable for the analysis of intact proteins from a range of biological substrates. Here we show that LESA mass spectrometry may be coupled with high field asymmetric waveform ion mobility spectrometry (FAIMS) for top-down protein analysis directly from thin tissue sections (mouse liver, mouse brain) and from bacterial colonies (Escherichia coli) growing on agar. Incorporation of FAIMS results in significant improvements in signal-to-noise and reduced analysis time. Abundant protein signals are observed in single scan mass spectra. In addition, FAIMS enables gas-phase separation of molecular classes, for example, lipids and proteins, enabling improved analysis of both sets of species from a single LESA extraction.

  9. Protein profiling of preeclampsia placental tissues.

    PubMed

    Shu, Chang; Liu, Zitao; Cui, Lifeng; Wei, Chengguo; Wang, Shuwen; Tang, Jian Jenny; Cui, Miao; Lian, Guodong; Li, Wei; Liu, Xiufen; Xu, Hongmei; Jiang, Jing; Lee, Peng; Zhang, David Y; He, Jin; Ye, Fei

    2014-01-01

    Preeclampsia is a multi-system disorder involved in pregnancy without an effective treatment except delivery. The precise pathogenesis of this complicated disorder is still not completely understood. The objective of this study is to evaluate the alterations of protein expression and phosphorylations that are important in regulating placental cell function in preterm and term preeclampsia. Using the Protein Pathway Array, 38 proteins in placental tissues were found to be differentially expressed between preterm preeclampsia and gestational age matched control, while 25 proteins were found to be expressed differentially between term preeclampsia and matched controls. Among these proteins, 16 proteins and their associated signaling pathways overlapped between preterm and term preeclampsia, suggesting the common pathogenesis of two subsets of disease. On the other hand, many proteins are uniquely altered in either preterm or term preeclampsia and correlated with severity of clinical symptoms and outcomes, therefore, providing molecular basis for these two subsets of preeclampsia. Furthermore, the expression levels of some of these proteins correlated with neonatal small for gestational age (PAI-1 and PAPP-A) and adverse outcomes (Flt-1) in women with preterm preeclampsia. These proteins could potentially be used as candidate biomarkers for predicting outcomes of preeclampsia.

  10. Differences of protein profile before and after orthodontic treatment

    NASA Astrophysics Data System (ADS)

    Nasri, Farah Amirah Mohd; Wahab, Rohaya Megat Abdul; Karsani, Saiful Anuar; Ariffin, Shahrul Hisham Zainal

    2016-11-01

    Mechanical forces in orthodontic treatment used to treat malocclusion can cause inflamed gingival tissue and the process of tooth movement may resorb dental root. Root resorption is an iatrogenic effect of orthodontic treatment but it can be monitored using protein biomarker. This study aims to investigate the differences of protein profile before and after orthodontic treatment using different staining methods. Human gingival crevicular fluid and saliva were collected from orthodontic patients before and after treatment. Protein profile were observed using SDS-PAGE. Our study shows down regulation of proteins after 3 months of treatment. Hence, there are potential values from this study to aid in investigation for specific biomarkers for root resorption.

  11. Protein aggregation profile of the human kinome

    PubMed Central

    Graña-Montes, Ricardo; Sant'Anna de Oliveira, Ricardo; Ventura, Salvador

    2012-01-01

    Protein aggregation into amyloid fibrils is associated with the onset of an increasing number of human disorders, including Alzheimer's disease, diabetes, and some types of cancer. The ability to form toxic amyloids appears to be a property of most polypeptides. Accordingly, it has been proposed that reducing aggregation and its effect in cell fitness is a driving force in the evolution of proteins sequences. This control of protein solubility should be especially important for regulatory hubs in biological networks, like protein kinases. These enzymes are implicated in practically all processes in normal and abnormal cell physiology, and phosphorylation is one of the most frequent protein modifications used to control protein activity. Here, we use the AGGRESCAN algorithm to study the aggregation propensity of kinase sequences. We compared them with the rest of globular proteins to decipher whether they display differential aggregation properties. In addition, we compared the human kinase complement with the kinomes of other organisms to see if we can identify any evolutionary trend in the aggregational properties of this protein superfamily. Our analysis indicates that kinase domains display significant aggregation propensity, a property that decreases with increasing organism complexity. PMID:23181023

  12. 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

  13. Prefractionation of intact proteins by reversed-phase and anion-exchange chromatography for the differential proteomic analysis of Saccharomyces cerevisiae.

    PubMed

    Stobaugh, Jordan T; Fague, Kaitlin M; Jorgenson, James W

    2013-02-01

    The need for multidimensional separations for bottom-up proteomic analyses has been well demonstrated by many over the past decade. The vast majority of reported approaches has focused primarily on improving the separation once the sample has already been digested. The work presented in this study shows an improvement in multidimensional approaches by prefractionation of intact proteins prior to digestion and separation of the peptides. Two modes of intact protein separation were compared, anion-exchange and reversed-phase, to assess the utility of each mode for the purpose of fractionation. Each of the samples was then enzymatically digested and analyzed by RP-UPLC-MS(E). To assess the validity of each approach, baker's yeast (Saccharomyces cerevisiae) was grown on two different carbon sources, glycerol and dextrose. More proteins were identified by the reversed-phase prefractionation approach (546) than were found by the anion-exchange method (262). As a result, there was much greater coverage of the metabolic pathways of interest for the reversed-phase method than for the anion-exchange method.

  14. Intact cell matrix-assisted laser desorption/ionization mass spectrometry as a tool to screen drugs in vivo for regulation of protein expression.

    PubMed

    Kulkarni, Mahesh J; Vinod, V P; Umasankar, P K; Patole, Milind S; Rao, Mala

    2006-01-01

    Here we demonstrate for the first time the application of intact cell matrix-assisted laser desorption/ionization mass spectrometry (ICM-MS) to study the regulation of protein expression. This technique can be extended to screen the drugs that inhibit protein synthesis in various diseases. We have used Escherichia coli cells expressing a recombinant glutathione-S-transferase (GST) gene under an arabinose-inducible promoter as a model system. Using ICM-MS analysis, we have detected a 28 kDa peak corresponding to the production of recombinant GST under the arabinose-induced condition. Furthermore, recombinant GST protein was purified by a single-step affinity purification using a glutathione Sepharose 4B affinity column from arabinose-induced E. coli cells. The purified GST protein was found to be a 28 kDa protein by MALDI analysis suggesting the arabinose-induced protein is indeed GST. The regulation of protein expression was studied using glucose as an alternative metabolite. The glucose-mediated regulation of the ara-operon was followed using the ICM-MS technique. All the results obtained from ICM-MS data were validated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The present technique can be extended for in vivo screening of drugs and it holds tremendous potential to discover novel drugs against specific protein expressions in different diseases.

  15. Protein profiles of hatchery egg shell membrane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Eggshells, which consist largely of calcareous outer shell and shell membranes, constitute a significant part of poultry hatchery waste. The shell membranes (ESM) not only contain proteins that originate from egg whites but also from the developing embryos and different contaminants of m...

  16. 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

  17. 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

  18. Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-Galpha q/11 association.

    PubMed

    Little, Kevin D; Hemler, Martin E; Stipp, Christopher S

    2004-05-01

    By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein-coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Galpha(q), Galpha(11), and Gbeta, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Galpha(q/11) complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Galpha(q/11) complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.

  19. Sensitive protein comparisons with profiles and hidden Markov models.

    PubMed

    Hofmann, K

    2000-05-01

    Sequence database searches have become an important tool for the life sciences in general and for gene discovery-driven biotechnology in particular. Both the functional assignment of newly found proteins and the mining of genome databases for functional candidates are equally important tasks typically addressed by database searches. Sensitivity and reliability of the search methods are of crucial importance. The overall performance of sequence alignments and database searches can be enhanced considerably, when profiles or hidden Markov models (HMMs) derived from protein families are used as query objects instead of single sequences. This review discusses the concept of profiles, generalised profiles and profile-HMMs, the methods how they are constructed and the scope of possible applications in gene discovery and gene functional assignment.

  20. 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

  1. Microfluidic chips for protein differential expression profiling.

    PubMed

    Armenta, Jenny M; Dawoud, Abdulilah A; Lazar, Iulia M

    2009-04-01

    Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics-MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF-7 breast cancer cellular extracts is reported in this manuscript. Loading approximately 0.1-1 microg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of approximately 40-100 proteins. The potential of an LC-ESI-MS chip for comparative proteomic analysis of isotopically labeled MCF-7 breast cancer cell extracts is explored for the first time.

  2. Phospholamban and troponin I are substrates for protein kinase C in vitro but not in intact beating guinea pig hearts

    SciTech Connect

    Edes, I.; Kranias, E.G. )

    1990-08-01

    The incorporation of (32P)inorganic phosphate into membranous, myofibrillar, and cytosolic proteins was studied in Langendorff-perfused guinea pig hearts treated with phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoylglycerol (D8G), which are potent activators of protein kinase C. Control hearts were perfused with an inactive phorbol ester (4 alpha-phorbol 12,13-didecanoate), which does not cause activation of protein kinase C. To ensure the blockade of different receptor systems, the perfusions were carried out in the presence of prazosin, propranolol, and atropine. Perfusion of hearts with either PMA (4 microM) or D8G (200 microM) was associated with a negative effect on left ventricular inotropy and relaxation. Examination of the 32P incorporation into various fractions revealed that there were no increases in the degree of phosphorylation of phospholamban in sarcoplasmic reticulum, and troponin I and C protein in the myofibrils, although these proteins were found to be substrates for protein kinase C in vitro. However, in the same hearts, there were significant changes in the 32P incorporation into a 28-kDa cytosolic-protein. Examination of the activity levels of protein kinase C in hearts perfused with PMA indicated a redistribution of this activity from the cytosolic to the membrane fraction, suggesting the activation of the enzyme in vivo. These findings indicate that cardiac regulatory phosphoproteins, which may be phosphorylated by protein kinase C in vitro, are not substrates for protein kinase C in beating hearts perfused with phorbol esters or diacylglycerol analogues.

  3. 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

  4. 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.

  5. An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara.

    PubMed

    Quinan, Bárbara R; Flesch, Inge E A; Pinho, Tânia M G; Coelho, Fabiana M; Tscharke, David C; da Fonseca, Flávio G

    2014-05-23

    Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.

  6. Twelve serum protein profiles in children with acute nonbacterial gastroenterocolitis.

    PubMed

    Wiermannová, D; Wiedermann, D; Kadlcáková, E

    1978-01-01

    In thirty children hospitalized with acute benign, short-duration gastroenterocolitis, no obligate pathogens were isolated from stools. Five bleedings were established from each patient in order to obtain the protein profiles of albumin, orosomucoid, haptoglobin, alpha2-macroglobulin, ceruloplasmin, transferrin, C3-component, C-reactive protein, immunglobulins IgG, IgA, IgM and IgD. The proteins were quantitated by the single radial immunodiffusion method. The initial drop in some of the proteins followed may be related to general protein loss, negative nitrogen balance or hemodilution. The absence of a significant increase in all the investigated immunoglobulin classes contrasted with remarkable increase in haptoglobin and orosomucoid, both reaching normal levels in late convalescence. C-reactive protein could be demonstrated in half of the children showing early normalization with disappearance of clinical symptoms. In contrast to ceruloplasmin and C3- component, alpha2-macroglobulin was not involved in the acute phase protein reaction.

  7. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

    PubMed Central

    2011-01-01

    Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain. PMID:21345212

  8. Protein kinase C epsilon induces systolic cardiac failure marked by exhausted inotropic reserve and intact Frank-Starling mechanism.

    PubMed

    Montgomery, David E; Rundell, Veronica L M; Goldspink, Paul H; Urboniene, Dalia; Geenen, David L; de Tombe, Pieter P; Buttrick, Peter M

    2005-11-01

    Myofilament dysfunction is a common point of convergence for many forms of heart failure. Recently, we showed that cardiac overexpression of PKC epsilon initially depresses myofilament activity and then leads to a progression of changes characteristic of human heart failure. Here, we examined the effects of PKC epsilon on contractile reserve, Starling mechanism, and myofilament activation in this model of end-stage dilated cardiomyopathy. Pressure-volume loop analysis and echocardiography showed that the PKC epsilon mice have markedly compromised systolic function and increased end-diastolic volumes. Dobutamine challenge resulted in a small increase in contractility in PKC epsilon mice but failed to enhance cardiac output. The PKC epsilon mice showed a normal length-dependent tension development in skinned cardiac muscle preparations, although Frank-Starling mechanism appeared to be compromised in the intact animal. Simultaneous measurement of tension and ATPase demonstrated that the maximum tension and ATPase were markedly lower in the PKC epsilon mice at any length or Ca2+ concentration. However, the tension cost was also lower indicating less energy expenditure. We conclude 1) that prolonged overexpression of PKC epsilon ultimately leads to a dilated cardiomyopathy marked by exhausted contractile reserve, 2) that PKC epsilon does not compromise the Frank-Starling mechanism at the myofilament level, and 3) that the Starling curve excursion is limited by the inotropic state of the heart. These results reflect the significance of the primary myofilament contractilopathy induced by phosphorylation and imply a role for PKC epsilon-mediated phosphorylation in myofilament physiology and the pathophysiology of decompensated cardiac failure.

  9. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    PubMed Central

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

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

    PubMed Central

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

    2012-01-01

    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/. PMID:22553364

  11. 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/.

  12. Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics*

    PubMed Central

    Subota, Ines; Julkowska, Daria; Vincensini, Laetitia; Reeg, Nele; Buisson, Johanna; Blisnick, Thierry; Huet, Diego; Perrot, Sylvie; Santi-Rocca, Julien; Duchateau, Magalie; Hourdel, Véronique; Rousselle, Jean-Claude; Cayet, Nadège; Namane, Abdelkader; Chamot-Rooke, Julia; Bastin, Philippe

    2014-01-01

    Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity. PMID:24741115

  13. Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.

    PubMed

    Subota, Ines; Julkowska, Daria; Vincensini, Laetitia; Reeg, Nele; Buisson, Johanna; Blisnick, Thierry; Huet, Diego; Perrot, Sylvie; Santi-Rocca, Julien; Duchateau, Magalie; Hourdel, Véronique; Rousselle, Jean-Claude; Cayet, Nadège; Namane, Abdelkader; Chamot-Rooke, Julia; Bastin, Philippe

    2014-07-01

    Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.

  14. Capillary electrophoresis-mass spectrometry of intact basic proteins using Polybrene-dextran sulfate-Polybrene-coated capillaries: system optimization and performance.

    PubMed

    Haselberg, Rob; de Jong, Gerhardus J; Somsen, Govert W

    2010-09-23

    A capillary electrophoresis-mass spectrometry (CE-MS) method using sheath liquid electrospray ionization interfacing was studied and optimized for the analysis of intact basic proteins. To prevent protein adsorption, capillaries with a noncovalent positively charged coating were utilized. Capillaries were coated by subsequent rinsing with solutions of Polybrene, dextran sulfate and Polybrene. The coating proved to be fully compatible with MS detection, causing no background signals and ionization suppression. The composition of the sheath liquid and BGE was optimized using the model proteins α-chymotrypsinogen A, ribonuclease A, lysozyme and cytochrome c. A sheath liquid of isopropanol-water-acetic acid (75:25:0.1, v/v/v) at 2 μL min(-1) resulted in optimal signal intensities for most proteins, but caused dissociation of the heme group of cytochrome c. Optimum protein responses were obtained with a BGE of 50 mM acetic acid (pH 3.0), which allowed a baseline separation of the test protein mixture. Several minor impurities present in the mixture could be detected and provisionally identified using accurate mass and a protein modification database. The selectivity of the CE-MS system was investigated by the analysis of acetylated lysozyme. Eight highly related species, identified as non-acetylated lysozyme and lysozyme acetylated in various degrees, could be distinguished. The CE-MS system showed good reproducibility yielding interday (three weeks period) RSDs for migration time and peak area within 2% and 10%, respectively. With the CE-MS system, determination coefficients (R(2)) for protein concentration and peak area were higher than 0.996, whereas detection limits were between 11 and 19 nM.

  15. 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.

  16. 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

  17. Protein profile of Lupinus texensis phloem sap exudates: searching for Fe- and Zn-containing proteins.

    PubMed

    Lattanzio, Giuseppe; Andaluz, Sofía; Matros, Andrea; Calvete, Juan José; Kehr, Julia; Abadía, Anunciación; Abadía, Javier; López-Millán, Ana-Flor

    2013-08-01

    The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI-MS and ESI-MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19-21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe-containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe-binding proteins in phloem sap: a metallothionein-like protein type 2B identified in the Fe-affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem-specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn-binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.

  18. Plasma profiling of intact isoflavone metabolites by high-performance liquid chromatography and mass spectrometric identification of flavone glycosides daidzin and genistin in human plasma after administration of kinako.

    PubMed

    Hosoda, Kaori; Furuta, Takashi; Yokokawa, Akitomo; Ogura, Kenichiro; Hiratsuka, Akira; Ishii, Kazuo

    2008-08-01

    The roles of isoflavones in the prevention of several hormone-dependent cancers and osteoporosis are of great interest. Despite many pharmacokinetics studies of the isoflavones, the actual types of conjugates circulating in the body and the position(s) of conjugation sites on the flavone skeleton are still uncertain because, in general, conjugated compounds in biological fluids have been evaluated by measuring the free aglycones obtained after selective enzymatic hydrolysis. Using an high-performance (HPLC)-UV-diode-array detector (DAD) method combined with solid-phase extraction, we have obtained HPLC profiles of isoflavone glycosides [daidzin (Din) and genistin (Gin)] and of intact isoflavone metabolites in human plasma: daidzein, genistein, daizein-7-glucuronide, daidzein-4'-glucuronide, genistein-7-glucuronide, genistein-4'-glucuronide, daidzein-7-sulfate, daidzein-4'-sulfate, genistein-7-sulfate, and genistein-4'-sulfate. We investigated the plasma profile of intact isoflavone metabolites in plasma obtained 1 to-7 h after orally administration of 50 g of kinako (baked soybean powder) to two healthy volunteers. The results of DAD analysis indicated that the main isoflavone metabolite peaks were identified on the HPLC chromatogram. Furthermore, the intact glycosides Din and Gin were detected in 1-h plasma samples by their positive electrospray ionization mass spectra, demonstrating that the glycosides Din and Gin can be absorbed from the gut.

  19. Distinguishing between respiratory syncytial virus subgroups by protein profile analysis.

    PubMed Central

    Walpita, P; Mufson, M A; Stanek, R J; Pfeifer, D; Connor, J D

    1992-01-01

    We subgrouped 75 strains of respiratory syncytial virus by a protein profile method (PPM) which relies on different mobilities of the phosphoprotein in one-dimensional polyacrylamide gel electrophoresis and does not require monoclonal antibodies. When compared with enzyme immunoassay, PPM correctly subgrouped 54 of 56 subgroup A and all 19 subgroup B strains. Images PMID:1572961

  20. Changes in serum protein profiles of chickens with tibial dyschondroplasia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Differences in serum protein profiles were analyzed to identify biomarkers associated with a poultry leg problem named tibial dyschondroplasia (TD) that can cause lameness. We used a bead-based affinity matrix containing a combinatorial library of hexapeptides (ProteoMinerTM) to deplete high abundan...

  1. 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.

  2. 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.

  3. Rapid discrimination of Bacillus anthracis from other members of the B. cereus group by mass and sequence of "intact" small acid soluble proteins (SASPs) using mass spectrometry.

    PubMed

    Castanha, Elisangela R; Fox, Alvin; Fox, Karen F

    2006-11-01

    The intentional contamination of buildings, e.g. anthrax in the bioterrorism attacks of 2001, demonstrated that the population can be affected rapidly and lethally if the appropriate treatment is not provided at the right time. Molecular approaches, primarily involving PCR, have proved useful in characterizing "white powders" used in these attacks as well as isolated organisms. However there is a need for a simpler approach, which does not involve temperamental reagents (e.g. enzymes and primers) which could potentially be used by first responders. It is demonstrated here that small acid-soluble proteins (SASPs), located in the core region of Bacillus spores, are reliable biomarkers for identification. The general strategy used in this study was to measure the molecular weight (MW) of an intact SASP by electrospray ionization mass spectrometry (ESI MS) followed by generation of sequence-specific information by ESI MS/MS (tandem mass spectrometry). A prominent SASP of mass 6679 was present in all B. anthracis strains. For B. cereus and B. thuringiensis strains the SASP had a mass of 6712. This represents a two amino acid substitution (serine to alanine; phenylalanine to tyrosine). The only SASP present in the B. anthracis genome consistent with this sequence is encoded by the gene ssB. This protein has a predicted mass of 6810, presumably post-translational processing leads to loss of methionine (mass 131) generating a SASP of mass 6679. This study showed that intact SASPs can be used as a biomarker for identification of B. anthracis; the protocol is simple and rapid. Extrapolation of this approach might prove important for real-time biodetection.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26424599

  5. 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.

  6. 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.

  7. Chemoproteomic profiling and discovery of protein electrophiles in human cells

    NASA Astrophysics Data System (ADS)

    Matthews, Megan L.; He, Lin; Horning, Benjamin D.; Olson, Erika J.; Correia, Bruno E.; Yates, John R.; Dawson, Philip E.; Cravatt, Benjamin F.

    2016-10-01

    Activity-based protein profiling (ABPP) serves as a chemical proteomic platform to discover and characterize functional amino acids in proteins on the basis of their enhanced reactivity towards small-molecule probes. This approach, to date, has mainly targeted nucleophilic functional groups, such as the side chains of serine and cysteine, using electrophilic probes. Here we show that 'reverse-polarity' (RP)-ABPP using clickable, nucleophilic hydrazine probes can capture and identify protein-bound electrophiles in cells. Using this approach, we demonstrate that the pyruvoyl cofactor of S-adenosyl-L-methionine decarboxylase (AMD1) is dynamically controlled by intracellular methionine concentrations. We also identify a heretofore unknown modification—an N-terminally bound glyoxylyl group—in the poorly characterized protein secernin-3. RP-ABPP thus provides a versatile method to monitor the metabolic regulation of electrophilic cofactors and discover novel types of electrophilic modifications on proteins in human cells.

  8. Structural Analysis of the Glycosylated Intact HIV-1 gp120-b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting.

    PubMed

    Li, Xiaoyan; Grant, Oliver C; Ito, Keigo; Wallace, Aaron; Wang, Shixia; Zhao, Peng; Wells, Lance; Lu, Shan; Woods, Robert J; Sharp, Joshua S

    2017-02-21

    Glycoprotein gp120 is a surface antigen and virulence factor of human immunodeficiency virus 1. Broadly neutralizing antibodies (bNAbs) that react to gp120 from a variety of HIV isolates offer hope for the development of broadly effective immunogens for vaccination purposes, if the interactions between gp120 and bNAbs can be understood. From a structural perspective, gp120 is a particularly difficult system because of its size, the presence of multiple flexible regions, and the large amount of glycosylation, all of which are important in gp120-bNAb interactions. Here, the interaction of full-length, glycosylated gp120 with bNAb b12 is probed using high-resolution hydroxyl radical protein footprinting (HR-HRPF) by fast photochemical oxidation of proteins. HR-HRPF allows for the measurement of changes in the average solvent accessible surface area of multiple amino acids without the need for measures that might alter the protein conformation, such as mutagenesis. HR-HRPF of the gp120-b12 complex coupled with computational modeling shows a novel extensive interaction of the V1/V2 domain, probably with the light chain of b12. Our data also reveal HR-HRPF protection in the C3 domain caused by interaction of the N330 glycan with the b12 light chain. In addition to providing information about the interactions of full-length, glycosylated gp120 with b12, this work serves as a template for the structural interrogation of full-length glycosylated gp120 with other bNAbs to better characterize the interactions that drive the broad specificity of the bNAb.

  9. 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

  10. Elevation of Intact and Proteolytic Fragments of Acute Phase Proteins Constitutes the Earliest Systemic Antiviral Response in HIV-1 Infection

    PubMed Central

    Kramer, Holger B.; Lavender, Kerry J.; Qin, Li; Stacey, Andrea R.; Liu, Michael K. P.; di Gleria, Katalin; Simmons, Alison; Gasper-Smith, Nancy; Haynes, Barton F.; McMichael, Andrew J.; Borrow, Persephone; Kessler, Benedikt M.

    2010-01-01

    The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, β-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5–7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha–1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies. PMID:20463814

  11. 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

    2015-10-06

    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.

  12. 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.

  13. 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

  14. 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.

  15. Distinctive serum protein profiles involving abundant proteins in lung cancer patients based upon antibody microarray analysis

    PubMed Central

    Gao, Wei-Min; Kuick, Rork; Orchekowski, Randal P; Misek, David E; Qiu, Ji; Greenberg, Alissa K; Rom, William N; Brenner, Dean E; Omenn, Gilbert S; Haab, Brian B; Hanash, Samir M

    2005-01-01

    Background Cancer serum protein profiling by mass spectrometry has uncovered mass profiles that are potentially diagnostic for several common types of cancer. However, direct mass spectrometric profiling has a limited dynamic range and difficulties in providing the identification of the distinctive proteins. We hypothesized that distinctive profiles may result from the differential expression of relatively abundant serum proteins associated with the host response. Methods Eighty-four antibodies, targeting a wide range of serum proteins, were spotted onto nitrocellulose-coated microscope slides. The abundances of the corresponding proteins were measured in 80 serum samples, from 24 newly diagnosed subjects with lung cancer, 24 healthy controls, and 32 subjects with chronic obstructive pulmonary disease (COPD). Two-color rolling-circle amplification was used to measure protein abundance. Results Seven of the 84 antibodies gave a significant difference (p < 0.01) for the lung cancer patients as compared to healthy controls, as well as compared to COPD patients. Proteins that exhibited higher abundances in the lung cancer samples relative to the control samples included C-reactive protein (CRP; a 13.3 fold increase), serum amyloid A (SAA; a 2.0 fold increase), mucin 1 and α-1-antitrypsin (1.4 fold increases). The increased expression levels of CRP and SAA were validated by Western blot analysis. Leave-one-out cross-validation was used to construct Diagonal Linear Discriminant Analysis (DLDA) classifiers. At a cutoff where all 56 of the non-tumor samples were correctly classified, 15/24 lung tumor patient sera were correctly classified. Conclusion Our results suggest that a distinctive serum protein profile involving abundant proteins may be observed in lung cancer patients relative to healthy subjects or patients with chronic disease and may have utility as part of strategies for detecting lung cancer. PMID:16117833

  16. 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.

  17. Expression of constitutively active Akt/protein kinase B signals GLUT4 translocation in the absence of an intact actin cytoskeleton.

    PubMed

    Eyster, Craig A; Duggins, Quwanza S; Olson, Ann Louise

    2005-05-06

    The actin cytoskeleton has been shown to be required for insulin-dependent GLUT4 translocation; however, the role that the actin network plays is unknown. Actin may play a role in formation of an active signaling complex, or actin may be required for movement of vesicles to the plasma membrane surface. To distinguish between these possibilities, we examined the ability of myr-Akt, a constitutively active form of Akt that signals GLUT4 translocation to the plasma membrane in the absence of insulin, to signal translocation of an HA-GLUT4-GFP reporter protein in the presence or absence of an intact cytoskeleton in 3T3-L1 adipocytes. Expression of myr-Akt signaled the redistribution of the GLUT4 reporter protein to the cell surface in the absence or presence of 10 microm latrunculin B, a concentration sufficient to completely inhibit insulin-dependent redistribution of the GLUT4 reporter to the cell surface. These data suggest that the actin network plays a primary role in organization of the insulin-signaling complex. To further support this conclusion, we measured the activation of known signaling proteins using a saturating concentration of insulin in cells pretreated without or with 10 microm latrunculin B. We found that latrunculin treatment did not affect insulin-dependent tyrosine phosphorylation of the insulin receptor beta-subunit and IRS-1 but completely inhibited activation of Akt/PKB enzymatic activity. Phosphorylation of Akt/PKB at Ser-473 and Thr-308 was inhibited by latrunculin B treatment, indicating that the defect in signaling lies prior to Akt/PKB activation. In summary, our data support the hypothesis that the actin network plays a role in organization of the insulin-signaling complex but is not required for vesicle trafficking and/or fusion.

  18. Parameters affecting the separation of intact proteins in gradient-elution reversed-phase chromatography using poly(styrene-co-divinylbenzene) monolithic capillary columns.

    PubMed

    Detobel, Frederik; Broeckhoven, Ken; Wellens, Joke; Wouters, Bert; Swart, Remco; Ursem, Mario; Desmet, Gert; Eeltink, Sebastiaan

    2010-04-30

    An experimental study was performed to investigate the effects of column parameters and gradient conditions on the separation of intact proteins using styrene-based monolithic columns. The effect of flow rate on peak width was investigated at constant gradient steepness by normalizing the gradient time for the column hold-up time. When operating the column at a temperature of 60 degrees C a small C-term effect was observed in a flow rate range of 1-4 microL/min. However, the C-term effect on peak width is not as strong as the decrease in peak width due to increasing flow rate. The peak capacity increased according to the square root of the column length. Decreasing the macropore size of the polymer monolith while maintaining the column length constant, resulted in an increase in peak capacity. A trade-off between peak capacity and total analysis time was made for 50, 100, and 250 mm long monolithic columns and a microparticulate column packed with 5 microm porous silica particles while operating at a flow rate of 2 microL/min. The peak capacity per unit time of the 50mm long monolithic column with small pore size was superior when the total analysis time is below 120 min, yielding a maximum peak capacity of 380. For more demanding separations the 250 mm long monolith provided the highest peak capacity in the shortest possible time frame.

  19. Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei

    PubMed Central

    Chandler, Julie M; Treece, Erin R; Trenary, Heather R; Brenneman, Jessica L; Flickner, Tressa J; Frommelt, Jonathan L; Oo, Zaw M; Patterson, Megan M; Rundle, William T; Valle, Olga V; Kim, Thomas D; Walker, Gary R; Cooper, Chester R

    2008-01-01

    Background Penicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37°C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei. Results Whole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein sequence, which contained the

  20. Specificity profiling of protein phosphatases toward phosphoseryl and phosphothreonyl peptides.

    PubMed

    Xiao, Qing; Luechapanichkul, Rinrada; Zhai, Yujing; Pei, Dehua

    2013-07-03

    A combinatorial library method was developed to systematically profile the substrate specificity of protein phosphatases toward phosphoseryl (pS) and phosphothreonyl (pT) peptides. Application of this method and a previously reported phosphotyrosyl (pY) library screening technique to dual-specificity phosphatase (DUSP) VH1 of vaccinia virus revealed that VH1 is highly active toward both pS/pT and pY peptides. VH1 exhibits different and more stringent sequence specificity toward pS/pT than pY substrates. Unlike previously characterized protein tyrosine phosphatases (PTPs), the activity and specificity of VH1 are primarily determined by the amino acid residues C-terminal to the pS, pT, or pY residue. In contrast, the mammalian VH1-related (VHR) DUSP has intrinsically low catalytic activity toward pS and pT substrates, suggesting that its primary physiological function is to dephosphorylate pY residues in substrate proteins. This method is applicable to other DUSPs and protein-serine/threonine phosphatases, and the substrate specificity data will be useful for identifying the physiological substrates of these enzymes.

  1. 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

    2015-09-08

    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.

  2. Post-polymerization photografting on methacrylate-based monoliths for separation of intact proteins and protein digests with comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry.

    PubMed

    Vonk, Rudy J; Wouters, Sam; Barcaru, Andrei; Vivó-Truyols, Gabriel; Eeltink, Sebastiaan; de Koning, Leo J; Schoenmakers, Peter J

    2015-05-01

    Post-polymerization photografting is a versatile tool to alter the surface chemistry of organic-based monoliths so as to obtain desired stationary phase properties. In this study, 2-acrylamido-2-methyl-1-propanesulfonic acid was grafted to a hydrophobic poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith to create a strong cation exchange stationary phase. Both single-step and two-step photografting were addressed, and the effects of grafting conditions were assessed. An experimental design has been applied in an attempt to optimize three of the key parameters of the two-step photografting chemistry, i.e. the grafting time of the initiator, the monomer concentration and the monomer irradiation time. The photografted columns were implemented in a comprehensive two-dimensional column liquid chromatography ( (t) LC ×  (t) LC) workflow and applied for the separation of intact proteins and peptides. A baseline separation of 11 intact proteins was obtained within 20 min by implementing a gradient across a limited RP composition window in the second dimension. (t) LC ×  (t) LC with UV detection was used for the separation of cytochrome c digest, bovine serum insulin digest and a digest of a complex protein mixture. A semi-quantitative estimation of the occupation of separation space, the orthogonality, of the (t) LC ×  (t) LC system yielded 75%. The (t) LC ×  (t) LC setup was hyphenated to a high-resolution Fourier transform ion cyclotron resonance mass spectrometer instrument to identify the bovine serum insulin tryptic peptides and to demonstrate the compatibility with MS analysis.

  3. 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.

  4. Mercury Alters B-Cell Protein Phosphorylation Profiles

    PubMed Central

    Carruthers, Nicholas J.; Stemmer, Paul M.; Shin, Namhee; Dombkowski, Alan; Caruso, Joseph A.; Gill, Randal; Rosenspire, Allen

    2014-01-01

    Environmental exposure to mercury is suggested to contribute to human immune dysfunction. To shed light on the mechanism we identified changes in the phosphoproteomic profile of the WEHI-231 B cell line after intoxication with Hg2+. These changes were compared to changes in the phosphoproteome that were induced by pervanadate or okadaic acid exposure. Both 250 μM HgCl2 and pervanadate, a known phosphotyrosine phosphatase inhibitor, caused an increase in the number of proteins identified after TiO2 affinity selection and LC-MS/MS analysis. Pervanadate treatment had a larger effect than Hg2+ on the number of Scansite motifs which were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling pathways activated, 4 with score > 5.0. However, Hg2+ had a more focused effect, primarily causing tyrosine-phosphorylation in SH2 domains in proteins that are in the B cell receptor signaling pathway. The finding that many of the changes induced by Hg2+ overlap with those of pervanadate, indicates that at high concentrations Hg2+ inhibits protein tyrosine phosphatases. PMID:24224561

  5. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    PubMed

    Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2011-01-01

    It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  6. Protein profiling and phosphoprotein analysis by isoelectric focusing.

    PubMed

    Maccarrone, Giuseppina; Filiou, Michaela D

    2015-01-01

    Protein profiling enables the qualitative characterization of a proteome of interest. Phosphorylation is a post-translational modification with regulatory functions in a plethora of cell processes. We present an experimental workflow for simultaneous analysis of the proteome and phosphoproteome with no additional enrichment for phosphoproteins/phosphopeptides. Our approach is based on isoelectric focusing (IEF) which allows the separation of peptide mixtures on an immobilized pH gradient (IPG) according to their isoelectric point. Due to the negative charge of the phosphogroup, most of the phosphopeptides migrate toward acidic pH values. Peptides and phosphopeptides are then identified by mass spectrometry (MS) and phosphopeptide spectra are manually checked for the assignment of phosphorylation sites. Here, we apply this methodology to investigate synaptosome extracts from whole mouse brain. IEF-based peptide separation is an efficient method for peptide and phosphopeptide identification.

  7. Using size exclusion chromatography-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling

    SciTech Connect

    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.

    2006-07-27

    Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated the capability for broad proteome coverage and good throughput, but is not ideally suited to the discovery and identification of modified proteins. Top-down proteomics (including subjecting intact protein ions to gas-phase dissociation) allows the study of modified proteins, but coverage, sensitivity and throughput are presently problematic. In this work, we describe the combination of bottom-up with intact protein analyses for the characterization of modified proteins. Fractionation at the intact protein level was employed to reduce complexity and increase measurement dynamic range. Bottom-up measurements were used to identify the subset of proteins that were present in each fraction. These identifications were then used in combination with high-accuracy Fourier-transform ion cyclotron resonance (FTICR)-mass spectrometry (MS) intact protein mass measurements to achieve protein and modified-protein identifications. The relative performance of size exclusion chromatography (SEC) fractionation combined with on-line reversed-phase liquid chromatography (RPLC)-FTICR-MS was compared with RPLC fractionation combined with capillary isoelectric focusing (CIEF)-FTICR-MS. Finally, the relative coverage provided by proteomic analyses based on tryptic peptides and intact proteins is considered.

  8. Computational Drug Target Screening through Protein Interaction Profiles

    PubMed Central

    Vilar, Santiago; Quezada, Elías; Uriarte, Eugenio; Costanzi, Stefano; Borges, Fernanda; Viña, Dolores; Hripcsak, George

    2016-01-01

    The development of computational methods to discover novel drug-target interactions on a large scale is of great interest. We propose a new method for virtual screening based on protein interaction profile similarity to discover new targets for molecules, including existing drugs. We calculated Target Interaction Profile Fingerprints (TIPFs) based on ChEMBL database to evaluate drug similarity and generated new putative compound-target candidates from the non-intersecting targets in each pair of compounds. A set of drugs was further studied in monoamine oxidase B (MAO-B) and cyclooxygenase-1 (COX-1) enzyme through molecular docking and experimental assays. The drug ethoxzolamide and the natural compound piperlongumine, present in Piper longum L, showed hMAO-B activity with IC50 values of 25 and 65 μM respectively. Five candidates, including lapatinib, SB-202190, RO-316233, GW786460X and indirubin-3′-monoxime were tested against human COX-1. Compounds SB-202190 and RO-316233 showed a IC50 in hCOX-1 of 24 and 25 μM respectively (similar range as potent inhibitors such as diclofenac and indomethacin in the same experimental conditions). Lapatinib and indirubin-3′-monoxime showed moderate hCOX-1 activity (19.5% and 28% of enzyme inhibition at 25 μM respectively). Our modeling constitutes a multi-target predictor for large scale virtual screening with potential in lead discovery, repositioning and drug safety. PMID:27845365

  9. 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.

  10. Target identification with quantitative activity based protein profiling (ABPP).

    PubMed

    Chen, Xiao; Wong, Yin Kwan; Wang, Jigang; Zhang, Jianbin; Lee, Yew-Mun; Shen, Han-Ming; Lin, Qingsong; Hua, Zi-Chun

    2017-02-01

    As many small bioactive molecules fulfill their functions through interacting with protein targets, the identification of such targets is crucial in understanding their mechanisms of action (MOA) and side effects. With technological advancements in target identification, it has become possible to accurately and comprehensively study the MOA and side effects of small molecules. While small molecules with therapeutic potential were derived solely from nature in the past, the remodeling and synthesis of such molecules have now been made possible. Presently, while some small molecules have seen successful application as drugs, the majority remain undeveloped, requiring further understanding of their MOA and side effects to fully tap into their potential. Given the typical promiscuity of many small molecules and the complexity of the cellular proteome, a high-flux and high-accuracy method is necessary. While affinity chromatography approaches combined with MS have had successes in target identification, limitations associated with nonspecific results remain. To overcome these complications, quantitative chemical proteomics approaches have been developed including metabolic labeling, chemical labeling, and label-free methods. These new approaches are adopted in conjunction with activity-based protein profiling (ABPP), allowing for a rapid process and accurate results. This review will briefly introduce the principles involved in ABPP, then summarize current advances in quantitative chemical proteomics approaches as well as illustrate with examples how ABPP coupled with quantitative chemical proteomics has been used to detect the targets of drugs and other bioactive small molecules including natural products.

  11. 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

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

    PubMed

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

    2016-02-15

    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.

  13. 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/.

  14. Translational Profiling of Clock Cells Reveals Circadianly Synchronized Protein Synthesis

    PubMed Central

    Huang, Yanmei; Ainsley, Joshua A.; Reijmers, Leon G.; Jackson, F. Rob

    2013-01-01

    Abstract Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a Drosophila cell-specific translational profiling approach to document the rhythmic “translatome” of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts—as assayed by mRNA ribosome association—occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes—metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others—are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes Tdc2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity. PMID:24348200

  15. Three-level prediction of protein function by combining profile-sequence search, profile-profile search, and domain co-occurrence networks.

    PubMed

    Wang, Zheng; Cao, Renzhi; Cheng, Jianlin

    2013-01-01

    Predicting protein function from sequence is useful for biochemical experiment design, mutagenesis analysis, protein engineering, protein design, biological pathway analysis, drug design, disease diagnosis, and genome annotation as a vast number of protein sequences with unknown function are routinely being generated by DNA, RNA and protein sequencing in the genomic era. However, despite significant progresses in the last several years, the accuracy of protein function prediction still needs to be improved in order to be used effectively in practice, particularly when little or no homology exists between a target protein and proteins with annotated function. Here, we developed a method that integrated profile-sequence alignment, profile-profile alignment, and Domain Co-Occurrence Networks (DCN) to predict protein function at different levels of complexity, ranging from obvious homology, to remote homology, to no homology. We tested the method blindingly in the 2011 Critical Assessment of Function Annotation (CAFA). Our experiments demonstrated that our three-level prediction method effectively increased the recall of function prediction while maintaining a reasonable precision. Particularly, our method can predict function terms defined by the Gene Ontology more accurately than three standard baseline methods in most situations, handle multi-domain proteins naturally, and make ab initio function prediction when no homology exists. These results show that our approach can combine complementary strengths of most widely used BLAST-based function prediction methods, rarely used in function prediction but more sensitive profile-profile comparison-based homology detection methods, and non-homology-based domain co-occurrence networks, to effectively extend the power of function prediction from high homology, to low homology, to no homology (ab initio cases).

  16. Three-Level Prediction of Protein Function by Combining Profile-Sequence Search, Profile-Profile Search, and Domain Co-Occurrence Networks

    PubMed Central

    2013-01-01

    Predicting protein function from sequence is useful for biochemical experiment design, mutagenesis analysis, protein engineering, protein design, biological pathway analysis, drug design, disease diagnosis, and genome annotation as a vast number of protein sequences with unknown function are routinely being generated by DNA, RNA and protein sequencing in the genomic era. However, despite significant progresses in the last several years, the accuracy of protein function prediction still needs to be improved in order to be used effectively in practice, particularly when little or no homology exists between a target protein and proteins with annotated function. Here, we developed a method that integrated profile-sequence alignment, profile-profile alignment, and Domain Co-Occurrence Networks (DCN) to predict protein function at different levels of complexity, ranging from obvious homology, to remote homology, to no homology. We tested the method blindingly in the 2011 Critical Assessment of Function Annotation (CAFA). Our experiments demonstrated that our three-level prediction method effectively increased the recall of function prediction while maintaining a reasonable precision. Particularly, our method can predict function terms defined by the Gene Ontology more accurately than three standard baseline methods in most situations, handle multi-domain proteins naturally, and make ab initio function prediction when no homology exists. These results show that our approach can combine complementary strengths of most widely used BLAST-based function prediction methods, rarely used in function prediction but more sensitive profile-profile comparison-based homology detection methods, and non-homology-based domain co-occurrence networks, to effectively extend the power of function prediction from high homology, to low homology, to no homology (ab initio cases). PMID:23514381

  17. Convergent evolution in structural elements of proteins investigated using cross profile analysis

    PubMed Central

    2012-01-01

    Background Evolutionary relations of similar segments shared by different protein folds remain controversial, even though many examples of such segments have been found. To date, several methods such as those based on the results of structure comparisons, sequence-based classifications, and sequence-based profile-profile comparisons have been applied to identify such protein segments that possess local similarities in both sequence and structure across protein folds. However, to capture more precise sequence-structure relations, no method reported to date combines structure-based profiles, and sequence-based profiles based on evolutionary information. The former are generally regarded as representing the amino acid preferences at each position of a specific conformation of protein segment. They might reflect the nature of ancient short peptide ancestors, using the results of structural classifications of protein segments. Results This report describes the development and use of "Cross Profile Analysis" to compare sequence-based profiles and structure-based profiles based on amino acid occurrences at each position within a protein segment cluster. Using systematic cross profile analysis, we found structural clusters of 9-residue and 15-residue segments showing remarkably strong correlation with particular sequence profiles. These correlations reflect structural similarities among constituent segments of both sequence-based and structure-based profiles. We also report previously undetectable sequence-structure patterns that transcend protein family and fold boundaries, and present results of the conformational analysis of the deduced peptide of a segment cluster. These results suggest the existence of ancient short-peptide ancestors. Conclusions Cross profile analysis reveals the polyphyletic and convergent evolution of β-hairpin-like structures, which were verified both experimentally and computationally. The results presented here give us new insights into the

  18. Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop.

    PubMed Central

    Kettunen, R; Tyystjärvi, E; Aro, E M

    1996-01-01

    Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves. PMID:8756500

  19. Exploring Proteins in Anopheles gambiae Male and Female Antennae through MALDI Mass Spectrometry Profiling

    PubMed Central

    Dani, Francesca R.; Francese, Simona; Mastrobuoni, Guido; Felicioli, Antonio; Caputo, Beniamino; Simard, Frederic; Pieraccini, Giuseppe; Moneti, Gloriano; Coluzzi, Mario; della Torre, Alessandra; Turillazzi, Stefano

    2008-01-01

    MALDI profiling and imaging mass spectrometry (IMS) are novel techniques for direct analysis of peptides and small proteins in biological tissues. In this work we applied them to the study of Anopheles gambiae antennae, with the aim of analysing expression of soluble proteins involved in olfaction perireceptor events. MALDI spectra obtained by direct profiling on single antennae and by the analysis of extracts, showed similar profiles, although spectra obtained through profiling had a richer ion population and higher signal to noise ratio. Male and female antennae showed distinct protein profiles. MALDI imaging experiments were also performed and differences were observed in the localization of some proteins. Two proteins were identified through high resolution measurement and top-down MS/MS experiments. A 8 kDa protein only present in the male antennae matched with an unannotated sequence of the An. gambiae genome, while the presence of odorant binding protein 9 (OBP-9) was confirmed through experiments of 2-DE, followed by MS and MS/MS analysis of digested spots. This work shows that MALDI MS profiling is a technique suitable for the analysis of proteins of small and medium MW in insect appendices, and allows obtaining data for several specimens which can be investigated for differences between groups. Proteins of interest can be identified through other complementary MS approaches. PMID:18665262

  20. Coaxial atomizer liquid intact lengths

    NASA Technical Reports Server (NTRS)

    Eroglu, Hasan; Chigier, Norman; Farago, Zoltan

    1991-01-01

    Average intact lengths of round liquid jets generated by airblast coaxial atomizer were measured from over 1500 photographs. The intact lengths were studied over a jet Reynolds number range of 18,000 and Weber number range of 260. Results are presented for two different nozzle geometries. The intact lengths were found to be strongly dependent on Re and We numbers. An empirical equation was derived as a function of these parameters. A comparison of the intact lengths for round jets and flat sheets shows that round jets generate shorter intact lengths.

  1. Label-free detection of proteins in ternary mixtures using surface-enhanced Raman scattering and protein melting profiles

    NASA Astrophysics Data System (ADS)

    Keskin, Sercan; Efeoğlu, Esen; Keçeci, Kaan; Çulha, Mustafa

    2013-03-01

    The multiplex detection of biologically important molecules such as proteins in complex mixtures has critical importance not only in disease diagnosis but also in other fields such as proteomics and biotechnology. Surface-enhanced Raman scattering (SERS) is a powerful technique for multiplex identification of molecular components in a mixture. We combined the multiplexing power of SERS and heat denaturation of proteins to identify proteins in ternary protein mixtures. The heat denaturation profiles of four model blood proteins, transferrin, human serum albumin, fibrinogen, and hemoglobin, were studied with SERS. Then, two ternary mixtures of these four proteins were used to test the feasibility of the approach. It was demonstrated that unique denaturation profiles of each protein could be used for their identification in the mixture.

  2. 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

  3. STRUCTFAST: protein sequence remote homology detection and alignment using novel dynamic programming and profile-profile scoring.

    PubMed

    Debe, Derek A; Danzer, Joseph F; Goddard, William A; Poleksic, Aleksandar

    2006-09-01

    STRUCTFAST is a novel profile-profile alignment algorithm capable of detecting weak similarities between protein sequences. The increased sensitivity and accuracy of the STRUCTFAST method are achieved through several unique features. First, the algorithm utilizes a novel dynamic programming engine capable of incorporating important information from a structural family directly into the alignment process. Second, the algorithm employs a rigorous analytical formula for profile-profile scoring to overcome the limitations of ad hoc scoring functions that require adjustable parameter training. Third, the algorithm employs Convergent Island Statistics (CIS) to compute the statistical significance of alignment scores independently for each pair of sequences. STRUCTFAST routinely produces alignments that meet or exceed the quality obtained by an expert human homology modeler, as evidenced by its performance in the latest CAFASP4 and CASP6 blind prediction benchmark experiments.

  4. Enhancing the Pharmacokinetic Profile of Protein-Based Drugs

    DTIC Science & Technology

    2014-06-12

    bioavailability when given subcutaneously. Extend Biosciences is developing proprietary carrier molecules that will allow proteins to access a...in the development of longer-lasting versions of bioscavenger proteins that could then be delivered subcutaneously and become bioavailable within...protein-based drugs have limited efficacy due to a short half-life or require intravenous delivery because of low bioavailability when given

  5. Factors affecting the reactivation of the oligomycin-sensitive adenosine 5'-triphosphatase and the release of ATPase inhibitor protein during the re-energization of intact mitochondria from ischemic cardiac muscle.

    PubMed

    Rouslin, W

    1987-03-15

    In the present study we examined factors affecting the reversal of the ischemia-induced protonic inhibition of the mitochondrial ATPase described earlier (Rouslin, W. (1983) J. Biol. Chem. 258, 9657-9661). It was found that ATPase reactivation and accompanying inhibitor protein release during the re-energization of intact mitochondria isolated from 20-min ischemic canine heart muscle could be blocked completely by either carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) or nigericin but was unaffected by valinomycin at 35 mM K+. At higher K+ concentrations, valinomycin also blocked ATPase reactivation but not quite as completely as did nigericin. These observations suggest that ATPase reactivation and inhibitor protein release are particularly dependent upon either the trans-inner membrane pH gradient (delta pH) or possibly upon matrix pH per se and slightly less dependent upon membrane potential (delta psi) in intact cardiac muscle mitochondria. The addition of FCCP at the end of the re-energization incubations limited partially the extent of both ATPase reactivation and inhibitor protein release. This latter effect appears to have been mediated by a partial reassociation of the inhibitor protein with the enzyme, and it was accentuated (when FCCP was added at the end of the incubations) or mimicked (when FCCP was absent) by lowering the pH of the re-energization medium. A close examination of the first 10 min of the time course of enzyme activation and of inhibitor protein release revealed that while the former process was essentially finished in 1 min or less, the latter required approximately 10 min for completion. This observation led to the proposal of a two-site model of enzyme-inhibitor interaction which is discussed.

  6. (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.

  7. Triton shells of intact erythrocytes.

    PubMed

    Sheetz, M P; Sawyer, D

    1978-01-01

    About 40% of human erythrocyte membrane protein is resistant to solubilization in 0.5% Triton X-114. These components comprise a structure called a Triton shell roughly similar in size and shape to the original erythrocyte and thus constitute a cytoskeleton. With increasing concentrations of Triton the lipid content of the Triton shell decreases dramatically, whereas the majority of the protein components remain constant. Exceptions to this rule include proteins contained in band 3, the presumed anion channel, and in band 4 which decrease with increasing Triton concentration. The Triton-insoluble complex includes spectrin (bands 1 and 2), actin (band 5), and bands 3' and 7. Component 3' has an apparent molecular weight of 88,000 daltons as does 3; but unlike 3, it is insensitive to protease treatment of the intact cell, has a low extinction coefficient at 280 nm, and is solubilized from the shells in alkaline water solutions. Component 7 also has a low extinction coefficient at 280 nm. Spectrin alone is solubilized from the Triton shells in isotonic media. The solubilized spectrin contains no bound Triton and coelectrophoreses with spectrin eluted in hypotonic solutions from ghosts. Electron micrographs of fixed Triton shells stained with uranyl acetate show the presence of numerous filaments which appear beaded and are 80--120 A in diameter. The filaments cannot be composed mainly af actin, but enough spectrin is present to form the filaments. Triton shells may provide an excellent source of material useful in the investigation of the erythrocyte cytoskeleton.

  8. Emory University: High-Throughput Protein-Protein Interaction Analysis for Hippo Pathway Profiling | Office of Cancer Genomics

    Cancer.gov

    The CTD2 Center at Emory University used high-throughput protein-protein interaction (PPI) mapping for Hippo signaling pathway profiling to rapidly unveil promising PPIs as potential therapeutic targets and advance functional understanding of signaling circuitry in cells. Read the abstract.

  9. 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.

  10. 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.

  11. 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

    2014-10-08

    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.

  12. Chemical Probes to Directly Profile Palmitoleoylation of Proteins.

    PubMed

    Zheng, Baohui; Jarugumilli, Gopala K; Chen, Baoen; Wu, Xu

    2016-11-03

    Palmitoleoylation is a unique fatty acylation of proteins in which a monounsaturated fatty acid, palmitoleic acid (C16:1), is covalently attached to a protein. Wnt proteins are known to be palmitoleoylated by cis-Δ9 palmitoleate at conserved serine residues. O-palmitoleoylation plays a critical role in regulating Wnt secretion, binding to the receptors, and in the dynamics of Wnt signaling. Therefore, protein palmitoleoylation is important in tissue homeostasis and tumorigenesis. Chemical probes based on saturated fatty acids, such as ω-alkynyl palmitic acid (Alk-14 or Alk-C16 ), have been used to study Wnt palmitoleoylation. However, such probes require prior conversion to the unsaturated fatty acid by stearoyl-CoA desaturase (SCD) in cells, significantly decreasing their selectivity and efficiency for studying protein palmitoleoylation. We synthesized and characterized ω-alkynyl cis- and trans-palmitoleic acids (cis- and trans-Alk-14:1) as chemical probes to directly study protein palmitoleoylation. We found that cis-Alk-14:1 could more efficiently label Wnt proteins in cells. Interestingly, the DHHC family of palmitoyl acyltransferases can charge both saturated and unsaturated fatty acids, potentially using both as acyl donors in protein palmitoylation and palmitoleoylation. Furthermore, proteomic analysis of targets labeled by these probes revealed new cis- and trans-palmitoleoylated proteins. Our studies provided new chemical tools and revealed new insights into palmitoleoylation in cell signaling.

  13. Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

    PubMed Central

    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. PMID:22474537

  14. Intact Cell MALDI-TOF MS on Sperm: A Molecular Test For Male Fertility Diagnosis.

    PubMed

    Soler, Laura; Labas, Valérie; Thélie, Aurore; Grasseau, Isabelle; Teixeira-Gomes, Ana-Paula; Blesbois, Elisabeth

    2016-06-01

    Currently, evaluation of sperm quality is primarily based on in vitro measures of sperm function such as motility, viability and/or acrosome reaction. However, results are often poorly correlated with fertility, and alternative diagnostic tools are therefore needed both in veterinary and human medicine. In a recent pilot study, we demonstrated that MS profiles from intact chicken sperm using MALDI-TOF profiles could detect significant differences between fertile/subfertile spermatozoa showing that such profiles could be useful for in vitro male fertility testing. In the present study, we performed larger standardized experimental procedures designed for the development of fertility- predictive mathematical models based on sperm cell MALDI-TOF MS profiles acquired through a fast, automated method. This intact cell MALDI-TOF MS-based method showed high diagnostic accuracy in identifying fertile/subfertile males in a large male population of known fertility from two distinct genetic lineages (meat and egg laying lines). We additionally identified 40% of the m/z peaks observed in sperm MS profiles through a top-down high-resolution protein identification analysis. This revealed that the MALDI-TOF MS spectra obtained from intact sperm cells contained a large proportion of protein degradation products, many implicated in important functional pathways in sperm such as energy metabolism, structure and movement. Proteins identified by our predictive model included diverse and important functional classes providing new insights into sperm function as it relates to fertility differences in this experimental system. Thus, in addition to the chicken model system developed here, with the use of appropriate models these methods should effectively translate to other animal taxa where similar tests for fertility are warranted.

  15. RNA Whole-Mount In Situ Hybridization Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

    PubMed

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

    2017-03-03

    Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein interactions as a consequence of the complexities associated with working with RNA. This unit describes a method for the adaptation of the In Situ Hybridization-Proximity Ligation Assay (ISH-PLA) to the study of RNA regulation (rISH-PLA). 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 heterogeneous 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. © 2017 by John Wiley & Sons, Inc.

  16. Correlations Between Single Cell Signaling Dynamics and Protein Expressions Profiles

    DTIC Science & Technology

    2005-08-16

    the need for fluorescent or radioactive labels. These deter- minations have been performed within picoliter volumes using microfluidic channels...developments are addressing this. Future efforts will fully integrate the microfluidic nanophysiometer, OCIBD analyte detection system, MALDI-TOF protein...upon full integration of the microfluidic nanophysiometer, OCIBD analyte detection system, MALDI-TOF protein traps, and cell loading (for internalization

  17. 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.

  18. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    PubMed Central

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-01-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing. PMID:28290550

  19. 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.

  20. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    NASA Astrophysics Data System (ADS)

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-03-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  1. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding.

    PubMed

    Shahi, Payam; Kim, Samuel C; Haliburton, John R; Gartner, Zev J; Abate, Adam R

    2017-03-14

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  2. 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.

  3. 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.

  4. Early Lung Cancer Detection via Global Protein Modification Profiles

    DTIC Science & Technology

    2013-12-01

    remission at 3 years (low risk) following diagnosis (Figure 2). The top graph shows the mean difference in the observed expression of the first 100...in patients with remission at 3 years. These preliminary results suggest that the PTM profiles of Lung cancer tumors with poor prognosis may be...highly divergent from that of tumors from patients that were in remission at 3 years following diagnosis and these differences can be detected using

  5. Proteomic analysis of Clostridium thermocellum core metabolism: relative protein expression profiles and growth phase-dependent changes in protein expression

    PubMed Central

    2012-01-01

    Background Clostridium thermocellum produces H2 and ethanol, as well as CO2, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase. Results Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase

  6. Ribosome profiling: a Hi-Def monitor for protein synthesis at the genome-wide scale

    PubMed Central

    Michel, Audrey M; Baranov, Pavel V

    2013-01-01

    Ribosome profiling or ribo-seq is a new technique that provides genome-wide information on protein synthesis (GWIPS) in vivo. It is based on the deep sequencing of ribosome protected mRNA fragments allowing the measurement of ribosome density along all RNA molecules present in the cell. At the same time, the high resolution of this technique allows detailed analysis of ribosome density on individual RNAs. Since its invention, the ribosome profiling technique has been utilized in a range of studies in both prokaryotic and eukaryotic organisms. Several studies have adapted and refined the original ribosome profiling protocol for studying specific aspects of translation. Ribosome profiling of initiating ribosomes has been used to map sites of translation initiation. These studies revealed the surprisingly complex organization of translation initiation sites in eukaryotes. Multiple initiation sites are responsible for the generation of N-terminally extended and truncated isoforms of known proteins as well as for the translation of numerous open reading frames (ORFs), upstream of protein coding ORFs. Ribosome profiling of elongating ribosomes has been used for measuring differential gene expression at the level of translation, the identification of novel protein coding genes and ribosome pausing. It has also provided data for developing quantitative models of translation. Although only a dozen or so ribosome profiling datasets have been published so far, they have already dramatically changed our understanding of translational control and have led to new hypotheses regarding the origin of protein coding genes. © 2013 John Wiley & Sons, Ltd. PMID:23696005

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

    2012-05-07

    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

  12. 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

  13. 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

  14. 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)

  15. 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.

  16. The polar profile of ancient proteins: a computational extrapolation from prebiotics to paleobiochemistry.

    PubMed

    Polanco, Carlos; Buhse, Thomas; Vizcaíno, Gloria; Picciotto, Jacobo Levy

    2017-01-01

    This paper addresses the polar profile of ancient proteins using a comparative study of amino acids found in 25 000 000-year-old shells described in Abelson's work. We simulated the polar profile with a computer platform that represented an evolutionary computational toy model that mimicked the generation of small proteins starting from a pool of monomeric amino acids and that included several dynamic properties, such as self-replication and fragmentation-recombination of the proteins. The simulations were taken up to 15 generations and produced a considerable number of proteins of 25 amino acids in length. The computational model included the amino acids found in the ancient shells, the thermal degradation factor, and the relative abundance of the amino acids observed in the Miller-Urey experimental simulation of the prebiotic amino acid formation. We found that the amino acid polar profiles of the ancient shells and those simulated and extrapolated from the Miller-Urey abundances are coincident.

  17. 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.

  18. Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

    PubMed

    Pertl-Obermeyer, Heidi; Wu, Xu Na; Schrodt, Jens; Müdsam, Christina; Obermeyer, Gerhard; Schulze, Waltraud X

    2016-09-01

    Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

  19. Milk protein profiles in response to Streptococcus agalactiae subclinical mastitis in dairy cows.

    PubMed

    Pongthaisong, Pongphol; Katawatin, Suporn; Thamrongyoswittayakul, Chaiyapas; Roytrakul, Sittiruk

    2016-01-01

    The objective of this study was to investigate the milk protein profiles of normal milk and those of milk during the course of subclinical mastitis, caused by natural Streptococcus agalactiae infection. Two-dimensional gel electrophoresis and liquid chromatography mass spectrometry were used to assess protein profiles and to identify the proteins. The results showed that S. agalactiae subclinical mastitis altered the protein profiles of milk. Following Mascot database matching, 11 and 12 protein types were identified in the milk collected from healthy and S. agalactiae subclinical mastitic udders, respectively. The distinct presence of the antibacterial protein cathelicidin-1 was detected in infected milk samples, which in turn was highly correlated to the severity of subclinical mastitis as represented by the milk somatic cell count (r = 0.616), but not the bacterial count. The protein profile of milk reveals changes in the host response to S. agalactiae intramammary infection; cathelicidin-1 could therefore serve as a biomarker for the detection of subclinical mastitis in dairy cows.

  20. Protein profile of exhaled breath condensate determined by high resolution mass spectrometry.

    PubMed

    Muccilli, Vera; Saletti, Rosaria; Cunsolo, Vincenzo; Ho, Jenny; Gili, Elisa; Conte, Enrico; Sichili, Stefania; Vancheri, Carlo; Foti, Salvatore

    2015-02-01

    A method based on liquid chromatography/high resolution tandem mass spectrometry coupled with electrophoretic separation, for determination and relative quantification of the protein composition of exhaled breath condensate (EBC), was developed. Application of the procedure to a sample of EBC, pooled from nine healthy subjects, resulted in the identification of 167 unique gene products, 113 of which not previously reported in EBC samples. The abundance of the protein identified was estimated by means of the exponentially modified protein abundance index protocol (emPAI). Cytokeratins were by far the most abundant proteins in EBC samples. Many of the identified proteins were associated with multiple cellular location with cytoplasm constituting the largest group. Cytosol, nucleus, membrane, cytoskeleton and extracellular were other abundantly represented locations. No amylase was detected, suggesting the absence of saliva protein contamination. The profile obtained represents the most comprehensive protein characterization of EBC so far reported and demonstrates that this approach provides a powerful tool for investigating the protein profile of EBC samples. Compared with analogous investigations, this study also shows that the protein profile of EBC is strongly affected by the sampling method adopted.

  1. Sorting competition with membrane-permeable peptides in intact epithelial cells revealed discrimination of transmembrane proteins not only at the trans-Golgi network but also at pre-Golgi stages.

    PubMed

    Soza, Andrea; Norambuena, Andrés; Cancino, Jorge; de la Fuente, Erwin; Henklein, Peter; González, Alfonso

    2004-04-23

    Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif.

  2. Profiling of Urine Using ProteinChip Technology

    DTIC Science & Technology

    2012-01-01

    Lindner, S., Meyer, M., Asif, A.R., Oellerich, M., Strutz, F. (2007) Characterization of Diabetic Nephropathy by Urinary Proteomic Analysis...Identifi cation of a Processed Ubiquitin Form as a Differentially Excreted Protein in Diabetic Nephropathy Patients. Clin Chem. 53 : 1636–1645 . 2...Thadhani, R. (2007) Prediction of Diabetic Nephropathy Using Urine Proteomic Profi ling 10 Years Prior to Development of Nephropathy . Diabetes Care. 30

  3. Evaluation of a new wide pore core-shell material (Aeris WIDEPORE) and comparison with other existing stationary phases for the analysis of intact proteins.

    PubMed

    Fekete, Szabolcs; Berky, Róbert; Fekete, Jenő; Veuthey, Jean-Luc; Guillarme, Davy

    2012-05-04

    The separation of large biomolecules such as proteins or monoclonal antibodies (mAbs) by RPLC can be drastically enhanced thanks to the use of columns packed with wide-pore porous sub-2 μm particles or shell particles. In this context, a new wide-pore core-shell material has been recently released under the trademark Aeris WIDEPORE. It is made of a 3.2 μm solid inner core surrounded by a 0.2 μm porous layer (total particle size of 3.6 μm). The aim of this study was to evaluate the performance of this new material, compare it to other recently developed and older conventional wide-pore columns and demonstrate its applicability to real-life separations of proteins and mAbs. At first, the traditional h(min) values of the Aeris WIDEPORE column were determined for small model compounds. The h(min) values were equal to 1.7-1.8 and 1.4 for the 2.1 and 4.6 mm I.D. columns, respectively, which are in agreement with the values reported for other core-shell materials. In the case of a small protein Insulin (5.7 kDa), the achievable lowest h value was below 2 and this impressive result confirms that the Aeris WIDEPORE material should be dedicated to protein analysis. This column was then compared with five other commercially available wide-pore and medium-pore stationary phases, in the gradient elution mode, using various flow rates, gradient steepness and model proteins of MW=5.7-66.8 kDa. The Aeris WIDEPORE material often provided the best performance, in terms of peak capacity, peak capacity per time and pressure unit (PPT) and also based on the gradient kinetic plot representation. Finally, real separations of filgrastim (18.8 kDa) and its oxidized and reduced forms were performed on the different columns and the Aeris WIDEPORE material provided the most impressive performance (peak capacity>100 for t(grad)<6 min). Last but not least, this new material was also evaluated on digested and reduced mAb and powerful, high-throughput separations were also attained.

  4. Tools for Interpreting Large-Scale Protein Profiling in Microbiology

    PubMed Central

    Hendrickson, E. L.; Lamont, R. J.; Hackett, M.

    2009-01-01

    Quantitative proteome analysis of microbial systems generates large datasets that can be difficult and time consuming to interpret. Fortunately, many of the data display and gene clustering tools developed to analyze large transcriptome microarray datasets are also applicable to proteomes. Plots of abundance ratio versus total signal or spectral counts can highlight regions of random error and putative change. Displaying data in the physical order of the genes in the genome sequence can highlight potential operons. At a basic level of transcriptional organization, identifying operons can give insights into regulatory pathways as well as provide corroborating evidence for proteomic results. Classification and clustering algorithms can group proteins together by their abundance changes under different conditions, helping to identify interesting expression patterns, but often work poorly with noisy data like that typically generated in a large-scale proteome analysis. Biological interpretation can be aided more directly by overlaying differential protein abundance data onto metabolic pathways, indicating pathways with altered activities. More broadly, ontology tools detect altered levels of protein abundance for different metabolic pathways, molecular functions and cellular localizations. In practice, pathway analysis and ontology are limited by the level of database curation associated with the organism of interest. PMID:18946006

  5. Structural Analysis of the Glycosylated Intact HIV-1 gp120–b12 Antibody Complex Using Hydroxyl Radical Protein Footprinting

    PubMed Central

    2017-01-01

    Glycoprotein gp120 is a surface antigen and virulence factor of human immunodeficiency virus 1. Broadly neutralizing antibodies (bNAbs) that react to gp120 from a variety of HIV isolates offer hope for the development of broadly effective immunogens for vaccination purposes, if the interactions between gp120 and bNAbs can be understood. From a structural perspective, gp120 is a particularly difficult system because of its size, the presence of multiple flexible regions, and the large amount of glycosylation, all of which are important in gp120–bNAb interactions. Here, the interaction of full-length, glycosylated gp120 with bNAb b12 is probed using high-resolution hydroxyl radical protein footprinting (HR-HRPF) by fast photochemical oxidation of proteins. HR-HRPF allows for the measurement of changes in the average solvent accessible surface area of multiple amino acids without the need for measures that might alter the protein conformation, such as mutagenesis. HR-HRPF of the gp120–b12 complex coupled with computational modeling shows a novel extensive interaction of the V1/V2 domain, probably with the light chain of b12. Our data also reveal HR-HRPF protection in the C3 domain caused by interaction of the N330 glycan with the b12 light chain. In addition to providing information about the interactions of full-length, glycosylated gp120 with b12, this work serves as a template for the structural interrogation of full-length glycosylated gp120 with other bNAbs to better characterize the interactions that drive the broad specificity of the bNAb. PMID:28102671

  6. 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.

  7. 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.

  8. Dynamic profiling of double-stranded RNA binding proteins

    PubMed Central

    Wang, Xinlei; Vukovic, Lela; Koh, Hye Ran; Schulten, Klaus; Myong, Sua

    2015-01-01

    Double-stranded (ds) RNA is a key player in numerous biological activities in cells, including RNA interference, anti-viral immunity and mRNA transport. The class of proteins responsible for recognizing dsRNA is termed double-stranded RNA binding proteins (dsRBP). However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 on six dsRNA substrates that vary in length and secondary structure. We combined single molecule pull-down (SiMPull), single molecule protein-induced fluorescence enhancement (smPIFE) and molecular dynamics (MD) simulations to investigate the dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificities and dynamic properties when in contact with different RNA substrates. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to highly structured RNA substrates. Upon interaction with RNA substrates, TRBP and Staufen1 exhibit dynamic sliding whereas two deaminases ADAR1 and ADAD2 mostly remain immobile when bound. MD simulations provide a detailed atomic interaction map that is largely consistent with the affinity differences observed experimentally. Collectively, our study highlights the diverse nature of substrate specificity and mobility exhibited by dsRBPs that may be critical for their cellular function. PMID:26184879

  9. Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations.

    PubMed

    Smith, Elizabeth M; Hennen, Jared; Chen, Yan; Mueller, Joachim D

    2015-07-01

    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. Furthermore, 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 delta 1 labeled with green fluorescent protein on 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.

  10. Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria.

    PubMed

    Cammarano, P; Mazzei, F; Londei, P; Teichner, A; de Rosa, M; Gambacorta, A

    1983-08-02

    Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release r

  11. Normal tear protein profiles and age-related changes.

    PubMed Central

    McGill, J I; Liakos, G M; Goulding, N; Seal, D V

    1984-01-01

    The specific and non-specific tear proteins have been analysed by means of the ELISA technique to establish the normal and age-related values. There is a linear and related decline of lysozyme and lactoferrin with age, and a similar but unrelated reduction in tear volume. IgA levels gradually decline, while caeruloplasmin and IgG both increase after the fifth decade. The results suggest that tear IgG and caeruloplasmin are probably transudates from the serum, that IgA is secreted independently of tear volume, and that lysozyme and lactoferrin are secreted at the same site but independently of tear volume. PMID:6712908

  12. 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.

  13. Effectiveness of intact capture media

    SciTech Connect

    Tsou, P.; Aubert, J.; Brownlee, D.; Hrubesh, L.; Williams, J.; Albee, A.

    1989-01-01

    The possibility of capturing cosmic dust at hypervelocity has been demonstrated in the laboratory and in the unintended Solar Max spacecraft. This technology will enable a comet coma sample return mission and be important for the earth orbital cosmic dust collection mission, i.e., the Space Station Cosmic Dust Collection Facility. Since the only controllable factor in an intact capture of cosmic dust is the capturing medium, characterizing the effectiveness and properties of available capture media would be very important in the development of the technique for capturing hypervelocity cosmic dust intact. We have evaluated various capture underdense media for the relative effectiveness for intact capture. 2 refs., 2 figs.

  14. Protein turnover, amino acid profile and amino acid flux in juvenile shrimp Litopenaeus vannamei: effects of dietary protein source.

    PubMed

    Mente, Eleni; Coutteau, Peter; Houlihan, Dominic; Davidson, Ian; Sorgeloos, Patrick

    2002-10-01

    The effect of dietary protein on protein synthesis and growth of juvenile shrimps Litopenaeus vannamei was investigated using three different diets with equivalent protein content. Protein synthesis was investigated by a flooding dose of tritiated phenylalanine. Survival, specific growth and protein synthesis rates were higher, and protein degradation was lower, in shrimps fed a fish/squid/shrimp meal diet, or a 50% laboratory diet/50% soybean meal variant diet, than in those fed a casein-based diet. The efficiency of retention of synthesized protein as growth was 94% for shrimps fed the fish meal diet, suggesting a very low protein turnover rate; by contrast, the retention of synthesized protein was only 80% for shrimps fed the casein diet. The amino acid profile of the casein diet was poorly correlated with that of the shrimps. 4 h after a single meal the protein synthesis rates increased following an increase in RNA activity. A model was developed for amino acid flux, suggesting that high growth rates involve a reduction in the turnover of proteins, while amino acid loss appears to be high.

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

    PubMed Central

    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 α2δ-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

  16. Anatomical profiling of G protein-coupled receptor expression

    PubMed Central

    Regard, Jean B.; Sato, Isaac T.; Coughlin, Shaun R.

    2008-01-01

    Summary G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane signaling molecules and regulate a host of physiological and disease processes. To better understand the functions of GPCRs in vivo, we quantified transcript levels of 353 non-odorant GPCRs in 41 adult mouse tissues. Cluster analysis placed many GPCRs into anticipated anatomical and functional groups and predicted novel roles for less studied receptors. From one such prediction, we showed that the Gpr91 ligand succinate can regulate lipolysis in white adipose tissue suggesting that signaling by this citric acid cycle intermediate may regulate energy homeostasis. We also showed that pairwise analysis of GPCR expression across tissues may help predict drug side effects. This resource will aid studies to understand GPCR function in vivo and may assist in the identification of therapeutic targets. PMID:18984166

  17. Changes in the 2-DE protein profile during zygotic embryogenesis in the Brazilian Pine (Araucaria angustifolia).

    PubMed

    Balbuena, Tiago S; Silveira, Vanildo; Junqueira, Magno; Dias, Leonardo L C; Santa-Catarina, Claudete; Shevchenko, Andrej; Floh, Eny I S

    2009-04-13

    Araucaria angustifolia is the only native conifer of economic importance in the Brazilian Atlantic Rainforest. Due to a clear-cutting form of exploitation this species has received the status of vulnerable. The aim of this work was to investigate and characterize changes in protein expression profile during seed development of this endangered species. For this, the proteome of developing seeds was characterized by 2-DE and LC-MS/MS. Ninety six proteins were confidently identified and classified according to their biological function and expression profile. Overaccumulated proteins in early seed development indicated a higher control on oxidative stress metabolism during this phase. In contrast, highly expressed proteins in late stages revealed an active metabolism, leading to carbon assimilation and storage compounds accumulation. Comprehensive protein expression profiles and identification of overaccumulated proteins provide new insights into the process of embryogenesis in this recalcitrant species. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed.

  18. Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

    PubMed Central

    Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

    2011-01-01

    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

  19. 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.

  20. 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.

  1. A (1)H NMR metabolic profiling to the assessment of protein tyrosine phosphatase 1B role in liver regeneration after partial hepatectomy.

    PubMed

    Samino, Sara; Revuelta-Cervantes, Jesús; Vinaixa, Maria; Rodríguez, Miguel Ángel; Valverde, Angela M; Correig, Xavier

    2013-04-01

    Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the tyrosine kinase growth factor signaling pathway, which is involved in major physiological mechanisms such as liver regeneration. We investigate early hepatic metabolic events produced by partial hepatectomy (PHx) for PTP1B deficient (PTP1B KO) and wild type (WT) mice using proton nuclear magnetic resonance spectroscopy. Metabolic response of the two genotypes produced 24 h upon PHx is compared using magic angle spinning high-resolution nuclear magnetic resonance ((1)H-HR-MAS-NMR) on intact liver tissues. In addition, genotype-associated metabolic profile changes were monitored during the first 48 h after PHx using high-resolution nuclear magnetic resonance ((1)H-HR-NMR) on liver extracts. A marked increase of lipid-related signals in regenerating livers was observed after 24 h PHx in either intact tissues or liver extracts studies. In spite of this common initial metabolic response, results obtained 48 h after PHx on liver extracts indicate a genotype-differential metabolic pattern. This metabolic pattern resulted in line with well known regenerative features such as more sustained cell proliferation, a better management of lipids as energy fuel and lessened liver injury for PTP1B KO mice as compared to WT. Taken together, these findings suggest the metabolic basis to the pivotal role of PTP1B in liver regeneration.

  2. 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.

  3. 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.

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

    PubMed Central

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

    2011-01-01

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

  5. Extracellular α-synuclein induces sphingosine 1-phosphate receptor subtype 1 uncoupled from inhibitory G-protein leaving β-arrestin signal intact

    PubMed Central

    Zhang, Lifang; Okada, Taro; Badawy, Shaymaa Mohamed Mohamed; Hirai, Chihoko; Kajimoto, Taketoshi; Nakamura, Shun-ichi

    2017-01-01

    Parkinson’s disease (PD) is the second most common neurodegenerative disorder. The presence of α-synuclein (α-Syn)-positive intracytoplasmic inclusions, known as Lewy bodies, is the cytopathological hallmark of PD. Increasing bodies of evidence suggest that cell-to-cell transmission of α-Syn plays a role in the progression of PD. Although extracellular α-Syn is known to cause abnormal cell motility, the precise mechanism remains elusive. Here we show that impairment of platelet-derived growth factor-induced cell motility caused by extracellular α-Syn is mainly attributed to selective inhibition of sphingosine 1-phosphate (S1P) signalling. Treatment of human neuroblastoma cells with recombinant α-Syn caused S1P type 1 (S1P1) receptor-selective uncoupling from inhibitory G-protein (Gi) as determined by both functional and fluorescence resonance energy transfer (FRET)-based structural analyses. By contrast, α-Syn caused little or no effect on S1P2 receptor-mediated signalling. Both wild-type and α-Syn(A53T), a mutant found in familiar PD, caused uncoupling of S1P1 receptor, although α-Syn(A53T) showed stronger potency in uncoupling. Moreover, S1P1 receptor-mediated β-arrestin signal was unaltered by α-Syn(A53T). These results suggest that exogenous α-Syn modulates S1P1 receptor-mediated signalling from both Gi and β-arrestin signals into β-arrestin-biased signal. These findings uncovered a novel function of exogenous α-Syn in the cells. PMID:28300069

  6. Schistosoma japonicum and S. mansoni cercariae: different effects of protein in medium, of mechanical stress, and of an intact complement system on in vitro transformation to schistosomula.

    PubMed

    Wang, Wenshi; Kirschfink, Michael; Ruppel, Andreas

    2006-08-01

    The cercariae of Schistosoma japonicum were subjected in vitro to treatments known for Schistosoma mansoni to generate schistosomula-like organisms. As a technical prerequisite to pipette or to otherwise handle the sticky cercariae of S. japonicum, the addition of protein to water or medium was found to abolish the stickiness of cercariae of this species. Shearing forces exerted in vitro by syringe (22 G) passage are known since long to fully transform S. mansoni cercariae, but this treatment was found to be much less efficient with S. japonicum. Thus, even with very narrow needles (27 G), complete transformation of cercariae was not obtained with S. japonicum. Complement, provided by fresh human serum, is also well known to induce rapid transformation of S. mansoni cercariae with subsequent killing of the schistosomula. This treatment of S. japonicum cercariae induced degeneration of the tails and strongly promoted the transformation to schistosomula-like organisms, but at a much slower pace. These effects were absent from sera either heat-inactivated or depleted of factor B or of complement component C8, but were restored after adding the purified respective complement components. The schistosomula-like organisms of S. japonicum were not susceptible to lysis after 1 day of in vitro culture in the presence of 50% fresh human serum, although both cercariae and schistosomula of S. mansoni were killed under these conditions. In conclusion, the dynamics of in vitro transformation of S. japonicum cercariae differ significantly from those of S. mansoni, and complement has a major transformation-promoting activity.

  7. 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

  8. An improved hypergeometric probability method for identification of functionally linked proteins using phylogenetic profiles.

    PubMed

    Kotaru, Appala Raju; Shameer, Khader; Sundaramurthy, Pandurangan; Joshi, Ramesh Chandra

    2013-01-01

    Predicting functions of proteins and alternatively spliced isoforms encoded in a genome is one of the important applications of bioinformatics in the post-genome era. Due to the practical limitation of experimental characterization of all proteins encoded in a genome using biochemical studies, bioinformatics methods provide powerful tools for function annotation and prediction. These methods also help minimize the growing sequence-to-function gap. Phylogenetic profiling is a bioinformatics approach to identify the influence of a trait across species and can be employed to infer the evolutionary history of proteins encoded in genomes. Here we propose an improved phylogenetic profile-based method which considers the co-evolution of the reference genome to derive the basic similarity measure, the background phylogeny of target genomes for profile generation and assigning weights to target genomes. The ordering of genomes and the runs of consecutive matches between the proteins were used to define phylogenetic relationships in the approach. We used Escherichia coli K12 genome as the reference genome and its 4195 proteins were used in the current analysis. We compared our approach with two existing methods and our initial results show that the predictions have outperformed two of the existing approaches. In addition, we have validated our method using a targeted protein-protein interaction network derived from protein-protein interaction database STRING. Our preliminary results indicates that improvement in function prediction can be attained by using coevolution-based similarity measures and the runs on to the same scale instead of computing them in different scales. Our method can be applied at the whole-genome level for annotating hypothetical proteins from prokaryotic genomes.

  9. An improved hypergeometric probability method for identification of functionally linked proteins using phylogenetic profiles

    PubMed Central

    Kotaru, Appala Raju; Shameer, Khader; Sundaramurthy, Pandurangan; Joshi, Ramesh Chandra

    2013-01-01

    Predicting functions of proteins and alternatively spliced isoforms encoded in a genome is one of the important applications of bioinformatics in the post-genome era. Due to the practical limitation of experimental characterization of all proteins encoded in a genome using biochemical studies, bioinformatics methods provide powerful tools for function annotation and prediction. These methods also help minimize the growing sequence-to-function gap. Phylogenetic profiling is a bioinformatics approach to identify the influence of a trait across species and can be employed to infer the evolutionary history of proteins encoded in genomes. Here we propose an improved phylogenetic profile-based method which considers the co-evolution of the reference genome to derive the basic similarity measure, the background phylogeny of target genomes for profile generation and assigning weights to target genomes. The ordering of genomes and the runs of consecutive matches between the proteins were used to define phylogenetic relationships in the approach. We used Escherichia coli K12 genome as the reference genome and its 4195 proteins were used in the current analysis. We compared our approach with two existing methods and our initial results show that the predictions have outperformed two of the existing approaches. In addition, we have validated our method using a targeted protein-protein interaction network derived from protein-protein interaction database STRING. Our preliminary results indicates that improvement in function prediction can be attained by using coevolution-based similarity measures and the runs on to the same scale instead of computing them in different scales. Our method can be applied at the whole-genome level for annotating hypothetical proteins from prokaryotic genomes. PMID:23750082

  10. Classification of Phylogenetic Profiles for Protein Function Prediction: An SVM Approach

    NASA Astrophysics Data System (ADS)

    Kotaru, Appala Raju; Joshi, Ramesh C.

    Predicting the function of an uncharacterized protein is a major challenge in post-genomic era due to problems complexity and scale. Having knowledge of protein function is a crucial link in the development of new drugs, better crops, and even the development of biochemicals such as biofuels. Recently numerous high-throughput experimental procedures have been invented to investigate the mechanisms leading to the accomplishment of a protein’s function and Phylogenetic profile is one of them. Phylogenetic profile is a way of representing a protein which encodes evolutionary history of proteins. In this paper we proposed a method for classification of phylogenetic profiles using supervised machine learning method, support vector machine classification along with radial basis function as kernel for identifying functionally linked proteins. We experimentally evaluated the performance of the classifier with the linear kernel, polynomial kernel and compared the results with the existing tree kernel. In our study we have used proteins of the budding yeast saccharomyces cerevisiae genome. We generated the phylogenetic profiles of 2465 yeast genes and for our study we used the functional annotations that are available in the MIPS database. Our experiments show that the performance of the radial basis kernel is similar to polynomial kernel is some functional classes together are better than linear, tree kernel and over all radial basis kernel outperformed the polynomial kernel, linear kernel and tree kernel. In analyzing these results we show that it will be feasible to make use of SVM classifier with radial basis function as kernel to predict the gene functionality using phylogenetic profiles.

  11. Comparative protein profiling of serum and plasma using an antibody suspension bead array approach.

    PubMed

    Schwenk, Jochen M; Igel, Ulrika; Kato, Bernet S; Nicholson, George; Karpe, Fredrik; Uhlén, Mathias; Nilsson, Peter

    2010-02-01

    In the pursuit towards a systematic analysis of human diseases, array-based approaches within antibody proteomics offer high-throughput strategies to discover protein biomarkers in serum and plasma. To investigate the influence of sample preparation on such discovery attempts, we report on a systematic effort to compare serum and plasma protein profiles determined with an antibody suspension bead array. The intensity levels were used to define protein profiles and no significant differences between serum and plasma were observed for 79% of the 174 antibodies (targeting 156 proteins). By excluding 36 antibodies giving rise to differential intensity levels, cluster analysis revealed donor-specific rather than preparation-dependent grouping. With a cohort from a clinically relevant medical condition, the metabolic syndrome, the influence of the sample type on a multiplexed biomarker discovery approach was further investigated. Independent comparisons of protein profiles in serum and plasma revealed an antibody targeting ADAMTSL-4, a protein that would qualify to be studied further in association with the condition. In general, the preparation type had an impact on the results of the applied antibody suspension bead array, and while the technical variability was equal, plasma offered a greater biological variability and allowed to give rise to more discoveries than serum.

  12. 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

  13. Fluorescence response profiling for small molecule sensors utilizing the green fluorescent protein chromophore and its derivatives.

    PubMed

    Lee, Jun-Seok; Baldridge, Anthony; Feng, Suihan; SiQiang, Yang; Kim, Yun Kyung; Tolbert, Laren M; Chang, Young-Tae

    2011-01-10

    Using a fluorescence response profile, a systematic examination was performed for synthetic chromophores of the green fluorescent protein (GFP) to discover new small molecule sensors. A group of 41 benzylideneimidazolinone compounds (BDI) was prepared and screened toward 94 biologically relevant analytes to generate fluorescence response profiles. From the response pattern, compounds containing aminobenzyl and heteroaromatic cyclic substructures revealed a pH dependent emission decrease effect, and unlike other fluorescence scaffolds, most BDIs showed fluorescence quenching when mixed with proteins. On the basis of the primary response profile, we obtained three selective fluorescence turn-on sensors for pH, human serum albumin (HSA), and total ribonucleic acid (RNA). Following analysis, a fluorescence response profile testing four nucleic acids revealed the alkyloxy (Ph-OR) functional group in the para position of benzyl analogues contributes to RNA selectivity. Among the primary hit compounds, BDI 2 showed outstanding selectivity toward total RNA with 5-fold emission enhancement. Finally, BDI 24 showed selective fluorescence increase to HSA (K(d) = 3.57 μM) with a blue-shifted emission max wavelength (Δλ(em) = 15 nm). These examples of fluorescence sensor discovery by large-scale fluorescence response profiling demonstrate the general applicability of this approach and the usefulness of the response profiles.

  14. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein

    PubMed Central

    Woellhaf, Michael W.; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M.

    2016-01-01

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker’s yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries. PMID:27582385

  15. Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein.

    PubMed

    Woellhaf, Michael W; Sommer, Frederik; Schroda, Michael; Herrmann, Johannes M

    2016-10-15

    Whereas the structure and function of cytosolic ribosomes are well characterized, we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC-based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial ribosome, most of which play a role in translation or ribosomal biogenesis. One of these proteins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated from the composite precursor protein Atp25 upon internal cleavage by the matrix processing peptidase MPP, and in this respect, it differs from all other characterized mitochondrial proteins of baker's yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries.

  16. Differential expression profile of membrane proteins in zebrafish (Danio rerio) brain exposed to methyl parathion.

    PubMed

    Huang, Qing-Yu; Huang, He-Qing

    2011-09-01

    Methyl parathion (MP) is a widely used organophosphorus pesticide, which has been related to a broad spectrum of toxic effects on environmental organisms. The present study investigated the changes in the protein profile of enriched membrane fraction from zebrafish (Danio rerio) brain exposed to three concentrations (0.5, 1 and 2 mg/L) of MP. 2-DE revealed that the abundance of 21 protein spots was significantly changed by MP stress. By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and database search, 16 protein spots were identified as membrane proteins, among which 8 were down-regulated, while 8 were up-regulated. These proteins are mainly involved in oxidative stress response, signal transduction, metabolism, protein synthesis and degradation, neuroplasticity and regeneration as well as synaptic transmission. These results may aid our understanding of the mechanism of MP-induced neurotoxicity and provide the possibility of the establishment of candidate biomarkers of MP.

  17. 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.

  18. 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

  19. 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.

  20. 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...

  1. Protein Electrophoretic Profiles and the Origin of the B Genome of Wheat

    PubMed Central

    Johnson, B. Lennart

    1972-01-01

    Protein electrophoretic profiles cast doubt upon the prevalent theory that the B genome of the polyploid wheats was derived from a species of Aegilops. They suggest, instead, that the wild tetraploid wheats comprise a complex, whose components were derived from various combinations of diploid Triticum types, which evidently include the B-genome type. Images PMID:4504349

  2. 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...

  3. Effects of gamma irradiation on chickpea seeds vis-a-vis total seed storage proteins, antioxidant activity and protein profiling.

    PubMed

    Bhagyawant, S S; Gupta, N; Shrivastava, N

    2015-10-23

    The present work describes radiation—induced effects on seed composition vis—à—vis total seed proteins, antioxidant levels and protein profiling employing two dimensional gel electrophoresis (2D—GE) in kabuli and desi chickpea varities. Seeds were exposed to the radiation doses of 1,2,3,4 and 5 kGy. The total protein concentrations decreased and antioxidant levels were increased with increasing dose compared to control seed samples. Radiation induced effects were dose dependent to these seed parameters while it showed tolerance to 1 kGy dose. Increase in the dose was complimented with increase in antioxidant levels, like 5 kGy enhanced % scavenging activities in all the seed extracts. Precisely, the investigations reflected that the dose range from 2 to 5 kGy was effective for total seed storage proteins, as depicted quantitatively and qualitative 2D—GE means enhance antioxidant activities in vitro.

  4. 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.

  5. Advancing the Accuracy of Protein Fold Recognition by Utilizing Profiles From Hidden Markov Models.

    PubMed

    Lyons, James; Dehzangi, Abdollah; Heffernan, Rhys; Yang, Yuedong; Zhou, Yaoqi; Sharma, Alok; Paliwal, Kuldip

    2015-10-01

    Protein fold recognition is an important step towards solving protein function and tertiary structure prediction problems. Among a wide range of approaches proposed to solve this problem, pattern recognition based techniques have achieved the best results. The most effective pattern recognition-based techniques for solving this problem have been based on extracting evolutionary-based features. Most studies have relied on the Position Specific Scoring Matrix (PSSM) to extract these features. However it is known that profile-profile sequence alignment techniques can identify more remote homologs than sequence-profile approaches like PSIBLAST. In this study we use a profile-profile sequence alignment technique, namely HHblits, to extract HMM profiles. We will show that unlike previous studies, using the HMM profile to extract evolutionary information can significantly enhance the protein fold prediction accuracy. We develop a new pattern recognition based system called HMMFold which extracts HMM based evolutionary information and captures remote homology information better than previous studies. Using HMMFold we achieve up to 93.8% and 86.0% prediction accuracies when the sequential similarity rates are less than 40% and 25%, respectively. These results are up to 10% better than previously reported results for this task. Our results show significant enhancement especially for benchmarks with sequential similarity as low as 25% which highlights the effectiveness of HMMFold to address this problem and its superiority over previously proposed approaches found in the literature. The HMMFold is available online at: http://sparks-lab.org/pmwiki/download/index.php?Download =HMMFold.tar.bz2.

  6. 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.

  7. 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.

  8. Direct prediction of profiles of sequences compatible to a protein structure by neural networks with fragment-based local and energy-based nonlocal profiles

    PubMed Central

    Li, Zhixiu; Yang, Yuedong; Faraggi, Eshel; Zhan, Jian; Zhou, Yaoqi

    2014-01-01

    Locating sequences compatible to a protein structural fold is the well-known inverse protein-folding problem. While significant progress has been made, the success rate of protein design remains low. As a result, a library of designed sequences or profile of sequences is currently employed for guiding experimental screening or directed evolution. Sequence profiles can be computationally predicted by iterative mutations of a random sequence to produce energy-optimized sequences, or by combining sequences of structurally similar fragments in a template library. The latter approach is computationally more efficient but yields less accurate profiles than the former because of lacking tertiary structural information. Here we present a method called SPIN that predicts Sequence Profiles by Integrated Neural network based on fragment-derived sequence profiles and structure-derived energy profiles. SPIN improves over the fragment-derived profile by 6.7% (from 23.6% to 30.3%) in sequence identity between predicted and wild-type sequences. The method also reduces the number of residues in low complex regions by 15.7% and has a significant better balance of hydrophilic and hydrophobic residues at protein surfaces. The accuracy of sequence profiles obtained is comparable to those generated from the protein design program RosettaDesign 3.5. This highly efficient method for predicting sequence profiles from structures will be useful as a single-body scoring term for improving scoring functions used in protein design and fold recognition. It also complements protein design programs in guiding experimental design of the sequence library for screening and directed evolution of designed sequences. The SPIN server is available at http://sparks-lab.org. PMID:24898915

  9. Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus.

    PubMed

    Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Asensio, Miguel A; Núñez, Félix

    2015-10-01

    Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study.

  10. 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.

  11. Fast and efficient MCR-based synthesis of clickable rhodamine tags for protein profiling.

    PubMed

    Brauch, Sebastian; Henze, Michael; Osswald, Bianca; Naumann, Kai; Wessjohann, Ludger A; van Berkel, Sander S; Westermann, Bernhard

    2012-02-07

    Protein profiling probes are important tools for studying the composition of the proteome and as such have contributed greatly to the understanding of various complex biological processes in higher organisms. For this purpose the application of fluorescently labeled activity or affinity probes is highly desirable. Especially for in vivo detection of low abundant target proteins, otherwise difficult to analyse by standard blotting techniques, fluorescently labeled profiling probes are of high value. Here, a one-pot protocol for the synthesis of activated fluorescent labels (i.e. azide, alkynyl or NHS), based on the Ugi-4-component reaction (Ugi-4CR), is presented. As a result of the peptoidic structure formed, the fluorescent properties of the products are pH insensitive. Moreover, the applicability of these probes, as exemplified by the labeling of model protein BSA, will be discussed.

  12. 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.

  13. Systematic Comparative Protein Expression Profiling of Clear Cell Renal Cell Carcinoma

    PubMed Central

    Lichtenfels, Rudolf; Dressler, Sven P.; Zobawa, Monica; Recktenwald, Christian V.; Ackermann, Angelika; Atkins, Derek; Kersten, Michael; Hesse, Andrea; Puttkammer, Maria; Lottspeich, Friedrich; Seliger, Barbara

    2009-01-01

    Proteome-based technologies represent powerful tools for the analysis of protein expression profiles, including the identification of potential cancer candidate biomarkers. Thus, here we provide a comprehensive protein expression map for clear cell renal cell carcinoma established by systematic comparative two-dimensional gel electrophoresis-based protein expression profiling of 16 paired tissue systems comprising clear cell renal cell carcinoma lesions and corresponding tumor-adjacent renal epithelium using overlapping narrow pH gradients. This approach led to the mapping of 348 distinct spots corresponding to 248 different protein identities. By implementing restriction criteria concerning their detection frequency and overall regulation mode, 28 up- and 56 down-regulated single target spots were considered as potential candidate biomarkers. Based on their gene ontology information, these differentially expressed proteins were classified into distinct functional groups and according to their cellular distribution. Moreover, three representative members of this group, namely calbindin, gelsolin, and heart fatty acid-binding protein, were selected, and their expression pattern was analyzed by immunohistochemistry using tissue microarrays. Thus, this pilot study provides a significant update of the current renal cell carcinoma map and defines a number of differentially expressed proteins, but both their potential as candidate biomarkers and clinical relevance has to be further explored in tissues and for body fluids like serum and urine. PMID:19752005

  14. Protein Profiles Associated With Context Fear Conditioning and Their Modulation by Memantine*

    PubMed Central

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

    2014-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2008-01-01

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

  16. 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

  17. Proteomic profiling of proteins associated with methamphetamine-induced neurotoxicity in different regions of rat brain.

    PubMed

    Li, Xuefeng; Wang, Huijun; Qiu, Pingming; Luo, Hong

    2008-01-01

    It is well documented that methamphetamine (MA) can cause obvious damage to the brain, but the exact mechanism is still unknown. In the present study, proteomic methods of two-dimensional gel electrophoresis in combination with mass spectrometry analysis were used to identify global protein profiles associated with MA-induced neurotoxicity. For the first time, 30 protein spots have been found differentially expressed in different regions of rat brain, including 14 in striatum, 12 in hippocampus and 4 in frontal cortex. The proteins identified by tandem mass spectrometry were Cu, Zn superoxide dismutase, dimethylarginine dimethylaminohydrolase 1, alpha synuclein, ubiquitin-conjugating enzyme E2N, stathmin 1, calcineurin B, cystatin B, subunit of mitochondrial H-ATP synthase, ATP synthase D chain, mitochondrial, NADH dehydrogenase(ubiquinone) Fe-S protein 8, glia maturation factor, beta, Ash-m, neurocalcin delta, myotrophin, profiling IIa, D-dopachrome tautomerase, and brain lipid binding protein. The known functions of these proteins were related to the pathogenesis of MA-induced neurotoxicity, including oxidative stress, degeneration/apoptosis, mitochontrial/energy metabolism and others. Of these proteins, alpha-synuclein was up-regulated, and ATP synthase D chain, mitochondrial was down-regulated in all brain regions. Two proteins, Cu, Zn superoxide dismutase, subunit of mitochondrial H-ATPsynthase were down-regulated and Ubiquitin-conjugating enzyme E2N, NADH dehydrogenase (ubiquinone) Fe-S protein 8 were up-regulated simultaneously in striatum and hippocaltum. The expression of dimethylarginine dimethylaminohydrolase 1 (DDAH 1) increased both in striatum and frontal cortex. The parallel expression patterns of these proteins suggest that the pathogenesis of MA neurotoxicity in different brain regions may share some same pathways.

  18. Profiles.

    ERIC Educational Resources Information Center

    School Arts, 1979

    1979-01-01

    Profiles seven Black, Native American, and Chicano artists and art teachers: Hale A. Woodruff, Allan Houser, Luis Jimenez, Betrand D. Phillips, James E. Pate, I, and Fernando Navarro. This article is part of a theme issue on multicultural art. (SJL)

  19. 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 Central

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

    1982-01-01

    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. Images PMID:6180184

  20. Cassava interspecific hybrids with increased protein content and improved amino acid profiles.

    PubMed

    Gomes, P T C; Nassar, N M A

    2013-04-12

    Cassava (Manihot esculenta) is a principal food for large populations of poor people in the tropics and subtropics. Its edible roots are poor in protein and lack several essential amino acids. Interspecific hybrids may acquire high protein characteristics from wild species. We analyzed 19 hybrids of M. esculenta with its wild relative, M. oligantha, for crude protein, amino acid profile, and total cyanide. Some hybrids produced roots with high protein content of up to 5.7%, while the common cultivar that we examined had just 2.3% crude protein. The essential amino acids alanine, phenylalanine, and valine were detected in the hybrids. The sulfur-containing amino acids cysteine and methionine were found at relatively high concentrations in the roots of 4 hybrids. The proportion of lysine in one hybrid was 20 times higher than in the common cultivar. The levels of total cyanide ranged from 19.73 to 172.56 mg/kg and most of the roots analyzed were classified as "non-toxic" and "low toxic". Furthermore, 2 progenies showed reasonable levels of cyanide, but higher protein content and amino acid profile more advantageous than the common cassava.

  1. Hexapeptide libraries for enhanced protein PTM identification and relative abundance profiling in whole human saliva.

    PubMed

    Bandhakavi, Sricharan; Van Riper, Susan K; Tawfik, Pierre N; Stone, Matthew D; Haddad, Tufia; Rhodus, Nelson L; Carlis, John V; Griffin, Timothy J

    2011-03-04

    Dynamic range compression (DRC) by hexapeptide libraries increases MS/MS-based identification of lower-abundance proteins in complex mixtures. However, two unanswered questions impede fully realizing DRC's potential in shotgun proteomics. First, does DRC enhance identification of post-translationally modified proteins? Second, can DRC be incorporated into a workflow enabling relative protein abundance profiling? We sought to answer both questions analyzing human whole saliva. Addressing question one, we coupled DRC with covalent glycopeptide enrichment and MS/MS. With DRC we identified ∼2 times more N-linked glycoproteins and their glycosylation sites than without DRC, dramatically increasing the known salivary glycoprotein catalog. Addressing question two, we compared differentially stable isotope-labeled saliva samples pooled from healthy and metastatic breast cancer women using a multidimensional peptide fractionation-based workflow, analyzing in parallel one sample portion with DRC and one portion without. Our workflow categorizes proteins with higher absolute abundance, whose relative abundance ratios are altered by DRC, from proteins of lower absolute abundance detected only after DRC. Within each of these salivary protein categories, we identified novel abundance changes putatively associated with breast cancer, demonstrating feasibility and benefits of DRC for relative abundance profiling. Collectively, our results bring us closer to realizing the full potential of DRC for proteomic studies.

  2. Proposed second class of Haemophilus ducreyi strains show altered protein and lipooligosaccharide profiles.

    PubMed

    Post, Deborah M B; Gibson, Bradford W

    2007-09-01

    Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Previously we have shown that the protein profiles and lipooligosaccharide (LOS) structures from various strains of H. ducreyi are generally well conserved. Previous studies have demonstrated that at least one strain, 33921, has a variant protein profile and LOS structure. In this study, both the whole cell lysate and the membrane proteins from strain 33921 were further examined and compared to the prototypical strain 35000HP by 2-DE and by the 16-BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE two-detergent system, respectively. These comparisons demonstrated that a number of the proteins that could be identified from both strains had altered positions on the gels, both in their apparent molecular weight and pI values. Strain 33921 has been suggested to be a member of a second class of strains, termed class II strains. In this study, the proteomic profiles and the LOS structures from the five potential class II strains were examined and found to be similar to strain 33921.

  3. 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.

  4. 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.

  5. Dietary indispensable amino acids profile affects protein utilization and growth of Senegalese sole larvae.

    PubMed

    Canada, Paula; Engrola, Sofia; Richard, Nadège; Lopes, Ana Filipa; Pinto, Wilson; Valente, Luísa M P; Conceição, Luís E C

    2016-12-01

    In diet formulation for fish, it is critical to assure that all the indispensable amino acids (IAA) are available in the right quantities and ratios. This will allow minimizing dietary AA imbalances that will result in unavoidable AA losses for energy dissipation rather than for protein synthesis and growth. The supplementation with crystalline amino acids (CAA) is a possible solution to correct the dietary amino acid (AA) profile that has shown positive results for larvae of some fish species. This study tested the effect of supplementing a practical microdiet with encapsulated CAA as to balance the dietary IAA profile and to improve the capacity of Senegalese sole larvae to utilize AA and maximize growth potential. Larvae were reared at 19 °C under a co-feeding regime from mouth opening. Two microdiets were formulated and processed as to have as much as possible the same ingredients and proximate composition. The control diet (CTRL) formulation was based on commonly used protein sources. A balanced diet (BAL) was formulated as to meet the ideal IAA profile defined for Senegalese sole: the dietary AA profile was corrected by replacing 4 % of encapsulated protein hydrolysate by CAA. The in vivo method of controlled tube-feeding was used to assess the effect on the larvae capacity to utilize protein, during key developmental stages. Growth was monitored until 51 DAH. The supplementation of microdiets with CAA in order to balance the dietary AA had a positive short-term effect on the Senegalese sole larvae capacity to retain protein. However, that did not translate into increased growth. On the contrary, larvae fed a more imbalanced (CTRL group) diet attained a better performance. Further studies are needed to ascertain whether this was due to an effect on the voluntary feed intake as a compensatory response to the dietary IAA imbalance in the CTRL diet or due to the higher content of tryptophan in the BAL diet.

  6. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    PubMed

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology.

  7. Profiling the Humoral Immune Response of Acute and Chronic Q Fever by Protein Microarray*

    PubMed Central

    Vigil, Adam; Chen, Chen; Jain, Aarti; Nakajima-Sasaki, Rie; Jasinskas, Algimantas; Pablo, Jozelyn; Hendrix, Laura R.; Samuel, James E.; Felgner, Philip L.

    2011-01-01

    Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response. PMID:21817167

  8. One reporter for in-cell activity profiling of majority of protein kinase oncogenes.

    PubMed

    Gudernova, Iva; Foldynova-Trantirkova, Silvie; Ghannamova, Barbora El; Fafilek, Bohumil; Varecha, Miroslav; Balek, Lukas; Hruba, Eva; Jonatova, Lucie; Jelinkova, Iva; Kunova Bosakova, Michaela; Trantirek, Lukas; Mayer, Jiri; Krejci, Pavel

    2017-02-15

    In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The EGR1 promoter was engineered to enhance trans-activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies.

  9. One reporter for in-cell activity profiling of majority of protein kinase oncogenes

    PubMed Central

    Gudernova, Iva; Foldynova-Trantirkova, Silvie; Ghannamova, Barbora El; Fafilek, Bohumil; Varecha, Miroslav; Balek, Lukas; Hruba, Eva; Jonatova, Lucie; Jelinkova, Iva; Bosakova, Michaela Kunova; Trantirek, Lukas; Mayer, Jiri; Krejci, Pavel

    2017-01-01

    In-cell profiling enables the evaluation of receptor tyrosine activity in a complex environment of regulatory networks that affect signal initiation, propagation and feedback. We used FGF-receptor signaling to identify EGR1 as a locus that strongly responds to the activation of a majority of the recognized protein kinase oncogenes, including 30 receptor tyrosine kinases and 154 of their disease-associated mutants. The EGR1 promoter was engineered to enhance trans-activation capacity and optimized for simple screening assays with luciferase or fluorescent reporters. The efficacy of the developed, fully synthetic reporters was demonstrated by the identification of novel targets for two clinically used tyrosine kinase inhibitors, nilotinib and osimertinib. A universal reporter system for in-cell protein kinase profiling will facilitate repurposing of existing anti-cancer drugs and identification of novel inhibitors in high-throughput screening studies. DOI: http://dx.doi.org/10.7554/eLife.21536.001 PMID:28199182

  10. Using Azido Analogue of S-Adenosyl-L-methionine for Bioorthogonal Profiling of Protein Methylation (BPPM)

    PubMed Central

    Blum, Gil; Islam, Kabirul; Luo, Minkui

    2013-01-01

    Protein methyltransferases (PMTs) utilize S-adenosyl-L-methionine as a cofactor and deliver its sulfonium methyl moiety to diverse substrates. These methylation events can lead to meaningful biological outcomes from transcriptional activation/silencing to cell cycle regulation. With the long-term goal of elucidating the substrates and defining the functions of PMTs, our laboratory recently developed technology based on protein engineering in tandem with SAM analogue cofactors and bioorthogonal click chemistry to unambiguously profile the substrates of a specific PMT. The following protocols encapsulate the logic and methods of selectively profiling the substrates of a candidate PMT by (1) engineering the selected PMT to accommodate a bulky SAM analogue; (2) generating the proteome containing the engineered PMT; (3) visualizing the proteome-wide substrates of the designated PMT via bioorthogonal labeling with a fluorescent tag and finally (4) pulling down the proteome-wide substrates for mass spectrometric analysis. PMID:23667794

  11. Protein profiling of mouse livers with peroxisome proliferator-activated receptor alpha activation.

    PubMed

    Chu, Ruiyin; Lim, Hanjo; Brumfield, Laura; Liu, Hong; Herring, Chris; Ulintz, Peter; Reddy, Janardan K; Davison, Matthew

    2004-07-01

    Peroxisome proliferator-activated receptor alpha (PPARalpha) is important in the induction of cell-specific pleiotropic responses, including the development of liver tumors, when it is chronically activated by structurally diverse synthetic ligands such as Wy-14,643 or by unmetabolized endogenous ligands resulting from the disruption of the gene encoding acyl coenzyme A (CoA) oxidase (AOX). Alterations in gene expression patterns in livers with PPARalpha activation were delineated by using a proteomic approach to analyze liver proteins of Wy-14,643-treated and AOX(-/-) mice. We identified 46 differentially expressed proteins in mouse livers with PPARalpha activation. Up-regulated proteins, including acetyl-CoA acetyltransferase, farnesyl pyrophosphate synthase, and carnitine O-octanoyltransferase, are involved in fatty acid metabolism, whereas down-regulated proteins, including ketohexokinase, formiminotransferase-cyclodeaminase, fructose-bisphosphatase aldolase B, sarcosine dehydrogenase, and cysteine sulfinic acid decarboxylase, are involved in carbohydrate and amino acid metabolism. Among stress response and xenobiotic metabolism proteins, selenium-binding protein 2 and catalase showed a dramatic approximately 18-fold decrease in expression and a modest approximately 6-fold increase in expression, respectively. In addition, glycine N-methyltransferase, pyrophosphate phosphohydrolase, and protein phosphatase 1D were down-regulated with PPARalpha activation. These observations establish proteomic profiles reflecting a common and predictable pattern of differential protein expression in livers with PPARalpha activation. We conclude that livers with PPARalpha activation are transcriptionally geared towards fatty acid combustion.

  12. Protein expression profile in the striatum of rats with methamphetamine-induced behavioral sensitization.

    PubMed

    Iwazaki, Takeshi; McGregor, Iain S; Matsumoto, Izuru

    2007-04-01

    Repeated administration of methamphetamine (MAP) results in an increased behavioral response to the drug during subsequent exposure. This phenomenon is called behavioral sensitization. Sensitization is an enduring phenomenon, and suggests chronic alterations in neuronal plasticity. MAP-induced sensitization has been proposed and widely investigated as an animal model of MAP psychosis and schizophrenia. However, little is known about the molecular mechanisms underlying MAP-induced sensitization. 2-DE-based proteomics allows us to examine global changes in protein expression in complex biological systems and to propose hypotheses concerning the mechanisms underlying various pathological conditions. In the present study, we examined protein expression profiles in the striatum of MAP-sensitized rats using 2-DE-based proteomics. Repeated administration of MAP (4.0 mg/kg, once a day, intraperitoneal (i.p.)) for 10 days significantly augmented the locomotor response to an MAP challenge injection (1.0 mg/kg, i.p.) on day 11. This enhanced activity was maintained even after a week of drug abstinence. 2-DE analysis revealed 42 protein spots were differentially regulated in the striatum of MAP-sensitized rats compared to control. Thirty-one protein spots were identified using MALDI-TOF, including synapsin II, synaptosomal-associated protein 25 (SNAP-25), adenylyl cyclase-associated protein 1 (CAP1), and dihydropyrimidinase-related protein 2 (DRP2). These proteins can be related to underlying mechanisms of MAP-induced behavioral sensitization, indicating cytoskeletal modification, and altered synaptic function.

  13. Alterations of protein profile in zebrafish liver cells exposed to methyl parathion: a membrane proteomics approach.

    PubMed

    Huang, Qingyu; Huang, He-Qing

    2012-03-01

    Methyl parathion (MP) is an extensively used organophosphorus pesticide, which has been associated with a wide spectrum of toxic effects on environmental organisms. The aim of this study is to investigate the alterations of membrane protein profiles in zebrafish liver (ZFL) cell line exposed to MP for 24 h using proteomic approaches. Two-dimensional gel electrophoresis revealed a total of 13 protein spots, whose expression levels were significantly altered by MP. These differential proteins were subjected to matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry analysis, and nine proteins were identified to be membrane proteins, among which seven were up-regulated, while two were down-regulated. In addition, the mRNA levels corresponding to these differential membrane proteins were further analyzed by quantitative real-time PCR. And the differential expression of arginase-2 was specially validated via Western blotting. Regarding the physiological functions, these proteins are involved in molecular chaperon, cytoskeleton system, cell metabolism, signal transduction, transport and hormone receptor respectively, suggesting the complexity of MP-mediated toxicity to ZFL cell. These data could provide useful insights for better understanding the hepatotoxic mechanisms of MP and develop novel protein biomarkers for effectively monitoring MP contamination level in aquatic environment.

  14. Change of protein profiles in the induction of the viable but nonculturable state of Vibrio parahaemolyticus.

    PubMed

    Lai, Chiou-Jour; Chen, Shau-Yan; Lin, I-Hsuan; Chang, Chuan-Hsiung; Wong, Hin-Chung

    2009-10-31

    This work reports on the metabolic response in the induction of the viable but nonculturable (VBNC) state of the seafood enteropathogen Vibrio parahaemolyticus, as determined by analyzing the corresponding change in protein profiles. V. parahaemolyticus ST550 was incubated at 4 degrees C in the Morita mineral salt-0.5% NaCl medium to induce the VBNC state in six weeks. Starving the cells by incubation at 25 degrees C for 24 h prior to 4 degrees C incubation inhibited the cells from entering VBNC state. Protein profiles were determined by two-dimensional polyacrylamide gel electrophoresis and the proteins which were enhanced in the VBNC induction/VBNC state or strongly down-regulated in the starved cells were identified by mass spectrophotometry. The 13 up-regulated proteins are known to be associated with transcription (two homologues of alpha subunit DNA-directed RNA polymerase, phosphoribosylaminoimidazole carboxamide formyltransferase/IMP cyclohydrolase), translation (ribosomal protein S1, two homologues of elongation factor TU, elongation factor EF-G), ATP synthase (F1 alpha subunit), gluconeogenesis-related metabolism (dihydrolipoamide acetyltransferase, glyceraldehyde 3-phosphate dehydrogenase), antioxidants (2 homologues of peroxiredoxins, AhpC/Tsa family) and a conserved hypothetical protein with unknown function. Expressions of the genes encoding four of these proteins were at high levels in the second week of VBNC induction; declined afterwards, and were down-regulated in the starved cells. These proteins may play important roles in the induction or maintenance of VBNC V. parahaemolyticus. The results of this investigation improve our understanding of the metabolic activities in the VBNC state of bacteria.

  15. Prediction of mitochondrial proteins of malaria parasite using split amino acid composition and PSSM profile.

    PubMed

    Verma, Ruchi; Varshney, Grish C; Raghava, G P S

    2010-06-01

    The rate of human death due to malaria is increasing day-by-day. Thus the malaria causing parasite Plasmodium falciparum (PF) remains the cause of concern. With the wealth of data now available, it is imperative to understand protein localization in order to gain deeper insight into their functional roles. In this manuscript, an attempt has been made to develop prediction method for the localization of mitochondrial proteins. In this study, we describe a method for predicting mitochondrial proteins of malaria parasite using machine-learning technique. All models were trained and tested on 175 proteins (40 mitochondrial and 135 non-mitochondrial proteins) and evaluated using five-fold cross validation. We developed a Support Vector Machine (SVM) model for predicting mitochondrial proteins of P. falciparum, using amino acids and dipeptides composition and achieved maximum MCC 0.38 and 0.51, respectively. In this study, split amino acid composition (SAAC) is used where composition of N-termini, C-termini, and rest of protein is computed separately. The performance of SVM model improved significantly from MCC 0.38 to 0.73 when SAAC instead of simple amino acid composition was used as input. In addition, SVM model has been developed using composition of PSSM profile with MCC 0.75 and accuracy 91.38%. We achieved maximum MCC 0.81 with accuracy 92% using a hybrid model, which combines PSSM profile and SAAC. When evaluated on an independent dataset our method performs better than existing methods. A web server PFMpred has been developed for predicting mitochondrial proteins of malaria parasites ( http://www.imtech.res.in/raghava/pfmpred/).

  16. Charge-based separation of proteins and peptides by electrically induced dynamic pH profiles.

    PubMed

    Brod, E; S Ben-Yosef, V; Bandhakavi, S; Sivan, U

    2016-01-29

    A new method for generating complex, dynamic pH profiles in an ampholyte-free separation channel is presented together with the theory behind its operation. The pH is modulated by an array of proton and hydroxide ion injectors placed along the separation channel. The ions generated in-situ by electrically driven water splitting across a bipolar membrane are injected to the channel in the presence of a longitudinal electric field, leading to the formation of a multi-step pH profile. Real-time control over the pH profile along the channel facilitates new dynamic separation strategies as well as steering and harvesting of focused molecules, which are both impossible with conventional separation methods. These freedoms are particularly attractive for Lab-on-a-Chip applications. The pH step-like profile alleviates one of the main hurdles of conventional isoelectric separation methods, namely, the slowing down of focused molecules as they approach their focusing spot. As a result, separation is completed within minutes for both peptides and proteins, even with low applied electric fields. We demonstrate protein and peptide separation within minutes, and resolution of ΔpI=0.2. Novel separation strategies based on spatio-temporal pH control are demonstrated as well.

  17. Changes of protein profile of human urine after long-term orbital flights.

    PubMed

    Pastushkova, L Kh; Valeeva, O A; Kononikhin, A S; Nikolaev, E N; Larina, I M; Dobrokhotov, I V; Popov, I A; Pochuev, V I; Kireev, K S

    2013-12-01

    We analyzed protein profile of urine samples obtained from 7 Russian cosmonauts (age 35-51 years), participants of space flights on the International Space Station lasting for 169-199 days. Gradient chromatography with linear increase of eluent proportion was carried out in a system consisting of an Agilent 1100 chromatograph (Agilent Technologies Inc.) and a hybrid mass-spectrometer LTQ-FT Ultra (Thermo). The obtained results help to understand changes in the human body induced by space flight factors.

  18. eProS--a database and toolbox for investigating protein sequence-structure-function relationships through energy profiles.

    PubMed

    Heinke, Florian; Schildbach, Stefan; Stockmann, Daniel; Labudde, Dirk

    2013-01-01

    Gaining information about structural and functional features of newly identified proteins is often a difficult task. This information is crucial for understanding sequence-structure-function relationships of target proteins and, thus, essential in comprehending the mechanisms and dynamics of the molecular systems of interest. Using protein energy profiles is a novel approach that can contribute in addressing such problems. An energy profile corresponds to the sequence of energy values that are derived from a coarse-grained energy model. Energy profiles can be computed from protein structures or predicted from sequences. As shown, correspondences and dissimilarities in energy profiles can be applied for investigations of protein mechanics and dynamics. We developed eProS (energy profile suite, freely available at http://bioservices.hs-mittweida.de/Epros/), a database that provides ∼76 000 pre-calculated energy profiles as well as a toolbox for addressing numerous problems of structure biology. Energy profiles can be browsed, visualized, calculated from an uploaded structure or predicted from sequence. Furthermore, it is possible to align energy profiles of interest or compare them with all entries in the eProS database to identify significantly similar energy profiles and, thus, possibly relevant structural and functional relationships. Additionally, annotations and cross-links from numerous sources provide a broad view of potential biological correspondences.

  19. 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.

  20. Protein threading with profiles and distance constraints using clique based algorithms.

    PubMed

    Dukka, Bahadur K C; Tomita, Etsuji; Suzuki, Jun'ichi; Horimoto, Katsuhisa; Akutsu, Tatsuya

    2006-02-01

    With the advent of experimental technologies like chemical cross-linking, it has become possible to obtain distances between specific residues of a newly sequenced protein. These types of experiments usually are less time consuming than X-ray crystallography or NMR. Consequently, it is highly desired to develop a method that incorporates this distance information to improve the performance of protein threading methods. However, protein threading with profiles in which constraints on distances between residues are given is known to be NP-hard. By using the notion of a maximum edge-weight clique finding algorithm, we introduce a more efficient method called FTHREAD for profile threading with distance constraints that is 18 times faster than its predecessor CLIQUETHREAD. Moreover, we also present a novel practical algorithm NTHREAD for profile threading with Non-strict constraints. The overall performance of FTHREAD on a data set shows that although our algorithm uses a simple threading function, our algorithm performs equally well as some of the existing methods. Particularly, when there are some unsatisfied constraints, NTHREAD (Non-strict constraints threading algorithm) performs better than threading with FTHREAD (Strict constraints threading algorithm). We have also analyzed the effects of using a number of distance constraints. This algorithm helps the enhancement of alignment quality between the query sequence and template structure, once the corresponding template structure is determined for the target sequence.

  1. Water penetration profile at the protein-lipid interface in Na,K-ATPase membranes.

    PubMed

    Bartucci, Rosa; Guzzi, Rita; Esmann, Mikael; Marsh, Derek

    2014-09-16

    The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from (2)H-hyperfine interaction with D2O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D2O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D2O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D2O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D2O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.

  2. 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

  3. Total nitrogen vs. amino-acid profile as indicator of protein content of beef.

    PubMed

    Hall, Nicolette G; Schönfeldt, Hettie C

    2013-10-01

    In most cited food composition studies and tables, the proximate system measures protein as total nitrogen (N) (determined by Kjeldahl or Dumas method) multiplied by a specific factor. A factor of 6.25 is used for determining total protein from total N (Jones, Munsey, & Walker, 1942). Although more expensive, it is considered more accurate to base protein content of foods on amino acid data (Greenfield & Southgate, 2003). A study on the nutrient composition of beef analysed the full amino-acid profile of fifteen retail cuts from three age groups and six fat codes, as well as determined total nitrogen content to determine proximate protein composition. For all cuts, the correlation coefficient of total amino acids to protein (N×6.25) was 0.635. This indicates a poor correlation for predicting actual protein content (as determined by total amino acid count), based on the nitrogen factor of 6.25. On average, the sum of amino acids per cut amounted to 91% of total determined protein (N×6.25) for the same cut.

  4. Altered protein profile in chronic myeloid leukemia chronic phase identified by a comparative proteomic study.

    PubMed

    Pizzatti, Luciana; Sá, Lílian Ayres; de Souza, Jamison Menezes; Bisch, Paulo Mascarello; Abdelhay, Eliana

    2006-05-01

    Chronic myeloid leukemia is a hematological disorder in which the Ph chromosome is a marker of the disease, detected virtually in all cases. The chimeric transcripts encode a 210-kDa chimeric protein with altered tyrosine kinase activity, responsible for the disease phenotype. In this work, we tried to identify which are the molecular changes common to chronic phase patients, those that represent the chronic phase molecular phenotype. To address this problem we analyzed through a comparative proteomic approach, several CML bone marrow cells protein profile from patients in chronic phase and healthy bone marrow donors. From these results, we identified 31 differentially expressed proteins. Among these proteins, we pointed out c-Myc binding protein 1, 53BP1, Mdm4, OSBP-related protein 3 and Mortalin as putative candidates to BCR-ABL targets in chronic phase. Moreover, we describe for the first time the cytoplasmic protein map from bone marrow cells that helped in the elucidation of the changes we were looking for.

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

    PubMed

    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

  6. Monitoring Intact Viruses Using Aptamers.

    PubMed

    Kumar, Penmetcha K R

    2016-08-04

    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.

  7. 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

  8. Label-free protein profiling of adipose-derived human stem cells under hyperosmotic treatment.

    PubMed

    Oswald, Elizabeth S; Brown, Lewis M; Bulinski, J Chloë; Hung, Clark T

    2011-07-01

    Our previous work suggested that treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. Here, we used label-free proteomic profiling to evaluate the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. Spectra were recorded in a data-independent fashion at alternate low (precursor) and high (product) fragmentation voltages (MS(E)). This method was supplemented with data mining of accurate mass and retention time matches in precursor ion spectra across the experiment. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase and transgelin, have been previously linked to cell differentiation state. This study demonstrates that label-free profiling can serve as a useful tool in characterizing cellular responses to chondrogenic treatment regimes, recommending its use in optimization of cell priming protocols for cartilage tissue engineering.

  9. Dietary live yeast alters metabolic profiles, protein biosynthesis and thermal stress tolerance of Drosophila melanogaster.

    PubMed

    Colinet, Hervé; Renault, David

    2014-04-01

    The impact of nutritional factors on insect's life-history traits such as reproduction and lifespan has been excessively examined; however, nutritional determinant of insect's thermal tolerance has not received a lot of attention. Dietary live yeast represents a prominent source of proteins and amino acids for laboratory-reared drosophilids. In this study, Drosophila melanogaster adults were fed on diets supplemented or not with live yeast. We hypothesized that manipulating nutritional conditions through live yeast supplementation would translate into altered physiology and stress tolerance. We verified how live yeast supplementation affected body mass characteristics, total lipids and proteins, metabolic profiles and cold tolerance (acute and chronic stress). Females fed with live yeast had increased body mass and contained more lipids and proteins. Using GC/MS profiling, we found distinct metabolic fingerprints according to nutritional conditions. Metabolite pathway enrichment analysis corroborated that live yeast supplementation was associated with amino acid and protein biosyntheses. The cold assays revealed that the presence of dietary live yeast greatly promoted cold tolerance. Hence, this study conclusively demonstrates a significant interaction between nutritional conditions and thermal tolerance.

  10. Correlation between protein accumulation profiles and conventional toxicological findings using a model antiandrogenic compound, flutamide.

    PubMed

    Friry-Santini, Claire; Rouquié, David; Kennel, Philippe; Tinwell, Helen; Benahmed, Mohamed; Bars, Rémi

    2007-05-01

    In conventional rodent toxicity studies the characterization of the adverse effects of a chemical relies primarily on gravimetric, and histopathological data. The aim of this study was to evaluate if the use of two-dimensional gel electrophoresis could generate protein accumulation profiles, which were in accordance with conventional toxicological findings by investigating a model antiandrogen, flutamide (FM), whose toxic effects, as measured using standard approaches, are well characterized. Male Sprague-Dawley rats were orally exposed to FM (0, 6, 30, and 150 mg/kg/day) for 28 days. The expected inhibition of androgen-dependent tissue stimulation, increased luteinizing hormone and testosterone plasma levels, and Leydig cell hyperplasia were observed. Changes in testicular protein accumulation profiles were evaluated in rats exposed to 150 mg/kg/day FM. Several proteins involved in steroidogenesis (e.g., StAR, ApoE, Hmgcs1, Idi1), cell cycle, and cancer (e.g., Ddx1, Hspd1) were modulated by FM, and these data provided molecular evidence for the hormonal and testicular histopathology changes recorded. Changes in proteins associated with spermatogenesis were also recorded, and these are discussed within the context of the testicular phenotype observed following FM treatment (i.e., normal spermatogenesis but Leydig cell hyperplasia). Overall, our data indicate that the combination of conventional toxicology measurements with omic observations has the potential to improve our global understanding of the toxicity of a compound.

  11. 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.

  12. Specificity Profiling of Protein-Binding Domains Using One-Bead-One-Compound Peptide Libraries

    PubMed Central

    Kunys, Andrew R.; Lian, Wenlong; Pei, Dehua

    2013-01-01

    One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need of any special equipment and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains) and subsequently using the specificity information to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains and the related data analysis. PMID:23788558

  13. Protein Profiling in Hepatocellular Carcinoma by Label-Free Quantitative Proteomics in Two West African Populations

    PubMed Central

    Fye, Haddy K. S.; Wright-Drakesmith, Cynthia; Kramer, Holger B.; Camey, Suzi; da Costa, Andre Nogueira; Jeng, Adam; Bah, Alasana; Kirk, Gregory D.; Sharif, Mohamed I. F.; Ladep, Nimzing G.; Okeke, Edith; Hainaut, Pierre; Taylor-Robinson, Simon D.; Kessler, Benedikt M.; Mendy, Maimuna E.

    2013-01-01

    Background Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC. Methods Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis. Results Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages. Conclusions The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of

  14. Influence of fermentable carbohydrates or protein on large intestinal and urinary metabolomic profiles in piglets.

    PubMed

    Pieper, R; Neumann, K; Kröger, S; Richter, J F; Wang, J; Martin, L; Bindelle, J; Htoo, J K; Vahjen, V; Van Kessel, A G; Zentek, J

    2012-12-01

    It was recently shown that variations in the ratio of dietary fermentable carbohydrates (fCHO) and fermentable protein (fCP) differentially affect large intestinal microbial ecology and the mucosal response. Here we investigated the use of mass spectrometry to profile changes in metabolite composition in colon and urine associated with variation in dietary fCHO and fCP composition and mucosal physiology. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP and low fCHO, low fCP and high fCHO, high fCP and low fCHO, and high fCP and high fCHO. After 21 to 23 d, all pigs were euthanized and colon digesta and urine metabolite profiles were obtained by mass spectrometry. Analysis of mass spectra by partial least squares approach indicated a clustering of both colonic and urinary profiles for each pig by feeding group. Metabolite identification and annotation using the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways revealed increased abundance of metabolites associated with arachidonic acid metabolism in colon of pigs fed a high concentration of fCP irrespective of dietary fCHO. Urinary metabolites did not show as clear patterns. Mass spectrometry can effectively differentiate metabolite profiles in colon contents and urine associated with changes in dietary composition. Whether metabolite profiling is an effective tool to identify specific metabolites (biomarkers) or metabolite profiles associated with gut function and integrity needs further elucidation.

  15. Proteomic Profiling Comparing the Effects of Different Heat Treatments on Camel (Camelus dromedarius) Milk Whey Proteins.

    PubMed

    Benabdelkamel, Hicham; Masood, Afshan; Alanazi, Ibrahim O; Alzahrani, Dunia A; Alrabiah, Deema K; AlYahya, Sami A; Alfadda, Assim A

    2017-03-28

    Camel milk is consumed in the Middle East because of its high nutritional value. Traditional heating methods and the duration of heating affect the protein content and nutritional quality of the milk. We examined the denaturation of whey proteins in camel milk by assessing the effects of temperature on the whey protein profile at room temperature (RT), moderate heating at 63 °C, and at 98 °C, for 1 h. The qualitative and quantitative variations in the whey proteins before and after heat treatments were determined using quantitative 2D-difference in gel electrophoresis (DIGE)-mass spectrometry. Qualitative gel image analysis revealed a similar spot distribution between samples at RT and those heated at 63 °C, while the spot distribution between RT and samples heated at 98 °C differed. One hundred sixteen protein spots were determined to be significantly different (p < 0.05 and a fold change of ≥1.2) between the non-heated and heated milk samples. Eighty protein spots were decreased in common in both the heat-treated samples and an additional 25 spots were further decreased in the 98 °C sample. The proteins with decreased abundance included serum albumin, lactadherin, fibrinogen β and γ chain, lactotransferrin, active receptor type-2A, arginase-1, glutathione peroxidase-1 and, thiopurine S, etc. Eight protein spots were increased in common to both the samples when compared to RT and included α-lactalbumin, a glycosylation-dependent cell adhesion molecule. Whey proteins present in camel milk were less affected by heating at 63 °C than at 98 °C. This experimental study showed that denaturation increased significantly as the temperature increased from 63 to 98 °C.

  16. Protein Profile in Corpus Luteum during Pregnancy in Korean Native Cows

    PubMed Central

    Chung, H. J.; Kim, K. W.; Han, D. W.; Lee, H. C.; Yang, B. C.; Chung, H. K.; Shim, M. R.; Choi, M. S.; Jo, E. B.; Jo, Y. M.; Oh, M. Y.; Jo, S. J.; Hong, S. K.; Park, J. K.; Chang, W. K.

    2012-01-01

    Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism, but the profile of proteins associated with progesterone synthesis in cyclic and pregnant corpus luteum (CL) is not well-known in cattle. In Experiment 1, plasma progesterone level was monitored in cyclic cows (n = 5) and pregnant cows (n = 6; until d-90). A significant decline in the plasma progesterone level occurred at d-19 of cyclic cows. Progesterone level in abbatoir-derived luteal tissues was also determined at d 1 to 5, 6 to 13 and 14 to 20 of cyclic cows, and d-60 and -90 of pregnant cows (n = 5 each). Progesterone level in d-60 CL was not different from those in d 6 to 13 CL and d-90 CL, although the difference between d 6 to 13 and d-90 was significant. In Experiment 2, protein expression pattern in CL at d-90 (n = 4) was compared with that in CL of cyclic cows at d 6 to 13 (n = 5). Significant changes in the level of protein expression were detected in 32 protein spots by two-dimensional polyacrylamide gel electrophoresis (2-DE), and 23 of them were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six proteins were found only in pregnant CL, while the other 17 proteins were found only in cyclic CL. Among the above 6 proteins, vimentin which is involved in the regulation of post-implantation development was included. Thus, the protein expression pattern in CL was disorientated from cyclic luteal phase to mid pregnancy, and alterations in specific CL protein expression may contribute to the maintenance of pregnancy in Korean native cows. PMID:25049514

  17. 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.

  18. 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.

  19. A rapid, automated surface protein profiling of single circulating exosomes in human blood

    PubMed Central

    Kibria, Golam; Ramos, Erika K.; Lee, Katelyn E.; Bedoyan, Sarah; Huang, Simo; Samaeekia, Ravand; Athman, Jaffre J.; Harding, Clifford V.; Lötvall, Jan; Harris, Lyndsay; Thompson, Cheryl L.; Liu, Huiping

    2016-01-01

    Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30–150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids. PMID:27819324

  20. Identification of plasma protein profiles associated with risk groups of prostate cancer patients

    PubMed Central

    Nordström, Malin; Wingren, Christer; Rose, Carsten; Bjartell, Anders; Becker, Charlotte; Lilja, Hans; Borrebaeck, Carl AK

    2015-01-01

    Purpose Early detection of prostate cancer (PC) using prostate-specific antigen (PSA) in blood reduces PC-death among unscreened men. However, due to modest specificity of PSA at commonly used cut-offs, there are urgent needs for additional biomarkers contributing enhanced risk classification among men with modestly elevated PSA. Experimental design Recombinant antibody microarrays were applied for protein expression profiling of 80 plasma samples from routine PSA-measurements, a priori divided into four risk groups, based on levels of total and %free PSA. Results The results demonstrated that plasma protein profiles could be identified that pinpointed PC (a malignant biomarker signature) and most importantly that showed moderate to high correlation with biochemically defined PC risk groups. Notably, the data also implied that the risk group with mid-range PSA and low %free PSA, a priori known to be heterogeneous, could be further stratified into two subgroups, more resembling the lowest and highest risk groups, respectively. Conclusions and clinical relevance In this pilot study, we have shown that plasma protein biomarker signatures, associated with risk groups of PC, could be identified from crude plasma samples using affinity proteomics. This approach could in the longer perspective provide novel opportunities for improved risk classification of PC patients. PMID:25196118

  1. 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

    2014-03-07

    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.

  2. 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.

  3. Itinerary profiling to analyze a large number of protein-folding trajectories

    PubMed Central

    Ota, Motonori; Ikeguchi, Mitsunori; Kidera, Akinori

    2016-01-01

    Understanding how proteins fold through a vast number of unfolded states is a major subject in the study of protein folding. Herein, we present itinerary profiling as a simple method to analyze molecular dynamics trajectories, and apply this method to Trp-cage. In itinerary profiling, structural clusters included in a trajectory are represented by a bit sequence, and a number of trajectories, as well as the structural clusters, can be compared and classified. As a consequence, the structural clusters that characterize the foldability of trajectories were able to be identified. The connections between the clusters were then illustrated as a network and the structural features of the clusters were examined. We found that in the true folding funnel, Trp-cage formed a left-handed main-chain topology and the Trp6 side-chain was located at the front of the main-chain ring, even in the initial unfolded states. In contrast, in the false folding funnel of the pseudo-native states, in which the Trp6 side-chain is upside down in the protein core, Trp-cage had a right-handed main-chain topology and the Trp side-chain was at the back. The initial topological partition, as determined by the main-chain handedness and the location of the Trp residue, predetermines Trp-cage foldability and the destination of the trajectory to the native state or the pseudo-native states.

  4. Protein expression profiling in head fragments during planarian regeneration after amputation.

    PubMed

    Chen, Xiaoguang; Xu, Cunshuan

    2015-04-01

    Following amputation, a planarian tail fragment can regrow into a complete organism including a well-organized brain within about 2-3 weeks, thus restoring the structure and function to presurgical levels. Despite the enormous potential of these animals for regenerative medicine, our understanding of the exact mechanism of planarian regeneration is incomplete. To better understand the molecular nature of planarian head regeneration, we applied two-dimensional electrophoresis (2-DE)/matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/time-of-flight mass spectrometry (TOF MS) technique to analyze the dynamic proteomic expression profiles over the course of 6 to 168 h post-decapitation. This approach identified a total of 141 differentially expressed proteins, 47 of which exhibited exceptionally high fold changes (≥3-fold change). Of these, Rx protein, an important regulator of head and brain development, was considered to be closely related to planarian head regeneration because of its exceptional high expression almost throughout the time course of regeneration process. Functional annotation analysis classified the 141 proteins into eight categories: (1) signaling, (2) Ca(2+) binding and translocation, (3) transcription and translation, (4) cytoskeleton, (5) metabolism, (6) cell protection, (7) tissue differentiation, and (8) cell cycle. Signaling pathway analysis indicated that Wnt1/Ca(2+) signaling pathway was activated during head regeneration. Integrating the analyses of proteome expression profiling, functional annotation, and signaling pathway, amputation-induced head reformation requires some mechanisms to promote cell proliferation and differentiation, including differential regulation of proapoptotic and antiapoptotic proteins, and the regulation of proliferation and differentiation-related proteins. Importantly, Wnt1/Ca(2+) signaling pathway upregulates Rx expression, finally facilitating the differentiation of neoblasts into various

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

    SciTech Connect

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

    2002-01-01

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

  6. Protein profiles of Escherichia coli and Staphylococcus warneri are altered by photosensitization with cationic porphyrins.

    PubMed

    Alves, Eliana; Esteves, Ana Cristina; Correia, António; Cunha, Ângela; Faustino, Maria A F; Neves, Maria G P M S; Almeida, Adelaide

    2015-06-01

    Oxidative stress induced by photodynamic treatment of microbial cells causes irreversible damages to vital cellular components such as proteins. Photodynamic inactivation (PDI) of bacteria, a promising therapeutic approach for the treatment of superficial and localized skin and oral infections, can be achieved by exciting a photosensitizing agent with visible light in an oxygenated environment. Although some studies have addressed the oxidative alterations of PDI in bacterial proteins, the present study is the first to compare the electrophoretic profiles of proteins of Gram-positive and Gram-negative bacteria, having two structurally different porphyrins, with different kinetics of photoinactivation. The cationic porphyrins 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide (Tri-Py(+)-Me-PF) and 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py(+)-Me) were used to photosensitize Escherichia coli and Staphylococcus warneri upon white light irradiation at an irradiance of 4.0 mW cm(-2). After different photosensitization periods, proteins were extracted from bacteria and analyzed using one-dimensional SDS-PAGE. Apparent molecular weights and band intensities were determined after an irradiation period corresponding to a reduction of 4 log10 in cell viability. After photodynamic treatment, there was a general loss of bacterial proteins, assigned to large-scale protein degradation. Protein loss was more pronounced after PDI with Tri-Py(+)-Me-PF in both bacteria. There was also an increase in the concentration of some proteins as well as an increase in the molecular weight of other proteins. We show that proteins of E. coli and S. warneri are important targets of PDI. Although there is an attempt of cellular response to the PDI-induced damage by overexpression of a limited number of proteins, the damage is lethal. Our results show that changes occurring in the protein pattern during photodynamic treatment are

  7. Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling

    PubMed Central

    Freiberger, Elyse C.; Richards, Alicia L.; Jochem, Adam; Rush, Matthew J. P.; Ulbrich, Arne; Robinson, Kyle P.; Hutchins, Paul D.; Veling, Mike T.; Guo, Xiao; Kemmerer, Zachary A.; Connors, Kyle J.; Trujillo, Edna A.; Sokol, Jacob; Marx, Harald; Westphall, Michael S.; Hebert, Alexander S.; Pagliarini, David J.; Coon, Joshua J.

    2016-01-01

    Mitochondrial dysfunction is associated with many human diseases, including cancer and neurodegeneration, that are often linked to proteins and pathways that are not well-characterized. To begin defining the functions of such poorly characterized proteins, we used mass spectrometry to map the proteomes, lipidomes and metabolomes of 174 yeast strains, each lacking a single gene related to mitochondrial biology. 144 of these genes have human homologs, 60 of which are associated with disease and 39 of which are uncharacterized. We present a multi-omic data analysis and visualization tool that we use to find covariance networks that can predict molecular functions, correlations between profiles of related gene deletions, gene-specific perturbations that reflect protein functions, and a global respiration deficiency response. Using this multi-omic approach, we link seven proteins including Hfd1p and its human homolog ALDH3A1 to mitochondrial coenzyme Q (CoQ) biosynthesis, an essential pathway disrupted in many human diseases. This Resource should provide broad molecular insights into mitochondrial protein functions. PMID:27669165

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26132554

  9. 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.

  10. 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.

  11. 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.

  12. Multiple protein extract microarray for profiling human food-specific immunoglobulins A, M, G and E.

    PubMed

    Renault, N K; Gaddipati, S R; Wulfert, F; Falcone, F H; Mirotti, L; Tighe, P J; Wright, V; Alcocer, M J C

    2011-02-01

    Existing food immunoglobulin (Ig) tests require large volumes of serum, are limited to one immunoglobulin class, are not amenable to high throughput analysis and only give a limited picture of the immunological response to food antigens. Conversely a new generation of Component Resolved Diagnostic systems using pure proteins is highly specific and totally dependent on the availability of the protein in its recombinant or natural origin form. Here we demonstrate a proof-of-concept of a microarray test based on protein extracts of food components. Our approach relies on innovations on three different fronts: the novelty of using arrayed food samples sequentially extracted with detergent and chaotropic agents, the ability to measure four different Ig classes simultaneously and the ability to analyse the generated data via a suitable bioinformatics/statistical analysis interface. This approach combines high numerical power of microarrays with automation, high throughput analysis and enables detailed investigation of the Ig profiles to food antigens. The prototype shown contains extracts of approximately 350 food ingredients that cover most of the food products found in the UK. Here we showed that the use of a sequential extraction technique to solubilise and then denature food samples has its benefits in the assessment of variations in antigenicity when tested with human sera. A patient dependent degree of class specificity was observed with human sera (IgG specificity correlates well with IgA>IgM>IgE). Besides generating a simultaneous profile for IgA, IgM, IgG and IgE the array system has shown good discrimination between challenge responders in atopic and non-atopic individuals. Poly- and mono-specific IgE responders were easily identified. The mathematical modelling of specific IgE content showed good correlations when compared with established IgE antibody testing assay (UniCAP). Although in its proof-of-principle stages, the immune profiling technique described

  13. 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

  14. Using size exclusion chromatography-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling

    SciTech Connect

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

    2006-07-01

    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.

  15. 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.

  16. Protein Kinase Target Discovery From Genome-Wide Messenger RNA Expression Profiling

    PubMed Central

    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. PMID:20687179

  17. 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.

  18. Brain death induces the alteration of liver protein expression profiles in rabbits.

    PubMed

    Du, Bing; Li, Ling; Zhong, Zhibiao; Fan, Xiaoli; Qiao, Bingbing; He, Chongxiang; Fu, Zhen; Wang, Yanfeng; Ye, Qifa

    2014-08-01

    At present, there is no accurate method for evaluating the quality of liver transplant from a brain-dead donor. Proteomics are used to investigate the mechanisms involved in brain death‑induced liver injury and to identify sensitive biomarkers. In the present study, age‑ and gender‑matched rabbits were randomly divided into the brain death and sham groups. The sham served as the control. A brain‑death model was established using an intracranial progressive pressurized method. The differentially expressed proteins extracted from the liver tissues of rabbits that were brain‑dead for 6 h in the two groups were determined by two‑dimensional gel electrophoresis and matrix‑assisted laser desorption ionization time of flight mass spectrometry. Although there was no obvious functional and morphological difference in 2, 4 and 6 h after brain death, results of the proteomics analysis revealed 973±34 and 987±38 protein spots in the control and brain death groups, respectively. Ten proteins exhibited a ≥2‑fold alteration. The downregulated proteins were: aldehyde dehydrogenase, runt‑related transcription factor 1 (RUNX1), inorganic pyrophosphatase, glutamate‑cysteine ligase regulatory subunit and microsomal cytochrome B5. By contrast, the expression of dihydropyrimidinase-related protein 4, peroxiredoxin‑6, 3‑phosphoinositide‑dependent protein kinase‑1, 3-mercaptopyruvate and alcohol dehydrogenase were clearly upregulated. Immunohistochemistry and western blot analysis results revealed that the expression of RUNX1 was gradually increased in a time‑dependent manner in 2, 4, and 6 h after brain death. In conclusion, alteration of the liver protein expression profile induced by brain death indicated the occurrence of complex pathological changes even if no functional or morphological difference was identified. Thus, RUNX1 may be a sensitive predict factor for evaluating the quality of brain death donated liver.

  19. 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.

  20. Ethanol utilization regulatory protein: profile alignments give no evidence of origin through aldehyde and alcohol dehydrogenase gene fusion.

    PubMed Central

    Nicholas, H. B.; Persson, B.; Jörnvall, H.; Hempel, J.

    1995-01-01

    The suggestion that the ethanol regulatory protein from Aspergillus has its evolutionary origin in a gene fusion between aldehyde and alcohol dehydrogenase genes (Hawkins AR, Lamb HK, Radford A, Moore JD, 1994, Gene 146:145-158) has been tested by profile analysis with aldehyde and alcohol dehydrogenase family profiles. We show that the degree and kind of similarity observed between these profiles and the ethanol regulatory protein sequence is that expected from random sequences of the same composition. This level of similarity fails to support the suggested gene fusion. PMID:8580855

  1. Imbalanced Expression of Vcan mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

    PubMed Central

    Burns, Tara A.; Dours-Zimmermann, Maria T.; Zimmermann, Dieter R.; Krug, Edward L.; Comte-Walters, Susana; Reyes, Leticia; Davis, Monica A.; Schey, Kevin L.; Schwacke, John H.; Kern, Christine B.; Mjaatvedt, Corey H.

    2014-01-01

    The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles. PMID:24586547

  2. 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.

  3. Serum protein N-glycans profiling for the discovery of potential biomarkers for nonalcoholic steatohepatitis.

    PubMed

    Chen, Cuiying; Schmilovitz-Weiss, Hemda; Liu, Xue-en; Pappo, Orit; Halpern, Marisa; Sulkes, Jaqueline; Braun, Marius; Cohen, Maya; Barak, Nir; Tur-Kaspa, Ran; Vanhooren, Valerie; Van Vlierberghe, Hans; Libert, Claude; Contreras, Roland; Ben-Ari, Ziv

    2009-02-01

    The hepatic histology in nonalcoholic fatty liver disease can vary from isolated hepatic steatosis to steatohepatitis can progress to cirrhosis and liver-related death. The aim was to evaluate the use of blood serum N-glycan fingerprinting as a tool for differential diagnosis of nonalcoholic steatohepatitis from steatosis. A group of 47 patients with NAFLD was diagnosed by clinical laboratory analysis and ultrasonography, and was studied histologically using the Brunt's scoring system. The control group included 13 healthy individuals. N-glycan profiles of serum proteins were determined by DNA sequencer-based carbohydrate analytical profiling. We have found that the concentrations of two glycans (NGA2F and NA2) and their logarithm ratio of NGA2F versus NA2 (named GlycoNashTest) were associated with the degree of NASH-related fibrosis, but had no correlation with the grade of inflammation nor steatosis severity. When used to screen NAFLD patients, GlycoNashTest could identify advanced NASH-related fibrosis (F3-F4) with the diagnosis sensitivity of 89.5% and specificity of 71.4%. The serum N-glycan profile is a promising noninvasive method for detecting NASH or NASH-related fibrosis in NAFLD patients, which could be a valuable supplement to other markers currently used in diagnosis of NASH.

  4. Profiling of Protein N-Termini and Their Modifications in Complex Samples.

    PubMed

    Demir, Fatih; Niedermaier, Stefan; Kizhakkedathu, Jayachandran N; Huesgen, Pitter F

    2017-01-01

    Protein N termini are a unique window to the functional state of the proteome, revealing translation initiation sites, co-translation truncation and modification, posttranslational maturation, and further proteolytic processing into different proteoforms with distinct functions. As a direct readout of proteolytic activity, protein N termini further reveal proteolytic regulation of diverse biological processes and provide a route to determine specific substrates and hence the physiological functions for any protease of interest. Here, we describe our current protocol of the successful Terminal Amine Isotope Labeling of Substrates (TAILS) technique, which enriches protein N-terminal peptides from complex proteome samples by negative selection. Genome-encoded N termini, protease-generated neo-N termini, and endogenously modified N termini are all enriched simultaneously. Subsequent mass spectrometric analysis therefore profiles all protein N termini and their modifications present in a complex sample in a single experiment. We further provide a detailed protocol for the TAILS-compatible proteome preparation from plant material and discuss specific considerations for N terminome data analysis and annotation.

  5. 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.

  6. 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

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

    PubMed

    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; Monaghan, Tanya M

    2015-09-01

    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.

  8. 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.

  9. 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

  10. 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.

  11. Protein Profiling and Histone Deacetylation Activities in Somaclonal Variants of Oil Palm (Elaeis guineensis Jacq.)

    PubMed Central

    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. Conformational transition free energy profiles of an adsorbed, lattice model protein by multicanonical Monte Carlo simulation

    NASA Astrophysics Data System (ADS)

    Castells, Victoria; Van Tassel, Paul R.

    2005-02-01

    Proteins often undergo changes in internal conformation upon interacting with a surface. We investigate the thermodynamics of surface induced conformational change in a lattice model protein using a multicanonical Monte Carlo method. The protein is a linear heteropolymer of 27 segments (of types A and B) confined to a cubic lattice. The segmental order and nearest neighbor contact energies are chosen to yield, in the absence of an adsorbing surface, a unique 3×3×3 folded structure. The surface is a plane of sites interacting either equally with A and B segments (equal affinity surface) or more strongly with the A segments (A affinity surface). We use a multicanonical Monte Carlo algorithm, with configuration bias and jump walking moves, featuring an iteratively updated sampling function that converges to the reciprocal of the density of states 1/Ω(E), E being the potential energy. We find inflection points in the configurational entropy, S(E)=klnΩ(E), for all but a strongly adsorbing equal affinity surface, indicating the presence of free energy barriers to transition. When protein-surface interactions are weak, the free energy profiles F(E)=E-TS(E) qualitatively resemble those of a protein in the absence of a surface: a free energy barrier separates a folded, lowest energy state from globular, higher energy states. The surface acts in this case to stabilize the globular states relative to the folded state. When the protein surface interactions are stronger, the situation differs markedly: the folded state no longer occurs at the lowest energy and free energy barriers may be absent altogether.

  13. Molecular characterization and expression profiling of the protein disulfide isomerase gene family in Brachypodium distachyon L.

    PubMed

    Zhu, Chong; Luo, Nana; He, Miao; Chen, Guanxing; Zhu, Jiantang; Yin, Guangjun; Li, Xiaohui; Hu, Yingkao; Li, Jiarui; Yan, Yueming

    2014-01-01

    Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene

  14. Intact capture of hypervelocity projectiles

    NASA Technical Reports Server (NTRS)

    Tsou, P.

    1990-01-01

    The ability to capture projectiles intact at hypervelocities opens new applications in science and technology that would either not be possible or would be very costly by other means. This capability has been demonstrated in the laboratory for aluminum projectiles of 1.6 mm diameter, captured at 6 km/s, in one unmelted piece, and retaining up to 95% of the original mass. Furthermore, capture was accomplished passively using microcellular underdense polymer foam. Another advantage of capturing projectiles in an underdense medium is the ability of such a medium to preserve a record of the projectile's original velocity components of speed and direction. A survey of these experimental results is described in terms of a dozen parameters which characterize the amount of capture and the effect on the projectile due to different capture media.

  15. Intact capture of hypervelocity projectiles.

    PubMed

    Tsou, P

    1990-01-01

    The ability to capture projectiles intact at hypervelocities opens new applications in science and technology that would either not be possible or would be very costly by other means. This capability has been demonstrated in the laboratory for aluminum projectiles of 1.6 mm diameter, captured at 6 km/s, in one unmelted piece, and retaining up to 95% of the original mass. Furthermore, capture was accomplished passively using microcellular underdense polymer foam. Another advantage of capturing projectiles in an underdense medium is the ability of such a medium to preserve a record of the projectile's original velocity components of speed and direction. A survey of these experimental results is described in terms of a dozen parameters which characterize the amount of capture and the effect on the projectile due to different capture media.

  16. Comparison of protein profiles between acetonitrile- and non-acetonitrile-treated sera from patients with nasopharyngeal carcinomas.

    PubMed

    Huang, Yuan-Jiao; Deng, Kai-Feng; Xuan, Chao; Zhang, Bei-Bei; Zhou, Yi; Yang, Xiaoli; He, Ming

    2011-05-01

    Serum proteins may be abnormally increased or decreased during the occurrence and development of nasopharyngeal carcinoma (NPC). However, currently there are no simple or effective methods to collect and differentiate these abnormally secreted proteins from abundant serum proteins. In this study, acetonitrile was used to remove the majority of high-abundance proteins from serum samples obtained from patients with NPC. The samples were subjected to surface-enhanced laser desorption/ionization time-of-flight mass spectrometry with a CM10 (weak cation exchange) ProteinChip, and the resulting protein profiles were compared with those of non-acetonitrile-treated serum samples. The results showed that the protein profiles differed between the acetonitrile- and non-acetonitrile-treated sera from patients with NPC. A large proportion of the non-acetonitrile-treated NPC serum protein peaks were <6000 kDa, while the detection rate of protein peaks >6000 kDa was relatively higher in the acetonitrile-treated NPC sera, accounting for more than half of all protein peaks (26.2+37.5%). Few differentially upregulated proteins were lost, and the peak value density increased after acetonitrile treatment. In conclusion, acetonitrile treatment of serum samples is effective in removing high-abundance macromolecular proteins. Therefore, acetonitrile treatment can be applied for the investigation of serum proteomics and may aid in the identification of differentially expressed proteins.

  17. Identification and expression profile analysis of odorant binding protein and chemosensory protein genes in Bemisia tabaci MED by head transcriptome

    PubMed Central

    Zhang, Wei; Zhang, Xiaoman; Qu, Cheng; Tetreau, Guillaume; Sun, Lujuan; Zhou, Jingjiang

    2017-01-01

    Odorant binding proteins (OBPs) and chemosensory proteins (CSPs) of arthropods are thought to be involved in chemical recognition which regulates pivotal behaviors including host choice, copulation and reproduction. In insects, OBPs and CSPs located mainly in the antenna but they have not been systematically characterized yet in Bemisia tabaci which is a cryptic species complex and could damage more than 600 plant species. In this study, among the 106,893 transcripts in the head assembly, 8 OBPs and 13 CSPs were identified in B. tabaci MED based on head transcriptomes of adults. Phylogenetic analyses were conducted to investigate the relationships of B. tabaci OBPs and CSPs with those from several other important Hemipteran species, and the motif-patterns between Hemiptera OBPs and CSPs were also compared by MEME. The expression profiles of the OBP and CSP genes in different tissues of B. tabaci MED adults were analyzed by real-time qPCR. Seven out of the 8 OBPs found in B. tabaci MED were highly expressed in the head. Conversely, only 4 CSPs were enriched in the head, while the other nine CSPs were specifically expressed in other tissues. Our findings pave the way for future research on chemical recognition of B. tabaci at the molecular level. PMID:28166541

  18. Protein profiling of Haemonchus contortus found in sheep of Kashmir valley.

    PubMed

    Tak, Irfan-Ur-Rauf; Chishti, M Z; Ahmad, Fayaz

    2015-12-01

    Economic losses due to helminth parasites in sheep throughout the world are considerable. Haemonchus contortus is a blood sucking intestinal helminth that lives in the abomasum of small ruminants worldwide. This parasite can be devastating to producers as it causes decreased production levels due to clinical signs such as anaemia, edema and death. For isolation of the proteins of the parasite, a well defined methodology was adopted. The abomasae of sheep in which this parasite resides were collected from abattoirs of various districts and were then carried to laboratory for screening. In case of collection sites falling in far areas, the organs were screened on spot. The parasites were collected in normal saline, washed and stored in 0.05 M PBS with pH of 7.4 at 0 °C. After refrigeration, frozen nematodes were thawed, homogenized and centrifuged at 1,000-15,000 rpm for 15 min. The supernatant was thus collected as a protein mixture and stored at -20 °C. Protein concentration of the samples was estimated by Lowry method. The samples were then analyzed through PAGE and then through SDS-PAGE. Protein estimation of the samples was estimated to be 4.2 mg/ml. The processed parasite samples were then subjected to PAGE and SDS-PAGE to determine the presence of the proteins. It showed high concentration of proteins in its whole protein profile. The proteins were seen as continuous bands intermixing with each other in PAGE analysis. The present study revealed two bands of molecular weights-55 and 33 kDa in PAGE analysis. The proteins when analyzed through SDS-PAGE were mostly found in the range of 25-70 kDa. The SDS-PAGE analysis showed four prominent bands. These bands were of the molecular weights of 66, 40, 33 and 26 kDa. The present work was a challenging one since only a single study was conducted in this region on this aspect and thus obviously was a big task to peep into the field where scanty input was available.

  19. Structural and molecular interrogation of intact biological systems

    PubMed Central

    Chung, Kwanghun; Wallace, Jenelle; Kim, Sung-Yon; Kalyanasundaram, Sandhiya; Andalman, Aaron S.; Davidson, Thomas J.; Mirzabekov, Julie J.; Zalocusky, Kelly A.; Mattis, Joanna; Denisin, Aleksandra K.; Pak, Sally; Bernstein, Hannah; Ramakrishnan, Charu; Grosenick, Logan; Gradinaru, Viviana; Deisseroth, Karl

    2014-01-01

    Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease. PMID:23575631

  20. Development and Validation of a Method for Profiling Post-Translational Modification Activities Using Protein Microarrays

    PubMed Central

    Widschwendter, Martin; Sun, Dahui; Sieburg, Hans B.; Spruck, Charles

    2010-01-01

    Background Post-translational modifications (PTMs) impact on the stability, cellular location, and function of a protein thereby achieving a greater functional diversity of the proteome. To fully appreciate how PTMs modulate signaling networks, proteome-wide studies are necessary. However, the evaluation of PTMs on a proteome-wide scale has proven to be technically difficult. To facilitate these analyses we have developed a protein microarray-based assay that is capable of profiling PTM activities in complex biological mixtures such as whole-cell extracts and pathological specimens. Methodology/Principal Findings In our assay, protein microarrays serve as a substrate platform for in vitro enzymatic reactions in which a recombinant ligase, or extracts prepared from whole cells or a pathological specimen is overlaid. The reactions include labeled modifiers (e.g., ubiquitin, SUMO1, or NEDD8), ATP regenerating system, and other required components (depending on the assay) that support the conjugation of the modifier. In this report, we apply this methodology to profile three molecularly complex PTMs (ubiquitylation, SUMOylation, and NEDDylation) using purified ligase enzymes and extracts prepared from cultured cell lines and pathological specimens. We further validate this approach by confirming the in vivo modification of several novel PTM substrates identified by our assay. Conclusions/Significance This methodology offers several advantages over currently used PTM detection methods including ease of use, rapidity, scale, and sample source diversity. Furthermore, by allowing for the intrinsic enzymatic activities of cell populations or pathological states to be directly compared, this methodology could have widespread applications for the study of PTMs in human diseases and has the potential to be directly applied to most, if not all, basic PTM research. PMID:20596523

  1. 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.

  2. 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.

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

    PubMed

    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; Walt, David R

    2014-03-21

    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 noninvasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics.

  4. SVM-PB-Pred: SVM based protein block prediction method using sequence profiles and secondary structures.

    PubMed

    Suresh, V; Parthasarathy, S

    2014-01-01

    We developed a support vector machine based web server called SVM-PB-Pred, to predict the Protein Block for any given amino acid sequence. The input features of SVM-PB-Pred include i) sequence profiles (PSSM) and ii) actual secondary structures (SS) from DSSP method or predicted secondary structures from NPS@ and GOR4 methods. There were three combined input features PSSM+SS(DSSP), PSSM+SS(NPS@) and PSSM+SS(GOR4) used to test and train the SVM models. Similarly, four datasets RS90, DB433, LI1264 and SP1577 were used to develop the SVM models. These four SVM models developed were tested using three different benchmarking tests namely; (i) self consistency, (ii) seven fold cross validation test and (iii) independent case test. The maximum possible prediction accuracy of ~70% was observed in self consistency test for the SVM models of both LI1264 and SP1577 datasets, where PSSM+SS(DSSP) input features was used to test. The prediction accuracies were reduced to ~53% for PSSM+SS(NPS@) and ~43% for PSSM+SS(GOR4) in independent case test, for the SVM models of above two same datasets. Using our method, it is possible to predict the protein block letters for any query protein sequence with ~53% accuracy, when the SP1577 dataset and predicted secondary structure from NPS@ server were used. The SVM-PB-Pred server can be freely accessed through http://bioinfo.bdu.ac.in/~svmpbpred.

  5. Oak protein profile alterations upon root colonization by an ectomycorrhizal fungus.

    PubMed

    Sebastiana, Mónica; Martins, Joana; Figueiredo, Andreia; Monteiro, Filipa; Sardans, Jordi; Peñuelas, Josep; Silva, Anabela; Roepstorff, Peter; Pais, Maria Salomé; Coelho, Ana Varela

    2017-02-01

    An increased knowledge on the real impacts of ectomycorrhizal symbiosis in forest species is needed to optimize forest sustainable productivity and thus to improve forest services and their capacity to act as carbon sinks. In this study, we investigated the response of an oak species to ectomycorrhizae formation using a proteomics approach complemented by biochemical analysis of carbohydrate levels. Comparative proteome analysis between mycorrhizal and nonmycorrhizal cork oak plants revealed no differences at the foliar level. However, the protein profile of 34 unique oak proteins was altered in the roots. Consistent with the results of the biochemical analysis, the proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose for the metabolic activity of mycorrhizal roots which is consistent with an increased allocation of carbohydrates from the plant to the fungus in order to sustain the symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of defense reactions, increased nutrient mobilization from the plant-fungus interface (N and P), as well as cytoskeleton rearrangements and induction of plant cell wall loosening for fungal root accommodation in colonized roots are also suggested by the results. The suggested improvement in root capacity to take up nutrients accompanied by an increase of root biomass without apparent changes in aboveground biomass strongly re-enforces the potential of mycorrhizal inoculation to improve cork oak forest resistance capacity to cope with coming climate change.

  6. [Protein profile and iron deficiency: value of the study of the albumin-transferrin couple].

    PubMed

    Cacoub, P; Thiolières, J M; Alexandre, J A; Foglietti, M J; Giraudet, P; Godeau, P

    1996-01-01

    From a clinical standpoint, the search for iron deficiency is based upon serum ferritin. However, serumferritin values may be pathologic in other numerous pathological conditions such as inflammation, liver diseases, malignant hematologic disorders, hemolysis, etc. Proteic profile combines the analyze of proteins variations: protein results are converted in percent of normal values referenced for the technique used. It has been suggested that on the protein profile, an increase in serum transferrin level compared to a normal serum albumin level (DAT: difference albumin-transferrin), appears early in the course of iron deficiency. In order to know the value of a pathologic DAT > or = 28% in the diagnosis of iron deficiency, we prospectively studied 156 patients consecutively hospitalized in an internal medicine department. Iron deficiency was defined by a low serum ferritin level. Diagnosis performance (sensitivity, specificity, positive and negative predictive values) of different biologic markers of iron deficiency (serum iron, saturation of total iron-binding capacity, low mean erythrocyte volume) and DAT was compared to the performance of low serum ferritin values. With the exception of low serum ferritin (which have by definition a specificity and a positive predictive value of 100%), pathologic DAT appeared as the best index of iron deficiency with the highest sensitivity (67.4%), specificity (97.3%), positive predictive value (91.2%), negative predicitive value (87.7%) and diagnosis efficacy (sensitivity x specificity = 0.66). A pathologic DAT associated to a low serum ferritin level increased the diagnosis performance of both tests to 0.72. Diagnosis efficacy of DAT was not changed (0.66) in 83 patients with a confounding factor for serum ferritin analysis (inflammation, liver diseases, malignant hematologic disorders, hemolysis) when diagnosis efficacy of all other tests decreased. There was a negative correlation between serum ferritin level and DAT level

  7. 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.

  8. Allergen profiles of natural rubber latex (NRL) proteins on gloves and glove powders.

    PubMed

    Tomazic-Jezic, Vesna J; Sanchez, B A

    2005-01-01

    The contributing role of glove powder in sensitization to natural rubber latex (NRL) proteins has been well documented in laboratory studies and through clinical evaluations. However, the quantitative relationship of the respiratory and topical exposures in the sensitization process remains unknown because the relative levels of protein on the glove powders in relation to the total levels of protein on NRL gloves have not been determined. In NRL allergens--Hev b 1, Hev b 3, Hev b 5, and Hev b 6.02--on randomly selected surgical and examination NRL gloves. We also examined the binding pattern of the four allergens to several glove powders that showed a different affinity to NRL proteins. The level of powder-bound protein was determined by the ELISA Inhibition Assay (ASTM D6499 standard method). Two cross-linked corn starch powders, one sample of cooking corn starch and one oat starch sample, were exposed to ammoniated (AL) or nonammoniated (NAL) raw NRL protein extracts. The levels of individual allergens were determined using the NRL allergen kit. In the NRL glove extracts we observed a wide range in the total allergen levels and a great diversity in the proportion of the four allergens. On the other hand, the evaluated starches had similar ratios of four individual allergens, regardless of the differences in their total allergen levels. The exposure of starches to NRL proteins with different allergen profiles did not affect the allergen ratio. All samples demonstrated a selective affinity for binding Hev b 1 and Hev b 5 allergens and a lesser affinity for the Hev b 6.02 allergen. Allergen Hev b 6.02 made up about 60% of the total allergen in the NAL extract, but only 12-30% of Hev b 6.02 was bound to starches. In contrast, there was only 3-7% of Hev b 1 allergen in the NAL extract, but powders had 35-45% of Hev b 1. These findings indicate that allergenic properties of NRL gloves and respective glove powders may be different.

  9. 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

  10. Differential Expression of In Vivo and In Vitro Protein Profile of Outer Membrane of Acidovorax avenae Subsp. avenae

    PubMed Central

    Qiu, Hui; Li, Bin; Jabeen, Amara; Li, Liping; Liu, He; Kube, Michael; Xie, Guanlin; Wang, Yanli; Sun, Guochang

    2012-01-01

    Outer membrane (OM) proteins play a significant role in bacterial pathogenesis. In this work, we examined and compared the expression of the OM proteins of the rice pathogen Acidovorax avenae subsp. avenae strain RS-1, a Gram-negative bacterium, both in an in vitro culture medium and in vivo rice plants. Global proteomic profiling of A. avenae subsp. avenae strain RS-1 comparing in vivo and in vitro conditions revealed the differential expression of proteins affecting the survival and pathogenicity of the rice pathogen in host plants. The shotgun proteomics analysis of OM proteins resulted in the identification of 97 proteins in vitro and 62 proteins in vivo by mass spectrometry. Among these OM proteins, there is a high number of porins, TonB-dependent receptors, lipoproteins of the NodT family, ABC transporters, flagellins, and proteins of unknown function expressed under both conditions. However, the major proteins such as phospholipase and OmpA domain containing proteins were expressed in vitro, while the proteins such as the surface anchored protein F, ATP-dependent Clp protease, OmpA and MotB domain containing proteins were expressed in vivo. This may indicate that these in vivo OM proteins have roles in the pathogenicity of A. avenae subsp. avenae strain RS-1. In addition, the LC-MS/MS identification of OmpA and MotB validated the in silico prediction of the existance of Type VI secretion system core components. To the best of our knowledge, this is the first study to reveal the in vitro and in vivo protein profiles, in combination with LC-MS/MS mass spectra, in silico OM proteome and in silico genome wide analysis, of pathogenicity or plant host required proteins of a plant pathogenic bacterium. PMID:23166741

  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.

  12. The spinal precerebellar nuclei: calcium binding proteins and gene expression profile in the mouse.

    PubMed

    Fu, YuHong; Sengul, Gulgun; Paxinos, George; Watson, Charles

    2012-06-19

    We have localized the spinocerebellar neuron groups in C57BL/6J mice by injecting the retrograde neuronal tracer Fluoro-Gold into the cerebellum and examined the distribution of SMI 32 and the calcium-binding proteins (CBPs), calbindin-D-28K (Cb), calretinin (Cr), and parvalbumin (Pv) in the spinal precerebellar nuclei. The spinal precerebellar neuron clusters identified were the dorsal nucleus, central cervical nucleus, lumbar border precerebellar nucleus, lumbar precerebellar nucleus, and sacral precerebellar nucleus. Some dispersed neurons in the deep dorsal horn and spinal laminae 6-8 also projected to the cerebellum. Cb, Cr, Pv, and SMI 32 were present in all major spinal precerebellar nuclei and Pv was the most commonly observed CBP. A number of genes expressed in hindbrain precerebellar nuclei are also expressed in spinal precerebellar groups, but there were some differences in gene expression profile between the different spinal precerebellar nuclei, pointing to functional diversity amongst them.

  13. 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.

  14. Glucocorticoids regulate arrestin gene expression and redirect the signaling profile of G protein-coupled receptors.

    PubMed

    Oakley, Robert H; Revollo, Javier; Cidlowski, John A

    2012-10-23

    G protein-coupled receptors (GPCRs) compose the largest family of cell surface receptors and are the most common target of therapeutic drugs. The nonvisual arrestins, β-arrestin-1 and β-arrestin-2, are multifunctional scaffolding proteins that play critical roles in GPCR signaling. On binding of activated GPCRs at the plasma membrane, β-arrestins terminate G protein-dependent responses (desensitization) and stimulate β-arrestin-dependent signaling pathways. Alterations in the cellular complement of β-arrestin-1 and β-arrestin-2 occur in many human diseases, and their genetic ablation in mice has severe consequences. Surprisingly, however, the factors that control β-arrestin gene expression are poorly understood. We demonstrate that glucocorticoids differentially regulate β-arrestin-1 and β-arrestin-2 gene expression in multiple cell types. Glucocorticoids act via the glucocorticoid receptor (GR) to induce the synthesis of β-arrestin-1 and repress the expression of β-arrestin-2. Glucocorticoid-dependent regulation involves the recruitment of ligand-activated glucocorticoid receptors to conserved and functional glucocorticoid response elements in intron-1 of the β-arrestin-1 gene and intron-11 of the β-arrestin-2 gene. In human lung adenocarcinoma cells, the increased expression of β-arrestin-1 after glucocorticoid treatment impairs G protein-dependent activation of inositol phosphate signaling while enhancing β-arrestin-1-dependent stimulation of the MAPK pathway by protease activated receptor 1. These studies demonstrate that glucocorticoids redirect the signaling profile of GPCRs via alterations in β-arrestin gene expression, revealing a paradigm for cross-talk between nuclear and cell surface receptors and a mechanism by which glucocorticoids alter the clinical efficacy of GPCR-based drugs.

  15. 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

  16. Prediction of RNA binding sites in a protein using SVM and PSSM profile.

    PubMed

    Kumar, Manish; Gromiha, M Michael; Raghava, G P S

    2008-04-01

    RNA-binding proteins (RBPs) play key roles in post-transcriptional control of gene expression, which, along with transcriptional regulation, is a major way to regulate patterns of gene expression during development. Thus, the identification and prediction of RNA binding sites is an important step in comprehensive understanding of how RBPs control organism development. Combining evolutionary information and support vector machine (SVM), we have developed an improved method for predicting RNA binding sites or RNA interacting residues in a protein sequence. The prediction models developed in this study have been trained and tested on 86 RNA binding protein chains and evaluated using fivefold cross validation technique. First, a SVM model was developed that achieved a maximum Matthew's correlation coefficient (MCC) of 0.31. The performance of this SVM model further improved the MCC from 0.31 to 0.45, when multiple sequence alignment in the form of PSSM profiles was used as input to the SVM, which is far better than the maximum MCC achieved by previous methods (0.41) on the same dataset. In addition, SVM models were also developed on an alternative dataset that contained 107 RBP chains. Utilizing PSSM as input information to the SVM, the training/testing on this alternate dataset achieved a maximum MCC of 0.32. Conclusively, the prediction performance of SVM models developed in this study is better than the existing methods on the same datasets. A web server 'Pprint' was also developed for predicting RNA binding residues in a protein sequence which is freely available at http://www.imtech.res.in/raghava/pprint/.

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

    PubMed

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

    2015-08-01

    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.

  18. 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.

  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. 24-Hour Glucose Profiles on Diets Varying in Protein Content and Glycemic Index

    PubMed Central

    van Baak, Marleen A.

    2014-01-01

    Evidence is increasing that the postprandial state is an important factor contributing to the risk of chronic diseases. Not only mean glycemia, but also glycemic variability has been implicated in this effect. In this exploratory study, we measured 24-h glucose profiles in 25 overweight participants in a long-term diet intervention study (DIOGENES study on Diet, Obesity and Genes), which had been randomized to four different diet groups consuming diets varying in protein content and glycemic index. In addition, we compared 24-h glucose profiles in a more controlled fashion, where nine other subjects followed in random order the same four diets differing in carbohydrate content by 10 energy% and glycemic index by 20 units during three days. Meals were provided in the lab and had to be eaten at fixed times during the day. No differences in mean glucose concentration or glucose variability (SD) were found between diet groups in the DIOGENES study. In the more controlled lab study, mean 24-h glucose concentrations were also not different. Glucose variability (SD and CONGA1), however, was lower on the diet combining a lower carbohydrate content and GI compared to the diet combining a higher carbohydrate content and GI. These data suggest that diets with moderate differences in carbohydrate content and GI do not affect mean 24-h or daytime glucose concentrations, but may result in differences in the variability of the glucose level in healthy normal weight and overweight individuals. PMID:25093276

  1. 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.

  2. Plasma Protein Biomarkers for Depression and Schizophrenia by Multi Analyte Profiling of Case-Control Collections

    PubMed Central

    Domenici, Enrico; Willé, David R.; Tozzi, Federica; Prokopenko, Inga; Miller, Sam; McKeown, Astrid; Brittain, Claire; Rujescu, Dan; Giegling, Ina; Turck, Christoph W.; Holsboer, Florian; Bullmore, Edward T.; Middleton, Lefkos; Merlo-Pich, Emilio; Alexander, Robert C.; Muglia, Pierandrea

    2010-01-01

    Despite significant research efforts aimed at understanding the neurobiological underpinnings of psychiatric disorders, the diagnosis and the evaluation of treatment of these disorders are still based solely on relatively subjective assessment of symptoms. Therefore, biological markers which could improve the current classification of psychiatry disorders, and in perspective stratify patients on a biological basis into more homogeneous clinically distinct subgroups, are highly needed. In order to identify novel candidate biological markers for major depression and schizophrenia, we have applied a focused proteomic approach using plasma samples from a large case-control collection. Patients were diagnosed according to DSM criteria using structured interviews and a number of additional clinical variables and demographic information were assessed. Plasma samples from 245 depressed patients, 229 schizophrenic patients and 254 controls were submitted to multi analyte profiling allowing the evaluation of up to 79 proteins, including a series of cytokines, chemokines and neurotrophins previously suggested to be involved in the pathophysiology of depression and schizophrenia. Univariate data analysis showed more significant p-values than would be expected by chance and highlighted several proteins belonging to pathways or mechanisms previously suspected to be involved in the pathophysiology of major depression or schizophrenia, such as insulin and MMP-9 for depression, and BDNF, EGF and a number of chemokines for schizophrenia. Multivariate analysis was carried out to improve the differentiation of cases from controls and identify the most informative panel of markers. The results illustrate the potential of plasma biomarker profiling for psychiatric disorders, when conducted in large collections. The study highlighted a set of analytes as candidate biomarker signatures for depression and schizophrenia, warranting further investigation in independent collections. PMID

  3. Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis

    PubMed Central

    Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie

    2015-01-01

    Introduction Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Materials and methods Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N = 66) and prostatitis patients (N = 36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Results Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (P < 0.001). Urinary bacteria count allowed for discriminating prostatitis patients from HV (P < 0.001). Total amount of biantennary structures (urinary 2A/MA marker) was significantly lower in prostatitis patients compared to HV (P < 0.001). Combining the urinary 2A/MA marker and urinary WBC count resulted in an AUC of 0.79, 95% confidence interval (CI) = (0.70–0.89) which was significantly better than urinary WBC count (AUC = 0.70, 95% CI = [0.59–0.82], P = 0.042) as isolated test. Conclusions We have demonstrated the diagnostic value of urinary N-glycosylation profiling, which shows great potential as biomarker for prostatitis. Further research is required to unravel the developmental course of prostatic inflammation. PMID:26526330

  4. Serum protein profile study of normal and cervical cancer subjects by high performance liquid chromatography with laser-induced fluorescence.

    PubMed

    Sujatha; Rai, Lavanya; Kumar, Pratap; Mahato, Krishna K; Kartha, Vasudevan B; Santhosh, Chidangil

    2008-01-01

    High performance liquid chromatography with high sensitivity laser-induced fluorescence detection is used to study the protein profiles of serum samples from healthy volunteers and cervical cancer subjects. The protein profiles are subjected to principal component analysis (PCA). PCA shows that the large number of chromatograms of a given class of serum samples--say normal/malignant--can be expressed in terms of a small number of factors (principal components). Three parameters--scores of the factors, squared residuals, and Mahalanobis distance--are derived from PCA. The parameters are observed to have a narrow range for protein profiles of standard calibration sets formed from groups of clinically confirmed normal/malignant classes. Limit tests using match/no match of the parameters of any test sample with parameters derived for the standard calibration sets give very good discrimination between malignant and normal samples with high sensitivity (approximately 100%) aand specificity (approximately 94%).

  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. 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.

  7. 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.

  8. Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

    PubMed Central

    Masuho, Ikuo; Ostrovskaya, Olga; Kramer, Grant M.; Jones, Christopher D.; Xie, Keqiang; Martemyanov, Kirill A.

    2016-01-01

    Members of the G protein coupled receptor (GPCR) family play key roles in many physiological functions and have been extensively exploited pharmacologically to treat diseases. Individual GPCRs exert diverse and distinct effects on cellular physiology and transduce signals by activating heterotrimeric G proteins. Mammalian genomes encode 16 different G protein alpha subunits, and each one of them has distinct properties. Here, we developed a single-platform, optical strategy for the direct monitoring of G protein activation in live cells, and using it we profiled the activities of individual GPCRs across a range of different G proteins, simultaneously quantifying both magnitude of their signaling and activation rates. We report that GPCRs engage multiple G proteins with varying efficacy and kinetics, generating fingerprint-like profiles that define individual receptors. We found that 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 expression 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 with varying signal magnitudes and activation kinetics, properties that may be exploited pharmacologically. PMID:26628681

  9. Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria.

    PubMed

    Min, Fangui; Pan, Jinchun; Wu, Ruike; Chen, Meiling; Kuang, Huiwen; Zhao, Weibo

    2016-01-01

    Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection.

  10. 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

  11. Activity-Based Protein Profiling Reveals Broad Reactivity of the Nerve Agent Sarin.

    PubMed

    Tuin, Adriaan W; Mol, Marijke A E; van den Berg, Roland M; Fidder, A; van der Marel, Gijs A; Overkleeft, Herman S; Noort, Daan

    2009-04-01

    Elucidation of noncholinesterase protein targets of organophosphates, and nerve agents in particular, may reveal additional mechanisms for their high toxicity as well as clues for novel therapeutic approaches toward intoxications with these agents. Within this framework, we here describe the synthesis of the activity-based probe 3, which contains a phosphonofluoridate moiety, a P-Me moiety, and a biotinylated O-alkyl group, and its use in activity-based protein profiling with two relevant biological samples, that is, rhesus monkey liver and cultured human A549 lung cells. In this way, we have unearthed eight serine hydrolases (fatty acid synthase, acylpeptide hydrolase, dipeptidyl peptidase 9, prolyl oligopeptidase, carboxylesterase, long-chain acyl coenzyme A thioesterase, PAF acetylhydrolase 1b, and esterase D/S-formyl glutathione hydrolase) as targets that are modified by the nerve agent sarin. It is also shown that the newly developed probe 3 might find its way into the development of alternative, less laborious purification protocols for human butyrylcholinesterase, a potent bioscavenger currently under clinical investigation as a prophylactic/therapeutic for nerve agent intoxications.

  12. Profiling serum antibodies to Mycobacterium tuberculosis proteins in rhesus monkeys with nontuberculous Mycobacteria

    PubMed Central

    Min, Fangui; Pan, Jinchun; Wu, Ruike; Chen, Meiling; Kuang, Huiwen; Zhao, Weibo

    2015-01-01

    Recent evidence indicates that the prevalence of diseases caused by nontuberculous mycobacteria (NTM) has been increasing in both human and animals. In this study, antibody profiles of NTM in rhesus monkeys (Macaca mulatta) were determined and compared with those of monkeys infected with Mycobacterium tuberculosis complex (MTBC). Antibodies against 10 M. tuberculosis proteins, purified protein derivative (PPD), and mammalian old tuberculin (MOT) were detected in 14 monkeys naturally infected with NTM by indirect ELISA. Sera from 10 monkeys infected with MTBC and 10 healthy monkeys were set as controls. All antigens showed high serological reactivities to MTBC infections and low reactivities in healthy monkeys. NTM infections showed strong antibody responses to MOT and PPD; moderate antibody responses to 16kDa, U1, MPT64L, 14kDa, and TB16.3; and low antibody responses to 38kDa, Ag85b, CFP10, ESAT-6, and CFP10-ESAT-6. According to the criteria of MTBC, only CFP10, ESAT-6, and CFP10-ESAT-6 showed negative antibody responses in all NTM infections. Taken together, these results suggest that positive results of a PPD/MOT-based ELISA in combination with results of antibodies to M. tuberculosis-specific antigens, such as CFP10 and ESAT-6, could discriminate NTM and MTBC infections. Two positive results indicate an MTBC infection, and a negative result for an M. tuberculosis-specific antigen may preliminarily predict an NTM infection. PMID:26437786

  13. Reaction rate and collisional efficiency of the rhodopsin-transducin system in intact retinal rods.

    PubMed Central

    Kahlert, M; Hofmann, K P

    1991-01-01

    A model of transducin activation is constructed from its partial reactions (formation of metarhodopsin II, association, and dissociation of the rhodopsin-transducin complex). The kinetic equations of the model are solved both numerically and, for small photoactivation, analytically. From data on the partial reactions in vitro, rate and activation energy profile of amplified transducin turnover are modeled and compared with measured light-scattering signals of transducin activation in intact retinal rods. The data leave one free parameter, the rate of association between transducin and rhodopsin. Best fit is achieved for an activation energy of 35 kJ/mol, indicating lateral membrane diffusion of the proteins as its main determinant. The absolute value of the association rate is discussed in terms of the success of collisions to form the catalytic complex. It is greater than 30% for the intact retina and 10 times lower after permeabilization with staphylococcus aureus alpha-toxin. Dissociation rates for micromolar guanosinetriphosphale (GTP) (Kohl, B., and K. P. Hofmann, 1987. Biophys. J. 52:271-277) must be extrapolated linearly up to the millimolar range to explain the rapid transducin turnover in situ. This is interpreted by an unstable rhodopsin-transducin-GTP transient state. At the time of maximal turnover after a flash, the rate of activation is determined as 30, 120, 800, 2,500, and 4,000 activated transducins per photoactivated rhodopsin and second at 5, 10, 20, 30, 37 degrees C, respectively. PMID:1901231

  14. Expression profile of ribosomal protein L10a throughout gonadal development in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Makkapan, Walaiporn; Yoshizaki, Goro; Tashiro, Masami; Chotigeat, Wilaiwan

    2014-08-01

    Ribosomal protein L10a (RpL10A) has been previously established as a stimulator during the early stages of ovarian development in both the banana prawn and the fruit fly. In order to develop a greater understanding of the role of this protein in vertebrates, the present study aimed to characterize the expression profile of rpl10a during gonadal development in fish. It was determined that the expression of rpl10a within genital ridges increased during embryonic development. Although rpl10a expression was observed in both gonadal somatic cells and primordial germ cells, higher levels of both transcript and protein expression were detected in somatic cells. rpl10a transcripts were observed in all of the adult tissues examined. Cellular level expression of rpl10a was subsequently characterized across various maturational stages using in situ hybridization and immunohistochemistry of both testes and ovaries. Analysis of tissue derived from the testis showed high levels of rpl10a expression within spermatogonia and the Sertoli cells attached to them. In ovarian tissue, rpl10a was strongly expressed in chromatin-nucleolus-stage and peri-nucleolus-stage oocytes. The relationship between rpl10a expression and regulation of gonadal development was confirmed using real-time PCR, which was performed in order to analyze rpl10a expression in testicular and ovarian tissues subsequent to incubation with salmon pituitary extract and various sex steroids for 24 h. Among them, 11-ketotestosterone at 100 ng/mL effectively up-regulated expression of rpl10a in testicular tissues, while 17β-estradiol down-regulated rpl10a expression in ovarian tissues. These results suggested that rpl10a played a role in the regulation of gonadal development in fish.

  15. 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.

  16. Profiling of drug binding proteins by monolithic affinity chromatography in combination with liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Xuepei; Wang, Tongdan; Zhang, Hanzhi; Han, Bing; Wang, Lishun; Kang, Jingwu

    2014-09-12

    A new approach for proteome-wide profiling drug binding proteins by using monolithic capillary affinity chromatography in combination with HPLC-MS/MS is reported. Two immunosuppresive drugs, namely FK506 and cyclosporin A, were utilized as the experimental models for proof-of-concept. The monolithic capillary affinity columns were prepared through a single-step copolymerization of the drug derivatives with glycidyl methacrylate and ethylene dimethacrylate. The capillary chromatography with the affinity monolithic column facilitates the purification of the drug binding proteins from the cell lysate. By combining the capillary affinity column purification and the shot-gun proteomic analysis, totally 33 FK506- and 32 CsA-binding proteins including all the literature reported target proteins of these two drugs were identified. Among them, two proteins, namely voltage-dependent anion-selective channel protein 1 and serine/threonine-protein phosphatase PGAM5 were verified by using the recombinant proteins. The result supports that the monolithic capillary affinity chromatography is likely to become a valuable tool for profiling of binding proteins of small molecular drugs as well as bioactive compounds.

  17. 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

  18. 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

    2016-04-13

    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.

  19. Construction of protein profile classification model and screening of proteomic signature of acute leukemia

    PubMed Central

    Xu, Yun; Zhuo, Jiacai; Duan, Yonggang; Shi, Benhang; Chen, Xuhong; Zhang, Xiaoli; Xiao, Liang; Lou, Jin; Huang, Ruihong; Zhang, Qiongli; Du, Xin; Li, Ming; Wang, Daping; Shi, Dunyun

    2014-01-01

    The French-American-British (FAB) and WHO classifications provide important guidelines for the diagnosis, treatment, and prognostic prediction of acute leukemia, but are incapable of accurately differentiating all subtypes, and not well correlated with the clinical outcomes. In this study, we performed the protein profiling of the bone marrow mononuclear cells from the patients with acute leukemia and the health volunteers (control) by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI_TOF_MS). The patients with acute leukemia were analyzed as unitary by the profiling that were grouped into acute promyelocytic leukemia (APL), acute myeloid leukemia-granulocytic (AML-Gran), acute myeloid leukemia-monocytic (AML-Mon) acute lymphocytic leukemia (ALL), and control. Based on 109 proteomic signatures, the classification models of acute leukemia were constructed to screen the predictors by the improvement of the proteomic signatures and to detect their expression characteristics. According to the improvement and the expression characteristics of the predictors, the proteomic signatures (M3829, M1593, M2121, M2536, M1016) characterized successively each group (CON, APL, AML-Gra, AML-Mon, ALL) were screened as target molecules for identification. Meanwhile, the proteomic-based class of determinant samples could be made by the classification models. The credibility of the proteomic-based classification passed the evaluation of Biomarker Patterns Software 5.0 (BPS 5.0) scoring and validated application in clinical practice. The results suggested that the proteomic signatures characterized by different blasts were potential for developing new treatment and monitoring approaches of leukemia blasts. Moreover, the classification model was potential in serving as new diagnose approach of leukemia. PMID:25337199

  20. 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

  1. 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...

  2. 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...

  3. 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.

  4. 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.

  5. 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.

  6. Analysis of the expression protein profiles of lung squamous carcinoma cell using shot-gun proteomics strategy.

    PubMed

    Nan, Yandong; Yang, Shuanying; Tian, Yingxuan; Zhang, Wei; Zhou, Bin; Bu, Lina; Huo, Shufen

    2009-01-01

    The aim of this study is to globally screen and identify the expression protein profiles of lung squamous carcinoma cell (SqCC) using shot-gun proteomics strategy and to further analyze function of individual proteins by bioinformatics, which may likely result in the identification of new biomarkers and provide helpful clues for pathogenesis, early diagnosis, and progression of lung SqCC. The specific tumor cells were isolated and collected from the tissues of six patients with lung SqCC by laser capture microdissection (LCM). Total proteins from the LCM cells were extracted, digested with trypsin. The sequence information of resulting peptides was acquired by high-performance liquid chromatography (HPLC) and tandem mass spectrometry (TMS). The global protein profiles of lung SqCC cell were identified with BioworksTM software in IPI human protein database. Cellular component, molecular function, and biological process of the all proteins were analyzed using gene ontology (GO). About 720,000 tumor cells were satisfactorily collected from tissues of six patients with lung SqCC by LCM and the homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. The high resolution profiles including HPLC, full mass spectrum, and tandem mass spectrum were successfully obtained. Database searching of the resulting bimolecular sequence information identified 1982 proteins in all samples. The bioinformatics of these proteins, including amino acids sequence, fraction of coverage, molecular weight, isoelectric point, etc., were analyzed in detail. Among them, the function of most proteins was recognized by using GO. Five candidate proteins, Prohibitin (PHB), Mitogen-activated protein kinase (MAPK), Heat shock protein27 (HSP27), Annexin A1(ANXA1), and High mobility group protein B1 (HMGB1), might play an important role in SqCC genesis, progression, recurrence, and metastasis according to relative literatures. We have successfully isolated

  7. 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

  8. 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.

  9. Major intrinsic proteins repertoire of Morus notabilis and their expression profiles in different species.

    PubMed

    Baranwal, Vinay Kumar; Khurana, Paramjit

    2017-02-01

    Leaf moisture content in Morus is a significant trait regulating the yield of silk production. Studies have shown that fresh leaves or leaves with high water content are preferably eaten by silk worm. Water and certain other molecules transport in plants is known to be regulated by aquaporins or Major Intrinsic Proteins (MIPs). Members of the MIP gene family have also been implicated in plant development and stress responsiveness. To understand how members of MIP gene family are regulated and evolved, we carried out an extensive analysis of the gene family. We identified a total of 36 non redundant MIPs in Morus notabilis genome, belonging to five subfamilies PIPs, TIPs, NIPs, XIPs and SIPs) have been identified. We performed a Gene ontology (GO) term enrichment analysis and looked at distribution of cis elements in their 2K upstream regulatory region to reveal their putative roles in various stresses and developmental aspects. Expression analysis in developmental stages revealed their tissue preferential expression pattern in diverse vegetative and reproductive tissues. Comparison of expression profiles in the leaves of three species including Morus notabilis, Morus serrata and Morus laevigata led to identification of differential expression in these species. In all, this study elaborates a basic insight into the structure, function and evolutionary analysis of MIP gene family in Morus which is hitherto unavailable. Our analysis will provide a ready reference to the mulberry research community involved in the Morus improvement program.

  10. The Eucalyptus Tonoplast Intrinsic Protein (TIP) Gene Subfamily: Genomic Organization, Structural Features, and Expression Profiles

    PubMed Central

    Rodrigues, Marcela I.; Takeda, Agnes A. S.; Bravo, Juliana P.; Maia, Ivan G.

    2016-01-01

    Plant aquaporins are water channels implicated in various physiological processes, including growth, development and adaptation to stress. In this study, the Tonoplast Intrinsic Protein (TIP) gene subfamily of Eucalyptus, an economically important woody species, was investigated and characterized. A genome-wide survey of the Eucalyptus grandis genome revealed the presence of eleven putative TIP genes (referred as EgTIP), which were individually assigned by phylogeny to each of the classical TIP1–5 groups. Homology modeling confirmed the presence of the two highly conserved NPA (Asn-Pro-Ala) motifs in the identified EgTIPs. Residue variations in the corresponding selectivity filters, that might reflect differences in EgTIP substrate specificity, were observed. All EgTIP genes, except EgTIP5.1, were transcribed and the majority of them showed organ/tissue-enriched expression. Inspection of the EgTIP promoters revealed the presence of common cis-regulatory elements implicated in abiotic stress and hormone responses pointing to an involvement of the identified genes in abiotic stress responses. In line with these observations, additional gene expression profiling demonstrated increased expression under polyethylene glycol-imposed osmotic stress. Overall, the results obtained suggest that these novel EgTIPs might be functionally implicated in eucalyptus adaptation to stress. PMID:27965702

  11. Immunoproteomic Profiling of Antiviral Antibodies in New-Onset Type 1 Diabetes Using Protein Arrays

    PubMed Central

    Bian, Xiaofang; Wallstrom, Garrick; Davis, Amy; Wang, Jie; Park, Jin; Throop, Andrea; Steel, Jason; Yu, Xiaobo; Wasserfall, Clive; Schatz, Desmond; Atkinson, Mark

    2016-01-01

    The rapid rise in the incidence of type 1 diabetes (T1D) suggests the involvement of environmental factors including viral infections. We evaluated the association between viral infections and T1D by profiling antiviral antibodies using a high-throughput immunoproteomics approach in patients with new-onset T1D. We constructed a viral protein array comprising the complete proteomes of seven viruses associated with T1D and open reading frames from other common viruses. Antibody responses to 646 viral antigens were assessed in 42 patients with T1D and 42 age- and sex-matched healthy control subjects (mean age 12.7 years, 50% males). Prevalence of antiviral antibodies agreed with known infection rates for the corresponding virus based on epidemiological studies. Antibody responses to Epstein-Barr virus (EBV) were significantly higher in case than control subjects (odds ratio 6.6; 95% CI 2.0–25.7), whereas the other viruses showed no differences. The EBV and T1D association was significant in both sex and age subgroups (≤12 and >12 years), and there was a trend toward early EBV infections among the case subjects. These results suggest a potential role for EBV in T1D development. We believe our innovative immunoproteomics platform is useful for understanding the role of viral infections in T1D and other disorders where associations between viral infection and disease are unclear. PMID:26450993

  12. Profile of skin barrier proteins and cytokines in adults with atopic dermatitis.

    PubMed

    Orfali, Raquel L; Zaniboni, Mariana C; Aoki, Valeria

    2017-04-01

    Atopic dermatitis (AD), an inflammatory skin disorder with chronic course and characterized by intense pruritus, is a dermatosis of high prevalence of childhood. However, persistence of the disease in adolescents and adults may occur, and more studies regarding the interactions of the complex triggering factors, especially between the adaptive and innate immune alterations and skin barrier defects are needed. In this review the authors summarize the major novel findings of a dysfunctional skin barrier in AD, with emphasis on tight junction components, such as claudins and on proteins of the keratinocyte differentiation, such as filaggrin. This review also provides an update on the characterization of immune response in adults with atopic dermatitis. The adaptive immune dysfunction in AD, classically known as a Th2/Th1 model, has changed its profile, with recent reported cytokines such as interleukins 17, 22, and 31; as for the innate immune system scenario in AD, the characterization of skin microbiome opens new frontiers for the understanding of such a complex inflammatory disease.

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

    DOE PAGES

    Liu, Yun; Fredrickson, James K.; Sadler, Natalie C.; ...

    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

  14. 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

  15. Principal Component Analysis Reveals Age-Related and Muscle-Type-Related Differences in Protein Carbonyl Profiles of Muscle Mitochondria

    PubMed Central

    Feng, Juan; Navratil, Marian; Thompson, LaDora V.; Arriaga, Edgar A.

    2011-01-01

    Carbonyl-modified proteins are considered markers of oxidative damage caused by oxidative stress, aging, and disease. Here we use a previously developed capillary electrophoretic method for detecting femtomole (10−15 mole) carbonyl levels in mitochondrial proteins that are size separated and profiled. For protein labeling, carbonyls were tagged with Alexa 488 hydrazine and amine groups in proteins with 3-(2-furoyl)quinoline-2-carboxaldehyde. Total mitochondrial protein carbonyl levels were statistically higher in fast- than in slow-twitch muscle of young Fischer 344 rats, and statistically higher in old than in young slow-twitch muscle. Even when some statistical comparisons of the total protein carbonyl levels would not reveal differences, principal component analysis (PCA) classified the carbonyl profiles into four distinct sample groups of different age and muscle types. In addition, PCA was used to predict that most age-related or muscle-type-related changes in carbonyl levels occur in proteins with a molecular weight between 9.8 and 11.7 kD. PMID:19126840

  16. 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

    2013-06-07

    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 ).

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

    PubMed

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

    2014-12-01

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

  18. Protein profiling in the gut of Penaeus monodon gavaged with oral WSSV-vaccines and live white spot syndrome virus.

    PubMed

    Kulkarni, Amod D; Kiron, Viswanath; Rombout, Jan H W M; Brinchmann, Monica F; Fernandes, Jorge M O; Sudheer, Naduvilamuriparampu S; Singh, Bright I S

    2014-07-01

    White spot syndrome virus (WSSV) is a pathogen that causes considerable mortality of the farmed shrimp, Penaeus monodon. Candidate 'vaccines', WSSV envelope protein VP28 and formalin-inactivated WSSV, can provide short-lived protection against the virus. In this study, P. monodon was orally intubated with the aforementioned vaccine candidates, and protein expression in the gut of immunised shrimps was profiled. The alterations in protein profiles in shrimps infected orally with live-WSSV were also examined. Seventeen of the identified proteins in the vaccine and WSSV-intubated shrimps varied significantly compared to those in the control shrimps. These proteins, classified under exoskeletal, cytoskeletal, immune-related, intracellular organelle part, intracellular calcium-binding or energy metabolism, are thought to directly or indirectly affect shrimp's immunity. The changes in the expression levels of crustacyanin, serine proteases, myosin light chain, and ER protein 57 observed in orally vaccinated shrimp may probably be linked to immunoprotective responses. On the other hand, altered expression of proteins linked to exoskeleton, calcium regulation and energy metabolism in WSSV-intubated shrimps is likely to symbolise disturbances in calcium homeostasis and energy metabolism.

  19. Effects of different protein and glycemic index diets on metabolic profiles and substrate partitioning in lean healthy males.

    PubMed

    Munsters, Marjet J; Geraedts, Maartje C; Saris, Wim H

    2013-11-01

    Dietary glycemic index (GI) and protein affects postprandial insulin responses and consequently 24 h glucose metabolism and therefore substrate partitioning. This study investigated the mechanistic effects of different protein and GI diets on 24 h profiles of metabolic markers and substrate partitioning. After 3 days of diet and physical activity standardization, 10 healthy male subjects (BMI: 22.5 ± 0.6 kg/m(2)) stayed in a respiration chamber 4 times for 36 h each time to measure substrate partitioning. All subjects randomly received four isoenergetic diets: a normal (15En%) dairy protein and low GI (<40 units) (NDP-LGI) diet; a high (25En%) dairy protein and low GI (HDP-LGI) diet; a normal vegetable protein and low GI (NVP-LGI) diet; or a normal dairy protein and high GI (>60 units) (NDP-HGI) diet. During the day, blood was sampled at fixed time points for the measurement of metabolic markers and satiety hormones. The HDP-LGI diet increased 24 h protein oxidation and sleeping metabolic rate (SMR) compared with the NDP-LGI diet (p < 0.002). No significant differences in 24 h carbohydrate and fat oxidation (day and night) were found between all intervention diets. Net incremental area under the curve (net iAUC) of 24 h plasma glucose decreased in the HDP-LGI diet compared with the NDP-LGI diet (p < 0.01), but no effect was observed on insulin levels. No difference in appetite profiles were observed between all intervention diets. The lower 24 h glycemic profile as a result of a high dairy protein diet did not lead to changes in 24 h substrate partitioning in lean healthy subjects with a normal insulin sensitivity.

  20. 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.

  1. Comparative proteomic study for profiling differentially expressed proteins between Chinese left- and right-sided colon cancers.

    PubMed

    Shen, Hong; Huang, Jinlin; Pei, Haiping; Zeng, Shan; Tao, Yiming; Shen, Liangfang; Zeng, Liang; Zhu, Hong

    2013-01-01

    The aim of the present study is to profile differentially expressed protein markers between left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC). Fresh tumor tissue samples from LSCC (n = 7) and RSCC (n = 7) groups were analyzed by two-dimensional electrophoresis coupled with MALDI-TOF-MS, followed by Western blotting. In 50 paraffin embedded samples from each group, levels of four differentially expressed proteins (identified by proteomics analysis) were measured by tissue microarray with immunohistochemistry staining to compare the different protein markers between LSCC and RSCC. Sixteen proteins were found to be differentially expressed between LSCC and RSCC. Ten proteins including HSP-60 and PDIA1 were identified to be highly expressed in LSCC (P < 0.01 or P < 0.05), while the expression of six proteins including EEF1D and HSP-27 were higher in RSCC (P < 0.01 or P < 0.05). Virtually all of the indentified proteins were involved in cellular energy metabolism, protein folding/unfolding, and/or oxidative stress. Human colon tumors at various locations have different proteomic biomarkers. Differentially expressed proteins associated with energy metabolism, protein folding/unfolding and oxidative stress contribute to different tumorigenesis, tumor progression, and prognosis between left- and right-sided colon cancer.

  2. 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.

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

    EPA Science Inventory

    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 te...

  4. 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.

  5. Outer Membrane Proteins and DNA Profiles in Strains of Haemophilus parasuis Recovered from Systemic and Respiratory Sites

    PubMed Central

    Ruiz, Alvaro; Oliveira, Simone; Torremorell, Montserrat; Pijoan, Carlos

    2001-01-01

    Polyserositis caused by Haemophilus parasuis is an important disease that affects mostly weaned pigs. Recent studies have shown that virulence can differ among strains recovered from distinct body sites and also that it may be related to the presence of certain outer membrane proteins (OMPs). The objective of this study was to compare the OMP and DNA profiles of H. parasuis strains isolated from systemic and respiratory sites from diseased and healthy pigs. Strains evaluated in this study were processed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and repetitive-PCR techniques. Two experiments were conducted in order to better define the relationship among genotype, phenotype, and site of isolation. Experiment 1 included 53 H. parasuis isolates recovered from healthy and diseased pigs from unrelated herds. Experiment 2 included 31 isolates of H. parasuis obtained from diseased pigs involved in an outbreak in a large, multifarm system. Results showed that strains recovered from systemic sites had more homogeneous OMP and DNA profiles than those isolated from respiratory sites. Evaluation of isolates involved in the multifarm outbreak showed that only two H. parasuis strains were causing disease. These strains had homogeneous OMP and DNA profiles. However, it was noted that these two parameters were unrelated, since strains classified in the same genotype group expressed different OMP profiles. The homogeneity of OMP and DNA profiles of strains isolated from systemic sites strongly suggests the existence of clonal relationships between virulent strains and also suggests that expression of certain OMP profiles may be related to virulence. PMID:11325986

  6. The Calcium Binding Protein, S100B, is Increased in the Amniotic Fluid of Women with Intra-Amniotic Infection/Inflammation Following Preterm Labor with Intact or Ruptured Membranes

    PubMed Central

    Friel, Lara A.; Romero, Roberto; Edwin, Sam; Nien, Jyh Kae; Gomez, Ricardo; Chaiworapongsa, Tinnakorn; Kusanovic, Juan Pedro; Tolosa, Jorge E.; Hassan, Sonia S.; Espinoza, Jimmy

    2008-01-01

    Objective S100B is produced by glia of the central and peripheral nervous systems and is considered a marker of neurologic injury in the perinatal period. Indeed, increased neonatal urine S100B concentration is associated with adverse neurological outcomes including intraventricular hemorrhage and hypoxic-ischemic encephalopathy, while elevated adult serum concentrations are associated with infectious diseases/sepsis. The objective of this study was to determine whether amniotic fluid (AF) S100B concentrations change with advancing gestational age and intra-amniotic infection (IAI). Study Design S100B concentration was measured in the AF of women in midtrimester, at term, and in pregnancies with preterm labor and intact membranes (PTL) or preterm premature rupture of membranes (PPROM), with and without IAI. Placental pathology was performed and neonatal outcomes were analyzed. Results (1) AF S100B concentration did not change during gestation; (2) patients with IAI had significantly higher AF S100B concentration than those without IAI following an episode of PTL or PPROM and; (3) neonates who had morbidity/mortality had had an elevated AF S100B concentration; however, this could be explained by the association with intra-amniotic infection/inflammation. Thus, AF S100B concentration was not an independent predictor of neonatal morbidity or fetal/neonatal death. Conclusions An elevated concentration of AF S100B may reflect intra-amniotic infection/inflammation and not necessarily fetal neurologic damage. PMID:17624933

  7. Protein Profiling Reveals Novel Proteins in Pollen and Pistil of W22 (ga1; Ga1) in Maize

    PubMed Central

    Yu, Jin; Roy, Swapan Kumar; Kamal, Abu Hena Mostafa; Cho, Kun; Kwon, Soo-Jeong; Cho, Seong-Woo; So, Yoon-Sup; Holland, James B.; Woo, Sun Hee

    2014-01-01

    Gametophytic factors mediate pollen-pistil interactions in maize (Zea mays L.) and play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1); which did not carry a gametophytic factor and W22 (Ga1), a near iso-genic line, were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1) and W22 (Ga1). A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1) and W22 (Ga1) whereas 20 differentially expressed proteins were identified from the pistil of W22 (ga1) and W22 (Ga1). However, in pollen, 2 proteins were identified only in the W22 (ga1) and 12 proteins only in the W22 (Ga1) whereas 10 proteins were confirmed from the both of W22 (ga1) and W22 (Ga1). In contrary, 10 proteins were appeared only in the pistil of W22 (ga1) and 7 proteins from W22 (Ga1) while 3 proteins confirmed in the both of W22 (ga1) and W22 (Ga1). Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize. PMID:28250381

  8. Binding of Clostridium botulinum C3 exoenzyme to intact cells.

    PubMed

    Rohrbeck, Astrid; von Elsner, Leonie; Hagemann, Sandra; Just, Ingo

    2014-06-01

    C3 from Clostridium botulinum (C3) specifically modifies Rho GTPases RhoA, RhoB, and RhoC by mono-ADP-ribosylation. The confined substrate profile of C3 is the basis for its use as pharmacological tool in cell biology to study cellular functions of Rho GTPases. Although C3 exoenzyme does not possess a cell-binding/-translocation domain, C3 is taken up by intact cells via an unknown mechanism. In the present work, binding of C3 to the hippocampus-derived HT22 cells and J774A.1 macrophages was characterized. C3 bound concentration-dependent to HT22 and J774A.1 cells. Pronase treatment of intact cells significantly reduced both C3 binding and C3 cell entry. Removal of sugar residues by glycosidase F treatment resulted in an increased binding of C3, but a reduced cell entry. To explore the involvement of phosphorylation in the binding process of C3, intact HT22 and J774A.1 cells were pre-treated with vanadate prior to incubation with C3. Inhibition of de-phosphorylation by vanadate resulted in an increased binding of C3. To differentiate between intracellular and extracellular phosphorylation, intact cells were treated with CIP (calf intestine phosphatase) to remove extracellular phosphate residues. The removal of phosphate residues resulted in a strong reduction in binding of C3 to cells. In sum, the C3 membranous binding partner is proteinaceous, and the glycosylation as well as the phosphorylation state is critical for efficient binding of C3.

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

    SciTech Connect

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.; Löffler, F. E.

    2016-01-29

    Nitrosomonas europaeais 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 O2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein 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

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

    PubMed

    Bennett, Kristen; Sadler, Natalie C; Wright, Aaron T; Yeager, Chris; Hyman, Michael R

    2016-04-01

    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, andde novoprotein 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.

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

    PubMed

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

    2015-01-01

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

  12. Sake Protein Supplementation Affects Exercise Performance and Biochemical Profiles in Power-Exercise-Trained Mice

    PubMed Central

    Chen, Yi-Ming; Lin, Che-Li; Wei, Li; Hsu, Yi-Ju; Chen, Kuan-Neng; Huang, Chi-Chang; Kao, Chin-Hsung

    2016-01-01

    Exercise and fitness training programs have attracted the public’s attention in recent years. Sports nutrition supplementation is an important issue in the global sports market. Purpose: In this study, we designed a power exercise training (PET) program with a mouse model based on a strength and conditional training protocol for humans. We tested the effect of supplementation with functional branched-chain amino acid (BCAA)-rich sake protein (SP) to determine whether the supplement had a synergistic effect during PET and enhanced athletic performance and resistance to fatigue. Methods: Male ICR mice were divided into three groups (n = 8 per group) for four-week treatment: sedentary controls with vehicle (SC), and PET and PET groups with SP supplementation (3.8 g/kg, PET + SP). Exercise performance was evaluated by forelimb grip strength and exhaustive swimming time as well as changes in body composition and anti-fatigue activity levels of serum lactate, ammonia, glucose, and creatine kinase (CK) after a 15-min swimming exercise. The biochemical parameters were measured at the end of the experiment. Results: four-week PET significantly increased grip strength and exhaustive swimming time and decreased epididymal fat pad (EFP) weight and area. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, and uric acid (UA) were significantly increased. PET + SP supplementation significantly decreased serum lactate, ammonia and CK levels after the 15-min swimming exercise. The resting serum levels of AST, ALT, CREA and UA were all significantly decreased with PET + SP. Conclusion: The PET program could increase the exercise performance and modulate the body composition of mice. PET with SP conferred better anti-fatigue activity, improved biochemical profiles, and may be an effective ergogenic aid in strength training. PMID:26907336

  13. Identification and Expression Profile Analysis of Odorant Binding Proteins in the Oriental Fruit Fly Bactrocera dorsalis

    PubMed Central

    Zheng, Weiwei; Peng, Wei; Zhu, Chipan; Zhang, Qun; Saccone, Giuseppe; Zhang, Hongyu

    2013-01-01

    Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs) function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna) of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs (BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10) are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies. PMID:23867609

  14. Volatile profile, lipid oxidation and protein oxidation of irradiated ready-to-eat cured turkey meat products

    NASA Astrophysics Data System (ADS)

    Feng, Xi; Ahn, Dong Uk

    2016-10-01

    Irradiation had little effects on the thiobarbituric acid reactive substances (TBARS) values in ready-to-eat (RTE) turkey meat products, while it increased protein oxidation at 4.5 kGy. The volatile profile analyses indicated that the amount of sulfur compounds increased linearly as doses increased in RTE turkey meat products. By correlation analysis, a positive correlation was found between benzene/ benzene derivatives and alcohols with lipid oxidation, while aldehydes, ketones and alkane, alkenes and alkynes were positively correlated with protein oxidation. Principle component analysis showed that irradiated meat samples can be discriminated by two categories of volatile compounds: Strecker degradation products and radiolytic degradation products. The cluster analysis of volatile data demonstrated that low-dose irradiation had minor effects on the volatile profile of turkey sausages (<1.5 kGy). However, as the doses increased, the differences between the irradiated and non-irradiated cured turkey products became significant.

  15. Comparative exoprotein profiling of different Staphylococcus epidermidis strains reveals potential link between nonclassical protein export and virulence.

    PubMed

    Siljamäki, Pia; Varmanen, Pekka; Kankainen, Matti; Sukura, Antti; Savijoki, Kirsi; Nyman, Tuula A

    2014-07-03

    Staphylococcus epidermidis (SE) includes commensal and pathogenic strains capable of infecting humans and animals. This study reports global exoproteome profiling of bovine mastitis strain PM221 and two human strains, commensal-type ATCC12228 and sepsis-associated RP62A. We identified 451, 395, and 518 proteins from culture supernatants of PM221, ATCC12228, and RP62A, respectively. Comparison of the identified exoproteomes revealed several strain-specific differences related to secreted antigens and adhesins, higher virulence capability for RP62A, and similarities between the PM221 and RP62A exoproteomes. The majority of the identified proteins (∼80%) were predicted to be cytoplasmic, including proteins known to be associated in membrane vesicles (MVs) in Staphylococcus aureus and immunogenic/adhesive moonlighting proteins. Enrichment of MV fractions from culture supernatants and analysis of their protein composition indicated that this nonclassical protein secretion pathway was being exploited under the conditions used and that there are strain-specific differences in nonclassical protein export. In addition, several predicted cell-surface proteins were identified in the culture media. In summary, the present study is the first in-depth exoproteome analysis of SE highlighting strain-specific factors able to contribute to virulence and adaptation.

  16. Impact of exposure to low concentrations of nitric oxide on protein profile in murine and human pancreatic islet cells

    PubMed Central

    Tapia-Limonchi, Rafael; Díaz, Irene; Cahuana, Gladys M; Bautista, Mario; Martín, Franz; Soria, Bernat; Tejedo, Juan R; Bedoya, Francisco J

    2014-01-01

    Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic β cells, but the protein profile of this action remains to be determined. We have applied a 2 dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediated oxidative stress response pathways and eNOS signaling. The results indicate that exposure to exogenous NO counteracts the impact of serum deprivation on pancreatic β cell proteome. Species differences in the proteins involved are apparent. PMID:25658244

  17. Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

    PubMed Central

    2009-01-01

    Background Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins