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Sample records for accurate protein identification

  1. Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

    Directory of Open Access Journals (Sweden)

    Kevin R Ramkissoon

    Full Text Available The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

  2. Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

    Science.gov (United States)

    Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

    2013-01-01

    The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

  3. Accurate in silico identification of protein succinylation sites using an iterative semi-supervised learning technique.

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    Zhao, Xiaowei; Ning, Qiao; Chai, Haiting; Ma, Zhiqiang

    2015-06-07

    As a widespread type of protein post-translational modifications (PTMs), succinylation plays an important role in regulating protein conformation, function and physicochemical properties. Compared with the labor-intensive and time-consuming experimental approaches, computational predictions of succinylation sites are much desirable due to their convenient and fast speed. Currently, numerous computational models have been developed to identify PTMs sites through various types of two-class machine learning algorithms. These methods require both positive and negative samples for training. However, designation of the negative samples of PTMs was difficult and if it is not properly done can affect the performance of computational models dramatically. So that in this work, we implemented the first application of positive samples only learning (PSoL) algorithm to succinylation sites prediction problem, which was a special class of semi-supervised machine learning that used positive samples and unlabeled samples to train the model. Meanwhile, we proposed a novel succinylation sites computational predictor called SucPred (succinylation site predictor) by using multiple feature encoding schemes. Promising results were obtained by the SucPred predictor with an accuracy of 88.65% using 5-fold cross validation on the training dataset and an accuracy of 84.40% on the independent testing dataset, which demonstrated that the positive samples only learning algorithm presented here was particularly useful for identification of protein succinylation sites. Besides, the positive samples only learning algorithm can be applied to build predictors for other types of PTMs sites with ease. A web server for predicting succinylation sites was developed and was freely accessible at http://59.73.198.144:8088/SucPred/. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

    Science.gov (United States)

    Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

    2016-11-18

    O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

  5. HIPPI: highly accurate protein family classification with ensembles of HMMs

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    Nam-phuong Nguyen

    2016-11-01

    Full Text Available Abstract Background Given a new biological sequence, detecting membership in a known family is a basic step in many bioinformatics analyses, with applications to protein structure and function prediction and metagenomic taxon identification and abundance profiling, among others. Yet family identification of sequences that are distantly related to sequences in public databases or that are fragmentary remains one of the more difficult analytical problems in bioinformatics. Results We present a new technique for family identification called HIPPI (Hierarchical Profile Hidden Markov Models for Protein family Identification. HIPPI uses a novel technique to represent a multiple sequence alignment for a given protein family or superfamily by an ensemble of profile hidden Markov models computed using HMMER. An evaluation of HIPPI on the Pfam database shows that HIPPI has better overall precision and recall than blastp, HMMER, and pipelines based on HHsearch, and maintains good accuracy even for fragmentary query sequences and for protein families with low average pairwise sequence identity, both conditions where other methods degrade in accuracy. Conclusion HIPPI provides accurate protein family identification and is robust to difficult model conditions. Our results, combined with observations from previous studies, show that ensembles of profile Hidden Markov models can better represent multiple sequence alignments than a single profile Hidden Markov model, and thus can improve downstream analyses for various bioinformatic tasks. Further research is needed to determine the best practices for building the ensemble of profile Hidden Markov models. HIPPI is available on GitHub at https://github.com/smirarab/sepp .

  6. Accurate Identification of Cancerlectins through Hybrid Machine Learning Technology.

    Science.gov (United States)

    Zhang, Jieru; Ju, Ying; Lu, Huijuan; Xuan, Ping; Zou, Quan

    2016-01-01

    Cancerlectins are cancer-related proteins that function as lectins. They have been identified through computational identification techniques, but these techniques have sometimes failed to identify proteins because of sequence diversity among the cancerlectins. Advanced machine learning identification methods, such as support vector machine and basic sequence features (n-gram), have also been used to identify cancerlectins. In this study, various protein fingerprint features and advanced classifiers, including ensemble learning techniques, were utilized to identify this group of proteins. We improved the prediction accuracy of the original feature extraction methods and classification algorithms by more than 10% on average. Our work provides a basis for the computational identification of cancerlectins and reveals the power of hybrid machine learning techniques in computational proteomics.

  7. [A accurate identification method for Chinese materia medica--systematic identification of Chinese materia medica].

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    Wang, Xue-Yong; Liao, Cai-Li; Liu, Si-Qi; Liu, Chun-Sheng; Shao, Ai-Juan; Huang, Lu-Qi

    2013-05-01

    This paper put forward a more accurate identification method for identification of Chinese materia medica (CMM), the systematic identification of Chinese materia medica (SICMM) , which might solve difficulties in CMM identification used the ordinary traditional ways. Concepts, mechanisms and methods of SICMM were systematically introduced and possibility was proved by experiments. The establishment of SICMM will solve problems in identification of Chinese materia medica not only in phenotypic characters like the mnorphous, microstructure, chemical constituents, but also further discovery evolution and classification of species, subspecies and population in medical plants. The establishment of SICMM will improve the development of identification of CMM and create a more extensive study space.

  8. An Overview of Practical Applications of Protein Disorder Prediction and Drive for Faster, More Accurate Predictions.

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    Deng, Xin; Gumm, Jordan; Karki, Suman; Eickholt, Jesse; Cheng, Jianlin

    2015-07-07

    Protein disordered regions are segments of a protein chain that do not adopt a stable structure. Thus far, a variety of protein disorder prediction methods have been developed and have been widely used, not only in traditional bioinformatics domains, including protein structure prediction, protein structure determination and function annotation, but also in many other biomedical fields. The relationship between intrinsically-disordered proteins and some human diseases has played a significant role in disorder prediction in disease identification and epidemiological investigations. Disordered proteins can also serve as potential targets for drug discovery with an emphasis on the disordered-to-ordered transition in the disordered binding regions, and this has led to substantial research in drug discovery or design based on protein disordered region prediction. Furthermore, protein disorder prediction has also been applied to healthcare by predicting the disease risk of mutations in patients and studying the mechanistic basis of diseases. As the applications of disorder prediction increase, so too does the need to make quick and accurate predictions. To fill this need, we also present a new approach to predict protein residue disorder using wide sequence windows that is applicable on the genomic scale.

  9. An Overview of Practical Applications of Protein Disorder Prediction and Drive for Faster, More Accurate Predictions

    Directory of Open Access Journals (Sweden)

    Xin Deng

    2015-07-01

    Full Text Available Protein disordered regions are segments of a protein chain that do not adopt a stable structure. Thus far, a variety of protein disorder prediction methods have been developed and have been widely used, not only in traditional bioinformatics domains, including protein structure prediction, protein structure determination and function annotation, but also in many other biomedical fields. The relationship between intrinsically-disordered proteins and some human diseases has played a significant role in disorder prediction in disease identification and epidemiological investigations. Disordered proteins can also serve as potential targets for drug discovery with an emphasis on the disordered-to-ordered transition in the disordered binding regions, and this has led to substantial research in drug discovery or design based on protein disordered region prediction. Furthermore, protein disorder prediction has also been applied to healthcare by predicting the disease risk of mutations in patients and studying the mechanistic basis of diseases. As the applications of disorder prediction increase, so too does the need to make quick and accurate predictions. To fill this need, we also present a new approach to predict protein residue disorder using wide sequence windows that is applicable on the genomic scale.

  10. Fast and Accurate Identification of Cross-Linked Peptides for the Structural Analysis of Large Protein Complexes and Elucidation of Interaction Networks. / Tahir, Salman; Bukowski-Wills, Jimi-Carlo; Rasmussen, Morten; Rappsilber, Juri

    DEFF Research Database (Denmark)

    Rasmussen, Morten

    to investigate protein structure and protein-protein interactions. When applied to single proteins or small purified protein complexes, this methodology works well. However certain challenges arise when applied to more complex samples. One of the main problems is the combinatorial increase in the search space...... simplify a spectrum because we remove all peaks that are accounted for by the fragmentation of peptide one. This approach is highly sensitive and scales well as revealed by searching our data of synthetic cross-links against a large sequence database. Currently, against a protein database of >1300 proteins...... a spectrum is searched in 0.35 seconds - a vast improvement when compared to the exhaustive search method of combining every potential cross-link for each spectrum(60 hours). In fact the search time is comparable, if not better, than existing linear search engines. Furthermore, we auto-validate the results...

  11. Computational methods for protein identification from mass spectrometry data.

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    Leo McHugh

    2008-02-01

    Full Text Available Protein identification using mass spectrometry is an indispensable computational tool in the life sciences. A dramatic increase in the use of proteomic strategies to understand the biology of living systems generates an ongoing need for more effective, efficient, and accurate computational methods for protein identification. A wide range of computational methods, each with various implementations, are available to complement different proteomic approaches. A solid knowledge of the range of algorithms available and, more critically, the accuracy and effectiveness of these techniques is essential to ensure as many of the proteins as possible, within any particular experiment, are correctly identified. Here, we undertake a systematic review of the currently available methods and algorithms for interpreting, managing, and analyzing biological data associated with protein identification. We summarize the advances in computational solutions as they have responded to corresponding advances in mass spectrometry hardware. The evolution of scoring algorithms and metrics for automated protein identification are also discussed with a focus on the relative performance of different techniques. We also consider the relative advantages and limitations of different techniques in particular biological contexts. Finally, we present our perspective on future developments in the area of computational protein identification by considering the most recent literature on new and promising approaches to the problem as well as identifying areas yet to be explored and the potential application of methods from other areas of computational biology.

  12. Post-Electrophoretic Identification of Oxidized Proteins

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    Conrad, Craig C; Talent, John M; Malakowsky, Christina A

    1999-01-01

    The oxidative modification of proteins has been shown to play a major role in a number of human diseases. However, the ability to identify specific proteins that are most susceptible to oxidative modifications is difficult. Separation of proteins using polyacrylamide gel electrophoresis (PAGE) offers the analytical potential for the recovery, amino acid sequencing, and identification of thousands of individual proteins from cells and tissues. We have developed a method to allow underivatized proteins to be electroblotted onto PVDF membranes before derivatization and staining. Since both the protein and oxidation proteins are quantifiable, the specific oxidation index of each protein can be determined. The optimal sequence and conditions for the staining process are (a) electrophoresis, (b) electroblotting onto PVDF membranes, (c) derivatization of carbonyls with 2,4-DNP, (d) immunostaining with anti DNP antibody, and (e) protein staining with colloidal gold. PMID:12734585

  13. Accurate Lithium-ion battery parameter estimation with continuous-time system identification methods

    International Nuclear Information System (INIS)

    Xia, Bing; Zhao, Xin; Callafon, Raymond de; Garnier, Hugues; Nguyen, Truong; Mi, Chris

    2016-01-01

    Highlights: • Continuous-time system identification is applied in Lithium-ion battery modeling. • Continuous-time and discrete-time identification methods are compared in detail. • The instrumental variable method is employed to further improve the estimation. • Simulations and experiments validate the advantages of continuous-time methods. - Abstract: The modeling of Lithium-ion batteries usually utilizes discrete-time system identification methods to estimate parameters of discrete models. However, in real applications, there is a fundamental limitation of the discrete-time methods in dealing with sensitivity when the system is stiff and the storage resolutions are limited. To overcome this problem, this paper adopts direct continuous-time system identification methods to estimate the parameters of equivalent circuit models for Lithium-ion batteries. Compared with discrete-time system identification methods, the continuous-time system identification methods provide more accurate estimates to both fast and slow dynamics in battery systems and are less sensitive to disturbances. A case of a 2"n"d-order equivalent circuit model is studied which shows that the continuous-time estimates are more robust to high sampling rates, measurement noises and rounding errors. In addition, the estimation by the conventional continuous-time least squares method is further improved in the case of noisy output measurement by introducing the instrumental variable method. Simulation and experiment results validate the analysis and demonstrate the advantages of the continuous-time system identification methods in battery applications.

  14. Protein kinase substrate identification on functional protein arrays

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    Zhou Fang

    2008-02-01

    Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

  15. The SPECIES and ORGANISMS Resources for Fast and Accurate Identification of Taxonomic Names in Text

    DEFF Research Database (Denmark)

    Pafilis, Evangelos; Pletscher-Frankild, Sune; Fanini, Lucia

    2013-01-01

    The exponential growth of the biomedical literature is making the need for efficient, accurate text-mining tools increasingly clear. The identification of named biological entities in text is a central and difficult task. We have developed an efficient algorithm and implementation of a dictionary......-based approach to named entity recognition, which we here use to identify names of species and other taxa in text. The tool, SPECIES, is more than an order of magnitude faster and as accurate as existing tools. The precision and recall was assessed both on an existing gold-standard corpus and on a new corpus...

  16. Accurate and sensitive quantification of protein-DNA binding affinity.

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    Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

    2018-04-17

    Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

  17. MM-ISMSA: An Ultrafast and Accurate Scoring Function for Protein-Protein Docking.

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    Klett, Javier; Núñez-Salgado, Alfonso; Dos Santos, Helena G; Cortés-Cabrera, Álvaro; Perona, Almudena; Gil-Redondo, Rubén; Abia, David; Gago, Federico; Morreale, Antonio

    2012-09-11

    An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

  18. Final Progress Report: Isotope Identification Algorithm for Rapid and Accurate Determination of Radioisotopes Feasibility Study

    International Nuclear Information System (INIS)

    Rawool-Sullivan, Mohini; Bounds, John Alan; Brumby, Steven P.; Prasad, Lakshman; Sullivan, John P.

    2012-01-01

    This is the final report of the project titled, 'Isotope Identification Algorithm for Rapid and Accurate Determination of Radioisotopes,' PMIS project number LA10-HUMANID-PD03. The goal of the work was to demonstrate principles of emulating a human analysis approach towards the data collected using radiation isotope identification devices (RIIDs). It summarizes work performed over the FY10 time period. The goal of the work was to demonstrate principles of emulating a human analysis approach towards the data collected using radiation isotope identification devices (RIIDs). Human analysts begin analyzing a spectrum based on features in the spectrum - lines and shapes that are present in a given spectrum. The proposed work was to carry out a feasibility study that will pick out all gamma ray peaks and other features such as Compton edges, bremsstrahlung, presence/absence of shielding and presence of neutrons and escape peaks. Ultimately success of this feasibility study will allow us to collectively explain identified features and form a realistic scenario that produced a given spectrum in the future. We wanted to develop and demonstrate machine learning algorithms that will qualitatively enhance the automated identification capabilities of portable radiological sensors that are currently being used in the field.

  19. [Progress in the spectral library based protein identification strategy].

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    Yu, Derui; Ma, Jie; Xie, Zengyan; Bai, Mingze; Zhu, Yunping; Shu, Kunxian

    2018-04-25

    Exponential growth of the mass spectrometry (MS) data is exhibited when the mass spectrometry-based proteomics has been developing rapidly. It is a great challenge to develop some quick, accurate and repeatable methods to identify peptides and proteins. Nowadays, the spectral library searching has become a mature strategy for tandem mass spectra based proteins identification in proteomics, which searches the experiment spectra against a collection of confidently identified MS/MS spectra that have been observed previously, and fully utilizes the abundance in the spectrum, peaks from non-canonical fragment ions, and other features. This review provides an overview of the implement of spectral library search strategy, and two key steps, spectral library construction and spectral library searching comprehensively, and discusses the progress and challenge of the library search strategy.

  20. CASD-NMR 2: robust and accurate unsupervised analysis of raw NOESY spectra and protein structure determination with UNIO

    International Nuclear Information System (INIS)

    Guerry, Paul; Duong, Viet Dung; Herrmann, Torsten

    2015-01-01

    UNIO is a comprehensive software suite for protein NMR structure determination that enables full automation of all NMR data analysis steps involved—including signal identification in NMR spectra, sequence-specific backbone and side-chain resonance assignment, NOE assignment and structure calculation. Within the framework of the second round of the community-wide stringent blind NMR structure determination challenge (CASD-NMR 2), we participated in two categories of CASD-NMR 2, namely using either raw NMR spectra or unrefined NOE peak lists as input. A total of 15 resulting NMR structure bundles were submitted for 9 out of 10 blind protein targets. All submitted UNIO structures accurately coincided with the corresponding blind targets as documented by an average backbone root mean-square deviation to the reference proteins of only 1.2 Å. Also, the precision of the UNIO structure bundles was virtually identical to the ensemble of reference structures. By assessing the quality of all UNIO structures submitted to the two categories, we find throughout that only the UNIO–ATNOS/CANDID approach using raw NMR spectra consistently yielded structure bundles of high quality for direct deposition in the Protein Data Bank. In conclusion, the results obtained in CASD-NMR 2 are another vital proof for robust, accurate and unsupervised NMR data analysis by UNIO for real-world applications

  1. Identification of NAD interacting residues in proteins

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    Raghava Gajendra PS

    2010-03-01

    Full Text Available Abstract Background Small molecular cofactors or ligands play a crucial role in the proper functioning of cells. Accurate annotation of their target proteins and binding sites is required for the complete understanding of reaction mechanisms. Nicotinamide adenine dinucleotide (NAD+ or NAD is one of the most commonly used organic cofactors in living cells, which plays a critical role in cellular metabolism, storage and regulatory processes. In the past, several NAD binding proteins (NADBP have been reported in the literature, which are responsible for a wide-range of activities in the cell. Attempts have been made to derive a rule for the binding of NAD+ to its target proteins. However, so far an efficient model could not be derived due to the time consuming process of structure determination, and limitations of similarity based approaches. Thus a sequence and non-similarity based method is needed to characterize the NAD binding sites to help in the annotation. In this study attempts have been made to predict NAD binding proteins and their interacting residues (NIRs from amino acid sequence using bioinformatics tools. Results We extracted 1556 proteins chains from 555 NAD binding proteins whose structure is available in Protein Data Bank. Then we removed all redundant protein chains and finally obtained 195 non-redundant NAD binding protein chains, where no two chains have more than 40% sequence identity. In this study all models were developed and evaluated using five-fold cross validation technique on the above dataset of 195 NAD binding proteins. While certain type of residues are preferred (e.g. Gly, Tyr, Thr, His in NAD interaction, residues like Ala, Glu, Leu, Lys are not preferred. A support vector machine (SVM based method has been developed using various window lengths of amino acid sequence for predicting NAD interacting residues and obtained maximum Matthew's correlation coefficient (MCC 0.47 with accuracy 74.13% at window length 17

  2. A scalable and accurate method for classifying protein-ligand binding geometries using a MapReduce approach.

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    Estrada, T; Zhang, B; Cicotti, P; Armen, R S; Taufer, M

    2012-07-01

    We present a scalable and accurate method for classifying protein-ligand binding geometries in molecular docking. Our method is a three-step process: the first step encodes the geometry of a three-dimensional (3D) ligand conformation into a single 3D point in the space; the second step builds an octree by assigning an octant identifier to every single point in the space under consideration; and the third step performs an octree-based clustering on the reduced conformation space and identifies the most dense octant. We adapt our method for MapReduce and implement it in Hadoop. The load-balancing, fault-tolerance, and scalability in MapReduce allow screening of very large conformation spaces not approachable with traditional clustering methods. We analyze results for docking trials for 23 protein-ligand complexes for HIV protease, 21 protein-ligand complexes for Trypsin, and 12 protein-ligand complexes for P38alpha kinase. We also analyze cross docking trials for 24 ligands, each docking into 24 protein conformations of the HIV protease, and receptor ensemble docking trials for 24 ligands, each docking in a pool of HIV protease receptors. Our method demonstrates significant improvement over energy-only scoring for the accurate identification of native ligand geometries in all these docking assessments. The advantages of our clustering approach make it attractive for complex applications in real-world drug design efforts. We demonstrate that our method is particularly useful for clustering docking results using a minimal ensemble of representative protein conformational states (receptor ensemble docking), which is now a common strategy to address protein flexibility in molecular docking. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. An accurate and efficient identification of children with psychosocial problems by means of computerized adaptive testing

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    Reijneveld Symen A

    2011-08-01

    Full Text Available Abstract Background Questionnaires used by health services to identify children with psychosocial problems are often rather short. The psychometric properties of such short questionnaires are mostly less than needed for an accurate distinction between children with and without problems. We aimed to assess whether a short Computerized Adaptive Test (CAT can overcome the weaknesses of short written questionnaires when identifying children with psychosocial problems. Method We used a Dutch national data set obtained from parents of children invited for a routine health examination by Preventive Child Healthcare with 205 items on behavioral and emotional problems (n = 2,041, response 84%. In a random subsample we determined which items met the requirements of an Item Response Theory (IRT model to a sufficient degree. Using those items, item parameters necessary for a CAT were calculated and a cut-off point was defined. In the remaining subsample we determined the validity and efficiency of a Computerized Adaptive Test using simulation techniques, with current treatment status and a clinical score on the Total Problem Scale (TPS of the Child Behavior Checklist as criteria. Results Out of 205 items available 190 sufficiently met the criteria of the underlying IRT model. For 90% of the children a score above or below cut-off point could be determined with 95% accuracy. The mean number of items needed to achieve this was 12. Sensitivity and specificity with the TPS as a criterion were 0.89 and 0.91, respectively. Conclusion An IRT-based CAT is a very promising option for the identification of psychosocial problems in children, as it can lead to an efficient, yet high-quality identification. The results of our simulation study need to be replicated in a real-life administration of this CAT.

  4. Accurate protein structure modeling using sparse NMR data and homologous structure information.

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    Thompson, James M; Sgourakis, Nikolaos G; Liu, Gaohua; Rossi, Paolo; Tang, Yuefeng; Mills, Jeffrey L; Szyperski, Thomas; Montelione, Gaetano T; Baker, David

    2012-06-19

    While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining (1)H(N), (13)C, and (15)N backbone and (13)Cβ chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2-1.9 Å relative to the conventional determined NMR ensembles and of 0.9-1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.

  5. Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

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    Stovgaard Kasper

    2010-08-01

    Full Text Available Abstract Background Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference. Results We present a method for the efficient calculation of accurate SAXS curves based on the Debye formula and a set of scattering form factors for dummy atom representations of amino acids. Such a method avoids the computationally costly iteration over all atoms. We estimated the form factors using generated data from a set of high quality protein structures. No ad hoc scaling or correction factors are applied in the calculation of the curves. Two coarse-grained representations of protein structure were investigated; two scattering bodies per amino acid led to significantly better results than a single scattering body. Conclusion We show that the obtained point estimates allow the calculation of accurate SAXS curves from coarse-grained protein models. The resulting curves are on par with the current state-of-the-art program CRYSOL, which requires full atomic detail. Our method was also comparable to CRYSOL in recognizing native structures among native-like decoys. As a proof-of-concept, we combined the coarse-grained Debye calculation with a previously described probabilistic model of protein structure, TorusDBN. This resulted in a significant improvement in the decoy recognition performance. In conclusion, the presented method shows great promise for

  6. Using context to improve protein domain identification

    Directory of Open Access Journals (Sweden)

    Llinás Manuel

    2011-03-01

    Full Text Available Abstract Background Identifying domains in protein sequences is an important step in protein structural and functional annotation. Existing domain recognition methods typically evaluate each domain prediction independently of the rest. However, the majority of proteins are multidomain, and pairwise domain co-occurrences are highly specific and non-transitive. Results Here, we demonstrate how to exploit domain co-occurrence to boost weak domain predictions that appear in previously observed combinations, while penalizing higher confidence domains if such combinations have never been observed. Our framework, Domain Prediction Using Context (dPUC, incorporates pairwise "context" scores between domains, along with traditional domain scores and thresholds, and improves domain prediction across a variety of organisms from bacteria to protozoa and metazoa. Among the genomes we tested, dPUC is most successful at improving predictions for the poorly-annotated malaria parasite Plasmodium falciparum, for which over 38% of the genome is currently unannotated. Our approach enables high-confidence annotations in this organism and the identification of orthologs to many core machinery proteins conserved in all eukaryotes, including those involved in ribosomal assembly and other RNA processing events, which surprisingly had not been previously known. Conclusions Overall, our results demonstrate that this new context-based approach will provide significant improvements in domain and function prediction, especially for poorly understood genomes for which the need for additional annotations is greatest. Source code for the algorithm is available under a GPL open source license at http://compbio.cs.princeton.edu/dpuc/. Pre-computed results for our test organisms and a web server are also available at that location.

  7. Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

    Science.gov (United States)

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

  8. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  9. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning.

    Science.gov (United States)

    Pärnamaa, Tanel; Parts, Leopold

    2017-05-05

    High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy. Copyright © 2017 Parnamaa and Parts.

  10. Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Karosiene, Edita; Rasmussen, Michael

    2015-01-01

    with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped...

  11. Improved method for rapid and accurate isolation and identification of Streptococcus mutans and Streptococcus sobrinus from human plaque samples.

    Science.gov (United States)

    Villhauer, Alissa L; Lynch, David J; Drake, David R

    2017-08-01

    Mutans streptococci (MS), specifically Streptococcus mutans (SM) and Streptococcus sobrinus (SS), are bacterial species frequently targeted for investigation due to their role in the etiology of dental caries. Differentiation of S. mutans and S. sobrinus is an essential part of exploring the role of these organisms in disease progression and the impact of the presence of either/both on a subject's caries experience. Of vital importance to the study of these organisms is an identification protocol that allows us to distinguish between the two species in an easy, accurate, and timely manner. While conducting a 5-year birth cohort study in a Northern Plains American Indian tribe, the need for a more rapid procedure for isolating and identifying high volumes of MS was recognized. We report here on the development of an accurate and rapid method for MS identification. Accuracy, ease of use, and material and time requirements for morphological differentiation on selective agar, biochemical tests, and various combinations of PCR primers were compared. The final protocol included preliminary identification based on colony morphology followed by PCR confirmation of species identification using primers targeting regions of the glucosyltransferase (gtf) genes of SM and SS. This method of isolation and identification was found to be highly accurate, more rapid than the previous methodology used, and easily learned. It resulted in more efficient use of both time and material resources. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

    DEFF Research Database (Denmark)

    Stovgaard, Kasper; Andreetta, Christian; Ferkinghoff-Borg, Jesper

    2010-01-01

    , which is paramount for structure determination based on statistical inference. Results: We present a method for the efficient calculation of accurate SAXS curves based on the Debye formula and a set of scattering form factors for dummy atom representations of amino acids. Such a method avoids......DBN. This resulted in a significant improvement in the decoy recognition performance. In conclusion, the presented method shows great promise for use in statistical inference of protein structures from SAXS data....

  13. Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry.

    Science.gov (United States)

    Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y; Drake, Steven K; Gucek, Marjan; Sacks, David B; Yu, Yi-Kuo

    2018-06-05

    Rapid and accurate identification and classification of microorganisms is of paramount importance to public health and safety. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is complicating correct microbial identification even in a simple sample due to the large number of candidates present. To properly untwine candidate microbes in samples containing one or more microbes, one needs to go beyond apparent morphology or simple "fingerprinting"; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptide-centric representations of microbes to better separate them and by augmenting our earlier analysis method that yields accurate statistical significance. Here, we present an updated analysis workflow that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000 MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. We have also shown that the proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html . Graphical Abstract ᅟ.

  14. Neural network and SVM classifiers accurately predict lipid binding proteins, irrespective of sequence homology.

    Science.gov (United States)

    Bakhtiarizadeh, Mohammad Reza; Moradi-Shahrbabak, Mohammad; Ebrahimi, Mansour; Ebrahimie, Esmaeil

    2014-09-07

    Due to the central roles of lipid binding proteins (LBPs) in many biological processes, sequence based identification of LBPs is of great interest. The major challenge is that LBPs are diverse in sequence, structure, and function which results in low accuracy of sequence homology based methods. Therefore, there is a need for developing alternative functional prediction methods irrespective of sequence similarity. To identify LBPs from non-LBPs, the performances of support vector machine (SVM) and neural network were compared in this study. Comprehensive protein features and various techniques were employed to create datasets. Five-fold cross-validation (CV) and independent evaluation (IE) tests were used to assess the validity of the two methods. The results indicated that SVM outperforms neural network. SVM achieved 89.28% (CV) and 89.55% (IE) overall accuracy in identification of LBPs from non-LBPs and 92.06% (CV) and 92.90% (IE) (in average) for classification of different LBPs classes. Increasing the number and the range of extracted protein features as well as optimization of the SVM parameters significantly increased the efficiency of LBPs class prediction in comparison to the only previous report in this field. Altogether, the results showed that the SVM algorithm can be run on broad, computationally calculated protein features and offers a promising tool in detection of LBPs classes. The proposed approach has the potential to integrate and improve the common sequence alignment based methods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E.; Korgsdam, A.-M.; Jørgensen, H.F.

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  16. Optimization of the GBMV2 implicit solvent force field for accurate simulation of protein conformational equilibria.

    Science.gov (United States)

    Lee, Kuo Hao; Chen, Jianhan

    2017-06-15

    Accurate treatment of solvent environment is critical for reliable simulations of protein conformational equilibria. Implicit treatment of solvation, such as using the generalized Born (GB) class of models arguably provides an optimal balance between computational efficiency and physical accuracy. Yet, GB models are frequently plagued by a tendency to generate overly compact structures. The physical origins of this drawback are relatively well understood, and the key to a balanced implicit solvent protein force field is careful optimization of physical parameters to achieve a sufficient level of cancellation of errors. The latter has been hampered by the difficulty of generating converged conformational ensembles of non-trivial model proteins using the popular replica exchange sampling technique. Here, we leverage improved sampling efficiency of a newly developed multi-scale enhanced sampling technique to re-optimize the generalized-Born with molecular volume (GBMV2) implicit solvent model with the CHARMM36 protein force field. Recursive optimization of key GBMV2 parameters (such as input radii) and protein torsion profiles (via the CMAP torsion cross terms) has led to a more balanced GBMV2 protein force field that recapitulates the structures and stabilities of both helical and β-hairpin model peptides. Importantly, this force field appears to be free of the over-compaction bias, and can generate structural ensembles of several intrinsically disordered proteins of various lengths that seem highly consistent with available experimental data. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Fast and accurate protein substructure searching with simulated annealing and GPUs

    Directory of Open Access Journals (Sweden)

    Stivala Alex D

    2010-09-01

    Full Text Available Abstract Background Searching a database of protein structures for matches to a query structure, or occurrences of a structural motif, is an important task in structural biology and bioinformatics. While there are many existing methods for structural similarity searching, faster and more accurate approaches are still required, and few current methods are capable of substructure (motif searching. Results We developed an improved heuristic for tableau-based protein structure and substructure searching using simulated annealing, that is as fast or faster and comparable in accuracy, with some widely used existing methods. Furthermore, we created a parallel implementation on a modern graphics processing unit (GPU. Conclusions The GPU implementation achieves up to 34 times speedup over the CPU implementation of tableau-based structure search with simulated annealing, making it one of the fastest available methods. To the best of our knowledge, this is the first application of a GPU to the protein structural search problem.

  18. Seed Storage Proteins as a System for Teaching Protein Identification by Mass Spectrometry in Biochemistry Laboratory

    Science.gov (United States)

    Wilson, Karl A.; Tan-Wilson, Anna

    2013-01-01

    Mass spectrometry (MS) has become an important tool in studying biological systems. One application is the identification of proteins and peptides by the matching of peptide and peptide fragment masses to the sequences of proteins in protein sequence databases. Often prior protein separation of complex protein mixtures by 2D-PAGE is needed,…

  19. Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS

    DEFF Research Database (Denmark)

    Kudirkiene, Egle; Welker, Martin; Knudsen, Nanna Reumert

    2015-01-01

    phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspecies.......3±7.5%). This result may be attributed to the highly clonal population structure of SEE, as opposed to the diversity of SEZ seen in horses. Importantly strains with atypical colony appearance both within SEE and SEZ did not affect correct identification of the strains by MALDI-TOF MS. Atypical colony variants...... are often associated with a higher persistence or virulence of S. equi, thus their correct identification using the current method strengthens its potential use in routine clinical diagnostics. In conclusion, reliable identification of S. equi subspecies was achieved by combining a MALDI-TOF MS method...

  20. Sad people are more accurate at expression identification with a smaller own-ethnicity bias than happy people.

    Science.gov (United States)

    Hills, Peter J; Hill, Dominic M

    2017-07-12

    Sad individuals perform more accurately at face identity recognition (Hills, Werno, & Lewis, 2011), possibly because they scan more of the face during encoding. During expression identification tasks, sad individuals do not fixate on the eyes as much as happier individuals (Wu, Pu, Allen, & Pauli, 2012). Fixating on features other than the eyes leads to a reduced own-ethnicity bias (Hills & Lewis, 2006). This background indicates that sad individuals would not view the eyes as much as happy individuals and this would result in improved expression recognition and a reduced own-ethnicity bias. This prediction was tested using an expression identification task, with eye tracking. We demonstrate that sad-induced participants show enhanced expression recognition and a reduced own-ethnicity bias than happy-induced participants due to scanning more facial features. We conclude that mood affects eye movements and face encoding by causing a wider sampling strategy and deeper encoding of facial features diagnostic for expression identification.

  1. Rapid and accurate prediction and scoring of water molecules in protein binding sites.

    Directory of Open Access Journals (Sweden)

    Gregory A Ross

    Full Text Available Water plays a critical role in ligand-protein interactions. However, it is still challenging to predict accurately not only where water molecules prefer to bind, but also which of those water molecules might be displaceable. The latter is often seen as a route to optimizing affinity of potential drug candidates. Using a protocol we call WaterDock, we show that the freely available AutoDock Vina tool can be used to predict accurately the binding sites of water molecules. WaterDock was validated using data from X-ray crystallography, neutron diffraction and molecular dynamics simulations and correctly predicted 97% of the water molecules in the test set. In addition, we combined data-mining, heuristic and machine learning techniques to develop probabilistic water molecule classifiers. When applied to WaterDock predictions in the Astex Diverse Set of protein ligand complexes, we could identify whether a water molecule was conserved or displaced to an accuracy of 75%. A second model predicted whether water molecules were displaced by polar groups or by non-polar groups to an accuracy of 80%. These results should prove useful for anyone wishing to undertake rational design of new compounds where the displacement of water molecules is being considered as a route to improved affinity.

  2. Serum protein profile at remission can accurately assess therapeutic outcomes and survival for serous ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Jinhua Wang

    Full Text Available BACKGROUND: Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer. EXPERIMENTAL DESIGN: Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD, remission (RM and recurrence (RC. The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis. RESULTS: Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC have individually good area-under-the-curve (AUC values (AUC = 0.69-0.86 and more than 10 three-marker combinations have excellent AUC values (0.91-0.93 in distinguishing active cancer samples (PD & RC from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC. Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1 measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p<10(-3. CONCLUSION: We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.

  3. Efficient and accurate Greedy Search Methods for mining functional modules in protein interaction networks.

    Science.gov (United States)

    He, Jieyue; Li, Chaojun; Ye, Baoliu; Zhong, Wei

    2012-06-25

    Most computational algorithms mainly focus on detecting highly connected subgraphs in PPI networks as protein complexes but ignore their inherent organization. Furthermore, many of these algorithms are computationally expensive. However, recent analysis indicates that experimentally detected protein complexes generally contain Core/attachment structures. In this paper, a Greedy Search Method based on Core-Attachment structure (GSM-CA) is proposed. The GSM-CA method detects densely connected regions in large protein-protein interaction networks based on the edge weight and two criteria for determining core nodes and attachment nodes. The GSM-CA method improves the prediction accuracy compared to other similar module detection approaches, however it is computationally expensive. Many module detection approaches are based on the traditional hierarchical methods, which is also computationally inefficient because the hierarchical tree structure produced by these approaches cannot provide adequate information to identify whether a network belongs to a module structure or not. In order to speed up the computational process, the Greedy Search Method based on Fast Clustering (GSM-FC) is proposed in this work. The edge weight based GSM-FC method uses a greedy procedure to traverse all edges just once to separate the network into the suitable set of modules. The proposed methods are applied to the protein interaction network of S. cerevisiae. Experimental results indicate that many significant functional modules are detected, most of which match the known complexes. Results also demonstrate that the GSM-FC algorithm is faster and more accurate as compared to other competing algorithms. Based on the new edge weight definition, the proposed algorithm takes advantages of the greedy search procedure to separate the network into the suitable set of modules. Experimental analysis shows that the identified modules are statistically significant. The algorithm can reduce the

  4. Identification of outer membrane proteins of Yersinia pestis through biotinylation

    NARCIS (Netherlands)

    Smither, S.J.; Hill, J.; Baar, B.L.M. van; Hulst, A.G.; Jong, A.L. de; Titball, R.W.

    2007-01-01

    The outer membrane of Gram-negative bacteria contains proteins that might be good targets for vaccines, antimicrobials or detection systems. The identification of surface located proteins using traditional methods is often difficult. Yersinia pestis, the causative agent of plague, was labelled with

  5. Mass spectrometry allows direct identification of proteins in large genomes

    DEFF Research Database (Denmark)

    Küster, B; Mortensen, Peter V.; Andersen, Jens S.

    2001-01-01

    Proteome projects seek to provide systematic functional analysis of the genes uncovered by genome sequencing initiatives. Mass spectrometric protein identification is a key requirement in these studies but to date, database searching tools rely on the availability of protein sequences derived fro...

  6. An accurate and efficient identification of children with psychosocial problems by means of computerized adaptive testing

    NARCIS (Netherlands)

    Vogels, Antonius G. C.; Jacobusse, Gert W.; Reijneveld, Symen A.

    2011-01-01

    Background: Questionnaires used by health services to identify children with psychosocial problems are often rather short. The psychometric properties of such short questionnaires are mostly less than needed for an accurate distinction between children with and without problems. We aimed to assess

  7. Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris.

    Science.gov (United States)

    Kordalewska, Milena; Zhao, Yanan; Lockhart, Shawn R; Chowdhary, Anuradha; Berrio, Indira; Perlin, David S

    2017-08-01

    Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species. Copyright © 2017 Kordalewska et al.

  8. Electrostatics of proteins in dielectric solvent continua. I. An accurate and efficient reaction field description.

    Science.gov (United States)

    Bauer, Sebastian; Mathias, Gerald; Tavan, Paul

    2014-03-14

    We present a reaction field (RF) method which accurately solves the Poisson equation for proteins embedded in dielectric solvent continua at a computational effort comparable to that of an electrostatics calculation with polarizable molecular mechanics (MM) force fields. The method combines an approach originally suggested by Egwolf and Tavan [J. Chem. Phys. 118, 2039 (2003)] with concepts generalizing the Born solution [Z. Phys. 1, 45 (1920)] for a solvated ion. First, we derive an exact representation according to which the sources of the RF potential and energy are inducible atomic anti-polarization densities and atomic shielding charge distributions. Modeling these atomic densities by Gaussians leads to an approximate representation. Here, the strengths of the Gaussian shielding charge distributions are directly given in terms of the static partial charges as defined, e.g., by standard MM force fields for the various atom types, whereas the strengths of the Gaussian anti-polarization densities are calculated by a self-consistency iteration. The atomic volumes are also described by Gaussians. To account for covalently overlapping atoms, their effective volumes are calculated by another self-consistency procedure, which guarantees that the dielectric function ε(r) is close to one everywhere inside the protein. The Gaussian widths σ(i) of the atoms i are parameters of the RF approximation. The remarkable accuracy of the method is demonstrated by comparison with Kirkwood's analytical solution for a spherical protein [J. Chem. Phys. 2, 351 (1934)] and with computationally expensive grid-based numerical solutions for simple model systems in dielectric continua including a di-peptide (Ac-Ala-NHMe) as modeled by a standard MM force field. The latter example shows how weakly the RF conformational free energy landscape depends on the parameters σ(i). A summarizing discussion highlights the achievements of the new theory and of its approximate solution particularly by

  9. Electrostatics of proteins in dielectric solvent continua. I. An accurate and efficient reaction field description

    Energy Technology Data Exchange (ETDEWEB)

    Bauer, Sebastian; Mathias, Gerald; Tavan, Paul, E-mail: paul.tavan@physik.uni-muenchen.de [Lehrstuhl für BioMolekulare Optik, Ludwig–Maximilians Universität München, Oettingenstr. 67, 80538 München (Germany)

    2014-03-14

    We present a reaction field (RF) method which accurately solves the Poisson equation for proteins embedded in dielectric solvent continua at a computational effort comparable to that of an electrostatics calculation with polarizable molecular mechanics (MM) force fields. The method combines an approach originally suggested by Egwolf and Tavan [J. Chem. Phys. 118, 2039 (2003)] with concepts generalizing the Born solution [Z. Phys. 1, 45 (1920)] for a solvated ion. First, we derive an exact representation according to which the sources of the RF potential and energy are inducible atomic anti-polarization densities and atomic shielding charge distributions. Modeling these atomic densities by Gaussians leads to an approximate representation. Here, the strengths of the Gaussian shielding charge distributions are directly given in terms of the static partial charges as defined, e.g., by standard MM force fields for the various atom types, whereas the strengths of the Gaussian anti-polarization densities are calculated by a self-consistency iteration. The atomic volumes are also described by Gaussians. To account for covalently overlapping atoms, their effective volumes are calculated by another self-consistency procedure, which guarantees that the dielectric function ε(r) is close to one everywhere inside the protein. The Gaussian widths σ{sub i} of the atoms i are parameters of the RF approximation. The remarkable accuracy of the method is demonstrated by comparison with Kirkwood's analytical solution for a spherical protein [J. Chem. Phys. 2, 351 (1934)] and with computationally expensive grid-based numerical solutions for simple model systems in dielectric continua including a di-peptide (Ac-Ala-NHMe) as modeled by a standard MM force field. The latter example shows how weakly the RF conformational free energy landscape depends on the parameters σ{sub i}. A summarizing discussion highlights the achievements of the new theory and of its approximate solution

  10. Rapid detection, classification and accurate alignment of up to a million or more related protein sequences.

    Science.gov (United States)

    Neuwald, Andrew F

    2009-08-01

    The patterns of sequence similarity and divergence present within functionally diverse, evolutionarily related proteins contain implicit information about corresponding biochemical similarities and differences. A first step toward accessing such information is to statistically analyze these patterns, which, in turn, requires that one first identify and accurately align a very large set of protein sequences. Ideally, the set should include many distantly related, functionally divergent subgroups. Because it is extremely difficult, if not impossible for fully automated methods to align such sequences correctly, researchers often resort to manual curation based on detailed structural and biochemical information. However, multiply-aligning vast numbers of sequences in this way is clearly impractical. This problem is addressed using Multiply-Aligned Profiles for Global Alignment of Protein Sequences (MAPGAPS). The MAPGAPS program uses a set of multiply-aligned profiles both as a query to detect and classify related sequences and as a template to multiply-align the sequences. It relies on Karlin-Altschul statistics for sensitivity and on PSI-BLAST (and other) heuristics for speed. Using as input a carefully curated multiple-profile alignment for P-loop GTPases, MAPGAPS correctly aligned weakly conserved sequence motifs within 33 distantly related GTPases of known structure. By comparison, the sequence- and structurally based alignment methods hmmalign and PROMALS3D misaligned at least 11 and 23 of these regions, respectively. When applied to a dataset of 65 million protein sequences, MAPGAPS identified, classified and aligned (with comparable accuracy) nearly half a million putative P-loop GTPase sequences. A C++ implementation of MAPGAPS is available at http://mapgaps.igs.umaryland.edu. Supplementary data are available at Bioinformatics online.

  11. Proteomics: Protein Identification Using Online Databases

    Science.gov (United States)

    Eurich, Chris; Fields, Peter A.; Rice, Elizabeth

    2012-01-01

    Proteomics is an emerging area of systems biology that allows simultaneous study of thousands of proteins expressed in cells, tissues, or whole organisms. We have developed this activity to enable high school or college students to explore proteomic databases using mass spectrometry data files generated from yeast proteins in a college laboratory…

  12. Protein identification by peptide mass fingerprinting

    DEFF Research Database (Denmark)

    Hjernø, Karin

    2007-01-01

      Peptide mass fingerprinting is an effective way of identifying, e.g., gel-separated proteins, by matching experimentally obtained peptide mass data against large databases. However, several factors are known to influence the quality of the resulting matches, such as proteins contaminating the s...

  13. Automated selected reaction monitoring software for accurate label-free protein quantification.

    Science.gov (United States)

    Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

    2012-07-06

    Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

  14. Identification of Ina proteins from Fusarium acuminatum

    Science.gov (United States)

    Scheel, Jan Frederik; Kunert, Anna Theresa; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Freezing of water above -36° C is based on ice nucleation activity (INA) mediated by ice nucleators (IN) which can be of various origins. Beside mineral IN, biological particles are a potentially important source of atmospheric IN. The best-known biological IN are common plant-associated bacteria. The IN activity of these bacteria is induced by a surface protein on the outer cell membrane, which is fully characterized. In contrast, much less is known about the nature of fungal IN. The fungal genus Fusarium is widely spread throughout the earth. It belongs to the Ascomycota and is one of the most severe fungal pathogens. It can affect a variety of organisms from plants to animals including humans. INA of Fusarium was already described about 30 years ago and INA of Fusarium as well as other fungal genera is assumed to be mediated by proteins or at least to contain a proteinaceous compound. Although many efforts were made the precise INA machinery of Fusarium and other fungal species including the proteins and their corresponding genes remain unidentified. In this study preparations from living fungal samples of F. acuminatum were fractionated by liquid chromatography and IN active fractions were identified by freezing assays. SDS-page and de novo sequencing by mass spectrometry were used to identify the primary structure of the protein. Preliminary results show that the INA protein of F. acuminatum is contained in the early size exclusion chromatography fractions indicating a high molecular size. Moreover we could identify a single protein band from IN active fractions at 130-145 kDa corresponding to sizes of IN proteins from bacterial species. To our knowledge this is for the first time an isolation of a single protein from in vivo samples, which can be assigned as IN active from Fusarium.

  15. Accurate identification of RNA editing sites from primitive sequence with deep neural networks.

    Science.gov (United States)

    Ouyang, Zhangyi; Liu, Feng; Zhao, Chenghui; Ren, Chao; An, Gaole; Mei, Chuan; Bo, Xiaochen; Shu, Wenjie

    2018-04-16

    RNA editing is a post-transcriptional RNA sequence alteration. Current methods have identified editing sites and facilitated research but require sufficient genomic annotations and prior-knowledge-based filtering steps, resulting in a cumbersome, time-consuming identification process. Moreover, these methods have limited generalizability and applicability in species with insufficient genomic annotations or in conditions of limited prior knowledge. We developed DeepRed, a deep learning-based method that identifies RNA editing from primitive RNA sequences without prior-knowledge-based filtering steps or genomic annotations. DeepRed achieved 98.1% and 97.9% area under the curve (AUC) in training and test sets, respectively. We further validated DeepRed using experimentally verified U87 cell RNA-seq data, achieving 97.9% positive predictive value (PPV). We demonstrated that DeepRed offers better prediction accuracy and computational efficiency than current methods with large-scale, mass RNA-seq data. We used DeepRed to assess the impact of multiple factors on editing identification with RNA-seq data from the Association of Biomolecular Resource Facilities and Sequencing Quality Control projects. We explored developmental RNA editing pattern changes during human early embryogenesis and evolutionary patterns in Drosophila species and the primate lineage using DeepRed. Our work illustrates DeepRed's state-of-the-art performance; it may decipher the hidden principles behind RNA editing, making editing detection convenient and effective.

  16. Retrival experience as an accurate indicator of person identification in line-ups

    Directory of Open Access Journals (Sweden)

    María José Contreras

    2011-07-01

    Full Text Available Responses in eyewitness identification of a person in a line-up may be based on two types of recovery experiences, remember and know experiences. Remember responses involve eyewitness identification of the target person as an episodic memory task, because it implies retrieving information about the target person in the place and at the time of the event. Know responses, in contrast, engage recognition based on familiarity or perceptual facilitation, that is, as a semantic memory task. To explore the relation between retrieval experiences and recognition accuracy, 86 participants took part in a recognition task with two conditions: one with an interpolated target absent line-up and the other only with the target present line-up. Accuracy of recognition and retrieval experience was measured. The results showed that, having previously participated in a target-absent line-up, increased omissions, while the number of hits decreased. Furthermore, participants’ know responses were associated to false recognition, whilst remember responses were associated to hits in recognition. Thus, asking eyewitnesses to inform about the kind of retrieval experience in which they based their recognition responses, may serve as a reliable indicator of accuracy in recognition. Future studies are needed to investigate whether this is also the case in natural settings.

  17. DeepBound: accurate identification of transcript boundaries via deep convolutional neural fields

    KAUST Repository

    Shao, Mingfu; Ma, Jianzhu; Wang, Sheng

    2017-01-01

    Motivation: Reconstructing the full- length expressed transcripts (a. k. a. the transcript assembly problem) from the short sequencing reads produced by RNA-seq protocol plays a central role in identifying novel genes and transcripts as well as in studying gene expressions and gene functions. A crucial step in transcript assembly is to accurately determine the splicing junctions and boundaries of the expressed transcripts from the reads alignment. In contrast to the splicing junctions that can be efficiently detected from spliced reads, the problem of identifying boundaries remains open and challenging, due to the fact that the signal related to boundaries is noisy and weak.

  18. DeepBound: accurate identification of transcript boundaries via deep convolutional neural fields

    KAUST Repository

    Shao, Mingfu

    2017-04-20

    Motivation: Reconstructing the full- length expressed transcripts (a. k. a. the transcript assembly problem) from the short sequencing reads produced by RNA-seq protocol plays a central role in identifying novel genes and transcripts as well as in studying gene expressions and gene functions. A crucial step in transcript assembly is to accurately determine the splicing junctions and boundaries of the expressed transcripts from the reads alignment. In contrast to the splicing junctions that can be efficiently detected from spliced reads, the problem of identifying boundaries remains open and challenging, due to the fact that the signal related to boundaries is noisy and weak.

  19. A transition radiation detector for RHIC featuring accurate tracking and dE/dx particle identification

    Energy Technology Data Exchange (ETDEWEB)

    O`Brien, E.; Lissauer, D.; McCorkle, S.; Polychronakos, V.; Takai, H. [Brookhaven National Lab., Upton, NY (United States); Chi, C.Y.; Nagamiya, S.; Sippach, W.; Toy, M.; Wang, D.; Wang, Y.F.; Wiggins, C.; Willis, W. [Columbia Univ., New York, NY (United States); Cherniatin, V.; Dolgoshein, B. [Moscow Institute of Physics and Engineering, (Russian Federation); Bennett, M.; Chikanian, A.; Kumar, S.; Mitchell, J.T.; Pope, K. [Yale Univ., New Haven, CT (United States)

    1991-12-31

    We describe the results of a test ran involving a Transition Radiation Detector that can both distinguish electrons from pions which momenta greater titan 0.7 GeV/c and simultaneously track particles passing through the detector. The particle identification is accomplished through a combination of the detection of Transition Radiation from the electron and the differences in electron and pion energy loss (dE/dx) in the detector. The dE/dx particle separation is most, efficient below 2 GeV/c while particle ID utilizing Transition Radiation effective above 1.5 GeV/c. Combined, the electron-pion separation is-better than 5 {times} 10{sup 2}. The single-wire, track-position resolution for the TRD is {approximately}230 {mu}m.

  20. A transition radiation detector which features accurate tracking and dE/dx particle identification

    International Nuclear Information System (INIS)

    O'Brien, E.; Lissauer, D.; McCorkle, S.; Polychronakos, V.; Takai, H.; Chi, C.Y.; Nagamiya, S.; Sippach, W.; Toy, M.; Wang, D.; Wang, Y.F.; Wiggins, C.; Willis, W.; Cherniatin, V.; Dolgoshein, B.; Bennett, M.; Chikanian, A.; Kumar, S.; Mitchell, J.T.; Pope, K.

    1991-01-01

    We describe the results of a test run involving a Transition Radiation Detector that can both distinguish electrons from pions with momenta greater than 0.7 GeV/c and simultaneously track particles passing through the detector. The particle identification is accomplished through a combination of the detection of Transition Radiation from the electron and the differences in electron and pion energy loss (dE/dx) in the detector. The dE/dx particle separation is most efficient below 2 GeV/c while particle ID utilizing Transition Radiation is effective above 1.5 GeV/c. Combined, the electron-pion separation is better than 5 x l0 2 . The single-wire, track-position resolution for the TRD is ∼230μm

  1. Re-Ranking Sequencing Variants in the Post-GWAS Era for Accurate Causal Variant Identification

    Science.gov (United States)

    Faye, Laura L.; Machiela, Mitchell J.; Kraft, Peter; Bull, Shelley B.; Sun, Lei

    2013-01-01

    Next generation sequencing has dramatically increased our ability to localize disease-causing variants by providing base-pair level information at costs increasingly feasible for the large sample sizes required to detect complex-trait associations. Yet, identification of causal variants within an established region of association remains a challenge. Counter-intuitively, certain factors that increase power to detect an associated region can decrease power to localize the causal variant. First, combining GWAS with imputation or low coverage sequencing to achieve the large sample sizes required for high power can have the unintended effect of producing differential genotyping error among SNPs. This tends to bias the relative evidence for association toward better genotyped SNPs. Second, re-use of GWAS data for fine-mapping exploits previous findings to ensure genome-wide significance in GWAS-associated regions. However, using GWAS findings to inform fine-mapping analysis can bias evidence away from the causal SNP toward the tag SNP and SNPs in high LD with the tag. Together these factors can reduce power to localize the causal SNP by more than half. Other strategies commonly employed to increase power to detect association, namely increasing sample size and using higher density genotyping arrays, can, in certain common scenarios, actually exacerbate these effects and further decrease power to localize causal variants. We develop a re-ranking procedure that accounts for these adverse effects and substantially improves the accuracy of causal SNP identification, often doubling the probability that the causal SNP is top-ranked. Application to the NCI BPC3 aggressive prostate cancer GWAS with imputation meta-analysis identified a new top SNP at 2 of 3 associated loci and several additional possible causal SNPs at these loci that may have otherwise been overlooked. This method is simple to implement using R scripts provided on the author's website. PMID:23950724

  2. Accurate Identification of Fatty Liver Disease in Data Warehouse Utilizing Natural Language Processing.

    Science.gov (United States)

    Redman, Joseph S; Natarajan, Yamini; Hou, Jason K; Wang, Jingqi; Hanif, Muzammil; Feng, Hua; Kramer, Jennifer R; Desiderio, Roxanne; Xu, Hua; El-Serag, Hashem B; Kanwal, Fasiha

    2017-10-01

    Natural language processing is a powerful technique of machine learning capable of maximizing data extraction from complex electronic medical records. We utilized this technique to develop algorithms capable of "reading" full-text radiology reports to accurately identify the presence of fatty liver disease. Abdominal ultrasound, computerized tomography, and magnetic resonance imaging reports were retrieved from the Veterans Affairs Corporate Data Warehouse from a random national sample of 652 patients. Radiographic fatty liver disease was determined by manual review by two physicians and verified with an expert radiologist. A split validation method was utilized for algorithm development. For all three imaging modalities, the algorithms could identify fatty liver disease with >90% recall and precision, with F-measures >90%. These algorithms could be used to rapidly screen patient records to establish a large cohort to facilitate epidemiological and clinical studies and examine the clinic course and outcomes of patients with radiographic hepatic steatosis.

  3. Accurate microRNA target prediction correlates with protein repression levels

    Directory of Open Access Journals (Sweden)

    Simossis Victor A

    2009-09-01

    Full Text Available Abstract Background MicroRNAs are small endogenously expressed non-coding RNA molecules that regulate target gene expression through translation repression or messenger RNA degradation. MicroRNA regulation is performed through pairing of the microRNA to sites in the messenger RNA of protein coding genes. Since experimental identification of miRNA target genes poses difficulties, computational microRNA target prediction is one of the key means in deciphering the role of microRNAs in development and disease. Results DIANA-microT 3.0 is an algorithm for microRNA target prediction which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score, which correlates with protein production fold change. Specifically, for each predicted interaction the program reports a signal to noise ratio and a precision score which can be used as an indication of the false positive rate of the prediction. Conclusion Recently, several computational target prediction programs were benchmarked based on a set of microRNA target genes identified by the pSILAC method. In this assessment DIANA-microT 3.0 was found to achieve the highest precision among the most widely used microRNA target prediction programs reaching approximately 66%. The DIANA-microT 3.0 prediction results are available online in a user friendly web server at http://www.microrna.gr/microT

  4. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.

    2013-09-03

    Phosphoproteomics is a fast-growing field that aims at characterizing phosphorylated proteins in a cell or a tissue at a given time. Phosphorylation of proteins is an important regulatory mechanism in many cellular processes. Gel-free phosphoproteome technique involving enrichment of phosphopeptide coupled with mass spectrometry has proven to be invaluable to detect and characterize phosphorylated proteins. In this chapter, a gel-free quantitative approach involving 15N metabolic labelling in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through the identification and quantification of responsive phospho(proteins). © Springer Science+Business Media New York 2013.

  5. Accurate identification of layer number for few-layer WS2 and WSe2 via spectroscopic study.

    Science.gov (United States)

    Li, Yuanzheng; Li, Xinshu; Yu, Tong; Yang, Guochun; Chen, Heyu; Zhang, Cen; Feng, Qiushi; Ma, Jiangang; Liu, Weizhen; Xu, Haiyang; Liu, Yichun; Liu, Xinfeng

    2018-03-23

    Transition metal dichalcogenides (TMDs) with a typical layered structure are highly sensitive to their layer number in optical and electronic properties. Seeking a simple and effective method for layer number identification is very important to low-dimensional TMD samples. Herein, a rapid and accurate layer number identification of few-layer WS 2 and WSe 2 is proposed via locking their photoluminescence (PL) peak-positions. As the layer number of WS 2 /WSe 2 increases, it is found that indirect transition emission is more thickness-sensitive than direct transition emission, and the PL peak-position differences between the indirect and direct transitions can be regarded as fingerprints to identify their layer number. Theoretical calculation confirms that the notable thickness-sensitivity of indirect transition derives from the variations of electron density of states of W atom d-orbitals and chalcogen atom p-orbitals. Besides, the PL peak-position differences between the indirect and direct transitions are almost independent of different insulating substrates. This work not only proposes a new method for layer number identification via PL studies, but also provides a valuable insight into the thickness-dependent optical and electronic properties of W-based TMDs.

  6. Identification & Characterization of Fungal Ice Nucleation Proteins

    Science.gov (United States)

    Scheel, Jan Frederik; Kunert, Anna Theresa; Kampf, Christopher Johannes; Mauri, Sergio; Weidner, Tobias; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2016-04-01

    Freezing of water at relatively warm subfreezing temperatures is dependent on ice nucleation catalysis facilitated by ice nuclei (IN). These IN can be of various origins and although extensive research was done and progress was achieved, the nature and mechanisms leading to an effective IN are to date still poorly understood. Some of the most important processes of our geosphere like the water cycle are highly dependent on effective ice nucleation at temperatures between -2°C - -8°C, a temperature range which is almost exclusively covered by biological IN (BioIN). BioIN are usually macromolecular structures of biological polymers. Sugars as well as proteins have been reported to serve as IN and the best characterized BioIN are ice nucleation proteins (IN-P) from gram negative bacteria. Fungal strains from Fusarium spp. were described to be effective IN at subfreezing temperatures up to -2°C already 25 years ago and more and more fungal species are described to serve as efficient IN. Fungal IN are also thought to be proteins or at least contain a proteinaceous compound, but to date the fungal IN-P primary structure as well as their coding genetic elements of all IN active fungi are unknown. The aim of this study is a.) to identify the proteins and their coding genetic elements from IN active fungi (F. acuminatum, F. avenaceum, M. alpina) and b.) to characterize the mechanisms by which fungal IN serve as effective IN. We designed an interdisciplinary approach using biological, analytical and physical methods to identify fungal IN-P and describe their biological, chemical, and physical properties.

  7. Algorithm of Golgi protein 73 and liver stiffness accurately diagnoses significant fibrosis in chronic HBV infection.

    Science.gov (United States)

    Cao, Zhujun; Li, Ziqiang; Wang, Hui; Liu, Yuhan; Xu, Yumin; Mo, Ruidong; Ren, Peipei; Chen, Lichang; Lu, Jie; Li, Hong; Zhuang, Yan; Liu, Yunye; Wang, Xiaolin; Zhao, Gangde; Tang, Weiliang; Xiang, Xiaogang; Cai, Wei; Liu, Longgen; Bao, Shisan; Xie, Qing

    2017-11-01

    Serum Golgi protein 73 (GP73) is a potential biomarker for fibrosis assessment. We aimed to develop an algorithm based on GP73 and liver stiffness (LS) for further improvement of accuracy for significant fibrosis in patients with antiviral-naïve chronic hepatitis B virus (HBV) infection. Diagnostic accuracy evaluation of GP73 and development of GP73-LS algorithm was performed in training cohort (n = 267) with an independent cohort (n = 133) for validation. A stepwise increasing pattern of serum GP73 was observed across fibrosis stages in patients with antiviral-naïve chronic HBV infection. Serum GP73 significantly correlated (rho = 0.48, P 73, accuracy: 63.6%). Using GP73-LS algorithm, GP73 < 63 in agreement with LS < 8.5 provided accuracy of 81.7% to excluded significant fibrosis. GP73 ≥ 63 in agreement with LS ≥ 8.5 provided accuracy of 93.3% to confirm significant fibrosis. Almost 64% or 68% of patients in the training or validation cohort could be accurately classified. Serum GP73 is a robust biomarker for significant fibrosis diagnosis. GP73-LS algorithm provided better diagnostic accuracy than currently available approaches. More than 60% antiviral naïve CHB patients could use this algorithm without resorting to liver biopsy. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  8. Accurate and Reliable Prediction of the Binding Affinities of Macrocycles to Their Protein Targets.

    Science.gov (United States)

    Yu, Haoyu S; Deng, Yuqing; Wu, Yujie; Sindhikara, Dan; Rask, Amy R; Kimura, Takayuki; Abel, Robert; Wang, Lingle

    2017-12-12

    Macrocycles have been emerging as a very important drug class in the past few decades largely due to their expanded chemical diversity benefiting from advances in synthetic methods. Macrocyclization has been recognized as an effective way to restrict the conformational space of acyclic small molecule inhibitors with the hope of improving potency, selectivity, and metabolic stability. Because of their relatively larger size as compared to typical small molecule drugs and the complexity of the structures, efficient sampling of the accessible macrocycle conformational space and accurate prediction of their binding affinities to their target protein receptors poses a great challenge of central importance in computational macrocycle drug design. In this article, we present a novel method for relative binding free energy calculations between macrocycles with different ring sizes and between the macrocycles and their corresponding acyclic counterparts. We have applied the method to seven pharmaceutically interesting data sets taken from recent drug discovery projects including 33 macrocyclic ligands covering a diverse chemical space. The predicted binding free energies are in good agreement with experimental data with an overall root-mean-square error (RMSE) of 0.94 kcal/mol. This is to our knowledge the first time where the free energy of the macrocyclization of linear molecules has been directly calculated with rigorous physics-based free energy calculation methods, and we anticipate the outstanding accuracy demonstrated here across a broad range of target classes may have significant implications for macrocycle drug discovery.

  9. Identification Of Protein Vaccine Candidates Using Comprehensive Proteomic Analysis Strategies

    Science.gov (United States)

    2007-12-01

    that fascinating fungus known as Coccidioides. I also want to thank the UA Mass Spectrometry Facility and the UA Proteomics Consortium, especially...W. & N. N. Kav. 2006. The proteome of the phytopathogenic fungus Sclerotinia sclerotiorum. Proteomics 6: 5995-6007. 127. de Godoy, L. M., J. V...IDENTIFICATION OF PROTEIN VACCINE CANDIDATES USING COMPREHENSIVE PROTEOMIC ANALYSIS STRATEGIES by James G. Rohrbough

  10. Identification of surface proteins in Enterococcus faecalis V583

    Directory of Open Access Journals (Sweden)

    Eijsink Vincent GH

    2011-03-01

    Full Text Available Abstract Background Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. Results Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31 or to be secreted (5. Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. Conclusions The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5 and surface (31 proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.

  11. Identification and analysis of multi-protein complexes in placenta.

    Directory of Open Access Journals (Sweden)

    Fuqiang Wang

    Full Text Available Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE and Liquid chromatography-tandem mass spectrometry (LC-MS/MS were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders.

  12. Identification of Importin 8 (IPO8 as the most accurate reference gene for the clinicopathological analysis of lung specimens

    Directory of Open Access Journals (Sweden)

    Pio Ruben

    2008-11-01

    Full Text Available Abstract Background The accurate normalization of differentially expressed genes in lung cancer is essential for the identification of novel therapeutic targets and biomarkers by real time RT-PCR and microarrays. Although classical "housekeeping" genes, such as GAPDH, HPRT1, and beta-actin have been widely used in the past, their accuracy as reference genes for lung tissues has not been proven. Results We have conducted a thorough analysis of a panel of 16 candidate reference genes for lung specimens and lung cell lines. Gene expression was measured by quantitative real time RT-PCR and expression stability was analyzed with the softwares GeNorm and NormFinder, mean of |ΔCt| (= |Ct Normal-Ct tumor| ± SEM, and correlation coefficients among genes. Systematic comparison between candidates led us to the identification of a subset of suitable reference genes for clinical samples: IPO8, ACTB, POLR2A, 18S, and PPIA. Further analysis showed that IPO8 had a very low mean of |ΔCt| (0.70 ± 0.09, with no statistically significant differences between normal and malignant samples and with excellent expression stability. Conclusion Our data show that IPO8 is the most accurate reference gene for clinical lung specimens. In addition, we demonstrate that the commonly used genes GAPDH and HPRT1 are inappropriate to normalize data derived from lung biopsies, although they are suitable as reference genes for lung cell lines. We thus propose IPO8 as a novel reference gene for lung cancer samples.

  13. EpHLA software: a timesaving and accurate tool for improving identification of acceptable mismatches for clinical purposes.

    Science.gov (United States)

    Filho, Herton Luiz Alves Sales; da Mata Sousa, Luiz Claudio Demes; von Glehn, Cristina de Queiroz Carrascosa; da Silva, Adalberto Socorro; dos Santos Neto, Pedro de Alcântara; do Nascimento, Ferraz; de Castro, Adail Fonseca; do Nascimento, Liliane Machado; Kneib, Carolina; Bianchi Cazarote, Helena; Mayumi Kitamura, Daniele; Torres, Juliane Roberta Dias; da Cruz Lopes, Laiane; Barros, Aryela Loureiro; da Silva Edlin, Evelin Nildiane; de Moura, Fernanda Sá Leal; Watanabe, Janine Midori Figueiredo; do Monte, Semiramis Jamil Hadad

    2012-06-01

    The HLAMatchmaker algorithm, which allows the identification of “safe” acceptable mismatches (AMMs) for recipients of solid organ and cell allografts, is rarely used in part due to the difficulty in using it in the current Excel format. The automation of this algorithm may universalize its use to benefit the allocation of allografts. Recently, we have developed a new software called EpHLA, which is the first computer program automating the use of the HLAMatchmaker algorithm. Herein, we present the experimental validation of the EpHLA program by showing the time efficiency and the quality of operation. The same results, obtained by a single antigen bead assay with sera from 10 sensitized patients waiting for kidney transplants, were analyzed either by conventional HLAMatchmaker or by automated EpHLA method. Users testing these two methods were asked to record: (i) time required for completion of the analysis (in minutes); (ii) number of eplets obtained for class I and class II HLA molecules; (iii) categorization of eplets as reactive or non-reactive based on the MFI cutoff value; and (iv) determination of AMMs based on eplets' reactivities. We showed that although both methods had similar accuracy, the automated EpHLA method was over 8 times faster in comparison to the conventional HLAMatchmaker method. In particular the EpHLA software was faster and more reliable but equally accurate as the conventional method to define AMMs for allografts. The EpHLA software is an accurate and quick method for the identification of AMMs and thus it may be a very useful tool in the decision-making process of organ allocation for highly sensitized patients as well as in many other applications.

  14. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes.

    Directory of Open Access Journals (Sweden)

    Jiawei Luo

    Full Text Available Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins.In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC, based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID, of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification.Experimental results based on three different PPI(protein-protein interaction networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC.LIDC is more effective for the prediction of essential proteins than other recently developed methods.

  15. Stable and accurate methods for identification of water bodies from Landsat series imagery using meta-heuristic algorithms

    Science.gov (United States)

    Gamshadzaei, Mohammad Hossein; Rahimzadegan, Majid

    2017-10-01

    Identification of water extents in Landsat images is challenging due to surfaces with similar reflectance to water extents. The objective of this study is to provide stable and accurate methods for identifying water extents in Landsat images based on meta-heuristic algorithms. Then, seven Landsat images were selected from various environmental regions in Iran. Training of the algorithms was performed using 40 water pixels and 40 nonwater pixels in operational land imager images of Chitgar Lake (one of the study regions). Moreover, high-resolution images from Google Earth were digitized to evaluate the results. Two approaches were considered: index-based and artificial intelligence (AI) algorithms. In the first approach, nine common water spectral indices were investigated. AI algorithms were utilized to acquire coefficients of optimal band combinations to extract water extents. Among the AI algorithms, the artificial neural network algorithm and also the ant colony optimization, genetic algorithm, and particle swarm optimization (PSO) meta-heuristic algorithms were implemented. Index-based methods represented different performances in various regions. Among AI methods, PSO had the best performance with average overall accuracy and kappa coefficient of 93% and 98%, respectively. The results indicated the applicability of acquired band combinations to extract accurately and stably water extents in Landsat imagery.

  16. Computational identification of strain-, species- and genus-specific proteins

    Directory of Open Access Journals (Sweden)

    Thiagarajan Rathi

    2005-11-01

    Full Text Available Abstract Background The identification of unique proteins at different taxonomic levels has both scientific and practical value. Strain-, species- and genus-specific proteins can provide insight into the criteria that define an organism and its relationship with close relatives. Such proteins can also serve as taxon-specific diagnostic targets. Description A pipeline using a combination of computational and manual analyses of BLAST results was developed to identify strain-, species-, and genus-specific proteins and to catalog the closest sequenced relative for each protein in a proteome. Proteins encoded by a given strain are preliminarily considered to be unique if BLAST, using a comprehensive protein database, fails to retrieve (with an e-value better than 0.001 any protein not encoded by the query strain, species or genus (for strain-, species- and genus-specific proteins respectively, or if BLAST, using the best hit as the query (reverse BLAST, does not retrieve the initial query protein. Results are manually inspected for homology if the initial query is retrieved in the reverse BLAST but is not the best hit. Sequences unlikely to retrieve homologs using the default BLOSUM62 matrix (usually short sequences are re-tested using the PAM30 matrix, thereby increasing the number of retrieved homologs and increasing the stringency of the search for unique proteins. The above protocol was used to examine several food- and water-borne pathogens. We find that the reverse BLAST step filters out about 22% of proteins with homologs that would otherwise be considered unique at the genus and species levels. Analysis of the annotations of unique proteins reveals that many are remnants of prophage proteins, or may be involved in virulence. The data generated from this study can be accessed and further evaluated from the CUPID (Core and Unique Protein Identification system web site (updated semi-annually at http://pir.georgetown.edu/cupid. Conclusion CUPID

  17. Machine Learning Identification of Protein Properties Useful for Specific Applications

    KAUST Repository

    Khamis, Abdullah

    2016-03-31

    Proteins play critical roles in cellular processes of living organisms. It is therefore important to identify and characterize their key properties associated with their functions. Correlating protein’s structural, sequence and physicochemical properties of its amino acids (aa) with protein functions could identify some of the critical factors governing the specific functionality. We point out that not all functions of even well studied proteins are known. This, complemented by the huge increase in the number of newly discovered and predicted proteins, makes challenging the experimental characterization of the whole spectrum of possible protein functions for all proteins of interest. Consequently, the use of computational methods has become more attractive. Here we address two questions. The first one is how to use protein aa sequence and physicochemical properties to characterize a family of proteins. The second one focuses on how to use transcription factor (TF) protein’s domains to enhance accuracy of predicting TF DNA binding sites (TFBSs). To address the first question, we developed a novel method using computational representation of proteins based on characteristics of different protein regions (N-terminal, M-region and C-terminal) and combined these with the properties of protein aa sequences. We show that this description provides important biological insight about characterization of the protein functional groups. Using feature selection techniques, we identified key properties of proteins that allow for very accurate characterization of different protein families. We demonstrated efficiency of our method in application to a number of antimicrobial peptide families. To address the second question we developed another novel method that uses a combination of aa properties of DNA binding domains of TFs and their TFBS properties to develop machine learning models for predicting TFBSs. Feature selection is used to identify the most relevant characteristics

  18. Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

    Science.gov (United States)

    Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

    2017-01-01

    ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

  19. Identification of antigenic proteins of setaria cervi by immunoblotting technique

    International Nuclear Information System (INIS)

    Kaushal, N.A.; Kaushal, D.C.; Ghatak, S.

    1987-01-01

    Identification and characterization of antigenic proteins of Setaria cervi (bovine filarial parasite) adults and microfilariae was done by immunoblotting technique using hyperimmune rabbit sera against S. cervi and Brugia malayi. The antigens recognized by these sera were detected by using 125 I protein-A followed by autoradiography. Fifteen different antigens were observed to be common between adult and microfilarial stages of the parasite. Some stage specific antigens were also identified. Many antigens of S. cervi adults and microfilariae were also recognized by rabbit anti-B.malayi serum showing the existence of common antigenic determinants between the bovine and human filarial parasites

  20. Identification and characterization of N-glycosylated proteins using proteomics

    DEFF Research Database (Denmark)

    Selby, David S; Larsen, Martin R; Calvano, Cosima Damiana

    2008-01-01

    and analysis of glycoproteins and glycopeptides. Combinations of affinity-enrichment techniques, chemical and biochemical protocols, and advanced mass spectrometry facilitate detailed glycoprotein analysis in proteomics, from fundamental biological studies to biomarker discovery in biomedicine....... is a complex task and is currently achieved by mass spectrometry-based methods that enable identification of glycoproteins and localization, classification, and analysis of individual glycan structures on proteins. In this chapter we briefly introduce a range of analytical technologies for recovery...

  1. Identification of Tobacco Topping Responsive Proteins in Roots

    Directory of Open Access Journals (Sweden)

    Hongxiang eGuo

    2016-04-01

    Full Text Available Tobacco plant has many responses to topping, such as the increase in ability of nicotine synthesis and secondary growth of roots. Some topping responsive miRNAs and genes had been identified in our previous work, but it is not enough to elaborate mechanism of tobacco response to topping. Here, topping responsive proteins were screened from tobacco roots with two-dimensional electrophoresis. Of these proteins, calretulin (CRT and Auxin-responsive protein IAA9 were related to the secondary growth of roots, LRR disease resistance, heat shock protein 70 and farnesyl pyrophosphate synthase 1(FPPS)were involved in wounding stress response, and F-box protein played an important role in promoting the ability of nicotine synthesis after topping. In addition, there were five tobacco bHLH proteins (NtbHLH, NtMYC1a, NtMYC1b, NtMYC2a and NtMYC2b related to nicotine synthesis. It was suggested that NtMYC2 might be the main positive transcription factor and NtbHLH protein is a negative regulator in the JA-mediating activation of nicotine synthesis after topping. Tobacco topping activates some comprehensive biology processes involving IAA and JA signaling pathway, and the identification of these proteins will be helpful to understand the process of topping response.

  2. Development of an enzyme-linked-immunosorbent-assay technique for accurate identification of poorly preserved silks unearthed in ancient tombs.

    Science.gov (United States)

    Zheng, Qin; Wu, Xiaofeng; Zheng, Hailing; Zhou, Yang

    2015-05-01

    We report the preparation of a specific fibroin antibody and its use for the identification of unearthed ancient silk relics. Based on the 12-amino-acid repeat sequence "GAGAGSGAGAGS", which is found in fibroin of the silkworm Bombyx mori, a specific antibody against fibroin was prepared in rabbits through peptide synthesis and carrier-protein coupling. This antibody was highly specific for fibroin found in silk. Using this antibody we have successfully identified four silk samples from different time periods. Our results reveal, for the first time, a method capable of detecting silk from a few milligrams of archaeological fabric that has been buried for thousands of years, confirming that the ancient practice of wearing silk products while praying for rebirth dated back to at least 400 BCE. This method also complements current approaches in silk detection, especially for the characterization of poorly preserved silks, promoting the investigation of silk origins and of ancient clothing cultures.

  3. Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing.

    Directory of Open Access Journals (Sweden)

    Hansaim Lim

    2016-10-01

    Full Text Available Target-based screening is one of the major approaches in drug discovery. Besides the intended target, unexpected drug off-target interactions often occur, and many of them have not been recognized and characterized. The off-target interactions can be responsible for either therapeutic or side effects. Thus, identifying the genome-wide off-targets of lead compounds or existing drugs will be critical for designing effective and safe drugs, and providing new opportunities for drug repurposing. Although many computational methods have been developed to predict drug-target interactions, they are either less accurate than the one that we are proposing here or computationally too intensive, thereby limiting their capability for large-scale off-target identification. In addition, the performances of most machine learning based algorithms have been mainly evaluated to predict off-target interactions in the same gene family for hundreds of chemicals. It is not clear how these algorithms perform in terms of detecting off-targets across gene families on a proteome scale. Here, we are presenting a fast and accurate off-target prediction method, REMAP, which is based on a dual regularized one-class collaborative filtering algorithm, to explore continuous chemical space, protein space, and their interactome on a large scale. When tested in a reliable, extensive, and cross-gene family benchmark, REMAP outperforms the state-of-the-art methods. Furthermore, REMAP is highly scalable. It can screen a dataset of 200 thousands chemicals against 20 thousands proteins within 2 hours. Using the reconstructed genome-wide target profile as the fingerprint of a chemical compound, we predicted that seven FDA-approved drugs can be repurposed as novel anti-cancer therapies. The anti-cancer activity of six of them is supported by experimental evidences. Thus, REMAP is a valuable addition to the existing in silico toolbox for drug target identification, drug repurposing

  4. Large-Scale Off-Target Identification Using Fast and Accurate Dual Regularized One-Class Collaborative Filtering and Its Application to Drug Repurposing.

    Science.gov (United States)

    Lim, Hansaim; Poleksic, Aleksandar; Yao, Yuan; Tong, Hanghang; He, Di; Zhuang, Luke; Meng, Patrick; Xie, Lei

    2016-10-01

    Target-based screening is one of the major approaches in drug discovery. Besides the intended target, unexpected drug off-target interactions often occur, and many of them have not been recognized and characterized. The off-target interactions can be responsible for either therapeutic or side effects. Thus, identifying the genome-wide off-targets of lead compounds or existing drugs will be critical for designing effective and safe drugs, and providing new opportunities for drug repurposing. Although many computational methods have been developed to predict drug-target interactions, they are either less accurate than the one that we are proposing here or computationally too intensive, thereby limiting their capability for large-scale off-target identification. In addition, the performances of most machine learning based algorithms have been mainly evaluated to predict off-target interactions in the same gene family for hundreds of chemicals. It is not clear how these algorithms perform in terms of detecting off-targets across gene families on a proteome scale. Here, we are presenting a fast and accurate off-target prediction method, REMAP, which is based on a dual regularized one-class collaborative filtering algorithm, to explore continuous chemical space, protein space, and their interactome on a large scale. When tested in a reliable, extensive, and cross-gene family benchmark, REMAP outperforms the state-of-the-art methods. Furthermore, REMAP is highly scalable. It can screen a dataset of 200 thousands chemicals against 20 thousands proteins within 2 hours. Using the reconstructed genome-wide target profile as the fingerprint of a chemical compound, we predicted that seven FDA-approved drugs can be repurposed as novel anti-cancer therapies. The anti-cancer activity of six of them is supported by experimental evidences. Thus, REMAP is a valuable addition to the existing in silico toolbox for drug target identification, drug repurposing, phenotypic screening, and

  5. Development of a method for the accurate measurement of protein turnover in neoplastic cells grown in culture

    International Nuclear Information System (INIS)

    Silverman, J.A.

    1984-01-01

    In this study, it was shown that standard techniques for cell recovery and sample preparation for liquid scintillation counting led to underestimation of the radioactivity present in cell proteins by 20-40%. These techniques involved labeling with 3 He leucine or 14 C leucine, scraping the cells from the dish in a buffer, TCA precipitation of the cell proteins, solubilization in NaOH and counting in a liquid scintillation counter. Hydrolysis of the proteins with HCl or Pronase significantly increased the recovery of the labeled proteins. Also, solubilization in situ with NaOH or hydrolysis in situ with Pronase recovered 5-10% additional labeled proteins. The techniques developed here allow the accurate measurement of radioactivity in cell proteins. In addition, these techniques were used to study protein turnover in rat hepatoma cells grown in culture. These cells regulated their growth rate through changes in the protein synthesis rate as opposed to changes in the protein degradation rate. These data support the hypothesis that neoplastic cells, unlike normal cells, do not regulate proteolysis in growth control; normal cells under similar conditions have been shown to activate lysosomal proteolysis as they reach confluence. The physiologic implications of this observation are discussed

  6. Accurate, rapid identification of dislocation lines in coherent diffractive imaging via a min-max optimization formulation

    Energy Technology Data Exchange (ETDEWEB)

    Ulvestad, A. [Materials Science Division, Argonne National Laboratory, Lemont, IL 60439, USA; Menickelly, M. [Mathematics and Computer Science Division, Argonne National Laboratory, Lemont, IL 60439, USA; Wild, S. M. [Mathematics and Computer Science Division, Argonne National Laboratory, Lemont, IL 60439, USA

    2018-01-01

    Defects such as dislocations impact materials properties and their response during external stimuli. Imaging these defects in their native operating conditions to establish the structure-function relationship and, ultimately, to improve performance via defect engineering has remained a considerable challenge for both electron-based and x-ray-based imaging techniques. While Bragg coherent x-ray diffractive imaging (BCDI) is successful in many cases, nuances in identifying the dislocations has left manual identification as the preferred method. Derivative-based methods are also used, but they can be inaccurate and are computationally inefficient. Here we demonstrate a derivative-free method that is both more accurate and more computationally efficient than either derivative-or human-based methods for identifying 3D dislocation lines in nanocrystal images produced by BCDI. We formulate the problem as a min-max optimization problem and show exceptional accuracy for experimental images. We demonstrate a 227x speedup for a typical experimental dataset with higher accuracy over current methods. We discuss the possibility of using this algorithm as part of a sparsity-based phase retrieval process. We also provide MATLAB code for use by other researchers.

  7. CMASA: an accurate algorithm for detecting local protein structural similarity and its application to enzyme catalytic site annotation

    Directory of Open Access Journals (Sweden)

    Li Gong-Hua

    2010-08-01

    Full Text Available Abstract Background The rapid development of structural genomics has resulted in many "unknown function" proteins being deposited in Protein Data Bank (PDB, thus, the functional prediction of these proteins has become a challenge for structural bioinformatics. Several sequence-based and structure-based methods have been developed to predict protein function, but these methods need to be improved further, such as, enhancing the accuracy, sensitivity, and the computational speed. Here, an accurate algorithm, the CMASA (Contact MAtrix based local Structural Alignment algorithm, has been developed to predict unknown functions of proteins based on the local protein structural similarity. This algorithm has been evaluated by building a test set including 164 enzyme families, and also been compared to other methods. Results The evaluation of CMASA shows that the CMASA is highly accurate (0.96, sensitive (0.86, and fast enough to be used in the large-scale functional annotation. Comparing to both sequence-based and global structure-based methods, not only the CMASA can find remote homologous proteins, but also can find the active site convergence. Comparing to other local structure comparison-based methods, the CMASA can obtain the better performance than both FFF (a method using geometry to predict protein function and SPASM (a local structure alignment method; and the CMASA is more sensitive than PINTS and is more accurate than JESS (both are local structure alignment methods. The CMASA was applied to annotate the enzyme catalytic sites of the non-redundant PDB, and at least 166 putative catalytic sites have been suggested, these sites can not be observed by the Catalytic Site Atlas (CSA. Conclusions The CMASA is an accurate algorithm for detecting local protein structural similarity, and it holds several advantages in predicting enzyme active sites. The CMASA can be used in large-scale enzyme active site annotation. The CMASA can be available by the

  8. MFPred: Rapid and accurate prediction of protein-peptide recognition multispecificity using self-consistent mean field theory.

    Directory of Open Access Journals (Sweden)

    Aliza B Rubenstein

    2017-06-01

    Full Text Available Multispecificity-the ability of a single receptor protein molecule to interact with multiple substrates-is a hallmark of molecular recognition at protein-protein and protein-peptide interfaces, including enzyme-substrate complexes. The ability to perform structure-based prediction of multispecificity would aid in the identification of novel enzyme substrates, protein interaction partners, and enable design of novel enzymes targeted towards alternative substrates. The relatively slow speed of current biophysical, structure-based methods limits their use for prediction and, especially, design of multispecificity. Here, we develop a rapid, flexible-backbone self-consistent mean field theory-based technique, MFPred, for multispecificity modeling at protein-peptide interfaces. We benchmark our method by predicting experimentally determined peptide specificity profiles for a range of receptors: protease and kinase enzymes, and protein recognition modules including SH2, SH3, MHC Class I and PDZ domains. We observe robust recapitulation of known specificities for all receptor-peptide complexes, and comparison with other methods shows that MFPred results in equivalent or better prediction accuracy with a ~10-1000-fold decrease in computational expense. We find that modeling bound peptide backbone flexibility is key to the observed accuracy of the method. We used MFPred for predicting with high accuracy the impact of receptor-side mutations on experimentally determined multispecificity of a protease enzyme. Our approach should enable the design of a wide range of altered receptor proteins with programmed multispecificities.

  9. Are current atomistic force fields accurate enough to study proteins in crowded environments?

    Directory of Open Access Journals (Sweden)

    Drazen Petrov

    2014-05-01

    Full Text Available The high concentration of macromolecules in the crowded cellular interior influences different thermodynamic and kinetic properties of proteins, including their structural stabilities, intermolecular binding affinities and enzymatic rates. Moreover, various structural biology methods, such as NMR or different spectroscopies, typically involve samples with relatively high protein concentration. Due to large sampling requirements, however, the accuracy of classical molecular dynamics (MD simulations in capturing protein behavior at high concentration still remains largely untested. Here, we use explicit-solvent MD simulations and a total of 6.4 µs of simulated time to study wild-type (folded and oxidatively damaged (unfolded forms of villin headpiece at 6 mM and 9.2 mM protein concentration. We first perform an exhaustive set of simulations with multiple protein molecules in the simulation box using GROMOS 45a3 and 54a7 force fields together with different types of electrostatics treatment and solution ionic strengths. Surprisingly, the two villin headpiece variants exhibit similar aggregation behavior, despite the fact that their estimated aggregation propensities markedly differ. Importantly, regardless of the simulation protocol applied, wild-type villin headpiece consistently aggregates even under conditions at which it is experimentally known to be soluble. We demonstrate that aggregation is accompanied by a large decrease in the total potential energy, with not only hydrophobic, but also polar residues and backbone contributing substantially. The same effect is directly observed for two other major atomistic force fields (AMBER99SB-ILDN and CHARMM22-CMAP as well as indirectly shown for additional two (AMBER94, OPLS-AAL, and is possibly due to a general overestimation of the potential energy of protein-protein interactions at the expense of water-water and water-protein interactions. Overall, our results suggest that current MD force fields

  10. Enhanced detection method for corneal protein identification using shotgun proteomics

    Directory of Open Access Journals (Sweden)

    Schlager John J

    2009-06-01

    Full Text Available Abstract Background The cornea is a specialized transparent connective tissue responsible for the majority of light refraction and image focus for the retina. There are three main layers of the cornea: the epithelium that is exposed and acts as a protective barrier for the eye, the center stroma consisting of parallel collagen fibrils that refract light, and the endothelium that is responsible for hydration of the cornea from the aqueous humor. Normal cornea is an immunologically privileged tissue devoid of blood vessels, but injury can produce a loss of these conditions causing invasion of other processes that degrade the homeostatic properties resulting in a decrease in the amount of light refracted onto the retina. Determining a measure and drift of phenotypic cornea state from normal to an injured or diseased state requires knowledge of the existing protein signature within the tissue. In the study of corneal proteins, proteomics procedures have typically involved the pulverization of the entire cornea prior to analysis. Separation of the epithelium and endothelium from the core stroma and performing separate shotgun proteomics using liquid chromatography/mass spectrometry results in identification of many more proteins than previously employed methods using complete pulverized cornea. Results Rabbit corneas were purchased, the epithelium and endothelium regions were removed, proteins processed and separately analyzed using liquid chromatography/mass spectrometry. Proteins identified from separate layers were compared against results from complete corneal samples. Protein digests were separated using a six hour liquid chromatographic gradient and ion-trap mass spectrometry used for detection of eluted peptide fractions. The SEQUEST database search results were filtered to allow only proteins with match probabilities of equal or better than 10-3 and peptides with a probability of 10-2 or less with at least two unique peptides isolated within

  11. Fast and accurate resonance assignment of small-to-large proteins by combining automated and manual approaches.

    Science.gov (United States)

    Niklasson, Markus; Ahlner, Alexandra; Andresen, Cecilia; Marsh, Joseph A; Lundström, Patrik

    2015-01-01

    The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

  12. Fast and accurate resonance assignment of small-to-large proteins by combining automated and manual approaches.

    Directory of Open Access Journals (Sweden)

    Markus Niklasson

    2015-01-01

    Full Text Available The process of resonance assignment is fundamental to most NMR studies of protein structure and dynamics. Unfortunately, the manual assignment of residues is tedious and time-consuming, and can represent a significant bottleneck for further characterization. Furthermore, while automated approaches have been developed, they are often limited in their accuracy, particularly for larger proteins. Here, we address this by introducing the software COMPASS, which, by combining automated resonance assignment with manual intervention, is able to achieve accuracy approaching that from manual assignments at greatly accelerated speeds. Moreover, by including the option to compensate for isotope shift effects in deuterated proteins, COMPASS is far more accurate for larger proteins than existing automated methods. COMPASS is an open-source project licensed under GNU General Public License and is available for download from http://www.liu.se/forskning/foass/tidigare-foass/patrik-lundstrom/software?l=en. Source code and binaries for Linux, Mac OS X and Microsoft Windows are available.

  13. Accurate prediction of stability changes in protein mutants by combining machine learning with structure based computational mutagenesis.

    Science.gov (United States)

    Masso, Majid; Vaisman, Iosif I

    2008-09-15

    Accurate predictive models for the impact of single amino acid substitutions on protein stability provide insight into protein structure and function. Such models are also valuable for the design and engineering of new proteins. Previously described methods have utilized properties of protein sequence or structure to predict the free energy change of mutants due to thermal (DeltaDeltaG) and denaturant (DeltaDeltaG(H2O)) denaturations, as well as mutant thermal stability (DeltaT(m)), through the application of either computational energy-based approaches or machine learning techniques. However, accuracy associated with applying these methods separately is frequently far from optimal. We detail a computational mutagenesis technique based on a four-body, knowledge-based, statistical contact potential. For any mutation due to a single amino acid replacement in a protein, the method provides an empirical normalized measure of the ensuing environmental perturbation occurring at every residue position. A feature vector is generated for the mutant by considering perturbations at the mutated position and it's ordered six nearest neighbors in the 3-dimensional (3D) protein structure. These predictors of stability change are evaluated by applying machine learning tools to large training sets of mutants derived from diverse proteins that have been experimentally studied and described. Predictive models based on our combined approach are either comparable to, or in many cases significantly outperform, previously published results. A web server with supporting documentation is available at http://proteins.gmu.edu/automute.

  14. Aptamer-conjugated live human immune cell based biosensors for the accurate detection of C-reactive protein

    OpenAIRE

    Hwang, Jangsun; Seo, Youngmin; Jo, Yeonho; Son, Jaewoo; Choi, Jonghoon

    2016-01-01

    C-reactive protein (CRP) is a pentameric protein that is present in the bloodstream during inflammatory events, e.g., liver failure, leukemia, and/or bacterial infection. The level of CRP indicates the progress and prognosis of certain diseases; it is therefore necessary to measure CRP levels in the blood accurately. The normal concentration of CRP is reported to be 1?3?mg/L. Inflammatory events increase the level of CRP by up to 500 times; accordingly, CRP is a biomarker of acute inflammator...

  15. GalaxyDock BP2 score: a hybrid scoring function for accurate protein-ligand docking

    Science.gov (United States)

    Baek, Minkyung; Shin, Woong-Hee; Chung, Hwan Won; Seok, Chaok

    2017-07-01

    Protein-ligand docking is a useful tool for providing atomic-level understanding of protein functions in nature and design principles for artificial ligands or proteins with desired properties. The ability to identify the true binding pose of a ligand to a target protein among numerous possible candidate poses is an essential requirement for successful protein-ligand docking. Many previously developed docking scoring functions were trained to reproduce experimental binding affinities and were also used for scoring binding poses. However, in this study, we developed a new docking scoring function, called GalaxyDock BP2 Score, by directly training the scoring power of binding poses. This function is a hybrid of physics-based, empirical, and knowledge-based score terms that are balanced to strengthen the advantages of each component. The performance of the new scoring function exhibits significant improvement over existing scoring functions in decoy pose discrimination tests. In addition, when the score is used with the GalaxyDock2 protein-ligand docking program, it outperformed other state-of-the-art docking programs in docking tests on the Astex diverse set, the Cross2009 benchmark set, and the Astex non-native set. GalaxyDock BP2 Score and GalaxyDock2 with this score are freely available at http://galaxy.seoklab.org/softwares/galaxydock.html.

  16. Identification of protein binding in pictorial art Cuban

    International Nuclear Information System (INIS)

    Mendoza, Ariadna; Correa, Maurin; Maqueira, Isis

    2011-01-01

    In this paper were implemented microanalysis methodologies by histochemical analysis, and infrared spectroscopy to determine the nature of the binder in paintings and Gas Chromatography (GC) coupled to Mass Spectrometry (MS) for identification of protein binders of common use in tempera technique with the aim of having these methods as part of the identification of artistic materials in Cuban cultural heritage carried out by Archaeometry Laboratory of Havana city's Historian Cabinet. The methodologies implemented were evaluated using model samples of traditional painting techniques with variable protein binder: yolk, egg white, casein, nut oil and animal glue; ageing for 5 years. The models samples were correctly identified. It was determined the interference of pigments with the presence of nitrogen by histochemical analysis with Amido Black dye. IR spectroscopy technique allowed to differentiate between oily and mixed (oil plus protein) techniques and tempera with yolk. Oily technique was identified in wall paintings of the New San Francisco church (XIX century) and the Obrapia House (XVII century) and the technique of tempera with animal glue in the polychrome of the XVIII century which represents St. John the Evangelist belonging to the San Juan de Letran church

  17. Identification of Inhibitors of Biological Interactions Involving Intrinsically Disordered Proteins

    Directory of Open Access Journals (Sweden)

    Daniela Marasco

    2015-04-01

    Full Text Available Protein–protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered Proteins (IDPs are involved in cellular regulation, signaling and control: they bind to multiple partners and these high-specificity/low-affinity interactions play crucial roles in many human diseases. Disordered regions, terminal tails and flexible linkers are particularly abundant in DNA-binding proteins and play crucial roles in the affinity and specificity of DNA recognizing processes. Protein complexes involving IDPs are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to modulate them can produce important lead compounds: in this scenario peptides and/or peptidomimetics, deriving from structure-based, combinatorial or protein dissection approaches, can play a key role as hit compounds. Here, we propose a panoramic review of the structural features of IDPs and how they regulate molecular recognition mechanisms focusing attention on recently reported drug-design strategies in the field of IDPs.

  18. Rapid and accurate processing method for amide proton exchange rate measurement in proteins

    International Nuclear Information System (INIS)

    Koskela, Harri; Heikkinen, Outi; Kilpelaeinen, Ilkka; Heikkinen, Sami

    2007-01-01

    Exchange between protein backbone amide hydrogen and water gives relevant information about solvent accessibility and protein secondary structure stability. NMR spectroscopy provides a convenient tool to study these dynamic processes with saturation transfer experiments. Processing of this type of NMR spectra has traditionally required peak integration followed by exponential fitting, which can be tedious with large data sets. We propose here a computer-aided method that applies inverse Laplace transform in the exchange rate measurement. With this approach, the determination of exchange rates can be automated, and reliable results can be acquired rapidly without a need for manual processing

  19. Identification of Phosphorylated Proteins on a Global Scale.

    Science.gov (United States)

    Iliuk, Anton

    2018-05-31

    Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) has enabled researchers to analyze complex biological samples with unprecedented depth. It facilitates the identification and quantification of modifications within thousands of proteins in a single large-scale proteomic experiment. Analysis of phosphorylation, one of the most common and important post-translational modifications, has particularly benefited from such progress in the field. Here, detailed protocols are provided for a few well-regarded, common sample preparation methods for an effective phosphoproteomic experiment. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

  20. CYP450 phenotyping and accurate mass identification of metabolites of the 8-aminoquinoline, anti-malarial drug primaquine

    Directory of Open Access Journals (Sweden)

    Pybus Brandon S

    2012-08-01

    Full Text Available Abstract Background The 8-aminoquinoline (8AQ drug primaquine (PQ is currently the only approved drug effective against the persistent liver stage of the hypnozoite forming strains Plasmodium vivax and Plasmodium ovale as well as Stage V gametocytes of Plasmodium falciparum. To date, several groups have investigated the toxicity observed in the 8AQ class, however, exact mechanisms and/or metabolic species responsible for PQ’s haemotoxic and anti-malarial properties are not fully understood. Methods In the present study, the metabolism of PQ was evaluated using in vitro recombinant metabolic enzymes from the cytochrome P450 (CYP and mono-amine oxidase (MAO families. Based on this information, metabolite identification experiments were performed using nominal and accurate mass measurements. Results Relative activity factor (RAF-weighted intrinsic clearance values show the relative role of each enzyme to be MAO-A, 2C19, 3A4, and 2D6, with 76.1, 17.0, 5.2, and 1.7% contributions to PQ metabolism, respectively. CYP 2D6 was shown to produce at least six different oxidative metabolites along with demethylations, while MAO-A products derived from the PQ aldehyde, a pre-cursor to carboxy PQ. CYPs 2C19 and 3A4 produced only trace levels of hydroxylated species. Conclusions As a result of this work, CYP 2D6 and MAO-A have been implicated as the key enzymes associated with PQ metabolism, and metabolites previously identified as potentially playing a role in efficacy and haemolytic toxicity have been attributed to production via CYP 2D6 mediated pathways.

  1. Identification of osteocalcin as a permanent aging constituent of the bone matrix: basis for an accurate age at death determination.

    Science.gov (United States)

    Ritz, S; Turzynski, A; Schütz, H W; Hollmann, A; Rochholz, G

    1996-01-12

    Age at death determination based on aspartic acid racemization in dentin has been applied successfully in forensic odontology for several years now. An age-dependent accumulation of D-aspartic acid has also recently been demonstrated in bone osteocalcin, one of the most abundant noncollagenous proteins of the organic bone matrix. Evaluation of these initial data on in vivo racemization of aspartic acid in bone osteocalcin was taken a step further. After purification of osteocalcin from 53 skull bone specimens, the extent of aspartic acid racemization in this peptide was determined. The D-aspartic acid content of purified bone osteocalcin exhibited a very close relationship to age at death. This confirmed identification of bone osteocalcin as a permanent, 'aging' peptide of the organic bone matrix. Its D-aspartic acid content may be used as a measure of its age and hence that of the entire organism. The new biochemical approach to determination of age at death by analyzing bone is complex and demanding from a methodologic point of view, but appears to be superior in precision and reproducibility to most other methods applicable to bone.

  2. Efficient algorithms for accurate hierarchical clustering of huge datasets: tackling the entire protein space.

    Science.gov (United States)

    Loewenstein, Yaniv; Portugaly, Elon; Fromer, Menachem; Linial, Michal

    2008-07-01

    UPGMA (average linking) is probably the most popular algorithm for hierarchical data clustering, especially in computational biology. However, UPGMA requires the entire dissimilarity matrix in memory. Due to this prohibitive requirement, UPGMA is not scalable to very large datasets. We present a novel class of memory-constrained UPGMA (MC-UPGMA) algorithms. Given any practical memory size constraint, this framework guarantees the correct clustering solution without explicitly requiring all dissimilarities in memory. The algorithms are general and are applicable to any dataset. We present a data-dependent characterization of hardness and clustering efficiency. The presented concepts are applicable to any agglomerative clustering formulation. We apply our algorithm to the entire collection of protein sequences, to automatically build a comprehensive evolutionary-driven hierarchy of proteins from sequence alone. The newly created tree captures protein families better than state-of-the-art large-scale methods such as CluSTr, ProtoNet4 or single-linkage clustering. We demonstrate that leveraging the entire mass embodied in all sequence similarities allows to significantly improve on current protein family clusterings which are unable to directly tackle the sheer mass of this data. Furthermore, we argue that non-metric constraints are an inherent complexity of the sequence space and should not be overlooked. The robustness of UPGMA allows significant improvement, especially for multidomain proteins, and for large or divergent families. A comprehensive tree built from all UniProt sequence similarities, together with navigation and classification tools will be made available as part of the ProtoNet service. A C++ implementation of the algorithm is available on request.

  3. Sequence-specific capture of protein-DNA complexes for mass spectrometric protein identification.

    Directory of Open Access Journals (Sweden)

    Cheng-Hsien Wu

    Full Text Available The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1 promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.

  4. Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

    Science.gov (United States)

    Hong, Xinguo; Hao, Quan

    2009-01-01

    In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan (2D mode), with an interval as small as 1.5 times the beam size, at low temperature (e.g., 4 °C). A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio (better than the reported data on the same protein using a flow cell). The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

  5. Measurements of accurate x-ray scattering data of protein solutions using small stationary sample cells

    International Nuclear Information System (INIS)

    Hong Xinguo; Hao Quan

    2009-01-01

    In this paper, we report a method of precise in situ x-ray scattering measurements on protein solutions using small stationary sample cells. Although reduction in the radiation damage induced by intense synchrotron radiation sources is indispensable for the correct interpretation of scattering data, there is still a lack of effective methods to overcome radiation-induced aggregation and extract scattering profiles free from chemical or structural damage. It is found that radiation-induced aggregation mainly begins on the surface of the sample cell and grows along the beam path; the diameter of the damaged region is comparable to the x-ray beam size. Radiation-induced aggregation can be effectively avoided by using a two-dimensional scan (2D mode), with an interval as small as 1.5 times the beam size, at low temperature (e.g., 4 deg. C). A radiation sensitive protein, bovine hemoglobin, was used to test the method. A standard deviation of less than 5% in the small angle region was observed from a series of nine spectra recorded in 2D mode, in contrast to the intensity variation seen using the conventional stationary technique, which can exceed 100%. Wide-angle x-ray scattering data were collected at a standard macromolecular diffraction station using the same data collection protocol and showed a good signal/noise ratio (better than the reported data on the same protein using a flow cell). The results indicate that this method is an effective approach for obtaining precise measurements of protein solution scattering.

  6. Identification of Proteins with Potential Osteogenic Activity Present in the Water-Soluble Matrix Proteins from Crassostrea gigas Nacre Using a Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Daniel V. Oliveira

    2012-01-01

    Full Text Available Nacre, when implanted in vivo in bones of dogs, sheep, mice, and humans, induces a biological response that includes integration and osteogenic activity on the host tissue that seems to be activated by a set of proteins present in the nacre water-soluble matrix (WSM. We describe here an experimental approach that can accurately identify the proteins present in the WSM of shell mollusk nacre. Four proteins (three gigasin-2 isoforms and a cystatin A2 were for the first time identified in WSM of Crassostrea gigas nacre using 2DE and LC-MS/MS for protein identification. These proteins are thought to be involved in bone remodeling processes and could be responsible for the biocompatibility shown between bone and nacre grafts. These results represent a contribution to the study of shell biomineralization process and opens new perspectives for the development of new nacre biomaterials for orthopedic applications.

  7. GOSSIP: a method for fast and accurate global alignment of protein structures.

    Science.gov (United States)

    Kifer, I; Nussinov, R; Wolfson, H J

    2011-04-01

    The database of known protein structures (PDB) is increasing rapidly. This results in a growing need for methods that can cope with the vast amount of structural data. To analyze the accumulating data, it is important to have a fast tool for identifying similar structures and clustering them by structural resemblance. Several excellent tools have been developed for the comparison of protein structures. These usually address the task of local structure alignment, an important yet computationally intensive problem due to its complexity. It is difficult to use such tools for comparing a large number of structures to each other at a reasonable time. Here we present GOSSIP, a novel method for a global all-against-all alignment of any set of protein structures. The method detects similarities between structures down to a certain cutoff (a parameter of the program), hence allowing it to detect similar structures at a much higher speed than local structure alignment methods. GOSSIP compares many structures in times which are several orders of magnitude faster than well-known available structure alignment servers, and it is also faster than a database scanning method. We evaluate GOSSIP both on a dataset of short structural fragments and on two large sequence-diverse structural benchmarks. Our conclusions are that for a threshold of 0.6 and above, the speed of GOSSIP is obtained with no compromise of the accuracy of the alignments or of the number of detected global similarities. A server, as well as an executable for download, are available at http://bioinfo3d.cs.tau.ac.il/gossip/.

  8. Accurate determination of the diffusion coefficient of proteins by Fourier analysis with whole column imaging detection.

    Science.gov (United States)

    Zarabadi, Atefeh S; Pawliszyn, Janusz

    2015-02-17

    Analysis in the frequency domain is considered a powerful tool to elicit precise information from spectroscopic signals. In this study, the Fourier transformation technique is employed to determine the diffusion coefficient (D) of a number of proteins in the frequency domain. Analytical approaches are investigated for determination of D from both experimental and data treatment viewpoints. The diffusion process is modeled to calculate diffusion coefficients based on the Fourier transformation solution to Fick's law equation, and its results are compared to time domain results. The simulations characterize optimum spatial and temporal conditions and demonstrate the noise tolerance of the method. The proposed model is validated by its application for the electropherograms from the diffusion path of a set of proteins. Real-time dynamic scanning is conducted to monitor dispersion by employing whole column imaging detection technology in combination with capillary isoelectric focusing (CIEF) and the imaging plug flow (iPF) experiment. These experimental techniques provide different peak shapes, which are utilized to demonstrate the Fourier transformation ability in extracting diffusion coefficients out of irregular shape signals. Experimental results confirmed that the Fourier transformation procedure substantially enhanced the accuracy of the determined values compared to those obtained in the time domain.

  9. Accurate determination of interfacial protein secondary structure by combining interfacial-sensitive amide I and amide III spectral signals.

    Science.gov (United States)

    Ye, Shuji; Li, Hongchun; Yang, Weilai; Luo, Yi

    2014-01-29

    Accurate determination of protein structures at the interface is essential to understand the nature of interfacial protein interactions, but it can only be done with a few, very limited experimental methods. Here, we demonstrate for the first time that sum frequency generation vibrational spectroscopy can unambiguously differentiate the interfacial protein secondary structures by combining surface-sensitive amide I and amide III spectral signals. This combination offers a powerful tool to directly distinguish random-coil (disordered) and α-helical structures in proteins. From a systematic study on the interactions between several antimicrobial peptides (including LKα14, mastoparan X, cecropin P1, melittin, and pardaxin) and lipid bilayers, it is found that the spectral profiles of the random-coil and α-helical structures are well separated in the amide III spectra, appearing below and above 1260 cm(-1), respectively. For the peptides with a straight backbone chain, the strength ratio for the peaks of the random-coil and α-helical structures shows a distinct linear relationship with the fraction of the disordered structure deduced from independent NMR experiments reported in the literature. It is revealed that increasing the fraction of negatively charged lipids can induce a conformational change of pardaxin from random-coil to α-helical structures. This experimental protocol can be employed for determining the interfacial protein secondary structures and dynamics in situ and in real time without extraneous labels.

  10. Identification of transcriptional signals in Encephalitozoon cuniculi widespread among Microsporidia phylum: support for accurate structural genome annotation

    Directory of Open Access Journals (Sweden)

    Wincker Patrick

    2009-12-01

    Full Text Available Abstract Background Microsporidia are obligate intracellular eukaryotic parasites with genomes ranging in size from 2.3 Mbp to more than 20 Mbp. The extremely small (2.9 Mbp and highly compact (~1 gene/kb genome of the human parasite Encephalitozoon cuniculi has been fully sequenced. The aim of this study was to characterize noncoding motifs that could be involved in regulation of gene expression in E. cuniculi and to show whether these motifs are conserved among the phylum Microsporidia. Results To identify such signals, 5' and 3'RACE-PCR experiments were performed on different E. cuniculi mRNAs. This analysis confirmed that transcription overrun occurs in E. cuniculi and may result from stochastic recognition of the AAUAAA polyadenylation signal. Such experiments also showed highly reduced 5'UTR's (E. cuniculi genes presented a CCC-like motif immediately upstream from the coding start. To characterize other signals involved in differential transcriptional regulation, we then focused our attention on the gene family coding for ribosomal proteins. An AAATTT-like signal was identified upstream from the CCC-like motif. In rare cases the cytosine triplet was shown to be substituted by a GGG-like motif. Comparative genomic studies confirmed that these different signals are also located upstream from genes encoding ribosomal proteins in other microsporidian species including Antonospora locustae, Enterocytozoon bieneusi, Anncaliia algerae (syn. Brachiola algerae and Nosema ceranae. Based on these results a systematic analysis of the ~2000 E. cuniculi coding DNA sequences was then performed and brings to highlight that 364 translation initiation codons (18.29% of total CDSs had been badly predicted. Conclusion We identified various signals involved in the maturation of E. cuniculi mRNAs. Presence of such signals, in phylogenetically distant microsporidian species, suggests that a common regulatory mechanism exists among the microsporidia. Furthermore

  11. Working with Proteins in silico: A Review of Online Available Tools for Basic Identification of Proteins

    Directory of Open Access Journals (Sweden)

    Caner Yavuz

    2017-01-01

    Full Text Available Increase in online available bioinformatics tools for protein research creates an important opportunity for scientists to reveal characteristics of the protein of interest by only starting from the predicted or known amino acid sequence without fully depending on experimental approaches. There are many sophisticated tools used for diverse purposes; however, there are not enough reviews covering the tips and tricks in selecting and using the correct tools as the literature mainly state the promotion of the new ones. In this review, with the aim of providing young scientists with no specific experience on protein work a reliable starting point for in silico analysis of the protein of interest, we summarized tools for annotation, identification of motifs and domains, determination isoelectric point, molecular weight, subcellular localization, and post-translational modifications by focusing on the important points to be considered while selecting from online available tools.

  12. Towards accurate free energy calculations in ligand protein-binding studies.

    Science.gov (United States)

    Steinbrecher, Thomas; Labahn, Andreas

    2010-01-01

    Cells contain a multitude of different chemical reaction paths running simultaneously and quite independently next to each other. This amazing feat is enabled by molecular recognition, the ability of biomolecules to form stable and specific complexes with each other and with their substrates. A better understanding of this process, i.e. of the kinetics, structures and thermodynamic properties of biomolecule binding, would be invaluable in the study of biological systems. In addition, as the mode of action of many pharmaceuticals is based upon their inhibition or activation of biomolecule targets, predictive models of small molecule receptor binding are very helpful tools in rational drug design. Since the goal here is normally to design a new compound with a high inhibition strength, one of the most important thermodynamic properties is the binding free energy DeltaG(0). The prediction of binding constants has always been one of the major goals in the field of computational chemistry, because the ability to reliably assess a hypothetical compound's binding properties without having to synthesize it first would save a tremendous amount of work. The different approaches to this question range from fast and simple empirical descriptor methods to elaborate simulation protocols aimed at putting the computation of free energies onto a solid foundation of statistical thermodynamics. While the later methods are still not suited for the screenings of thousands of compounds that are routinely performed in computational drug design studies, they are increasingly put to use for the detailed study of protein ligand interactions. This review will focus on molecular mechanics force field based free energy calculations and their application to the study of protein ligand interactions. After a brief overview of other popular methods for the calculation of free energies, we will describe recent advances in methodology and a variety of exemplary studies of molecular dynamics

  13. Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

    Science.gov (United States)

    Borsuk, Sibele; Newcombe, Jane; Mendum, Tom A; Dellagostin, Odir A; McFadden, Johnjoe

    2009-11-01

    The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

  14. Fast and accurate non-sequential protein structure alignment using a new asymmetric linear sum assignment heuristic.

    Science.gov (United States)

    Brown, Peter; Pullan, Wayne; Yang, Yuedong; Zhou, Yaoqi

    2016-02-01

    The three dimensional tertiary structure of a protein at near atomic level resolution provides insight alluding to its function and evolution. As protein structure decides its functionality, similarity in structure usually implies similarity in function. As such, structure alignment techniques are often useful in the classifications of protein function. Given the rapidly growing rate of new, experimentally determined structures being made available from repositories such as the Protein Data Bank, fast and accurate computational structure comparison tools are required. This paper presents SPalignNS, a non-sequential protein structure alignment tool using a novel asymmetrical greedy search technique. The performance of SPalignNS was evaluated against existing sequential and non-sequential structure alignment methods by performing trials with commonly used datasets. These benchmark datasets used to gauge alignment accuracy include (i) 9538 pairwise alignments implied by the HOMSTRAD database of homologous proteins; (ii) a subset of 64 difficult alignments from set (i) that have low structure similarity; (iii) 199 pairwise alignments of proteins with similar structure but different topology; and (iv) a subset of 20 pairwise alignments from the RIPC set. SPalignNS is shown to achieve greater alignment accuracy (lower or comparable root-mean squared distance with increased structure overlap coverage) for all datasets, and the highest agreement with reference alignments from the challenging dataset (iv) above, when compared with both sequentially constrained alignments and other non-sequential alignments. SPalignNS was implemented in C++. The source code, binary executable, and a web server version is freely available at: http://sparks-lab.org yaoqi.zhou@griffith.edu.au. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. A Deep Learning Framework for Robust and Accurate Prediction of ncRNA-Protein Interactions Using Evolutionary Information.

    Science.gov (United States)

    Yi, Hai-Cheng; You, Zhu-Hong; Huang, De-Shuang; Li, Xiao; Jiang, Tong-Hai; Li, Li-Ping

    2018-06-01

    The interactions between non-coding RNAs (ncRNAs) and proteins play an important role in many biological processes, and their biological functions are primarily achieved by binding with a variety of proteins. High-throughput biological techniques are used to identify protein molecules bound with specific ncRNA, but they are usually expensive and time consuming. Deep learning provides a powerful solution to computationally predict RNA-protein interactions. In this work, we propose the RPI-SAN model by using the deep-learning stacked auto-encoder network to mine the hidden high-level features from RNA and protein sequences and feed them into a random forest (RF) model to predict ncRNA binding proteins. Stacked assembling is further used to improve the accuracy of the proposed method. Four benchmark datasets, including RPI2241, RPI488, RPI1807, and NPInter v2.0, were employed for the unbiased evaluation of five established prediction tools: RPI-Pred, IPMiner, RPISeq-RF, lncPro, and RPI-SAN. The experimental results show that our RPI-SAN model achieves much better performance than other methods, with accuracies of 90.77%, 89.7%, 96.1%, and 99.33%, respectively. It is anticipated that RPI-SAN can be used as an effective computational tool for future biomedical researches and can accurately predict the potential ncRNA-protein interacted pairs, which provides reliable guidance for biological research. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. DisoMCS: Accurately Predicting Protein Intrinsically Disordered Regions Using a Multi-Class Conservative Score Approach.

    Directory of Open Access Journals (Sweden)

    Zhiheng Wang

    Full Text Available The precise prediction of protein intrinsically disordered regions, which play a crucial role in biological procedures, is a necessary prerequisite to further the understanding of the principles and mechanisms of protein function. Here, we propose a novel predictor, DisoMCS, which is a more accurate predictor of protein intrinsically disordered regions. The DisoMCS bases on an original multi-class conservative score (MCS obtained by sequence-order/disorder alignment. Initially, near-disorder regions are defined on fragments located at both the terminus of an ordered region connecting a disordered region. Then the multi-class conservative score is generated by sequence alignment against a known structure database and represented as order, near-disorder and disorder conservative scores. The MCS of each amino acid has three elements: order, near-disorder and disorder profiles. Finally, the MCS is exploited as features to identify disordered regions in sequences. DisoMCS utilizes a non-redundant data set as the training set, MCS and predicted secondary structure as features, and a conditional random field as the classification algorithm. In predicted near-disorder regions a residue is determined as an order or a disorder according to the optimized decision threshold. DisoMCS was evaluated by cross-validation, large-scale prediction, independent tests and CASP (Critical Assessment of Techniques for Protein Structure Prediction tests. All results confirmed that DisoMCS was very competitive in terms of accuracy of prediction when compared with well-established publicly available disordered region predictors. It also indicated our approach was more accurate when a query has higher homologous with the knowledge database.The DisoMCS is available at http://cal.tongji.edu.cn/disorder/.

  17. Accurate Quantification of Cardiovascular Biomarkers in Serum Using Protein Standard Absolute Quantification (PSAQ™) and Selected Reaction Monitoring*

    Science.gov (United States)

    Huillet, Céline; Adrait, Annie; Lebert, Dorothée; Picard, Guillaume; Trauchessec, Mathieu; Louwagie, Mathilde; Dupuis, Alain; Hittinger, Luc; Ghaleh, Bijan; Le Corvoisier, Philippe; Jaquinod, Michel; Garin, Jérôme; Bruley, Christophe; Brun, Virginie

    2012-01-01

    Development of new biomarkers needs to be significantly accelerated to improve diagnostic, prognostic, and toxicity monitoring as well as therapeutic follow-up. Biomarker evaluation is the main bottleneck in this development process. Selected Reaction Monitoring (SRM) combined with stable isotope dilution has emerged as a promising option to speed this step, particularly because of its multiplexing capacities. However, analytical variabilities because of upstream sample handling or incomplete trypsin digestion still need to be resolved. In 2007, we developed the PSAQ™ method (Protein Standard Absolute Quantification), which uses full-length isotope-labeled protein standards to quantify target proteins. In the present study we used clinically validated cardiovascular biomarkers (LDH-B, CKMB, myoglobin, and troponin I) to demonstrate that the combination of PSAQ and SRM (PSAQ-SRM) allows highly accurate biomarker quantification in serum samples. A multiplex PSAQ-SRM assay was used to quantify these biomarkers in clinical samples from myocardial infarction patients. Good correlation between PSAQ-SRM and ELISA assay results was found and demonstrated the consistency between these analytical approaches. Thus, PSAQ-SRM has the capacity to improve both accuracy and reproducibility in protein analysis. This will be a major contribution to efficient biomarker development strategies. PMID:22080464

  18. Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

    Science.gov (United States)

    Pinne, Marija; Matsunaga, James; Haake, David A

    2012-11-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

  19. Combining structural modeling with ensemble machine learning to accurately predict protein fold stability and binding affinity effects upon mutation.

    Directory of Open Access Journals (Sweden)

    Niklas Berliner

    Full Text Available Advances in sequencing have led to a rapid accumulation of mutations, some of which are associated with diseases. However, to draw mechanistic conclusions, a biochemical understanding of these mutations is necessary. For coding mutations, accurate prediction of significant changes in either the stability of proteins or their affinity to their binding partners is required. Traditional methods have used semi-empirical force fields, while newer methods employ machine learning of sequence and structural features. Here, we show how combining both of these approaches leads to a marked boost in accuracy. We introduce ELASPIC, a novel ensemble machine learning approach that is able to predict stability effects upon mutation in both, domain cores and domain-domain interfaces. We combine semi-empirical energy terms, sequence conservation, and a wide variety of molecular details with a Stochastic Gradient Boosting of Decision Trees (SGB-DT algorithm. The accuracy of our predictions surpasses existing methods by a considerable margin, achieving correlation coefficients of 0.77 for stability, and 0.75 for affinity predictions. Notably, we integrated homology modeling to enable proteome-wide prediction and show that accurate prediction on modeled structures is possible. Lastly, ELASPIC showed significant differences between various types of disease-associated mutations, as well as between disease and common neutral mutations. Unlike pure sequence-based prediction methods that try to predict phenotypic effects of mutations, our predictions unravel the molecular details governing the protein instability, and help us better understand the molecular causes of diseases.

  20. Verification of Single-Peptide Protein Identifications by the Application of Complementary Database Search Algorithms

    National Research Council Canada - National Science Library

    Rohrbough, James G; Breci, Linda; Merchant, Nirav; Miller, Susan; Haynes, Paul A

    2005-01-01

    .... One such technique, known as the Multi-Dimensional Protein Identification Technique, or MudPIT, involves the use of computer search algorithms that automate the process of identifying proteins...

  1. Computational identification of MoRFs in protein sequences.

    Science.gov (United States)

    Malhis, Nawar; Gsponer, Jörg

    2015-06-01

    Intrinsically disordered regions of proteins play an essential role in the regulation of various biological processes. Key to their regulatory function is the binding of molecular recognition features (MoRFs) to globular protein domains in a process known as a disorder-to-order transition. Predicting the location of MoRFs in protein sequences with high accuracy remains an important computational challenge. In this study, we introduce MoRFCHiBi, a new computational approach for fast and accurate prediction of MoRFs in protein sequences. MoRFCHiBi combines the outcomes of two support vector machine (SVM) models that take advantage of two different kernels with high noise tolerance. The first, SVMS, is designed to extract maximal information from the general contrast in amino acid compositions between MoRFs, their surrounding regions (Flanks), and the remainders of the sequences. The second, SVMT, is used to identify similarities between regions in a query sequence and MoRFs of the training set. We evaluated the performance of our predictor by comparing its results with those of two currently available MoRF predictors, MoRFpred and ANCHOR. Using three test sets that have previously been collected and used to evaluate MoRFpred and ANCHOR, we demonstrate that MoRFCHiBi outperforms the other predictors with respect to different evaluation metrics. In addition, MoRFCHiBi is downloadable and fast, which makes it useful as a component in other computational prediction tools. http://www.chibi.ubc.ca/morf/. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. Disease candidate gene identification and prioritization using protein interaction networks

    Directory of Open Access Journals (Sweden)

    Aronow Bruce J

    2009-02-01

    Full Text Available Abstract Background Although most of the current disease candidate gene identification and prioritization methods depend on functional annotations, the coverage of the gene functional annotations is a limiting factor. In the current study, we describe a candidate gene prioritization method that is entirely based on protein-protein interaction network (PPIN analyses. Results For the first time, extended versions of the PageRank and HITS algorithms, and the K-Step Markov method are applied to prioritize disease candidate genes in a training-test schema. Using a list of known disease-related genes from our earlier study as a training set ("seeds", and the rest of the known genes as a test list, we perform large-scale cross validation to rank the candidate genes and also evaluate and compare the performance of our approach. Under appropriate settings – for example, a back probability of 0.3 for PageRank with Priors and HITS with Priors, and step size 6 for K-Step Markov method – the three methods achieved a comparable AUC value, suggesting a similar performance. Conclusion Even though network-based methods are generally not as effective as integrated functional annotation-based methods for disease candidate gene prioritization, in a one-to-one comparison, PPIN-based candidate gene prioritization performs better than all other gene features or annotations. Additionally, we demonstrate that methods used for studying both social and Web networks can be successfully used for disease candidate gene prioritization.

  3. Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Jinfeng Ti

    Full Text Available This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb 3G2 by indirect enzyme-linked immunosorbent assay (ELISA. In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.

  4. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  5. A systematic identification of species-specific protein succinylation sites using joint element features information

    Directory of Open Access Journals (Sweden)

    Hasan MM

    2017-08-01

    Full Text Available Md Mehedi Hasan,1 Mst Shamima Khatun,2 Md Nurul Haque Mollah,2 Cao Yong,3 Dianjing Guo1 1School of Life Sciences and the State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong, People’s Republic of China; 2Laboratory of Bioinformatics, Department of Statistics, University of Rajshahi, Rajshahi, Bangladesh; 3Department of Mechanical Engineering and Automation, Harbin Institute of Technology, Shenzhen Graduate School, Shenzhen, People’s Republic of China Abstract: Lysine succinylation, an important type of protein posttranslational modification, plays significant roles in many cellular processes. Accurate identification of succinylation sites can facilitate our understanding about the molecular mechanism and potential roles of lysine succinylation. However, even in well-studied systems, a majority of the succinylation sites remain undetected because the traditional experimental approaches to succinylation site identification are often costly, time-consuming, and laborious. In silico approach, on the other hand, is potentially an alternative strategy to predict succinylation substrates. In this paper, a novel computational predictor SuccinSite2.0 was developed for predicting generic and species-specific protein succinylation sites. This predictor takes the composition of profile-based amino acid and orthogonal binary features, which were used to train a random forest classifier. We demonstrated that the proposed SuccinSite2.0 predictor outperformed other currently existing implementations on a complementarily independent dataset. Furthermore, the important features that make visible contributions to species-specific and cross-species-specific prediction of protein succinylation site were analyzed. The proposed predictor is anticipated to be a useful computational resource for lysine succinylation site prediction. The integrated species-specific online tool of SuccinSite2.0 is publicly

  6. Efficiency of Database Search for Identification of Mutated and Modified Proteins via Mass Spectrometry

    OpenAIRE

    Pevzner, Pavel A.; Mulyukov, Zufar; Dancik, Vlado; Tang, Chris L

    2001-01-01

    Although protein identification by matching tandem mass spectra (MS/MS) against protein databases is a widespread tool in mass spectrometry, the question about reliability of such searches remains open. Absence of rigorous significance scores in MS/MS database search makes it difficult to discard random database hits and may lead to erroneous protein identification, particularly in the case of mutated or post-translationally modified peptides. This problem is especially important for high-thr...

  7. Rapid and Accurate Identification by Real-Time PCR of Biotoxin-Producing Dinoflagellates from the Family Gymnodiniaceae

    Directory of Open Access Journals (Sweden)

    Kirsty F. Smith

    2014-03-01

    Full Text Available The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR assays targeting the large subunit ribosomal RNA (LSU rRNA gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

  8. Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

    Science.gov (United States)

    Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

    2014-03-07

    The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

  9. Identification and accurate quantification of structurally related peptide impurities in synthetic human C-peptide by liquid chromatography-high resolution mass spectrometry.

    Science.gov (United States)

    Li, Ming; Josephs, Ralf D; Daireaux, Adeline; Choteau, Tiphaine; Westwood, Steven; Wielgosz, Robert I; Li, Hongmei

    2018-06-04

    Peptides are an increasingly important group of biomarkers and pharmaceuticals. The accurate purity characterization of peptide calibrators is critical for the development of reference measurement systems for laboratory medicine and quality control of pharmaceuticals. The peptides used for these purposes are increasingly produced through peptide synthesis. Various approaches (for example mass balance, amino acid analysis, qNMR, and nitrogen determination) can be applied to accurately value assign the purity of peptide calibrators. However, all purity assessment approaches require a correction for structurally related peptide impurities in order to avoid biases. Liquid chromatography coupled to high resolution mass spectrometry (LC-hrMS) has become the key technique for the identification and accurate quantification of structurally related peptide impurities in intact peptide calibrator materials. In this study, LC-hrMS-based methods were developed and validated in-house for the identification and quantification of structurally related peptide impurities in a synthetic human C-peptide (hCP) material, which served as a study material for an international comparison looking at the competencies of laboratories to perform peptide purity mass fraction assignments. More than 65 impurities were identified, confirmed, and accurately quantified by using LC-hrMS. The total mass fraction of all structurally related peptide impurities in the hCP study material was estimated to be 83.3 mg/g with an associated expanded uncertainty of 3.0 mg/g (k = 2). The calibration hierarchy concept used for the quantification of individual impurities is described in detail. Graphical abstract ᅟ.

  10. Accurate spectroscopic characterization of oxirane: A valuable route to its identification in Titan's atmosphere and the assignment of unidentified infrared bands

    Energy Technology Data Exchange (ETDEWEB)

    Puzzarini, Cristina [Dipartimento di Chimica " Giacomo Ciamician," Università di Bologna, Via Selmi 2, I-40126 Bologna (Italy); Biczysko, Malgorzata; Bloino, Julien; Barone, Vincenzo, E-mail: cristina.puzzarini@unibo.it [Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126 Pisa (Italy)

    2014-04-20

    In an effort to provide an accurate spectroscopic characterization of oxirane, state-of-the-art computational methods and approaches have been employed to determine highly accurate fundamental vibrational frequencies and rotational parameters. Available experimental data were used to assess the reliability of our computations, and an accuracy on average of 10 cm{sup –1} for fundamental transitions as well as overtones and combination bands has been pointed out. Moving to rotational spectroscopy, relative discrepancies of 0.1%, 2%-3%, and 3%-4% were observed for rotational, quartic, and sextic centrifugal-distortion constants, respectively. We are therefore confident that the highly accurate spectroscopic data provided herein can be useful for identification of oxirane in Titan's atmosphere and the assignment of unidentified infrared bands. Since oxirane was already observed in the interstellar medium and some astronomical objects are characterized by very high D/H ratios, we also considered the accurate determination of the spectroscopic parameters for the mono-deuterated species, oxirane-d1. For the latter, an empirical scaling procedure allowed us to improve our computed data and to provide predictions for rotational transitions with a relative accuracy of about 0.02% (i.e., an uncertainty of about 40 MHz for a transition lying at 200 GHz).

  11. Identification of novel components in microProtein signalling

    DEFF Research Database (Denmark)

    Rodrigues, Vandasue Lily

    characterization of smaller proteins. Using a computational approach, we identified putative microProteins that could target a diverse variety of protein classes. Using a synthetic microProtein approach, we demonstrate that miPs can target a diverse variety of target proteins, which makes them of interest...

  12. Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity.

    Science.gov (United States)

    Barkla, Bronwyn J

    2016-09-08

    Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised.

  13. Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity

    Directory of Open Access Journals (Sweden)

    Bronwyn J. Barkla

    2016-09-01

    Full Text Available Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised.

  14. Verification of protein biomarker specificity for the identification of biological stains by quadrupole time-of-flight mass spectrometry.

    Science.gov (United States)

    Legg, Kevin M; Powell, Roger; Reisdorph, Nichole; Reisdorph, Rick; Danielson, Phillip B

    2017-03-01

    Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of "candidate biomarkers" specific to each of five body fluids (i.e., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time-of-flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single-source samples of these human body fluids were accurately identified by the detection of one or more high-specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2-component mixtures of human body fluids, the multiplex assay accurately identified both components in a single-pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. ETHNOPRED: a novel machine learning method for accurate continental and sub-continental ancestry identification and population stratification correction

    Science.gov (United States)

    2013-01-01

    Background Population stratification is a systematic difference in allele frequencies between subpopulations. This can lead to spurious association findings in the case–control genome wide association studies (GWASs) used to identify single nucleotide polymorphisms (SNPs) associated with disease-linked phenotypes. Methods such as self-declared ancestry, ancestry informative markers, genomic control, structured association, and principal component analysis are used to assess and correct population stratification but each has limitations. We provide an alternative technique to address population stratification. Results We propose a novel machine learning method, ETHNOPRED, which uses the genotype and ethnicity data from the HapMap project to learn ensembles of disjoint decision trees, capable of accurately predicting an individual’s continental and sub-continental ancestry. To predict an individual’s continental ancestry, ETHNOPRED produced an ensemble of 3 decision trees involving a total of 10 SNPs, with 10-fold cross validation accuracy of 100% using HapMap II dataset. We extended this model to involve 29 disjoint decision trees over 149 SNPs, and showed that this ensemble has an accuracy of ≥ 99.9%, even if some of those 149 SNP values were missing. On an independent dataset, predominantly of Caucasian origin, our continental classifier showed 96.8% accuracy and improved genomic control’s λ from 1.22 to 1.11. We next used the HapMap III dataset to learn classifiers to distinguish European subpopulations (North-Western vs. Southern), East Asian subpopulations (Chinese vs. Japanese), African subpopulations (Eastern vs. Western), North American subpopulations (European vs. Chinese vs. African vs. Mexican vs. Indian), and Kenyan subpopulations (Luhya vs. Maasai). In these cases, ETHNOPRED produced ensembles of 3, 39, 21, 11, and 25 disjoint decision trees, respectively involving 31, 502, 526, 242 and 271 SNPs, with 10-fold cross validation accuracy of

  16. Accurate, safe, and rapid method of intraoperative tumor identification for totally laparoscopic distal gastrectomy: injection of mixed fluid of sodium hyaluronate and patent blue.

    Science.gov (United States)

    Nakagawa, Masatoshi; Ehara, Kazuhisa; Ueno, Masaki; Tanaka, Tsuyoshi; Kaida, Sachiko; Udagawa, Harushi

    2014-04-01

    In totally laparoscopic distal gastrectomy, determining the resection line with safe proximal margins is often difficult, particularly for tumors located in a relatively upper area. This is because, in contrast to open surgery, identifying lesions by palpating or opening the stomach is essentially impossible. This study introduces a useful method of tumor identification that is accurate, safe, and rapid. On the operation day, after inducing general anesthesia, a mixture of sodium hyaluronate and patent blue is injected into the submucosal layer of the proximal margin. When resecting stomach, all marker spots should be on the resected side. In all cases, the proximal margin is examined histologically by using frozen sections during the operation. From October 2009 to September 2011, a prospective study that evaluated this method was performed. A total of 34 patients who underwent totally laparoscopic distal gastrectomy were enrolled in this study. Approximately 5 min was required to complete the procedure. Proximal margins were negative in all cases, and the mean ± standard deviation length of the proximal margin was 23.5 ± 12.8 mm. No side effects, such as allergy, were encountered. As a method of tumor identification for totally laparoscopic distal gastrectomy, this procedure appears accurate, safe, and rapid.

  17. Identification of the chemical forms of selenium in soy protein

    International Nuclear Information System (INIS)

    Rodibaugh, R.

    1989-01-01

    Soybeans (Glycine max. L. Merr., Century) were grown hydroponically and intrinsically radiolabeled with 75 Se, an isotope of selenium (Se). The isotope was provided as 75 Se-Na 2 SeO 3 during the reproductive stage of growth until onset of senescence. Harvested seeds were processed into defatted soy meal. Soluble proteins were extracted in 20mM Tris-HCl buffer and fractionated into 11S, 7S, and 2S protein fractions by isoelectric precipitation. The 11S and 7S globulins, containing the glycinin and conglycinin storage proteins respectively, constitute the majority of extractable soy proteins. These storage proteins are the predominant proteins in soy protein isolate frequently used in food for human consumption. Approximately 24% of the defatted meal was soluble protein and accounted for 65% of the radioactivity associated with the soybean meal. The 11S fraction contained approximately 31% of the extracted protein and 27% of the extracted radioactivity. The 7S fraction contained approximately 32% and 35% of the extractable protein and radioactivity, respectively. The 2S fraction, containing the sulfur (S)-rich trypsin inhibitors, accounted for 17% of the protein and 27% of the radioactivity extracted from the defatted soy meal. Purification of the storage proteins by gel filtration and affinity chromatography showed higher levels of radioactivity associated with glycinin than conglycinin. Purified 11S proteins contained 1.09 ng Se per mg protein while 7S proteins contained 0.36 ng Se per mg protein

  18. Identification and characterization of the surface proteins of Clostridium difficile

    International Nuclear Information System (INIS)

    Dailey, D.C.

    1988-01-01

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated

  19. Identification and characterization of secreted proteins in Eimeria tenella

    Science.gov (United States)

    Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2015-09-01

    Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

  20. A novel method for accurate needle-tip identification in trans-rectal ultrasound-based high-dose-rate prostate brachytherapy.

    Science.gov (United States)

    Zheng, Dandan; Todor, Dorin A

    2011-01-01

    In real-time trans-rectal ultrasound (TRUS)-based high-dose-rate prostate brachytherapy, the accurate identification of needle-tip position is critical for treatment planning and delivery. Currently, needle-tip identification on ultrasound images can be subject to large uncertainty and errors because of ultrasound image quality and imaging artifacts. To address this problem, we developed a method based on physical measurements with simple and practical implementation to improve the accuracy and robustness of needle-tip identification. Our method uses measurements of the residual needle length and an off-line pre-established coordinate transformation factor, to calculate the needle-tip position on the TRUS images. The transformation factor was established through a one-time systematic set of measurements of the probe and template holder positions, applicable to all patients. To compare the accuracy and robustness of the proposed method and the conventional method (ultrasound detection), based on the gold-standard X-ray fluoroscopy, extensive measurements were conducted in water and gel phantoms. In water phantom, our method showed an average tip-detection accuracy of 0.7 mm compared with 1.6 mm of the conventional method. In gel phantom (more realistic and tissue-like), our method maintained its level of accuracy while the uncertainty of the conventional method was 3.4mm on average with maximum values of over 10mm because of imaging artifacts. A novel method based on simple physical measurements was developed to accurately detect the needle-tip position for TRUS-based high-dose-rate prostate brachytherapy. The method demonstrated much improved accuracy and robustness over the conventional method. Copyright © 2011 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.

  1. Identification of Heat Shock Protein families and J-protein types by incorporating Dipeptide Composition into Chou's general PseAAC.

    Science.gov (United States)

    Ahmad, Saeed; Kabir, Muhammad; Hayat, Maqsood

    2015-11-01

    Heat Shock Proteins (HSPs) are the substantial ingredients for cell growth and viability, which are found in all living organisms. HSPs manage the process of folding and unfolding of proteins, the quality of newly synthesized proteins and protecting cellular homeostatic processes from environmental stress. On the basis of functionality, HSPs are categorized into six major families namely: (i) HSP20 or sHSP (ii) HSP40 or J-proteins types (iii) HSP60 or GroEL/ES (iv) HSP70 (v) HSP90 and (vi) HSP100. Identification of HSPs family and sub-family through conventional approaches is expensive and laborious. It is therefore, highly desired to establish an automatic, robust and accurate computational method for prediction of HSPs quickly and reliably. Regard, a computational model is developed for the prediction of HSPs family. In this model, protein sequences are formulated using three discrete methods namely: Split Amino Acid Composition, Pseudo Amino Acid Composition, and Dipeptide Composition. Several learning algorithms are utilized to choice the best one for high throughput computational model. Leave one out test is applied to assess the performance of the proposed model. The empirical results showed that support vector machine achieved quite promising results using Dipeptide Composition feature space. The predicted outcomes of proposed model are 90.7% accuracy for HSPs dataset and 97.04% accuracy for J-protein types, which are higher than existing methods in the literature so far. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Identification of Proteins Involved in Salinity Tolerance in Salicornia bigelovii

    KAUST Repository

    Salazar Moya, Octavio Ruben

    2017-11-01

    With a global growing demand in food production, agricultural output must increase accordingly. An increased use of saline soils and brackish water would contribute to the required increase in world food production. Abiotic stresses, such as salinity and drought, are also major limiters of crop growth globally - most crops are relatively salt sensitive and are significantly affected when exposed to salt in the range of 50 to 200 mM NaCl. Genomic resources from plants that naturally thrive in highly saline environments have the potential to be valuable in the generation of salt tolerant crops; however, these resources have been largely unexplored. Salicornia bigelovii is a plant native to Mexico and the United States that grows in salt marshes and coastal regions. It can thrive in environments with salt concentrations higher than seawater. In contrast to most crops, S. bigelovii is able to accumulate very high concentrations (in the order of 1.5 M) of Na+ and Cl- in its photosynthetically active succulent shoots. Part of this tolerance is likely to include the storage of Na+ in the vacuoles of the shoots, making S. bigelovii a good model for understanding mechanisms of Na+ compartmentalization in the vacuoles and a good resource for gene discovery. In this research project, phenotypic, genomic, transcriptomic, and proteomic approaches have been used for the identification of candidate genes involved in salinity tolerance in S. bigelovii. The genomes and transcriptomes of three Salicornia species have been sequenced. This information has been used to support the characterization of the salt-induced transcriptome of S. bigelovii shoots and the salt-induced proteome of various organellar membrane enriched fractions from S. bigelovii shoots, which led to the creation of organellar membrane proteomes. Yeast spot assays at different salt concentrations revealed several proteins increasing or decreasing yeast salt tolerance. This work aims to create the basis for

  3. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species.

    Science.gov (United States)

    Alanio, A; Beretti, J-L; Dauphin, B; Mellado, E; Quesne, G; Lacroix, C; Amara, A; Berche, P; Nassif, X; Bougnoux, M-E

    2011-05-01

    New Aspergillus species have recently been described with the use of multilocus sequencing in refractory cases of invasive aspergillosis. The classical phenotypic identification methods routinely used in clinical laboratories failed to identify them adequately. Some of these Aspergillus species have specific patterns of susceptibility to antifungal agents, and misidentification may lead to inappropriate therapy. We developed a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based strategy to adequately identify Aspergillus species to the species level. A database including the reference spectra of 28 clinically relevant species from seven Aspergillus sections (five common and 23 unusual species) was engineered. The profiles of young and mature colonies were analysed for each reference strain, and species-specific spectral fingerprints were identified. The performance of the database was then tested on 124 clinical and 16 environmental isolates previously characterized by partial sequencing of the β-tubulin and calmodulin genes. One hundred and thirty-eight isolates of 140 (98.6%) were correctly identified. Two atypical isolates could not be identified, but no isolate was misidentified (specificity: 100%). The database, including species-specific spectral fingerprints of young and mature colonies of the reference strains, allowed identification regardless of the maturity of the clinical isolate. These results indicate that MALDI-TOF MS is a powerful tool for rapid and accurate identification of both common and unusual species of Aspergillus. It can give better results than morphological identification in clinical laboratories. © 2010 The Authors. Clinical Microbiology and Infection © 2010 European Society of Clinical Microbiology and Infectious Diseases.

  4. Identification and characterization of plastid-type proteins from sequence-attributed features using machine learning

    Science.gov (United States)

    2013-01-01

    Background Plastids are an important component of plant cells, being the site of manufacture and storage of chemical compounds used by the cell, and contain pigments such as those used in photosynthesis, starch synthesis/storage, cell color etc. They are essential organelles of the plant cell, also present in algae. Recent advances in genomic technology and sequencing efforts is generating a huge amount of DNA sequence data every day. The predicted proteome of these genomes needs annotation at a faster pace. In view of this, one such annotation need is to develop an automated system that can distinguish between plastid and non-plastid proteins accurately, and further classify plastid-types based on their functionality. We compared the amino acid compositions of plastid proteins with those of non-plastid ones and found significant differences, which were used as a basis to develop various feature-based prediction models using similarity-search and machine learning. Results In this study, we developed separate Support Vector Machine (SVM) trained classifiers for characterizing the plastids in two steps: first distinguishing the plastid vs. non-plastid proteins, and then classifying the identified plastids into their various types based on their function (chloroplast, chromoplast, etioplast, and amyloplast). Five diverse protein features: amino acid composition, dipeptide composition, the pseudo amino acid composition, Nterminal-Center-Cterminal composition and the protein physicochemical properties are used to develop SVM models. Overall, the dipeptide composition-based module shows the best performance with an accuracy of 86.80% and Matthews Correlation Coefficient (MCC) of 0.74 in phase-I and 78.60% with a MCC of 0.44 in phase-II. On independent test data, this model also performs better with an overall accuracy of 76.58% and 74.97% in phase-I and phase-II, respectively. The similarity-based PSI-BLAST module shows very low performance with about 50% prediction

  5. Identification of differentially expressed proteins in response to Pb ...

    African Journals Online (AJOL)

    In response to Pb, a total of 76 proteins, out of the 95 differentially expressed proteins, were subjected to MALDI-TOF-MS Of these, 46 identities were identified by PMF and 19 identities were identified by microsequencing. Basic metabolisms such as photosynthesis, photorespiration and protein biosynthesis in C. roseus ...

  6. Identification of a hypothetical membrane protein interactor of ...

    Indian Academy of Sciences (India)

    Unknown

    characterized earlier through co-precipitation studies us- ing antibodies against this conserved carboxyl-terminal region (Rich and Steitz 1987). Protein P0 is also involved at the eEF2 elongation factor-binding domain, as demon- strated in yeast (Justice et al 1999). The P0 protein, and not P1 and P2 proteins, is essential for ...

  7. Proteomics - a novel approach to the identification and characterisation of plasmodesmatal proteins

    International Nuclear Information System (INIS)

    Faulkner, C.R.; Blackman, L.M.; Lyon, B.R.; Overall, R.L.

    2001-01-01

    The development of proteomic methods, such as 2-dimensional gel electrophoresis (2-DE), has established a high resolution means of identifying and characterising proteins from a given protein mixture. The biochemical composition of plasmodesmata, the intercellular channels between plant cells, is poorly described despite extensive attempts to identify protemaceous plasmodesmatal components. These attempts have been confounded by the large number of proteins in the cell wall. We have exploited the anatomy of the alga Chara corallina to separate tissues with (nodal cells) and tissues without (internodal cells) plasmodesmata. Proteins specific to the cytoplasmic and wall protein extracts of nodal and internodal tissue were identified by comparison of 2-DE gels of these extracts. In particular, a 95 kDa protein was identified as specific to the nodal cells in both 1-dimensional and 2-dimensional comparisons of cytoplasmic nodal and internodal protein extracts. This protein was analysed by electron spray ionization time of flight tandem mass spectroscopy (ESI-TOF MS/MS) and the sequence obtained showed similarity to plant lipoxygenases. Further proteins of interest were identified in 2-DE resolution of extracts from the nodal cytoplasm, including two 49 kDa proteins and two 46 kDa proteins, and from the nodal cell walls, including a cluster of proteins around 30 kDa. Thus, a proteomic strategy for the identification and characterisation of proteins specific to different cell types in Chara corallina has been developed, with potential application to the identification and characterisation of plasmodesmatal proteins

  8. Identification of proteins that may directly interact with human RPA.

    Science.gov (United States)

    Nakaya, Ryou; Takaya, Junichiro; Onuki, Takeshi; Moritani, Mariko; Nozaki, Naohito; Ishimi, Yukio

    2010-11-01

    RPA, which consisted of three subunits (RPA1, 2 and 3), plays essential roles in DNA transactions. At the DNA replication forks, RPA binds to single-stranded DNA region to stabilize the structure and to assemble other replication proteins. Interactions between RPA and several replication proteins have been reported but the analysis is not comprehensive. We systematically performed the qualitative analysis to identify RPA interaction partners to understand the protein-protein interaction at the replication forks. We expressed in insect cells the three subunits of human RPA, together with one replication protein, which is present at the forks under normal conditions and/or under the replication stress conditions, to examine the interaction. Among 30 proteins examined in total, it was found that at least 14 proteins interacted with RPA. RPA interacted with MCM3-7, MCM-BP and CDC45 proteins among the proteins that play roles in the initiation and the elongation of the DNA replication. RPA bound with TIPIN, CLASPIN and RAD17, which are involved in the DNA replication checkpoint functions. RPA also bound with cyclin-dependent kinases and an amino-terminal fragment of Rb protein that negatively regulates DNA replication. These results suggest that RPA interacts with the specific proteins among those that play roles in the regulation of the replication fork progression.

  9. Identification of Sumoylated Proteins in the Silkworm Bombyx mori

    Science.gov (United States)

    Tang, Xudong; Fu, Xuliang; Hao, Bifang; Zhu, Feng; Xiao, Shengyan; Xu, Li; Shen, Zhongyuan

    2014-01-01

    Small ubiquitin-like modifier (SUMO) modification (SUMOylation) is an important and widely used reversible modification system in eukaryotic cells. It regulates various cell processes, including protein targeting, transcriptional regulation, signal transduction, and cell division. To understand its role in the model lepidoptera insect Bombyx mori, a recombinant baculovirus was constructed to express an enhanced green fluorescent protein (eGFP)-SUMO fusion protein along with ubiquitin carrier protein 9 of Bombyx mori (BmUBC9). SUMOylation substrates from Bombyx mori cells infected with this baculovirus were isolated by immunoprecipitation and identified by LC–ESI-MS/MS. A total of 68 candidate SUMOylated proteins were identified, of which 59 proteins were functionally categorized to gene ontology (GO) terms. Analysis of kyoto encyclopedia of genes and genomes (KEGG) pathways showed that 46 of the identified proteins were involved in 76 pathways that mainly play a role in metabolism, spliceosome and ribosome functions, and in RNA transport. Furthermore, SUMOylation of four candidates (polyubiquitin-C-like isoform X1, 3-hydroxyacyl-CoA dehydrogenase, cyclin-related protein FAM58A-like and GTP-binding nuclear protein Ran) were verified by co-immunoprecipitation in Drosophila schneide 2 cells. In addition, 74% of the identified proteins were predicted to have at least one SUMOylation site. The data presented here shed light on the crucial process of protein sumoylation in Bombyx mori. PMID:25470021

  10. Isolation and identification of the human homolog of a new p53-binding protein, Mdmx

    NARCIS (Netherlands)

    Shvarts, A.; Bazuine, M.; Dekker, P.; Ramos, Y. F.; Steegenga, W. T.; Merckx, G.; van Ham, R. C.; van der Houven van Oordt, W.; van der Eb, A. J.; Jochemsen, A. G.

    1997-01-01

    We recently reported the identification of a mouse cDNA encoding a new p53-associating protein that we called Mdmx because of its structural similarity to Mdm2, a well-known p53-binding protein. Here we report the isolation of a cDNA encoding the human homolog of Mdmx. The ORF of the cDNA encodes a

  11. Mass spectrometry based approach for identification and characterisation of fluorescent proteins from marine organisms

    DEFF Research Database (Denmark)

    Wojdyla, Katarzyna Iwona; Rogowska-Wrzesinska, Adelina; Wrzesinski, Krzysztof

    2011-01-01

    We present here a new analytical strategy for identification and characterisation of fluorescent proteins from marine organisms. By applying basic proteomics tools it is possible to screen large sample collections for fluorescent proteins of desired characteristics prior to gene cloning. Our...

  12. Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry

    DEFF Research Database (Denmark)

    Hjerrild, M.; Stensballe, A.; Rasmussen, T.E.

    2004-01-01

    Protein phosphorylation plays a key role in cell regulation and identification of phosphorylation sites is important for understanding their functional significance. Here, we present an artificial neural network algorithm: NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) that predicts protein...

  13. Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry

    DEFF Research Database (Denmark)

    Hjerrild, Majbrit; Stensballe, Allan; Rasmussen, Thomas E

    2011-01-01

    Protein phosphorylation plays a key role in cell regulation and identification of phosphorylation sites is important for understanding their functional significance. Here, we present an artificial neural network algorithm: NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) that predicts protein...

  14. Decision peptide-driven: a free software tool for accurate protein quantification using gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry.

    Science.gov (United States)

    Santos, Hugo M; Reboiro-Jato, Miguel; Glez-Peña, Daniel; Nunes-Miranda, J D; Fdez-Riverola, Florentino; Carvallo, R; Capelo, J L

    2010-09-15

    The decision peptide-driven tool implements a software application for assisting the user in a protocol for accurate protein quantification based on the following steps: (1) protein separation through gel electrophoresis; (2) in-gel protein digestion; (3) direct and inverse (18)O-labeling and (4) matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI analysis. The DPD software compares the MALDI results of the direct and inverse (18)O-labeling experiments and quickly identifies those peptides with paralleled loses in different sets of a typical proteomic workflow. Those peptides are used for subsequent accurate protein quantification. The interpretation of the MALDI data from direct and inverse labeling experiments is time-consuming requiring a significant amount of time to do all comparisons manually. The DPD software shortens and simplifies the searching of the peptides that must be used for quantification from a week to just some minutes. To do so, it takes as input several MALDI spectra and aids the researcher in an automatic mode (i) to compare data from direct and inverse (18)O-labeling experiments, calculating the corresponding ratios to determine those peptides with paralleled losses throughout different sets of experiments; and (ii) allow to use those peptides as internal standards for subsequent accurate protein quantification using (18)O-labeling. In this work the DPD software is presented and explained with the quantification of protein carbonic anhydrase. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  15. Proteomic identification of rhythmic proteins in rice seedlings.

    Science.gov (United States)

    Hwang, Heeyoun; Cho, Man-Ho; Hahn, Bum-Soo; Lim, Hyemin; Kwon, Yong-Kook; Hahn, Tae-Ryong; Bhoo, Seong Hee

    2011-04-01

    Many aspects of plant metabolism that are involved in plant growth and development are influenced by light-regulated diurnal rhythms as well as endogenous clock-regulated circadian rhythms. To identify the rhythmic proteins in rice, periodically grown (12h light/12h dark cycle) seedlings were harvested for three days at six-hour intervals. Continuous dark-adapted plants were also harvested for two days. Among approximately 3000 reproducible protein spots on each gel, proteomic analysis ascertained 354 spots (~12%) as light-regulated rhythmic proteins, in which 53 spots showed prolonged rhythm under continuous dark conditions. Of these 354 ascertained rhythmic protein spots, 74 diurnal spots and 10 prolonged rhythmic spots under continuous dark were identified by MALDI-TOF MS analysis. The rhythmic proteins were functionally classified into photosynthesis, central metabolism, protein synthesis, nitrogen metabolism, stress resistance, signal transduction and unknown. Comparative analysis of our proteomic data with the public microarray database (the Plant DIURNAL Project) and RT-PCR analysis of rhythmic proteins showed differences in rhythmic expression phases between mRNA and protein, suggesting that the clock-regulated proteins in rice are modulated by not only transcriptional but also post-transcriptional, translational, and/or post-translational processes. 2011 Elsevier B.V. All rights reserved.

  16. Identification Of Protein Vaccine Candidates Using Comprehensive Proteomic Analysis Strategies

    National Research Council Canada - National Science Library

    Rohrbough, James G

    2007-01-01

    Presented in this dissertation are proteomic analysis studies focused on identifying proteins to be used as vaccine candidates against Coccidioidomycosis, a potentially fatal human pulmonary disease...

  17. Identification of ultramodified proteins using top-down spectra

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiaowen; Hengel, Shawna M.; Wu, Si; Tolic, Nikola; Pasa-Tolic, Ljiljana; Pevzner, Pavel A.

    2013-04-10

    Post-translational modifications (PTMs) play an important role in various biological processes through changing protein structure and function. Some ultramodified proteins (like histones) have multiple PTMs forming PTM patterns that define the functionality of a protein. While bottom-up mass spectrometry (MS) has been successful in identifying individual PTMs within short peptides, it is unable to identify PTM patterns spread along entire proteins in a coordinated fashion. In contrast, top-down MS analyzes intact proteins and reveals PTM patterns along the entire proteins. However, while recent advances in instrumentation have made top-down MS accessible to many laboratories, most computational tools for top-down MS focus on proteins with few PTMs and are unable to identify complex PTM patterns. We propose a new algorithm, MS-Align-E, that identifies both expected and unexpected PTMs in ultramodified proteins. We demonstrate that MS-Align-E identifies many protein forms of histone H4 and benchmark it against the currently accepted software tools.

  18. Identification of Ultramodified Proteins Using Top-Down Mass Spectra

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiaowen; Hengel, Shawna M.; Wu, Si; Tolic, Nikola; Pasa-Tolic, Ljiljana; Pevzner, Pavel A.

    2013-11-05

    Post-translational modifications (PTMs) play an important role in various biological processes through changing protein structure and function. Some ultramodified proteins (like histones) have multiple PTMs forming PTM patterns that define the functionality of a protein. While bottom-up mass spectrometry (MS) has been successful in identifying individual PTMs within short peptides, it is unable to identify PTM patterns spread along entire proteins in a coordinated fashion. In contrast, top-down MS analyzes intact proteins and reveals PTM patterns along the entire proteins. However, while recent advances in instrumentation have made top-down MS accessible to many laboratories, most computational tools for top-down MS focus on proteins with few PTMs and are unable to identify complex PTM patterns. We propose a new algorithm, MS-Align-E, that identifies both expected and unexpected PTMs in ultramodified proteins. We demonstrate that MS-Align-E identifies many protein forms of histone H4 and benchmark it against the currently accepted software tools.

  19. Identification and quantitation of signal molecule-dependent protein phosphorylation

    KAUST Repository

    Groen, Arnoud J.; Thomas, Ludivine; Lilley, Kathryn S.; Marondedze, Claudius

    2013-01-01

    in combination with phosphopeptide enrichment by titanium dioxide (TiO2) and their identification by MS is described. This workflow can be used to gain insights into the role of signalling molecules such as cyclic nucleotides on regulatory networks through

  20. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

    Directory of Open Access Journals (Sweden)

    Gamal Wareth

    2016-04-01

    Full Text Available Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B. species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  1. Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts.

    Science.gov (United States)

    Wareth, Gamal; Eravci, Murat; Weise, Christoph; Roesler, Uwe; Melzer, Falk; Sprague, Lisa D; Neubauer, Heinrich; Murugaiyan, Jayaseelan

    2016-04-30

    Brucellosis is a debilitating zoonotic disease that affects humans and animals. The diagnosis of brucellosis is challenging, as accurate species level identification is not possible with any of the currently available serology-based diagnostic methods. The present study aimed at identifying Brucella (B.) species-specific proteins from the closely related species B. abortus and B. melitensis using sera collected from naturally infected host species. Unlike earlier reported investigations with either laboratory-grown species or vaccine strains, in the present study, field strains were utilized for analysis. The label-free quantitative proteomic analysis of the naturally isolated strains of these two closely related species revealed 402 differentially expressed proteins, among which 63 and 103 proteins were found exclusively in the whole cell extracts of B. abortus and B. melitensis field strains, respectively. The sera from four different naturally infected host species, i.e., cattle, buffalo, sheep, and goat were applied to identify the immune-binding protein spots present in the whole protein extracts from the isolated B. abortus and B. melitensis field strains and resolved on two-dimensional gel electrophoresis. Comprehensive analysis revealed that 25 proteins of B. abortus and 20 proteins of B. melitensis were distinctly immunoreactive. Dihydrodipicolinate synthase, glyceraldehyde-3-phosphate dehydrogenase and lactate/malate dehydrogenase from B. abortus, amino acid ABC transporter substrate-binding protein from B. melitensis and fumarylacetoacetate hydrolase from both species were reactive with the sera of all the tested naturally infected host species. The identified proteins could be used for the design of serological assays capable of detecting pan-Brucella, B. abortus- and B. melitensis-specific antibodies.

  2. Identification of proteins in hyperglycemia and stroke animal models.

    Science.gov (United States)

    Sung, Jin-Hee; Shah, Fawad-Ali; Gim, Sang-Ah; Koh, Phil-Ok

    2016-01-01

    Stroke is a major cause of disability and death in adults. Diabetes mellitus is a metabolic disorder that strongly increases the risk of severe vascular diseases. This study compared changes in proteins of the cerebral cortex during ischemic brain injury between nondiabetic and diabetic animals. Adult male rats were injected with streptozotocin (40 mg/kg) via the intraperitoneal route to induce diabetes and underwent surgical middle cerebral artery occlusion (MCAO) 4 wk after streptozotocin treatment. Cerebral cortex tissues were collected 24 h after MCAO and cerebral cortex proteins were analyzed by two-dimensional gel electrophoresis and mass spectrometry. Several proteins were identified as differentially expressed between nondiabetic and diabetic animals. Among the identified proteins, we focused on the following metabolism-related enzymes: isocitrate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, adenosylhomocysteinase, pyruvate kinase, and glucose-6-phosphate isomerase (neuroleukin). Expression of these proteins was decreased in animals that underwent MCAO. Moreover, protein expression was reduced to a greater extent in diabetic animals than in nondiabetic animals. Reverse transcription-polymerase chain reaction analysis confirmed that the diabetic condition exacerbates the decrease in expression of metabolism-related proteins after MCAO. These results suggest that the diabetic condition may exacerbate brain damage during focal cerebral ischemia through the downregulation of metabolism-related proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Identification and characterization of stable membrane protein complexes

    NARCIS (Netherlands)

    Spelbrink, R.E.J.

    2007-01-01

    Many membrane proteins exist as oligomers. Such oligomers play an important role in a broad variety of cellular processes such as ion transport, energy transduction, osmosensing and cell wall synthesis. We developed an electrophoresis-based method of identifying oligomeric membrane proteins that are

  4. Identification of the protein kinase C phosphorylation site in neuromodulin

    International Nuclear Information System (INIS)

    Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

    1990-01-01

    Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

  5. Identification of proteins regulated by curcumin in cerebral ischemia.

    Science.gov (United States)

    Shah, Fawad-Ali; Gim, Sang-Ah; Sung, Jin-Hee; Jeon, Seong-Jun; Kim, Myeong-Ok; Koh, Phil-Ok

    2016-03-01

    Curcumin is known to have a neuroprotective effect against cerebral ischemia. The objective of this study was to identify various proteins that are differentially expressed by curcumin treatment in focal cerebral ischemia using a proteomic approach. Adult male rats were treated with vehicle or curcumin 1 h after middle cerebral artery occlusion. Brain tissues were collected 24 h after the onset of middle cerebral artery occlusion, and cerebral cortices proteins were identified by two-dimensional gel electrophoresis and mass spectrometry. We detected several proteins with altered expression levels between vehicle- and curcumin-treated animals. Among these proteins, ubiquitin carboxy-terminal hydrolase L1, isocitrate dehydrogenase, adenosylhomocysteinase, and eukaryotic initiation factor 4A were decreased in the vehicle-treated animal, and curcumin treatment attenuated the injury-induced decreases of these proteins. Conversely, pyridoxal phosphate phosphatase was increased in the vehicle-treated animal, and curcumin treatment prevented decreases in this protein. The identified altered proteins are associated with cellular metabolism and differentiation. The results of this study suggest that curcumin exerts a neuroprotective effect by regulating the expression of various proteins in focal cerebral ischemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Identification and cloning of two insecticidal protein genes from ...

    African Journals Online (AJOL)

    Bacillus thuringiensis (Bt) is the most widely applied type of microbial pesticide due to its high specificity and environmental safety. The activity of Bt is largely attributed to the insecticidal crystal protein encoded by the cry genes. Different insecticidal crystal proteins of Bt have different bioactivity against distinct agricultural ...

  7. Serum protein identification and quantification of the corona of 5, 15 and 80 nm gold nanoparticles

    International Nuclear Information System (INIS)

    Schäffler, Martin; Semmler-Behnke, Manuela; Takenaka, Shinji; Wenk, Alexander; Schleh, Carsten; Johnston, Blair D; Kreyling, Wolfgang G; Sarioglu, Hakan; Hauck, Stefanie M

    2013-01-01

    When nanoparticles (NP) enter the body they come into contact with body fluids containing proteins which can adsorb to their surface. These proteins may influence the NP interactions with the biological vicinity, eventually determining their biological fate inside the body. Adsorption of the most abundantly binding proteins was studied after an in vitro 24 hr incubation of monodisperse, negatively charged 5, 15 and 80 nm gold spheres (AuNP) in mouse serum by a two-step analysis: proteomic protein identification and quantitative protein biochemistry. The adsorbed proteins were separated from non-adsorbed proteins by centrifugation and gel electrophoresis and identified using a MALDI-TOF-MS-Proteomics-Analyzer. Quantitative analysis of proteins in gel bands by protein densitometry, required the focus on predominantly binding serum proteins. Numerous proteins adsorbed to the AuNP depending on their size, e.g. apolipoproteins or complement C3. The qualitative and quantitative amount of adsorbed proteins differed between 5, 15 and 80 nm AuNP. Band intensities of adsorbed proteins decreased with increasing AuNP sizes based not only on their mass but also on their surface area. Summarizing, the AuNP surface is covered with serum proteins containing transport and immune related proteins among others. Hence, protein binding depends on the size, surface area and curvature of the AuNP. (paper)

  8. Improved gel electrophoresis matrix for hydrophobic protein separation and identification.

    Science.gov (United States)

    Tokarski, Caroline; Fillet, Marianne; Rolando, Christian

    2011-03-01

    We propose an improved acrylamide gel for the separation of hydrophobic proteins. The separation strategy is based on the incorporation of N-alkylated and N,N'-dialkylated acrylamide monomers in the gel composition in order to increase hydrophobic interactions between the gel matrix and the membrane proteins. Focusing on the most efficient monomer, N,N'-dimethylacrylamide, the potentiality of the new matrix was evaluated on membrane proteins of the human colon HCT-116 cell line. Protein analysis was performed using an adapted analytical strategy based on FT-ICR tandem mass spectrometry. As a result of this comparative study, including advanced reproducibility experiments, more hydrophobic proteins were identified in the new gel (average GRAVY: -0.085) than in the classical gel (average GRAVY: -0.411). Highly hydrophobic peptides were identified reaching a GRAVY value up to 1.450, therefore indicating their probable locations in the membrane. Focusing on predicted transmembrane domains, it can be pointed out that 27 proteins were identified in the hydrophobic gel containing up to 11 transmembrane domains; in the classical gel, only 5 proteins containing 1 transmembrane domain were successfully identified. For example, multiple ionic channels and receptors were characterized in the hydrophobic gel such as the sodium/potassium channel and the glutamate or the transferrin receptors whereas they are traditionally detected using specific enrichment techniques such as immunoprecipitation. In total, membrane proteins identified in the classical gel are well documented in the literature, while most of the membrane proteins only identified on the hydrophobic gel have rarely or never been described using a proteomic-based approach. 2010 Elsevier Inc. All rights reserved.

  9. Identification of proteins interacting with Arabidopsis ACD11

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; Joensen, Jan; McKinney, Lea V

    2009-01-01

    The Arabidopsis ACD11 gene encodes a sphingosine transfer protein and was identified by the accelerated cell death phenotype of the loss of function acd11 mutant, which exhibits heightened expression of genes involved in the disease resistance hypersensitive response (HR). We used ACD11 as bait...... in a yeast two-hybrid screen of an Arabidopsis cDNA library to identify ACD11 interacting proteins. One interactor identified is a protein of unknown function with an RNA recognition motif (RRM) designated BPA1 (binding partner of ACD11). Co-immunoprecipitation experiments confirmed the ACD11-BPA1...

  10. Machine Learning Identification of Protein Properties Useful for Specific Applications

    KAUST Repository

    Khamis, Abdullah M.

    2016-01-01

    Proteins play critical roles in cellular processes of living organisms. It is therefore important to identify and characterize their key properties associated with their functions. Correlating protein’s structural, sequence and physicochemical

  11. Proteomic identification of S-nitrosylated proteins in Arabidopsis

    DEFF Research Database (Denmark)

    Lindermayr, C.; Saalbach, G.; Durner, J.

    2005-01-01

    Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues...... to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S......-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were...

  12. Identification of Proteins in the Exosporium of Bacillus Anthracis

    National Research Council Canada - National Science Library

    Redmond, Caroline; Baillie, Leslie W. J; Hibbs, Stephen; Moir, Arthur J. G; Moir, Anne

    2004-01-01

    .... The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis...

  13. Simple method for identification of plasmid-coded proteins

    International Nuclear Information System (INIS)

    Sancar, A.; Hack, A.M.; Rupp, W.D.

    1979-01-01

    Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA

  14. Systematic identification of proteins that elicit drug side effects

    DEFF Research Database (Denmark)

    Kuhn, Michael; Al Banchaabouchi, Mumna; Campillos, Monica

    2013-01-01

    Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large-scale analysis to systematically predict and characterize proteins...... that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug-target relations to identify overrepresented protein-side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause......) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations....

  15. Identification of De Novo Synthesized and Relatively Older Proteins

    OpenAIRE

    Jaleel, Abdul; Henderson, Gregory C.; Madden, Benjamin J.; Klaus, Katherine A.; Morse, Dawn M.; Gopala, Srinivas; Nair, K. Sreekumaran

    2010-01-01

    OBJECTIVE The accumulation of old and damaged proteins likely contributes to complications of diabetes, but currently no methodology is available to measure the relative age of a specific protein alongside assessment of posttranslational modifications (PTM). To accomplish our goal of studying the impact of insulin deficiency and hyperglycemia in type 1 diabetes upon accumulation of old damaged isoforms of plasma apolipoprotein A-1 (ApoA-1), we sought to develop a novel methodology, which is r...

  16. Discovering functional interdependence relationship in PPI networks for protein complex identification.

    Science.gov (United States)

    Lam, Winnie W M; Chan, Keith C C

    2012-04-01

    Protein molecules interact with each other in protein complexes to perform many vital functions, and different computational techniques have been developed to identify protein complexes in protein-protein interaction (PPI) networks. These techniques are developed to search for subgraphs of high connectivity in PPI networks under the assumption that the proteins in a protein complex are highly interconnected. While these techniques have been shown to be quite effective, it is also possible that the matching rate between the protein complexes they discover and those that are previously determined experimentally be relatively low and the "false-alarm" rate can be relatively high. This is especially the case when the assumption of proteins in protein complexes being more highly interconnected be relatively invalid. To increase the matching rate and reduce the false-alarm rate, we have developed a technique that can work effectively without having to make this assumption. The name of the technique called protein complex identification by discovering functional interdependence (PCIFI) searches for protein complexes in PPI networks by taking into consideration both the functional interdependence relationship between protein molecules and the network topology of the network. The PCIFI works in several steps. The first step is to construct a multiple-function protein network graph by labeling each vertex with one or more of the molecular functions it performs. The second step is to filter out protein interactions between protein pairs that are not functionally interdependent of each other in the statistical sense. The third step is to make use of an information-theoretic measure to determine the strength of the functional interdependence between all remaining interacting protein pairs. Finally, the last step is to try to form protein complexes based on the measure of the strength of functional interdependence and the connectivity between proteins. For performance evaluation

  17. Identification of hierarchy of dynamic domains in proteins: comparison of HDWA and HCCP techniques

    Directory of Open Access Journals (Sweden)

    Yesylevskyy S. O.

    2010-07-01

    Full Text Available Aim. There are several techniques for the identification of hierarchy of dynamic domains in proteins. The goal of this work is to compare systematically two recently developed techniques, HCCP and HDWA,on a set of proteins from diverse structural classes. Methods. HDWA and HCCP techniques are used. The HDWA technique is designed to identify hierarchically organized dynamic domains in proteins using the Molecular Dynamics (MD trajectories, while HCCP utilizes the normal modes of simplified elastic network models. Results. It is shown that the dynamic domains found by HDWA are consistent with the domains identified by HCCP and other techniques. At the same time HDWA identifies flexible mobile loops of proteins correctly, which is hard to achieve with other model-based domain identification techniques. Conclusion. HDWA is shown to be a powerful method of analysis of MD trajectories, which can be used in various areas of protein science.

  18. Identification and characterization of the pseudorabies virus UL43 protein

    International Nuclear Information System (INIS)

    Klupp, Barbara G.; Altenschmidt, Jan; Granzow, Harald; Fuchs, Walter; Mettenleiter, Thomas C.

    2005-01-01

    Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants

  19. Identification and characterization of Euphorbia nivulia latex proteins.

    Science.gov (United States)

    Badgujar, Shamkant B; Mahajan, Raghunath T

    2014-03-01

    The protein profile of latex of Euphorbia nivulia Buch.-Ham. is established. Three new proteins viz., Nivulian-I, II and III have been purified to homogeneity from the latex. The relative molecular masses of Nivulian-I, II and III are 31,486.985, 43,670.846 and 52,803.470 Da respectively. Nivulian-I is a simple type of protein while Nivulian-II and III are glycoproteins. Peptide mass fingerprint analysis revealed peptides of these proteins match with Tubulin alpha-1 chain of Eleusine indica, Maturase K of Banksia quercifolia and hypothetical protein of Zea mays respectively. Tryptic digestion profile of Nivulian-I, II and III, infer the exclusive nature of latex origin proteins and may be new and are additive molecules in the dictionaries of phytoproteins or botany. This is the first of its kind, regarding characterization and validation of Nivulian-I, II and III with respect to peptide sequencing. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Biomarkers for ragwort poisoning in horses: identification of protein targets

    Directory of Open Access Journals (Sweden)

    Beynon Robert J

    2008-08-01

    Full Text Available Abstract Background Ingestion of the poisonous weed ragwort (Senecio jacobea by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. Results One dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin. Conclusion These findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock.

  1. Proteomic identification of secreted proteins of Propionibacterium acnes

    Directory of Open Access Journals (Sweden)

    Holland Carsten

    2010-08-01

    Full Text Available Abstract Background The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. Results Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS. A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, β-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. Conclusions Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence

  2. Identification of Anaplasma marginale type IV secretion system effector proteins.

    Directory of Open Access Journals (Sweden)

    Svetlana Lockwood

    Full Text Available Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS. The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141 of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.

  3. Identification of Redox and Glucose-Dependent Txnip Protein Interactions

    Directory of Open Access Journals (Sweden)

    Benjamin J. Forred

    2016-01-01

    Full Text Available Thioredoxin-interacting protein (Txnip acts as a negative regulator of thioredoxin function and is a critical modulator of several diseases including, but not limited to, diabetes, ischemia-reperfusion cardiac injury, and carcinogenesis. Therefore, Txnip has become an attractive therapeutic target to alleviate disease pathologies. Although Txnip has been implicated with numerous cellular processes such as proliferation, fatty acid and glucose metabolism, inflammation, and apoptosis, the molecular mechanisms underlying these processes are largely unknown. The objective of these studies was to identify Txnip interacting proteins using the proximity-based labeling method, BioID, to understand differential regulation of pleiotropic Txnip cellular functions. The BioID transgene fused to Txnip expressed in HEK293 identified 31 interacting proteins. Many protein interactions were redox-dependent and were disrupted through mutation of a previously described reactive cysteine (C247S. Furthermore, we demonstrate that this model can be used to identify dynamic Txnip interactions due to known physiological regulators such as hyperglycemia. These data identify novel Txnip protein interactions and demonstrate dynamic interactions dependent on redox and glucose perturbations, providing clarification to the pleiotropic cellular functions of Txnip.

  4. Identification and preliminary characterization of protein-cysteine farnesyltransferase

    International Nuclear Information System (INIS)

    Manne, V.; Roberts, D.; Tobin, A.; O'Rourke, E.; Barbacid, M.; De Virgilio, M.; Meyers, C.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, Hsiang-Fu

    1990-01-01

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2 Valendash19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway

  5. Identification of proteins similar to AvrE type III effector proteins from ...

    African Journals Online (AJOL)

    Stephen Opiyo

    GSE22274), and AraCyc databases, we highlighted 16 protein candidates from Arabidopsidis genome .... projection method similar to principal component analysis (PCA) .... RIN4 RIN4 (RPM1 INTERACTING PROTEIN 4); protein binding.

  6. Identification of novel direct protein-protein interactions by irradiating living cells with femtosecond UV laser pulses.

    Science.gov (United States)

    Itri, Francesco; Monti, Daria Maria; Chino, Marco; Vinciguerra, Roberto; Altucci, Carlo; Lombardi, Angela; Piccoli, Renata; Birolo, Leila; Arciello, Angela

    2017-10-07

    The identification of protein-protein interaction networks in living cells is becoming increasingly fundamental to elucidate main biological processes and to understand disease molecular bases on a system-wide level. We recently described a method (LUCK, Laser UV Cross-linKing) to cross-link interacting protein surfaces in living cells by UV laser irradiation. By using this innovative methodology, that does not require any protein modification or cell engineering, here we demonstrate that, upon UV laser irradiation of HeLa cells, a direct interaction between GAPDH and alpha-enolase was "frozen" by a cross-linking event. We validated the occurrence of this direct interaction by co-immunoprecipitation and Immuno-FRET analyses. This represents a proof of principle of the LUCK capability to reveal direct protein interactions in their physiological environment. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. An effective approach for identification of in vivo protein-DNA binding sites from paired-end ChIP-Seq data

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2010-02-01

    Full Text Available Abstract Background ChIP-Seq, which combines chromatin immunoprecipitation (ChIP with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites. Results Here, we present SIPeS (Site Identification from Paired-end Sequencing, a novel algorithm for precise identification of binding sites from short reads generated by paired-end solexa ChIP-Seq technology. In this paper we used ChIP-Seq data from the Arabidopsis basic helix-loop-helix transcription factor ABORTED MICROSPORES (AMS, which is expressed within the anther during pollen development, the results show that SIPeS has better resolution for binding site identification compared to two existing ChIP-Seq peak detection algorithms, Cisgenome and MACS. Conclusions When compared to Cisgenome and MACS, SIPeS shows better resolution for binding site discovery. Moreover, SIPeS is designed to calculate the mappable genome length accurately with the fragment length based on the paired-end reads. Dynamic baselines are also employed to effectively discriminate closely adjacent binding sites, for effective binding sites discovery, which is of particular value when working with high-density genomes.

  8. Identification of cancer protein biomarkers using proteomic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Mor, Gil G.; Ward, David C.; Bray-Ward, Patricia

    2016-10-18

    The claimed invention describes methods to diagnose or aid in the diagnosis of cancer. The claimed methods are based on the identification of biomarkers which are particularly well suited to discriminate between cancer subjects and healthy subjects. These biomarkers were identified using a unique and novel screening method described herein. The biomarkers identified herein can also be used in the prognosis and monitoring of cancer. The invention comprises the use of leptin, prolactin, OPN and IGF-II for diagnosing, prognosis and monitoring of ovarian cancer.

  9. Identification of O-GlcNAcylated proteins in Plasmodium falciparum.

    Science.gov (United States)

    Kupferschmid, Mattis; Aquino-Gil, Moyira Osny; Shams-Eldin, Hosam; Schmidt, Jörg; Yamakawa, Nao; Krzewinski, Frédéric; Schwarz, Ralph T; Lefebvre, Tony

    2017-11-29

    Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans. The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS). While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins. This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

  10. Protein social behavior makes a stronger signal for partner identification than surface geometry

    Science.gov (United States)

    Laine, Elodie

    2016-01-01

    ABSTRACT Cells are interactive living systems where proteins movements, interactions and regulation are substantially free from centralized management. How protein physico‐chemical and geometrical properties determine who interact with whom remains far from fully understood. We show that characterizing how a protein behaves with many potential interactors in a complete cross‐docking study leads to a sharp identification of its cellular/true/native partner(s). We define a sociability index, or S‐index, reflecting whether a protein likes or not to pair with other proteins. Formally, we propose a suitable normalization function that accounts for protein sociability and we combine it with a simple interface‐based (ranking) score to discriminate partners from non‐interactors. We show that sociability is an important factor and that the normalization permits to reach a much higher discriminative power than shape complementarity docking scores. The social effect is also observed with more sophisticated docking algorithms. Docking conformations are evaluated using experimental binding sites. These latter approximate in the best possible way binding sites predictions, which have reached high accuracy in recent years. This makes our analysis helpful for a global understanding of partner identification and for suggesting discriminating strategies. These results contradict previous findings claiming the partner identification problem being solvable solely with geometrical docking. Proteins 2016; 85:137–154. © 2016 Wiley Periodicals, Inc. PMID:27802579

  11. Identification of structural domains in proteins by a graph heuristic

    NARCIS (Netherlands)

    Wernisch, Lorenz; Hunting, M.M.G.; Wodak, Shoshana J.

    1999-01-01

    A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain

  12. Detection and partial identification of proteins in pearls formed in ...

    African Journals Online (AJOL)

    They were ground into a powder of >10,000 mesh followed by ultra-sonication and extraction in water for 4 h at room temperature. ... that one protein had significant sequence homology to a putative vitelline envelop receptor for lysine in the common marine mussel Mytilus edulis, and the other to the putative imaginal disc ...

  13. Identification of differentially expressed proteins in vitamin B 12

    Directory of Open Access Journals (Sweden)

    Swati Varshney

    2015-01-01

    Full Text Available Background: Vitamin B 12 (cobalamin is a water-soluble vitamin generally synthesized by microorganisms. Mammals cannot synthesize this vitamin but have evolved processes for absorption, transport and cellular uptake of this vitamin. Only about 30% of vitamin B 12 , which is bound to the protein transcobalamin (TC (Holo-TC [HoloTC] enters into the cell and hence is referred to as the biologically active form of vitamin B 12 . Vitamin B 12 deficiency leads to several complex disorders, including neurological disorders and anemia. We had earlier shown that vitamin B 12 deficiency is associated with coronary artery disease (CAD in Indian population. In the current study, using a proteomics approach we identified proteins that are differentially expressed in the plasma of individuals with low HoloTC levels. Materials and Methods: We used isobaric-tagging method of relative and absolute quantitation to identify proteins that are differently expressed in individuals with low HoloTC levels when compared to those with normal HoloTC level. Results: In two replicate isobaric tags for relative and absolute quantitation experiments several proteins involved in lipid metabolism, blood coagulation, cholesterol metabolic process, and lipoprotein metabolic process were found to be altered in individuals having low HoloTC levels. Conclusions: Our study indicates that low HoloTc levels could be a risk factor in the development of CAD.

  14. Biomarkers of Aspergillus spores: Strain typing and protein identification

    Czech Academy of Sciences Publication Activity Database

    Šulc, Miroslav; Pešlová, Kateřina; Žabka, Martin; Hajdúch, M.; Havlíček, Vladimír

    2009-01-01

    Roč. 280, 1-3 (2009), s. 162-168 ISSN 1387-3806 R&D Projects: GA MŠk LC07017; GA ČR GP203/05/P575 Institutional research plan: CEZ:AV0Z50200510 Keywords : aspergillus * spore * protein Subject RIV: EE - Microbiology, Virology Impact factor: 2.117, year: 2009

  15. Towards identification of oesophageal gland proteins in Globodera rostochiensis

    NARCIS (Netherlands)

    Boer, de J.M.

    1996-01-01


    Secretory proteins from the dorsal and subventral oesophageal glands of potato cyst- nematodes (Globodera rostochiensis and G.pallida ) are considered to play an important role in the induction and exploitation of the

  16. Identification of cell wall-associated proteins from Phytophthora ramorum

    NARCIS (Netherlands)

    Meijer, H.J.G.; Vondervoort, van de P.J.I.; Yin, Q.Y.; Koster, de C.G.; Klis, F.M.; Govers, F.; Groot, de P.W.J.

    2006-01-01

    The oomycete genus Phytophthora comprises a large group of fungal-like plant pathogens. Two Phytophthora genomes recently have been sequenced; one of them is the genome of Phytophthora ramorum, the causal agent of sudden oak death. During plant infection, extracellular proteins, either soluble

  17. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry

    DEFF Research Database (Denmark)

    Ho, Yuen; Gruhler, Albrecht; Heilbut, Adrian

    2002-01-01

    The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects...... as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were...... identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two...

  18. Identification of proteins similar to AvrE type III effector proteins from ...

    African Journals Online (AJOL)

    Type III effector proteins are injected into host cells through type III secretion systems. Some effectors are similar to host proteins to promote pathogenicity, while others lead to the activation of disease resistance. We used partial least squares alignment-free bioinformatics methods to identify proteins similar to AvrE proteins ...

  19. Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

    Science.gov (United States)

    Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

    2018-02-20

    Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

  20. Proteome scale identification, classification and structural analysis of iron-binding proteins in bread wheat.

    Science.gov (United States)

    Verma, Shailender Kumar; Sharma, Ankita; Sandhu, Padmani; Choudhary, Neha; Sharma, Shailaja; Acharya, Vishal; Akhter, Yusuf

    2017-05-01

    Bread wheat is one of the major staple foods of worldwide population and iron plays a significant role in growth and development of the plant. In this report, we are presenting the genome wide identification of iron-binding proteins in bread wheat. The wheat genome derived putative proteome was screened for identification of iron-binding sequence motifs. Out of 602 putative iron-binding proteins, 130 were able to produce reliable structural models by homology techniques and further analyzed for the presence of iron-binding structural motifs. The computationally identified proteins appear to bind to ferrous and ferric ions and showed diverse coordination geometries. Glu, His, Asp and Cys amino acid residues were found to be mostly involved in iron binding. We have classified these proteins on the basis of their localization in the different cellular compartments. The identified proteins were further classified into their protein folds, families and functional classes ranging from structure maintenance of cellular components, regulation of gene expression, post translational modification, membrane proteins, enzymes, signaling and storage proteins. This comprehensive report regarding structural iron binding proteome provides useful insights into the diversity of iron binding proteins of wheat plants and further utilized to study their roles in plant growth, development and physiology. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Identification and validation of novel small proteins in Pseudomonas putida

    DEFF Research Database (Denmark)

    Yang, Xiaochen; Ingemann Jensen, Sheila; Wulff, Tune

    2016-01-01

    Small proteins of fifty amino acids or less have been understudied due to difficulties that impede their annotation and detection. In order to obtain information on small open reading frames (sORFs) in P. putida, bioinformatic and proteomic approaches were used to identify putative small open...... reading frames (sORFs) in the well-characterized strain KT2440. A plasmid-based system was established for sORF validation, enabling expression of C-terminal sequential peptide affinity (SPA) tagged variants and their detection via protein immunoblotting. Out of 22 tested putative sORFs, the expression...... of fourteen sORFs was confirmed, where all except one are novel. All of the validated sORFs except one are located adjacent to annotated genes on the same strand and three are in close proximity to genes with known functions. These include an ABC transporter operon and the two transcriptional regulators Fis...

  2. Identification and Characterization of Perinucleolar Compartment-Associated Protein

    National Research Council Canada - National Science Library

    Leary, Daniel

    2002-01-01

    .... hSof1, like fibrillarin, localizes to both the nucleolus and nucleoplasm. However, unlike fibrillarin, hSof1 is also in the granular component of nucleoli and responds differently to the inhibition of the transcription of pre- rRNA. In addition, hSof1 -GFP also exhibits a higher nuclear mobility than fibrillarin-GFP and is a nucleocytoplasmic shuttling protein.

  3. LSM Proteins Provide Accurate Splicing and Decay of Selected Transcripts to Ensure Normal Arabidopsis Development[W

    Science.gov (United States)

    Perea-Resa, Carlos; Hernández-Verdeja, Tamara; López-Cobollo, Rosa; Castellano, María del Mar; Salinas, Julio

    2012-01-01

    In yeast and animals, SM-like (LSM) proteins typically exist as heptameric complexes and are involved in different aspects of RNA metabolism. Eight LSM proteins, LSM1 to 8, are highly conserved and form two distinct heteroheptameric complexes, LSM1-7 and LSM2-8,that function in mRNA decay and splicing, respectively. A search of the Arabidopsis thaliana genome identifies 11 genes encoding proteins related to the eight conserved LSMs, the genes encoding the putative LSM1, LSM3, and LSM6 proteins being duplicated. Here, we report the molecular and functional characterization of the Arabidopsis LSM gene family. Our results show that the 11 LSM genes are active and encode proteins that are also organized in two different heptameric complexes. The LSM1-7 complex is cytoplasmic and is involved in P-body formation and mRNA decay by promoting decapping. The LSM2-8 complex is nuclear and is required for precursor mRNA splicing through U6 small nuclear RNA stabilization. More importantly, our results also reveal that these complexes are essential for the correct turnover and splicing of selected development-related mRNAs and for the normal development of Arabidopsis. We propose that LSMs play a critical role in Arabidopsis development by ensuring the appropriate development-related gene expression through the regulation of mRNA splicing and decay. PMID:23221597

  4. Accurate protein structure annotation through competitive diffusion of enzymatic functions over a network of local evolutionary similarities.

    Directory of Open Access Journals (Sweden)

    Eric Venner

    Full Text Available High-throughput Structural Genomics yields many new protein structures without known molecular function. This study aims to uncover these missing annotations by globally comparing select functional residues across the structural proteome. First, Evolutionary Trace Annotation, or ETA, identifies which proteins have local evolutionary and structural features in common; next, these proteins are linked together into a proteomic network of ETA similarities; then, starting from proteins with known functions, competing functional labels diffuse link-by-link over the entire network. Every node is thus assigned a likelihood z-score for every function, and the most significant one at each node wins and defines its annotation. In high-throughput controls, this competitive diffusion process recovered enzyme activity annotations with 99% and 97% accuracy at half-coverage for the third and fourth Enzyme Commission (EC levels, respectively. This corresponds to false positive rates 4-fold lower than nearest-neighbor and 5-fold lower than sequence-based annotations. In practice, experimental validation of the predicted carboxylesterase activity in a protein from Staphylococcus aureus illustrated the effectiveness of this approach in the context of an increasingly drug-resistant microbe. This study further links molecular function to a small number of evolutionarily important residues recognizable by Evolutionary Tracing and it points to the specificity and sensitivity of functional annotation by competitive global network diffusion. A web server is at http://mammoth.bcm.tmc.edu/networks.

  5. Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

    International Nuclear Information System (INIS)

    Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki

    2006-01-01

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus

  6. Identification of a 5-protein biomarker molecular signature for predicting Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Martín Gómez Ravetti

    Full Text Available BACKGROUND: Alzheimer's disease (AD is a progressive brain disease with a huge cost to human lives. The impact of the disease is also a growing concern for the governments of developing countries, in particular due to the increasingly high number of elderly citizens at risk. Alzheimer's is the most common form of dementia, a common term for memory loss and other cognitive impairments. There is no current cure for AD, but there are drug and non-drug based approaches for its treatment. In general the drug-treatments are directed at slowing the progression of symptoms. They have proved to be effective in a large group of patients but success is directly correlated with identifying the disease carriers at its early stages. This justifies the need for timely and accurate forms of diagnosis via molecular means. We report here a 5-protein biomarker molecular signature that achieves, on average, a 96% total accuracy in predicting clinical AD. The signature is composed of the abundances of IL-1alpha, IL-3, EGF, TNF-alpha and G-CSF. METHODOLOGY/PRINCIPAL FINDINGS: Our results are based on a recent molecular dataset that has attracted worldwide attention. Our paper illustrates that improved results can be obtained with the abundance of only five proteins. Our methodology consisted of the application of an integrative data analysis method. This four step process included: a abundance quantization, b feature selection, c literature analysis, d selection of a classifier algorithm which is independent of the feature selection process. These steps were performed without using any sample of the test datasets. For the first two steps, we used the application of Fayyad and Irani's discretization algorithm for selection and quantization, which in turn creates an instance of the (alpha-beta-k-Feature Set problem; a numerical solution of this problem led to the selection of only 10 proteins. CONCLUSIONS/SIGNIFICANCE: the previous study has provided an extremely

  7. Identification of a putative protein-profile associating with tamoxifen therapy-resistance in breast cancer

    NARCIS (Netherlands)

    A. Umar (Arzu); J.W.M. Martens (John); J.A. Foekens (John); L. Paša-Tolić (Ljiljana); H. Kang; A.M. Timmermans (Mieke); M.P. Look (Maxime); M.E. Meijer van Gelder (Marion); N. Jaitly (Navdeep); M.A. den Bakker (Michael)

    2009-01-01

    textabstractTamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical parameters can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards

  8. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners.

    Science.gov (United States)

    Boulon, Séverine; Ahmad, Yasmeen; Trinkle-Mulcahy, Laura; Verheggen, Céline; Cobley, Andy; Gregor, Peter; Bertrand, Edouard; Whitehorn, Mark; Lamond, Angus I

    2010-05-01

    The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static "bead proteomes." The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.

  9. Performance of VITEK mass spectrometry V3.0 for rapid identification of clinical Aspergillus fumigatus in different culture conditions based on ribosomal proteins.

    Science.gov (United States)

    Zhou, Longrong; Chen, Yongquan; Xu, Yuanhong

    2017-01-01

    Fast and accurate discrimination of Aspergillus fumigatus is significant, since misidentification may lead to inappropriate clinical therapy. This study assessed VITEK mass spectrometry (MS) V3.0 for A. fumigatus identification using extracted fungal ribosomal proteins. A total of 52 isolates preliminarily identified as A. fumigatus by traditional morphological methods were inoculated in three different culture media and cultured at two different temperatures. The specific spectral fingerprints of different culture time points (48, 72, 96, and 120 h) were obtained. Of all strains, 88.5% (46/52) were discriminated as A. fumigatus , while the remaining 11.5% (6/52) produced results inconsistent with morphological analysis. Molecular sequencing, as a reference method for species identification, was used to validate the morphological analysis and matrix-assisted laser desorption/ionization time of flight MS. Chi-square tests ( χ 2 test, P =0.05) demonstrated that the culture medium and incubation temperature had no effects on identification accuracy; however, identification accuracy of the strains in the 48-h group was lower than that in other groups. In addition, we found that ribosomal proteins extracted from A. fumigatus can be stored in different environments for at least 1 week, with their profiles remaining stable and strain identification results showing no change. This is beneficial for medical institutions with no mass spectrometer at hand. Overall, this study showed the powerful ability of VITEK MS V 3.0 in identifying A. fumigatus .

  10. Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

    Directory of Open Access Journals (Sweden)

    Gao Fan

    2012-01-01

    Full Text Available Abstract Background To characterize the human humoral immune response against enterovirus 71 (EV71 infection and map human epitopes on the viral capsid proteins. Methods A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3 of BJ08 strain (genomic C4 were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl. Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel. Results A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3 were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15 was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71 were identified and mapped to VP1. Conclusion These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.

  11. Identification and characterization of immunogenic proteins of Mycoplasma genitalium

    DEFF Research Database (Denmark)

    Svenstrup, Helle Friis; Jensen, J.S.; Gevaert, K.

    2006-01-01

    serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed....... genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit...

  12. Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    Directory of Open Access Journals (Sweden)

    Daniel O Connor

    Full Text Available Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

  13. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein

    International Nuclear Information System (INIS)

    Dooley, J.S.G.; Trust, T.J.

    1988-01-01

    The surface protein composition of members of a serogroup of Aeromonas hydrophila was examined. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed a 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125 I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size required for A. hydrophila S-layer anchoring. The native S layer was permeable to 125 I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behavior characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein

  14. Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages

    NARCIS (Netherlands)

    Vlaming, M.L.; Duijn, E. van; Dillingh, M.R.; Brands, R.; Windhorst, A.D.; Hendrikse, N.H.; Bosgra, S.; Burggraaf, J.; Koning, M.C. de; Fidder, A.; Mocking, J.A.; Sandman, H.; Ligt, R.A. de; Fabriek, B.O.; Pasman, W.J.; Seinen, W.; Alves, T.; Carrondo, M.; Peixoto, C.; Peeters, P.A.; Vaes, W.H.

    2015-01-01

    Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of

  15. Small acid soluble proteins for rapid spore identification.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  16. Verification of Ribosomal Proteins of Aspergillus fumigatus for Use as Biomarkers in MALDI-TOF MS Identification.

    Science.gov (United States)

    Nakamura, Sayaka; Sato, Hiroaki; Tanaka, Reiko; Yaguchi, Takashi

    2016-01-01

    We have previously proposed a rapid identification method for bacterial strains based on the profiles of their ribosomal subunit proteins (RSPs), observed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method can perform phylogenetic characterization based on the mass of housekeeping RSP biomarkers, ideally calculated from amino acid sequence information registered in public protein databases. With the aim of extending its field of application to medical mycology, this study investigates the actual state of information of RSPs of eukaryotic fungi registered in public protein databases through the characterization of ribosomal protein fractions extracted from genome-sequenced Aspergillus fumigatus strains Af293 and A1163 as a model. In this process, we have found that the public protein databases harbor problems. The RSP names are in confusion, so we have provisionally unified them using the yeast naming system. The most serious problem is that many incorrect sequences are registered in the public protein databases. Surprisingly, more than half of the sequences are incorrect, due chiefly to mis-annotation of exon/intron structures. These errors could be corrected by a combination of in silico inspection by sequence homology analysis and MALDI-TOF MS measurements. We were also able to confirm conserved post-translational modifications in eleven RSPs. After these verifications, the masses of 31 expressed RSPs under 20,000 Da could be accurately confirmed. These RSPs have a potential to be useful biomarkers for identifying clinical isolates of A. fumigatus .

  17. RCK: accurate and efficient inference of sequence- and structure-based protein-RNA binding models from RNAcompete data.

    Science.gov (United States)

    Orenstein, Yaron; Wang, Yuhao; Berger, Bonnie

    2016-06-15

    Protein-RNA interactions, which play vital roles in many processes, are mediated through both RNA sequence and structure. CLIP-based methods, which measure protein-RNA binding in vivo, suffer from experimental noise and systematic biases, whereas in vitro experiments capture a clearer signal of protein RNA-binding. Among them, RNAcompete provides binding affinities of a specific protein to more than 240 000 unstructured RNA probes in one experiment. The computational challenge is to infer RNA structure- and sequence-based binding models from these data. The state-of-the-art in sequence models, Deepbind, does not model structural preferences. RNAcontext models both sequence and structure preferences, but is outperformed by GraphProt. Unfortunately, GraphProt cannot detect structural preferences from RNAcompete data due to the unstructured nature of the data, as noted by its developers, nor can it be tractably run on the full RNACompete dataset. We develop RCK, an efficient, scalable algorithm that infers both sequence and structure preferences based on a new k-mer based model. Remarkably, even though RNAcompete data is designed to be unstructured, RCK can still learn structural preferences from it. RCK significantly outperforms both RNAcontext and Deepbind in in vitro binding prediction for 244 RNAcompete experiments. Moreover, RCK is also faster and uses less memory, which enables scalability. While currently on par with existing methods in in vivo binding prediction on a small scale test, we demonstrate that RCK will increasingly benefit from experimentally measured RNA structure profiles as compared to computationally predicted ones. By running RCK on the entire RNAcompete dataset, we generate and provide as a resource a set of protein-RNA structure-based models on an unprecedented scale. Software and models are freely available at http://rck.csail.mit.edu/ bab@mit.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by

  18. Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein

    International Nuclear Information System (INIS)

    Lee, G.; Ronai, Z.A.; Pincus, M.R.; Brandt-Rauf, P.W.; Weinstein, I.B.; Murphy, R.B.; Delohery, T.M.; Nishimura, S.; Yamaizumi, Z.

    1989-01-01

    An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with modified p21 protein, the cells were pulsed with [ 35 S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21 - protein complexes. By using this technique, the authors found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. They suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes

  19. Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

    Science.gov (United States)

    Lee, G; Ronai, Z A; Pincus, M R; Brandt-Rauf, P W; Murphy, R B; Delohery, T M; Nishimura, S; Yamaizumi, Z; Weinstein, I B

    1989-11-01

    An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

  20. Computational Prediction of Human Salivary Proteins from Blood Circulation and Application to Diagnostic Biomarker Identification

    Science.gov (United States)

    Wang, Jiaxin; Liang, Yanchun; Wang, Yan; Cui, Juan; Liu, Ming; Du, Wei; Xu, Ying

    2013-01-01

    Proteins can move from blood circulation into salivary glands through active transportation, passive diffusion or ultrafiltration, some of which are then released into saliva and hence can potentially serve as biomarkers for diseases if accurately identified. We present a novel computational method for predicting salivary proteins that come from circulation. The basis for the prediction is a set of physiochemical and sequence features we found to be discerning between human proteins known to be movable from circulation to saliva and proteins deemed to be not in saliva. A classifier was trained based on these features using a support-vector machine to predict protein secretion into saliva. The classifier achieved 88.56% average recall and 90.76% average precision in 10-fold cross-validation on the training data, indicating that the selected features are informative. Considering the possibility that our negative training data may not be highly reliable (i.e., proteins predicted to be not in saliva), we have also trained a ranking method, aiming to rank the known salivary proteins from circulation as the highest among the proteins in the general background, based on the same features. This prediction capability can be used to predict potential biomarker proteins for specific human diseases when coupled with the information of differentially expressed proteins in diseased versus healthy control tissues and a prediction capability for blood-secretory proteins. Using such integrated information, we predicted 31 candidate biomarker proteins in saliva for breast cancer. PMID:24324552

  1. Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family

    NARCIS (Netherlands)

    Titorenko, Vladimir I.; Evers, Melchior E.; Diesel, Andre; Samyn, Bart; Beeumen, Josef van; Roggenkamp, Rainer; Kiel, Jan A.K.W.; Klei, Ida J. van der; Veenhuis, Marten

    We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was

  2. PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

    Science.gov (United States)

    Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

    2016-07-08

    The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Identification of proteins in the postsynaptic density fraction by mass spectrometry

    DEFF Research Database (Denmark)

    Walikonis, R S; Jensen, Ole Nørregaard; Mann, M

    2000-01-01

    Our understanding of the organization of postsynaptic signaling systems at excitatory synapses has been aided by the identification of proteins in the postsynaptic density (PSD) fraction, a subcellular fraction enriched in structures with the morphology of PSDs. In this study, we have completed...... not previously known to be constituents of the PSD fraction and 24 that had previously been associated with the PSD by other methods. The newly identified proteins include the heavy chain of myosin-Va (dilute myosin), a motor protein thought to be involved in vesicle trafficking, and the mammalian homolog...

  4. Efficient identification of critical residues based only on protein structure by network analysis.

    Directory of Open Access Journals (Sweden)

    Michael P Cusack

    2007-05-01

    Full Text Available Despite the increasing number of published protein structures, and the fact that each protein's function relies on its three-dimensional structure, there is limited access to automatic programs used for the identification of critical residues from the protein structure, compared with those based on protein sequence. Here we present a new algorithm based on network analysis applied exclusively on protein structures to identify critical residues. Our results show that this method identifies critical residues for protein function with high reliability and improves automatic sequence-based approaches and previous network-based approaches. The reliability of the method depends on the conformational diversity screened for the protein of interest. We have designed a web site to give access to this software at http://bis.ifc.unam.mx/jamming/. In summary, a new method is presented that relates critical residues for protein function with the most traversed residues in networks derived from protein structures. A unique feature of the method is the inclusion of the conformational diversity of proteins in the prediction, thus reproducing a basic feature of the structure/function relationship of proteins.

  5. Identification of Genetic on Blood Serum Protein of Prolific Ewes

    Science.gov (United States)

    Sutiyono; Ondho, Y. S.; Setiatin, E. T.; Sutopo; Laily, A. N.; Prasetyowati, D. E.; Noviani, F.

    2018-02-01

    The aim of the research was to identify the genetic specification of blood plasma protein in ewes that are prolific. The material of study of local sheep in Bawen and Jambu Sub-district of Semarang Regency is 132 which is determined by purposive sampling that have been give lambing three times. Ewes were divided into three groups that always has a single child (L1), ever had twins (L2) and twins more than two (LM2). Blood sampling was performed using dispossible syringe in jugular vein as much as 5 ml per ewe. Blood plasma was analyzed by Polyacrylamide Gel Electrophoresis-Thin Layer (PAGETLE) method in Biochemistry Laboratory of Veterinary Faculty of Gadjah Mada University. Data analysis is using descriptive statistics and the laws of equilibrium Hardy-Weberg. The research parameters were comparison type of ewes and frequency genetic of protein of blood serum. The results showed that the parent comparisons of L1, L2 and LM2 were 66 (50.00%), 49 (37.12%) and 17 (12.88%), respectively. The frequency genes haven a high propensity to relationship of prolificacy nature parent are Pal2, AlbB, CPF, TFB, PTFS and AmlB on pointes, 67.65, 55.88, 91.17, 70.59, 79.41 and 91.18%. Conclusion the mostly LM2 ewes have genotypes Pal1Pal2, AlbBAlbC, CpFCpF, TfATfB, PtfSPtfS and AmlBAmlB whit frequency are 52.94%, 52.94%, 88.24, 47.06, 64.71 and 88.24% respectively.

  6. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells

    Science.gov (United States)

    Lake, Michael P.; Bouchard, Louis-S.

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging. PMID:28636640

  7. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells.

    Science.gov (United States)

    Lake, Michael P; Bouchard, Louis-S

    2017-01-01

    Transmission electron microscopy (TEM) can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC) as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.

  8. SNP Data Quality Control in a National Beef and Dairy Cattle System and Highly Accurate SNP Based Parentage Verification and Identification

    Directory of Open Access Journals (Sweden)

    Matthew C. McClure

    2018-03-01

    Full Text Available A major use of genetic data is parentage verification and identification as inaccurate pedigrees negatively affect genetic gain. Since 2012 the international standard for single nucleotide polymorphism (SNP verification in Bos taurus cattle has been the ISAG SNP panels. While these ISAG panels provide an increased level of parentage accuracy over microsatellite markers (MS, they can validate the wrong parent at ≤1% misconcordance rate levels, indicating that more SNP are needed if a more accurate pedigree is required. With rapidly increasing numbers of cattle being genotyped in Ireland that represent 61 B. taurus breeds from a wide range of farm types: beef/dairy, AI/pedigree/commercial, purebred/crossbred, and large to small herd size the Irish Cattle Breeding Federation (ICBF analyzed different SNP densities to determine that at a minimum ≥500 SNP are needed to consistently predict only one set of parents at a ≤1% misconcordance rate. For parentage validation and prediction ICBF uses 800 SNP (ICBF800 selected based on SNP clustering quality, ISAG200 inclusion, call rate (CR, and minor allele frequency (MAF in the Irish cattle population. Large datasets require sample and SNP quality control (QC. Most publications only deal with SNP QC via CR, MAF, parent-progeny conflicts, and Hardy-Weinberg deviation, but not sample QC. We report here parentage, SNP QC, and a genomic sample QC pipelines to deal with the unique challenges of >1 million genotypes from a national herd such as SNP genotype errors from mis-tagging of animals, lab errors, farm errors, and multiple other issues that can arise. We divide the pipeline into two parts: a Genotype QC and an Animal QC pipeline. The Genotype QC identifies samples with low call rate, missing or mixed genotype classes (no BB genotype or ABTG alleles present, and low genotype frequencies. The Animal QC handles situations where the genotype might not belong to the listed individual by identifying: >1 non

  9. Fast and Accurate Protein False Discovery Rates on Large-Scale Proteomics Data Sets with Percolator 3.0

    Science.gov (United States)

    The, Matthew; MacCoss, Michael J.; Noble, William S.; Käll, Lukas

    2016-11-01

    Percolator is a widely used software tool that increases yield in shotgun proteomics experiments and assigns reliable statistical confidence measures, such as q values and posterior error probabilities, to peptides and peptide-spectrum matches (PSMs) from such experiments. Percolator's processing speed has been sufficient for typical data sets consisting of hundreds of thousands of PSMs. With our new scalable approach, we can now also analyze millions of PSMs in a matter of minutes on a commodity computer. Furthermore, with the increasing awareness for the need for reliable statistics on the protein level, we compared several easy-to-understand protein inference methods and implemented the best-performing method—grouping proteins by their corresponding sets of theoretical peptides and then considering only the best-scoring peptide for each protein—in the Percolator package. We used Percolator 3.0 to analyze the data from a recent study of the draft human proteome containing 25 million spectra (PM:24870542). The source code and Ubuntu, Windows, MacOS, and Fedora binary packages are available from http://percolator.ms/ under an Apache 2.0 license.

  10. Ribosomal proteins as biomarkers for bacterial identification by mass spectrometry in the clinical microbiology laboratory.

    Science.gov (United States)

    Suarez, Stéphanie; Ferroni, Agnès; Lotz, Aurélie; Jolley, Keith A; Guérin, Philippe; Leto, Julie; Dauphin, Brunhilde; Jamet, Anne; Maiden, Martin C J; Nassif, Xavier; Armengaud, Jean

    2013-09-01

    Whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640-12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates. © 2013 Elsevier B.V. All rights reserved.

  11. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    2010-07-01

    Full Text Available Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  12. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    Science.gov (United States)

    Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

    2010-07-26

    Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  13. Identification of Arsenic Direct-Binding Proteins in Acute Promyelocytic Leukaemia Cells

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    Tao Zhang

    2015-11-01

    Full Text Available The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS. More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1, heat shock 70 kDa protein 9 (HSPA9 and pyruvate kinase M2 (PKM2, were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL.

  14. PDTD: a web-accessible protein database for drug target identification

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    Gao Zhenting

    2008-02-01

    Full Text Available Abstract Background Target identification is important for modern drug discovery. With the advances in the development of molecular docking, potential binding proteins may be discovered by docking a small molecule to a repository of proteins with three-dimensional (3D structures. To complete this task, a reverse docking program and a drug target database with 3D structures are necessary. To this end, we have developed a web server tool, TarFisDock (Target Fishing Docking http://www.dddc.ac.cn/tarfisdock, which has been used widely by others. Recently, we have constructed a protein target database, Potential Drug Target Database (PDTD, and have integrated PDTD with TarFisDock. This combination aims to assist target identification and validation. Description PDTD is a web-accessible protein database for in silico target identification. It currently contains >1100 protein entries with 3D structures presented in the Protein Data Bank. The data are extracted from the literatures and several online databases such as TTD, DrugBank and Thomson Pharma. The database covers diverse information of >830 known or potential drug targets, including protein and active sites structures in both PDB and mol2 formats, related diseases, biological functions as well as associated regulating (signaling pathways. Each target is categorized by both nosology and biochemical function. PDTD supports keyword search function, such as PDB ID, target name, and disease name. Data set generated by PDTD can be viewed with the plug-in of molecular visualization tools and also can be downloaded freely. Remarkably, PDTD is specially designed for target identification. In conjunction with TarFisDock, PDTD can be used to identify binding proteins for small molecules. The results can be downloaded in the form of mol2 file with the binding pose of the probe compound and a list of potential binding targets according to their ranking scores. Conclusion PDTD serves as a comprehensive and

  15. Use of ribosomal proteins as biomarkers for identification of Flavobacterium psychrophilum by MALDI-TOF mass spectrometry.

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    Fernández-Álvarez, Clara; Torres-Corral, Yolanda; Santos, Ysabel

    2018-01-06

    Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a rapid methodology for identification of bacteria that is increasingly used in diagnostic laboratories. This work aimed at evaluating the potential of MALDI-TOF-MS for identification of the main serotypes of Flavobacterium psychrophilum isolated from salmonids, and its discrimination from closely related Flavobacterium spp. A mass spectra library was constructed by analysing 70 F. psychrophilum strains representing the serotypes O1, O2a, O2b and O3, including reference and clinical isolates. Peak mass lists were examined using the Mass-Up software for the detection of potential biomarkers, similarity and cluster analysis. Fourteen species-identifying biomarkers were detected in all the F. psychrophilum isolates tested, moreover, sets of serotype-identifying biomarkers ions were selected. F. psychrophilum-specific biomarkers were identified as ribosomal proteins by matching with protein databases. Furthermore, sequence variation corresponding to amino acid exchanges in several biomarker proteins were tentatively assigned. Closely related Flavobacterium species (F. flevense, F. succinicans, F. columnare, F. branchiophilum and F. johnsoniae) could be differentiated from F. psychrophilum by defining species identifying biomarkers and hierarchical cluster analysis. These results demonstrated that MALDI-TOF spectrometry represents a powerful tool for an accurate identification of the fish pathogen F. psychrophilum as well as for epidemiological studies. The results obtained in this study demonstrated that MALDI-TOF mass spectrometry represents a powerful tool that can be used by diagnostic laboratories for rapid identification of the fish pathogen Flavobacterium psychrophilum and its differentiation from other Flavobacterium-related species. Analysis of mass peak lists revealed the potential of the MALDI-TOF technique to identify epidemiologically important serotypes affecting

  16. Gene identification and protein classification in microbial metagenomic sequence data via incremental clustering

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    Li Weizhong

    2008-04-01

    Full Text Available Abstract Background The identification and study of proteins from metagenomic datasets can shed light on the roles and interactions of the source organisms in their communities. However, metagenomic datasets are characterized by the presence of organisms with varying GC composition, codon usage biases etc., and consequently gene identification is challenging. The vast amount of sequence data also requires faster protein family classification tools. Results We present a computational improvement to a sequence clustering approach that we developed previously to identify and classify protein coding genes in large microbial metagenomic datasets. The clustering approach can be used to identify protein coding genes in prokaryotes, viruses, and intron-less eukaryotes. The computational improvement is based on an incremental clustering method that does not require the expensive all-against-all compute that was required by the original approach, while still preserving the remote homology detection capabilities. We present evaluations of the clustering approach in protein-coding gene identification and classification, and also present the results of updating the protein clusters from our previous work with recent genomic and metagenomic sequences. The clustering results are available via CAMERA, (http://camera.calit2.net. Conclusion The clustering paradigm is shown to be a very useful tool in the analysis of microbial metagenomic data. The incremental clustering method is shown to be much faster than the original approach in identifying genes, grouping sequences into existing protein families, and also identifying novel families that have multiple members in a metagenomic dataset. These clusters provide a basis for further studies of protein families.

  17. Are neutral loss and internal product ions useful for top-down protein identification?

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

    2017-05-08

    Neutral loss and internal product ions have been found to be significant in both peptide and protein tandem mass spectra and they have been proposed to be included in database search and for protein identification. In addition to common canonical b/y ions in collision-based dissociation or c/z ions in electron-based dissociation, inclusion of neutral loss and internal product ions would certainly make better use of tandem mass spectra data; however, their ultimate utility for protein identification with false discovery rate control remains unclear. Here we report our proteome-level utility benchmarking of neutral loss and internal product ions with tandem mass spectra of intact E. coli proteome. Utility of internal product ions was further evaluated at the protein level using selected tandem mass spectra of individual E. coli proteins. We found that both neutral loss and internal products ions do not have direct utility for protein identification when they were used for scoring of P Score; but they do have indirect utility for provision of more canonical b/y ions when they are included in the database search and overlapping ions between different ion types are resolved. Tandem mass spectrometry has evolved to be a state-of-the-art method for characterization of protein primary structures (including amino acid sequence, post-translational modifications (PTMs) as well as their site location), where full study and utilization tandem mass spectra and product ions are indispensable. This primary structure information is essential for higher order structure and eventual function study of proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

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    Margaret Rohrbaugh

    Full Text Available Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

  19. Identification of physicochemical selective pressure on protein encoding nucleotide sequences

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    Sainudiin Raazesh

    2006-03-01

    Full Text Available Abstract Background Statistical methods for identifying positively selected sites in protein coding regions are one of the most commonly used tools in evolutionary bioinformatics. However, they have been limited by not taking the physiochemical properties of amino acids into account. Results We develop a new codon-based likelihood model for detecting site-specific selection pressures acting on specific physicochemical properties. Nonsynonymous substitutions are divided into substitutions that differ with respect to the physicochemical properties of interest, and those that do not. The substitution rates of these two types of changes, relative to the synonymous substitution rate, are then described by two parameters, γ and ω respectively. The new model allows us to perform likelihood ratio tests for positive selection acting on specific physicochemical properties of interest. The new method is first used to analyze simulated data and is shown to have good power and accuracy in detecting physicochemical selective pressure. We then re-analyze data from the class-I alleles of the human Major Histocompatibility Complex (MHC and from the abalone sperm lysine. Conclusion Our new method allows a more flexible framework to identify selection pressure on particular physicochemical properties.

  20. An approach to large scale identification of non-obvious structural similarities between proteins

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    Cherkasov, Artem; Jones, Steven JM

    2004-01-01

    Background A new sequence independent bioinformatics approach allowing genome-wide search for proteins with similar three dimensional structures has been developed. By utilizing the numerical output of the sequence threading it establishes putative non-obvious structural similarities between proteins. When applied to the testing set of proteins with known three dimensional structures the developed approach was able to recognize structurally similar proteins with high accuracy. Results The method has been developed to identify pathogenic proteins with low sequence identity and high structural similarity to host analogues. Such protein structure relationships would be hypothesized to arise through convergent evolution or through ancient horizontal gene transfer events, now undetectable using current sequence alignment techniques. The pathogen proteins, which could mimic or interfere with host activities, would represent candidate virulence factors. The developed approach utilizes the numerical outputs from the sequence-structure threading. It identifies the potential structural similarity between a pair of proteins by correlating the threading scores of the corresponding two primary sequences against the library of the standard folds. This approach allowed up to 64% sensitivity and 99.9% specificity in distinguishing protein pairs with high structural similarity. Conclusion Preliminary results obtained by comparison of the genomes of Homo sapiens and several strains of Chlamydia trachomatis have demonstrated the potential usefulness of the method in the identification of bacterial proteins with known or potential roles in virulence. PMID:15147578

  1. An approach to large scale identification of non-obvious structural similarities between proteins

    Directory of Open Access Journals (Sweden)

    Cherkasov Artem

    2004-05-01

    Full Text Available Abstract Background A new sequence independent bioinformatics approach allowing genome-wide search for proteins with similar three dimensional structures has been developed. By utilizing the numerical output of the sequence threading it establishes putative non-obvious structural similarities between proteins. When applied to the testing set of proteins with known three dimensional structures the developed approach was able to recognize structurally similar proteins with high accuracy. Results The method has been developed to identify pathogenic proteins with low sequence identity and high structural similarity to host analogues. Such protein structure relationships would be hypothesized to arise through convergent evolution or through ancient horizontal gene transfer events, now undetectable using current sequence alignment techniques. The pathogen proteins, which could mimic or interfere with host activities, would represent candidate virulence factors. The developed approach utilizes the numerical outputs from the sequence-structure threading. It identifies the potential structural similarity between a pair of proteins by correlating the threading scores of the corresponding two primary sequences against the library of the standard folds. This approach allowed up to 64% sensitivity and 99.9% specificity in distinguishing protein pairs with high structural similarity. Conclusion Preliminary results obtained by comparison of the genomes of Homo sapiens and several strains of Chlamydia trachomatis have demonstrated the potential usefulness of the method in the identification of bacterial proteins with known or potential roles in virulence.

  2. Performance of VITEK mass spectrometry V3.0 for rapid identification of clinical Aspergillus fumigatus in different culture conditions based on ribosomal proteins

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    Zhou L

    2017-12-01

    Full Text Available Longrong Zhou, Yongquan Chen, Yuanhong Xu Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Anhui, Hefei, People’s Republic of China Abstract: Fast and accurate discrimination of Aspergillus fumigatus is significant, since misidentification may lead to inappropriate clinical therapy. This study assessed VITEK mass spectrometry (MS V3.0 for A. fumigatus identification using extracted fungal ribosomal proteins. A total of 52 isolates preliminarily identified as A. fumigatus by traditional morphological methods were inoculated in three different culture media and cultured at two different temperatures. The specific spectral fingerprints of different culture time points (48, 72, 96, and 120 h were obtained. Of all strains, 88.5% (46/52 were discriminated as A. fumigatus, while the remaining 11.5% (6/52 produced results inconsistent with morphological analysis. Molecular sequencing, as a reference method for species identification, was used to validate the morphological analysis and matrix-assisted laser desorption/ionization time of flight MS. Chi-square tests (Χ2 test, P=0.05 demonstrated that the culture medium and incubation temperature had no effects on identification accuracy; however, identification accuracy of the strains in the 48-h group was lower than that in other groups. In addition, we found that ribosomal proteins extracted from A. fumigatus can be stored in different environments for at least 1 week, with their profiles remaining stable and strain identification results showing no change. This is beneficial for medical institutions with no mass spectrometer at hand. Overall, this study showed the powerful ability of VITEK MS V 3.0 in identifying A. fumigatus. Keywords: VITEK MS V 3.0, Aspergillus fumigatus, identification, ribosomal protein, spectral fingerprints, fungal, matrix assisted laser desorption ionization-time of flight mass spectrometry, MALDI-TOF MS

  3. Identification of membrane proteins by tandem mass spectrometry of protein ions

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    Carroll, Joe; Altman, Matthew C.; Fearnley, Ian M.; Walker, John E.

    2007-01-01

    The most common way of identifying proteins in proteomic analyses is to use short segments of sequence (“tags”) determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membrane-spanning α-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1–4 transmembrane α-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5–18 transmembrane α-helices) often contain fragment ions from N- and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase. PMID:17720804

  4. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

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    Alves-Ferreira Marcio

    2010-03-01

    Full Text Available Abstract Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR. Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references

  5. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.

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    Artico, Sinara; Nardeli, Sarah M; Brilhante, Osmundo; Grossi-de-Sa, Maria Fátima; Alves-Ferreira, Marcio

    2010-03-21

    Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1alpha5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhbetaTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in

  6. ModFOLD6: an accurate web server for the global and local quality estimation of 3D protein models.

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    Maghrabi, Ali H A; McGuffin, Liam J

    2017-07-03

    Methods that reliably estimate the likely similarity between the predicted and native structures of proteins have become essential for driving the acceptance and adoption of three-dimensional protein models by life scientists. ModFOLD6 is the latest version of our leading resource for Estimates of Model Accuracy (EMA), which uses a pioneering hybrid quasi-single model approach. The ModFOLD6 server integrates scores from three pure-single model methods and three quasi-single model methods using a neural network to estimate local quality scores. Additionally, the server provides three options for producing global score estimates, depending on the requirements of the user: (i) ModFOLD6_rank, which is optimized for ranking/selection, (ii) ModFOLD6_cor, which is optimized for correlations of predicted and observed scores and (iii) ModFOLD6 global for balanced performance. The ModFOLD6 methods rank among the top few for EMA, according to independent blind testing by the CASP12 assessors. The ModFOLD6 server is also continuously automatically evaluated as part of the CAMEO project, where significant performance gains have been observed compared to our previous server and other publicly available servers. The ModFOLD6 server is freely available at: http://www.reading.ac.uk/bioinf/ModFOLD/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Identification of lipopolysaccharide-interacting plasma membrane-type proteins in Arabidopsis thaliana.

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    Vilakazi, Cornelius S; Dubery, Ian A; Piater, Lizelle A

    2017-02-01

    Lipopolysaccharide (LPS) is an amphiphatic bacterial glycoconjugate found on the external membrane of Gram-negative bacteria. This endotoxin is considered as a microbe-associated molecular pattern (MAMP) molecule and has been shown to elicit defense responses in plants. Here, LPS-interacting proteins from Arabidopsis thaliana plasma membrane (PM)-type fractions were captured and identified in order to investigate those involved in LPS perception and linked to triggering of innate immune responses. A novel proteomics-based affinity-capture strategy coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for the enrichment and identification of LPS-interacting proteins. As such, LPS isolated from Burkholderia cepacia (LPS B.cep. ) was immobilized on three independent and distinct affinity-based matrices to serve as bait for interacting proteins from A. thaliana leaf and callus tissue. These were resolved by 1D electrophoresis and identified by mass spectrometry. Proteins specifically bound to LPS B.cep. have been implicated in membrane structure (e.g. COBRA-like and tubulin proteins), membrane trafficking and/or transport (e.g. soluble NSF attachment protein receptor (SNARE) proteins, patellin, aquaporin, PM instrinsic proteins (PIP) and H + -ATPase), signal transduction (receptor-like kinases and calcium-dependent protein kinases) as well as defense/stress responses (e.g. hypersensitive-induced response (HIR) proteins, jacalin-like lectin domain-containing protein and myrosinase-binding proteins). The novel affinity-capture strategy for the enrichment of LPS-interacting proteins proved to be effective, especially in the binding of proteins involved in plant defense responses, and can thus be used to elucidate LPS-mediated molecular recognition and disease mechanism(s). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. MASCOT HTML and XML parser: an implementation of a novel object model for protein identification data.

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    Yang, Chunguang G; Granite, Stephen J; Van Eyk, Jennifer E; Winslow, Raimond L

    2006-11-01

    Protein identification using MS is an important technique in proteomics as well as a major generator of proteomics data. We have designed the protein identification data object model (PDOM) and developed a parser based on this model to facilitate the analysis and storage of these data. The parser works with HTML or XML files saved or exported from MASCOT MS/MS ions search in peptide summary report or MASCOT PMF search in protein summary report. The program creates PDOM objects, eliminates redundancy in the input file, and has the capability to output any PDOM object to a relational database. This program facilitates additional analysis of MASCOT search results and aids the storage of protein identification information. The implementation is extensible and can serve as a template to develop parsers for other search engines. The parser can be used as a stand-alone application or can be driven by other Java programs. It is currently being used as the front end for a system that loads HTML and XML result files of MASCOT searches into a relational database. The source code is freely available at http://www.ccbm.jhu.edu and the program uses only free and open-source Java libraries.

  9. Processed Meat Protein and Heat-Stable Peptide Marker Identification Using Microwave-Assisted Tryptic Digestion

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    Magdalena Montowska

    2016-01-01

    Full Text Available New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages. Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed aft er 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

  10. A Robust Identification of the Protein Standard Bands in Two-Dimensional Electrophoresis Gel Images

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    Serackis Artūras

    2017-12-01

    Full Text Available The aim of the investigation presented in this paper was to develop a software-based assistant for the protein analysis workflow. The prior characterization of the unknown protein in two-dimensional electrophoresis gel images is performed according to the molecular weight and isoelectric point of each protein spot estimated from the gel image before further sequence analysis by mass spectrometry. The paper presents a method for automatic and robust identification of the protein standard band in a two-dimensional gel image. In addition, the method introduces the identification of the positions of the markers, prepared by using pre-selected proteins with known molecular mass. The robustness of the method was achieved by using special validation rules in the proposed original algorithms. In addition, a self-organizing map-based decision support algorithm is proposed, which takes Gabor coefficients as image features and searches for the differences in preselected vertical image bars. The experimental investigation proved the good performance of the new algorithms included into the proposed method. The detection of the protein standard markers works without modification of algorithm parameters on two-dimensional gel images obtained by using different staining and destaining procedures, which results in different average levels of intensity in the images.

  11. Identification of Differentially Abundant Proteins of Edwardsiella ictaluri during Iron Restriction.

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    Pradeep R Dumpala

    Full Text Available Edwardsiella ictaluri is a Gram-negative facultative anaerobe intracellular bacterium that causes enteric septicemia in channel catfish. Iron is an essential inorganic nutrient of bacteria and is crucial for bacterial invasion. Reduced availability of iron by the host may cause significant stress for bacterial pathogens and is considered a signal that leads to significant alteration in virulence gene expression. However, the precise effect of iron-restriction on E. ictaluri protein abundance is unknown. The purpose of this study was to identify differentially abundant proteins of E. ictaluri during in vitro iron-restricted conditions. We applied two-dimensional difference in gel electrophoresis (2D-DIGE for determining differentially abundant proteins and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS for protein identification. Gene ontology and pathway-based functional modeling of differentially abundant proteins was also conducted. A total of 50 unique differentially abundant proteins at a minimum of 2-fold (p ≤ 0.05 difference in abundance due to iron-restriction were detected. The numbers of up- and down-regulated proteins were 37 and 13, respectively. We noted several proteins, including EsrB, LamB, MalM, MalE, FdaA, and TonB-dependent heme/hemoglobin receptor family proteins responded to iron restriction in E. ictaluri.

  12. Identification of structural protein-protein interactions of herpes simplex virus type 1.

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    Lee, Jin H; Vittone, Valerio; Diefenbach, Eve; Cunningham, Anthony L; Diefenbach, Russell J

    2008-09-01

    In this study we have defined protein-protein interactions between the structural proteins of herpes simplex virus type 1 (HSV-1) using a LexA yeast two-hybrid system. The majority of the capsid, tegument and envelope proteins of HSV-1 were screened in a matrix approach. A total of 40 binary interactions were detected including 9 out of 10 previously identified tegument-tegument interactions (Vittone, V., Diefenbach, E., Triffett, D., Douglas, M.W., Cunningham, A.L., and Diefenbach, R.J., 2005. Determination of interactions between tegument proteins of herpes simplex virus type 1. J. Virol. 79, 9566-9571). A total of 12 interactions involving the capsid protein pUL35 (VP26) and 11 interactions involving the tegument protein pUL46 (VP11/12) were identified. The most significant novel interactions detected in this study, which are likely to play a role in viral assembly, include pUL35-pUL37 (capsid-tegument), pUL46-pUL37 (tegument-tegument) and pUL49 (VP22)-pUS9 (tegument-envelope). This information will provide further insights into the pathways of HSV-1 assembly and the identified interactions are potential targets for new antiviral drugs.

  13. Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

    Science.gov (United States)

    Fu, Zongming; Yan, Kun; Rosenberg, Avraham; Jin, Zhicheng; Crain, Barbara; Athas, Grace; Heide, Richard S Vander; Howard, Timothy; Everett, Allen D; Herrington, David; Van Eyk, Jennifer E

    2013-04-01

    Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Novel Accurate Bacterial Discrimination by MALDI-Time-of-Flight MS Based on Ribosomal Proteins Coding in S10-spc-alpha Operon at Strain Level S10-GERMS

    Science.gov (United States)

    Tamura, Hiroto; Hotta, Yudai; Sato, Hiroaki

    2013-08-01

    Matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is one of the most widely used mass-based approaches for bacterial identification and classification because of the simple sample preparation and extremely rapid analysis within a few minutes. To establish the accurate MALDI-TOF MS bacterial discrimination method at strain level, the ribosomal subunit proteins coded in the S 10-spc-alpha operon, which encodes half of the ribosomal subunit protein and is highly conserved in eubacterial genomes, were selected as reliable biomarkers. This method, named the S10-GERMS method, revealed that the strains of genus Pseudomonas were successfully identified and discriminated at species and strain levels, respectively; therefore, the S10-GERMS method was further applied to discriminate the pathovar of P. syringae. The eight selected biomarkers (L24, L30, S10, S12, S14, S16, S17, and S19) suggested the rapid discrimination of P. syringae at the strain (pathovar) level. The S10-GERMS method appears to be a powerful tool for rapid and reliable bacterial discrimination and successful phylogenetic characterization. In this article, an overview of the utilization of results from the S10-GERMS method is presented, highlighting the characterization of the Lactobacillus casei group and discrimination of the bacteria of genera Bacillus and Sphingopyxis despite only two and one base difference in the 16S rRNA gene sequence, respectively.

  15. Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

    Science.gov (United States)

    Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang; Groves, Jay T; Miller, Thomas; Shan, Shu-Ou

    2017-07-28

    The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

  16. Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis

    Energy Technology Data Exchange (ETDEWEB)

    Gaur, Vineet; Sethi, Dhruv K.; Salunke, Dinakar M., E-mail: dinakar@nii.res.in [National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067 (India)

    2008-01-01

    The purification, identification, crystallization and preliminary crystallographic studies of an allergy-related protein, Pru du amandin, from P. dulcis nuts are reported. Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P4{sub 1} (or P4{sub 3}), with unit-cell parameters a = b = 150.7, c = 164.9 Å.

  17. Identification and modification of dynamical regions in proteins for alteration of enzyme catalytic effect

    Science.gov (United States)

    Agarwal, Pratul K.

    2013-04-09

    A method for analysis, control, and manipulation for improvement of the chemical reaction rate of a protein-mediated reaction is provided. Enzymes, which typically comprise protein molecules, are very efficient catalysts that enhance chemical reaction rates by many orders of magnitude. Enzymes are widely used for a number of functions in chemical, biochemical, pharmaceutical, and other purposes. The method identifies key protein vibration modes that control the chemical reaction rate of the protein-mediated reaction, providing identification of the factors that enable the enzymes to achieve the high rate of reaction enhancement. By controlling these factors, the function of enzymes may be modulated, i.e., the activity can either be increased for faster enzyme reaction or it can be decreased when a slower enzyme is desired. This method provides an inexpensive and efficient solution by utilizing computer simulations, in combination with available experimental data, to build suitable models and investigate the enzyme activity.

  18. Proteomic platform for the identification of proteins in olive (Olea europaea) pulp.

    Science.gov (United States)

    Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; Piovesana, Susy; Samperi, Roberto; Stampachiacchiere, Serena; Laganà, Aldo

    2013-10-24

    The nutritional and cancer-protective properties of the oil extracted mechanically from the ripe fruits of Olea europaea trees are attracting constantly more attention worldwide. The preparation of high-quality protein samples from plant tissues for proteomic analysis poses many challenging problems. In this study we employed a proteomic platform based on two different extraction methods, SDS and CHAPS based protocols, followed by two precipitation protocols, TCA/acetone and MeOH precipitation, in order to increase the final number of identified proteins. The use of advanced MS techniques in combination with the Swissprot and NCBI Viridiplantae databases and TAIR10 Arabidopsis database allowed us to identify 1265 proteins, of which 22 belong to O. europaea. The application of this proteomic platform for protein extraction and identification will be useful also for other proteomic studies on recalcitrant plant/fruit tissues. Copyright © 2013. Published by Elsevier B.V.

  19. Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis

    International Nuclear Information System (INIS)

    Gaur, Vineet; Sethi, Dhruv K.; Salunke, Dinakar M.

    2007-01-01

    The purification, identification, crystallization and preliminary crystallographic studies of an allergy-related protein, Pru du amandin, from P. dulcis nuts are reported. Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P4 1 (or P4 3 ), with unit-cell parameters a = b = 150.7, c = 164.9 Å

  20. Investigation and identification of functional post-translational modification sites associated with drug binding and protein-protein interactions.

    Science.gov (United States)

    Su, Min-Gang; Weng, Julia Tzu-Ya; Hsu, Justin Bo-Kai; Huang, Kai-Yao; Chi, Yu-Hsiang; Lee, Tzong-Yi

    2017-12-21

    tools for exploring the structural characteristics of PTMs, is presented. In addition, all tertiary structures of PTM sites on proteins can be visualized using the JSmol program. Resolving the function of PTM sites is important for understanding the role that proteins play in biological mechanisms. Our work attempted to delineate the structural correlation between PTM sites and PPI or drug-target binding. CurxPTM could help scientists narrow the scope of their PTM research and enhance the efficiency of PTM identification in the face of big proteome data. CruxPTM is now available at http://csb.cse.yzu.edu.tw/CruxPTM/ .

  1. Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water

    International Nuclear Information System (INIS)

    Dalvit, Claudio; Pevarello, Paolo; Tato, Marco; Veronesi, Marina; Vulpetti, Anna; Sundstroem, Michael

    2000-01-01

    A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described. The method exploits efficiently the large reservoir of H 2 O magnetization. The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds. Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented

  2. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus

    Directory of Open Access Journals (Sweden)

    Beuy Joob

    2014-12-01

    Full Text Available The drug searching for combating the present outbreak of Ebola virus infection is the urgent activity at present. Finding the new effective drug at present must base on the molecular analysis of the pathogenic virus. The in-depth analysis of the viral protein to find the binding site, active pocket is needed. Here, the authors analyzed the envelope glycoprotein GP2 from Ebola virus. Identification of active pocket and protein druggability within envelope glycoprotein GP2 from Ebola virus was done. According to this assessment, 7 active pockets with varied druggability could be identified.

  3. Identification of Pentatricopeptide Repeat Proteins in the Model Organism Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Sam Manna

    2013-01-01

    Full Text Available Pentatricopeptide repeat (PPR proteins are RNA binding proteins with functions in organelle RNA metabolism. They are found in all eukaryotes but have been most extensively studied in plants. We report on the identification of 12 PPR-encoding genes in the genome of the protist Dictyostelium discoideum, with potential homologs in other members of the same lineage and some predicted novel functions for the encoded gene products in protists. For one of the gene products, we show that it localizes to the mitochondria, and we also demonstrate that antisense inhibition of its expression leads to slower growth, a phenotype associated with mitochondrial dysfunction.

  4. Identification of FUSE-binding proteins as interacting partners of TIA proteins

    International Nuclear Information System (INIS)

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

    2006-01-01

    TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

  5. Identification of Abiotic Stress Protein Biomarkers by Proteomic Screening of Crop Cultivar Diversity

    OpenAIRE

    Barkla, Bronwyn J.

    2016-01-01

    Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may cont...

  6. Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

    Science.gov (United States)

    Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2013-07-01

    Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

  7. AFM-based identification of the dynamic properties of globular proteins: simulation study

    International Nuclear Information System (INIS)

    Kim, Deok Ho; Park, Jung Yul; Kim, Moon K.; Hong, Keum Shik

    2008-01-01

    Nowadays a mathematical model-based computational approach is getting more attention as an effective tool for understanding the mechanical behaviors of biological systems. To find the mechanical properties of the proteins required to build such a model, this paper investigates a real-time identification method based on an AFM nanomanipulation system. First, an AFM-based bio-characterization system is introduced. Second, a second-order time-varying linear model representing the interaction between an AFM cantilever and globular proteins in a solvent is presented. Finally, we address a real-time estimation method in which the results of AFM experiments are designed to be inputs of the state estimator proposed here. Our attention is restricted to a theoretical feasibility analysis of the proposed methodology. We simply set the mechanical properties of the particular protein such as mass, stiffness, and damping coefficient in the system model prior to running the simulation. Simulation results show very good agreement with the preset properties. We anticipate that the realization of the AFM-based bio-characterization system will also provide an experimental validation of the proposed identification procedure in the future. This methodology can be used to determine a model of protein motion for the purpose of computer simulation and for a real-time modification of protein deformation

  8. Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila.

    Directory of Open Access Journals (Sweden)

    Wenhan Zhu

    2011-03-01

    Full Text Available A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.

  9. Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo.

    Science.gov (United States)

    Komar, Dorota N; Mouriz, Alfonso; Jarillo, José A; Piñeiro, Manuel

    2016-01-14

    Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

  10. Rapid and accurate identification of isolates of Candida species by melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA)

    NARCIS (Netherlands)

    Decat, E.; van Mechelen, E.; Saerens, B.; Vermeulen, S.J.T.; Boekhout, T.; de Blaiser, S.; Vaneechoutte, M.; Deschaght, P.

    2013-01-01

    Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA) as an identification

  11. Identification of cell wall proteins in the flax (Linum usitatissimum) stem.

    Science.gov (United States)

    Day, Arnaud; Fénart, Stéphane; Neutelings, Godfrey; Hawkins, Simon; Rolando, Christian; Tokarski, Caroline

    2013-03-01

    Sequential salt (CaCl2 , LiCl) extractions were used to obtain fractions enriched in cell wall proteins (CWPs) from the stem of 60-day-old flax (Linum usitatissimum) plants. High-resolution FT-ICR MS analysis and the use of recently published genomic data allowed the identification of 11 912 peptides corresponding to a total of 1418 different proteins. Subcellular localization using TargetP, Predotar, and WoLF PSORT led to the identification of 152 putative flax CWPs that were classified into nine different functional classes previously established for Arabidopsis thaliana. Examination of different functional classes revealed the presence of a number of proteins known to be involved in, or potentially involved in cell-wall metabolism in plants. The flax stem cell wall proteome was also compared with transcriptomic data previously obtained on comparable samples. This study represents a major contribution to the identification of CWPs in flax and will lead to a better understanding of cell wall biology in this species. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Preliminary identification of secreted proteins by Leptospira interrogans serovar Kennewicki strain Pomona Fromm

    International Nuclear Information System (INIS)

    Ricardi, L.M.P.; Portaro, F.C.; Abreu, P.A.E.; Barbosa, A.S.; Morais, Z.M.; Vasconcellos, S.A.

    2012-01-01

    Full text: This project aimed to identify secreted proteins by pathogenic Leptospira interrogans serovar Kennewicki strain Pomona Fromm (LPF) by proteomic analyses. The strain LPF, whose virulence was maintained by passages in hamsters, were cultured in EMJH medium. The supernatants were centrifuged, dialyzed and subjected to lyophilization. Protein samples were resolved first by IEF at pH 3 to 10, immobilized pH gradient 13-cm strips. Strips were then processed for the second-dimension separation on SDS-polyacrylamide gels. Proteins from gel spots were subjected to reduction, cysteine-alkylation, and in-gel tryptic digestion, and analyzed by LC/MS/MS spectrometry. Liquid chromatography-based separation followed by automated tandem mass spectrometry was also used to identify secreted proteins. In silico analyses were performed using the PSORTbV.3.0 program and SignalP server. One major obstacle to secretome studies is the difficulty to obtain extracts of secreted proteins without citoplasmatic contamination. In addition, the extraction of low concentration proteins from large volumes of culture media, which are rich in salts, BSA and other compounds, frequently interfere with most proteomics techniques. For these reasons, several experimental approaches were used to optimize the protocol applied. In spite of this fact, our analysis resulted in the identification of 200 proteins with high confidence. Only 5 of 63 secreted proteins predicted by in silico analysis were found. Other classes identified included proteins that possess signal peptide but whose cellular localization prediction is unknown or may have multiple localization sites, and proteins that lack signal peptide and are thus thought to be secreted via non conventional mechanisms or resulting from cytoplasmic contamination by cell lysis. Many of these are hypothetical proteins with no putative conserved domains detected. To our knowledge, this is the first study to identify secreted proteins by

  13. Preliminary identification of secreted proteins by Leptospira interrogans serovar Kennewicki strain Pomona Fromm

    Energy Technology Data Exchange (ETDEWEB)

    Ricardi, L.M.P.; Portaro, F.C.; Abreu, P.A.E.; Barbosa, A.S. [Instituto Butantan, Sao Paulo, SP (Brazil); Morais, Z.M.; Vasconcellos, S.A. [Universidade de Sao Paulo (USP), SP (Brazil)

    2012-07-01

    Full text: This project aimed to identify secreted proteins by pathogenic Leptospira interrogans serovar Kennewicki strain Pomona Fromm (LPF) by proteomic analyses. The strain LPF, whose virulence was maintained by passages in hamsters, were cultured in EMJH medium. The supernatants were centrifuged, dialyzed and subjected to lyophilization. Protein samples were resolved first by IEF at pH 3 to 10, immobilized pH gradient 13-cm strips. Strips were then processed for the second-dimension separation on SDS-polyacrylamide gels. Proteins from gel spots were subjected to reduction, cysteine-alkylation, and in-gel tryptic digestion, and analyzed by LC/MS/MS spectrometry. Liquid chromatography-based separation followed by automated tandem mass spectrometry was also used to identify secreted proteins. In silico analyses were performed using the PSORTbV.3.0 program and SignalP server. One major obstacle to secretome studies is the difficulty to obtain extracts of secreted proteins without citoplasmatic contamination. In addition, the extraction of low concentration proteins from large volumes of culture media, which are rich in salts, BSA and other compounds, frequently interfere with most proteomics techniques. For these reasons, several experimental approaches were used to optimize the protocol applied. In spite of this fact, our analysis resulted in the identification of 200 proteins with high confidence. Only 5 of 63 secreted proteins predicted by in silico analysis were found. Other classes identified included proteins that possess signal peptide but whose cellular localization prediction is unknown or may have multiple localization sites, and proteins that lack signal peptide and are thus thought to be secreted via non conventional mechanisms or resulting from cytoplasmic contamination by cell lysis. Many of these are hypothetical proteins with no putative conserved domains detected. To our knowledge, this is the first study to identify secreted proteins by

  14. PredPPCrys: accurate prediction of sequence cloning, protein production, purification and crystallization propensity from protein sequences using multi-step heterogeneous feature fusion and selection.

    Directory of Open Access Journals (Sweden)

    Huilin Wang

    Full Text Available X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed 'PredPPCrys' using the support vector machine (SVM. Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I. Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II, which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization

  15. Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

    Science.gov (United States)

    Hu, Shuiwang; Musante, Luca; Tataruch, Dorota; Xu, Xiaomeng; Kretz, Oliver; Henry, Michael; Meleady, Paula; Luo, Haihua; Zou, Hequn; Jiang, Yong; Holthofer, Harry

    2018-01-05

    Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally

  16. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    Science.gov (United States)

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  17. Identification of Newly Synthesized Proteins by Echinococcus granulosus Protoscoleces upon Induction of Strobilation.

    Directory of Open Access Journals (Sweden)

    João Antonio Debarba

    2015-09-01

    Full Text Available The proteins responsible for the key molecular events leading to the structural changes between the developmental stages of Echinococcus granulosus remain unknown. In this work, azidohomoalanine (AHA-specific labeling was used to identify proteins expressed by E. granulosus protoscoleces (PSCs upon the induction of strobilar development.The in vitro incorporation of AHA with different tags into newly synthesized proteins (NSPs by PSCs was analyzed using SDS-PAGE and confocal microscopy. The LC-MS/MS analysis of AHA-labeled NSPs by PSCs undergoing strobilation allowed for the identification of 365 proteins, of which 75 were differentially expressed in comparison between the presence or absence of strobilation stimuli and 51 were expressed exclusively in either condition. These proteins were mainly involved in metabolic, regulatory and signaling processes.After the controlled-labeling of proteins during the induction of strobilar development, we identified modifications in protein expression. The changes in the metabolism and the activation of control and signaling pathways may be important for the correct parasite development and be target for further studies.

  18. Bacillus anthracis secretome time course under host-simulated conditions and identification of immunogenic proteins

    Directory of Open Access Journals (Sweden)

    Whittington Jessica

    2007-07-01

    accumulation may be relevant in elucidation of the progression of pathogenicity, identification of therapeutics and diagnostic markers, and vaccine development. This study also adds to the continuously growing list of identified Bacillus anthracis secretome proteins.

  19. Multi-Segment Direct Inject nano-ESI-LTQ-FT-ICR-MS/MS For Protein Identification

    Directory of Open Access Journals (Sweden)

    Neal Rachel E

    2011-07-01

    Full Text Available Abstract Reversed phase high performance liquid chromatography (HPLC interfaced to electrospray tandem mass spectrometry (MS/MS is commonly used for the identification of peptides from proteolytically cleaved proteins embedded in a polyacrylamide gel matrix as well as for metabolomics screening. HPLC separations are time consuming (30-60 min average, costly (columns and mobile phase reagents, and carry the risk of column carry over between samples. The use of a chip-based nano-ESI platform (Advion NanoMate based on replaceable nano-tips for sample introduction eliminates sample cross-contamination, provides unchanging sample matrix, and enhances spray stability with attendant increases in reproducibility. Recent papers have established direct infusion nano-ESI-MS/MS utilizing the NanoMate for protein identification of gel spots based on full range MS scans with data dependent MS/MS. In a full range scan, discontinuous ion suppression due to sample matrix can impair identification of putative mass features of interest in both the proteomic and metabolomic workflows. In the current study, an extension of an established direct inject nano-ESI-MS/MS method is described that utilizes the mass filtering capability of an ion-trap for ion packet separation into four narrow mass ranges (50 amu overlap with segment specific dynamic data dependent peak inclusion for MS/MS fragmentation (total acquisition time of 3 minutes. Comparison of this method with a more traditional nanoLC-MS/MS based protocol utilizing solvent/sample stream splitting to achieve nanoflow demonstrated comparable results for protein identification from polyacrylamide gel matrices. The advantages of this method include full automation, lack of cross-contamination, low cost, and high throughput.

  20. Identification and characterization of RBM44 as a novel intercellular bridge protein.

    Directory of Open Access Journals (Sweden)

    Tokuko Iwamori

    2011-02-01

    Full Text Available Intercellular bridges are evolutionarily conserved structures that connect differentiating germ cells. We previously reported the identification of TEX14 as the first essential intercellular bridge protein, the demonstration that intercellular bridges are required for male fertility, and the finding that intercellular bridges utilize components of the cytokinesis machinery to form. Herein, we report the identification of RNA binding motif protein 44 (RBM44 as a novel germ cell intercellular bridge protein. RBM44 was identified by proteomic analysis after intercellular bridge enrichment using TEX14 as a marker protein. RBM44 is highly conserved between mouse and human and contains an RNA recognition motif of unknown function. RBM44 mRNA is enriched in testis, and immunofluorescence confirms that RBM44 is an intercellular bridge component. However, RBM44 only partially localizes to TEX14-positive intercellular bridges. RBM44 is expressed most highly in pachytene and secondary spermatocytes, but disappears abruptly in spermatids. We discovered that RBM44 interacts with itself and TEX14 using yeast two-hybrid, mammalian two-hybrid, and immunoprecipitation. To define the in vivo function of RBM44, we generated a targeted deletion of Rbm44 in mice. Rbm44 null male mice produce somewhat increased sperm, and show enhanced fertility of unknown etiology. Thus, although RBM44 localizes to intercellular bridges during meiosis, RBM44 is not required for fertility in contrast to TEX14.

  1. enDNA-Prot: Identification of DNA-Binding Proteins by Applying Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Ruifeng Xu

    2014-01-01

    Full Text Available DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97–9.52% in ACC and 0.08–0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83–16.63% in terms of ACC and 0.02–0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public.

  2. Proteomic Investigation of Falciparum and Vivax Malaria for Identification of Surrogate Protein Markers

    Science.gov (United States)

    Ray, Sandipan; Renu, Durairaj; Srivastava, Rajneesh; Gollapalli, Kishore; Taur, Santosh; Jhaveri, Tulip; Dhali, Snigdha; Chennareddy, Srinivasarao; Potla, Ankit; Dikshit, Jyoti Bajpai; Srikanth, Rapole; Gogtay, Nithya; Thatte, Urmila; Patankar, Swati; Srivastava, Sanjeeva

    2012-01-01

    This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM) (n = 20), vivax malaria (VM) (n = 17) and healthy controls (HC) (n = 20) were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC). Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05) serum proteins were identified in FM and VM respectively, and almost half (46.2%) of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC) were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC) curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates

  3. Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

    Science.gov (United States)

    Nongonierma, Alice B; FitzGerald, Richard J

    2018-06-01

    Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

  4. Proteomic investigation of falciparum and vivax malaria for identification of surrogate protein markers.

    Directory of Open Access Journals (Sweden)

    Sandipan Ray

    Full Text Available This study was conducted to analyze alterations in the human serum proteome as a consequence of infection by malaria parasites Plasmodium falciparum and P. vivax to obtain mechanistic insights about disease pathogenesis, host immune response, and identification of potential protein markers. Serum samples from patients diagnosed with falciparum malaria (FM (n = 20, vivax malaria (VM (n = 17 and healthy controls (HC (n = 20 were investigated using multiple proteomic techniques and results were validated by employing immunoassay-based approaches. Specificity of the identified malaria related serum markers was evaluated by means of analysis of leptospirosis as a febrile control (FC. Compared to HC, 30 and 31 differentially expressed and statistically significant (p<0.05 serum proteins were identified in FM and VM respectively, and almost half (46.2% of these proteins were commonly modulated due to both of the plasmodial infections. 13 proteins were found to be differentially expressed in FM compared to VM. Functional pathway analysis involving the identified proteins revealed the modulation of different vital physiological pathways, including acute phase response signaling, chemokine and cytokine signaling, complement cascades and blood coagulation in malaria. A panel of identified proteins consists of six candidates; serum amyloid A, hemopexin, apolipoprotein E, haptoglobin, retinol-binding protein and apolipoprotein A-I was used to build statistical sample class prediction models. By employing PLS-DA and other classification methods the clinical phenotypic classes (FM, VM, FC and HC were predicted with over 95% prediction accuracy. Individual performance of three classifier proteins; haptoglobin, apolipoprotein A-I and retinol-binding protein in diagnosis of malaria was analyzed using receiver operating characteristic (ROC curves. The discrimination of FM, VM, FC and HC groups on the basis of differentially expressed serum proteins demonstrates

  5. Identification of herpesvirus proteins that contribute to G1/S arrest.

    Science.gov (United States)

    Paladino, Patrick; Marcon, Edyta; Greenblatt, Jack; Frappier, Lori

    2014-04-01

    Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins

  6. MALDI-TOF MS is more accurate than VITEK II ANC card and API Rapid ID 32 A system for the identification of Clostridium species.

    Science.gov (United States)

    Kim, Young Jin; Kim, Si Hyun; Park, Hyun-Jung; Park, Hae-Geun; Park, Dongchul; Song, Sae Am; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kook, Joong-Ki; Kim, Hye Ran; Shin, Jeong Hwan

    2016-08-01

    All 50 Clostridium difficile strains were definitely identified by Vitek2 system, Rapid ID 32A system, and MALDI-TOF. For 18 non-difficile Clostridium strains, the identification results were correct in 0, 2, and 17 strains by Vitek2, Rapid ID 32A, and MALDI-TOF, respectively. MALDI-TOF could be used as the primary tool for identification of Clostridium species. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. The Identification and Validation of Novel Small Proteins in Pseudomonas Putida KT-2440

    DEFF Research Database (Denmark)

    Yang, Xiaochen; Long, Katherine

    2014-01-01

    and activities and may lead to the discovery of novel antimicrobial agents. Our project focuses on the identification, validation and characterization of novel s-­‐proteins in the bacterium Pseudomonas putida KT-­2440. As there is virtually no information on s-­‐proteins in pseudomonads, the first step......, total protein samples are prepared, fractionated, and analyzed with mass spectrometry (MS/MS). The MS/MS data are compared to a custom database containing >80000 putative sORF sequences to identify candidates for validation. A total of 56 and 22 putative sORFs were obtained from MS/MS data...... and bioinformatics prediction, respectively, where there is no overlap between the putative sORFs obtained from the two approaches. The sequences encoding the putative sORFs will be integrated onto the Tn7 site on the chromosome as well as on a plasmid expression vector for validation....

  8. In silico identification of essential proteins in Corynebacterium pseudotuberculosis based on protein

    DEFF Research Database (Denmark)

    Folador, Edson Luiz; de Carvalho, Paulo Vinícius Sanches Daltro; Silva, Wanderson Marques

    2016-01-01

    BACKGROUND: Corynebacterium pseudotuberculosis (Cp) is a gram-positive bacterium that is classified into equi and ovis serovars. The serovar ovis is the etiological agent of caseous lymphadenitis, a chronic infection affecting sheep and goats, causing economic losses due to carcass condemnation...... of the potential Cp interactome and to identify potentially essential proteins serving as putative drug targets. On average, we predict 16,669 interactions for each of the nine strains (with 15,495 interactions shared among all strains). An in silico sanity check suggests that the potential networks were...... not formed by spurious interactions but have a strong biological bias. With the inferred Cp networks we identify 181 essential proteins, among which 41 are non-host homologous. CONCLUSIONS: The list of candidate interactions of the Cp strains lay the basis for developing novel hypotheses and designing...

  9. Automated protein identification by the combination of MALDI MS and MS/MS spectra from different instruments.

    Science.gov (United States)

    Levander, Fredrik; James, Peter

    2005-01-01

    The identification of proteins separated on two-dimensional gels is most commonly performed by trypsin digestion and subsequent matrix-assisted laser desorption ionization (MALDI) with time-of-flight (TOF). Recently, atmospheric pressure (AP) MALDI coupled to an ion trap (IT) has emerged as a convenient method to obtain tandem mass spectra (MS/MS) from samples on MALDI target plates. In the present work, we investigated the feasibility of using the two methodologies in line as a standard method for protein identification. In this setup, the high mass accuracy MALDI-TOF spectra are used to calibrate the peptide precursor masses in the lower mass accuracy AP-MALDI-IT MS/MS spectra. Several software tools were developed to automate the analysis process. Two sets of MALDI samples, consisting of 142 and 421 gel spots, respectively, were analyzed in a highly automated manner. In the first set, the protein identification rate increased from 61% for MALDI-TOF only to 85% for MALDI-TOF combined with AP-MALDI-IT. In the second data set the increase in protein identification rate was from 44% to 58%. AP-MALDI-IT MS/MS spectra were in general less effective than the MALDI-TOF spectra for protein identification, but the combination of the two methods clearly enhanced the confidence in protein identification.

  10. Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

    Science.gov (United States)

    Manikwar, Prakash; Zimmerman, Tahl; Blanco, Francisco J; Williams, Todd D; Siahaan, Teruna J

    2011-07-20

    Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

  11. Proteomic identification of altered cerebral proteins in the complex regional pain syndrome animal model.

    Science.gov (United States)

    Nahm, Francis Sahngun; Park, Zee-Yong; Nahm, Sang-Soep; Kim, Yong Chul; Lee, Pyung Bok

    2014-01-01

    Complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP) model, a novel experimental model of CRPS. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

  12. Proteomic Identification of Altered Cerebral Proteins in the Complex Regional Pain Syndrome Animal Model

    Directory of Open Access Journals (Sweden)

    Francis Sahngun Nahm

    2014-01-01

    Full Text Available Background. Complex regional pain syndrome (CRPS is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP model, a novel experimental model of CRPS. Materials and Methods. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Conclusion. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS.

  13. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

    Science.gov (United States)

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-01-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  14. Identification of ZASP, a novel protein associated to Zona occludens-2

    Energy Technology Data Exchange (ETDEWEB)

    Lechuga, Susana; Alarcon, Lourdes; Solano, Jesus [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), Mexico, D.F. 07360 (Mexico); Huerta, Miriam; Lopez-Bayghen, Esther [Department of Genetics and Molecular Biology, Center for Research and Advanced Studies (Cinvestav), Mexico, D.F. 07360 (Mexico); Gonzalez-Mariscal, Lorenza, E-mail: lorenza@fisio.cinvestav.mx [Department of Physiology, Biophysics and Neuroscience, Center for Research and Advanced Studies (Cinvestav), Mexico, D.F. 07360 (Mexico)

    2010-11-15

    With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds.

  15. Identification of ZASP, a novel protein associated to Zona occludens-2.

    Science.gov (United States)

    Lechuga, Susana; Alarcón, Lourdes; Solano, Jesús; Huerta, Miriam; Lopez-Bayghen, Esther; González-Mariscal, Lorenza

    2010-11-15

    With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Identification of ZASP, a novel protein associated to Zona occludens-2

    International Nuclear Information System (INIS)

    Lechuga, Susana; Alarcon, Lourdes; Solano, Jesus; Huerta, Miriam; Lopez-Bayghen, Esther; Gonzalez-Mariscal, Lorenza

    2010-01-01

    With the aim of discovering new molecular interactions of the tight junction protein ZO-2, a two-hybrid screen was performed on a human kidney cDNA library using as bait the middle segment of ZO-2. Through this assay we identified a 24-kDa novel protein herein named ZASP for ZO-2 associated speckle protein. ZO-2/ZASP interaction further confirmed by pull down and immunoprecipitation experiments, requires the presence of the intact PDZ binding motif SQV of ZASP and the third PDZ domain of ZO-2. ZASP mRNA and protein are present in the kidney and in several epithelial cell lines. Endogenous ZASP is expressed primarily in nuclear speckles in co-localization with splicing factor SC-35. Nocodazole treatment and wash out reveals that ZASP disappears from the nucleus during mitosis in accordance with speckle disassembly during metaphase. ZASP amino acid sequence exhibits a canonical nuclear exportation signal and in agreement the protein exits the nucleus through a process mediated by exportin/CRM1. ZASP over-expression blocks the inhibitory activity of ZO-2 on cyclin D1 gene transcription and protein expression. The identification of ZASP helps to unfold the complex nuclear molecular arrays that form on ZO-2 scaffolds.

  17. Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense.

    Directory of Open Access Journals (Sweden)

    Peter Sperisen

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  18. Stealth Proteins: In Silico Identification of a Novel Protein Family Rendering Bacterial Pathogens Invisible to Host Immune Defense.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.

  19. Identification, characterization and antigenicity of the Plasmodium vivax rhoptry neck protein 1 (PvRON1

    Directory of Open Access Journals (Sweden)

    Patarroyo Manuel E

    2011-10-01

    Full Text Available Abstract Background Plasmodium vivax malaria remains a major health problem in tropical and sub-tropical regions worldwide. Several rhoptry proteins which are important for interaction with and/or invasion of red blood cells, such as PfRONs, Pf92, Pf38, Pf12 and Pf34, have been described during the last few years and are being considered as potential anti-malarial vaccine candidates. This study describes the identification and characterization of the P. vivax rhoptry neck protein 1 (PvRON1 and examine its antigenicity in natural P. vivax infections. Methods The PvRON1 encoding gene, which is homologous to that encoding the P. falciparum apical sushi protein (ASP according to the plasmoDB database, was selected as our study target. The pvron1 gene transcription was evaluated by RT-PCR using RNA obtained from the P. vivax VCG-1 strain. Two peptides derived from the deduced P. vivax Sal-I PvRON1 sequence were synthesized and inoculated in rabbits for obtaining anti-PvRON1 antibodies which were used to confirm the protein expression in VCG-1 strain schizonts along with its association with detergent-resistant microdomains (DRMs by Western blot, and its localization by immunofluorescence assays. The antigenicity of the PvRON1 protein was assessed using human sera from individuals previously exposed to P. vivax malaria by ELISA. Results In the P. vivax VCG-1 strain, RON1 is a 764 amino acid-long protein. In silico analysis has revealed that PvRON1 shares essential characteristics with different antigens involved in invasion, such as the presence of a secretory signal, a GPI-anchor sequence and a putative sushi domain. The PvRON1 protein is expressed in parasite's schizont stage, localized in rhoptry necks and it is associated with DRMs. Recombinant protein recognition by human sera indicates that this antigen can trigger an immune response during a natural infection with P. vivax. Conclusions This study shows the identification and characterization of

  20. Targeted nanodiamonds for identification of subcellular protein assemblies in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Michael P Lake

    Full Text Available Transmission electron microscopy (TEM can be used to successfully determine the structures of proteins. However, such studies are typically done ex situ after extraction of the protein from the cellular environment. Here we describe an application for nanodiamonds as targeted intensity contrast labels in biological TEM, using the nuclear pore complex (NPC as a model macroassembly. We demonstrate that delivery of antibody-conjugated nanodiamonds to live mammalian cells using maltotriose-conjugated polypropylenimine dendrimers results in efficient localization of nanodiamonds to the intended cellular target. We further identify signatures of nanodiamonds under TEM that allow for unambiguous identification of individual nanodiamonds from a resin-embedded, OsO4-stained environment. This is the first demonstration of nanodiamonds as labels for nanoscale TEM-based identification of subcellular protein assemblies. These results, combined with the unique fluorescence properties and biocompatibility of nanodiamonds, represent an important step toward the use of nanodiamonds as markers for correlated optical/electron bioimaging.

  1. Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

    Science.gov (United States)

    Chouchani, Edward T; James, Andrew M; Methner, Carmen; Pell, Victoria R; Prime, Tracy A; Erickson, Brian K; Forkink, Marleen; Lau, Gigi Y; Bright, Thomas P; Menger, Katja E; Fearnley, Ian M; Krieg, Thomas; Murphy, Michael P

    2017-09-01

    Nitrate (NO 3 - ) and nitrite (NO 2 - ) are known to be cardioprotective and to alter energy metabolism in vivo NO 3 - action results from its conversion to NO 2 - by salivary bacteria, but the mechanism(s) by which NO 2 - affects metabolism remains obscure. NO 2 - may act by S -nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S -nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S -nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO 2 - -dependent S -nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT ( S -nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO 2 - under normoxic conditions or exposure to ischemia alone results in minimal S -nitrosation of protein thiols. However, exposure to NO 2 - in conjunction with ischemia led to extensive S -nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S -nitrosated under these conditions, potentially contributing to the beneficial effects of NO 2 - on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO 2 , but not to NO 2 - , combined with the lack of S -nitrosation during anoxia alone or by NO 2 - during normoxia places constraints on how S -nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S -nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO 2 - exposure. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. A rapid and accurate method for determining protein content in dairy products based on asynchronous-injection alternating merging zone flow-injection spectrophotometry.

    Science.gov (United States)

    Liang, Qin-Qin; Li, Yong-Sheng

    2013-12-01

    An accurate and rapid method and a system to determine protein content using asynchronous-injection alternating merging zone flow-injection spectrophotometry based on reaction between coomassie brilliant blue G250 (CBBG) and protein was established. Main merit of our approach is that it can avoid interferences of other nitric-compounds in samples, such as melamine and urea. Optimized conditions are as follows: Concentrations of CBBG, polyvinyl alcohol (PVA), NaCl and HCl are 150 mg/l, 30 mg/l, 0.1 mol/l and 1.0% (v/v), respectively; volumes of the sample and reagent are 150 μl and 30 μl, respectively; length of a reaction coil is 200 cm; total flow rate is 2.65 ml/min. The linear range of the method is 0.5-15 mg/l (BSA), its detection limit is 0.05 mg/l, relative standard deviation is less than 1.87% (n=11), and analytical speed is 60 samples per hour. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Identification of Protein-Protein Interactions Involved in Pectin Biosynthesis in the golgi Apparatus

    DEFF Research Database (Denmark)

    Lund, Christian Have

    for instance as food additives, nutraceutical, for paper and energy production. Pectin is a cell wall glycan that crucial for every plant growing on land. Pectin is said to be one of the most complex glycans on earth and it is hypothesized that at least 67 enzymatic reactions are involved in its biosynthesis......The plant cell wall surrounds every plant cell and is an essential component that is involved in diverse functions including plant development, morphology, resistance towards plant pathogens etc. The plant cell wall is not only important for the plant. The cell wall has many industrial applications...... the diverse pectin structures for industrial, agronomic and biomedical uses. Increasing evidence suggests that complex formation is important in governing functional coordination of proteins involved in cell wall biosynthesis. In Arabidopsis thaliana, a homogalacturonan (HG) synthase core complex between...

  4. Identification of Protein Complexes from Tandem Affinity Purification/Mass Spectrometry Data via Biased Random Walk.

    Science.gov (United States)

    Cai, Bingjing; Wang, Haiying; Zheng, Huiru; Wang, Hui

    2015-01-01

    Systematic identification of protein complexes from protein-protein interaction networks (PPIs) is an important application of data mining in life science. Over the past decades, various new clustering techniques have been developed based on modelling PPIs as binary relations. Non-binary information of co-complex relations (prey/bait) in PPIs data derived from tandem affinity purification/mass spectrometry (TAP-MS) experiments has been unfairly disregarded. In this paper, we propose a Biased Random Walk based algorithm for detecting protein complexes from TAP-MS data, resulting in the random walk with restarting baits (RWRB). RWRB is developed based on Random walk with restart. The main contribution of RWRB is the incorporation of co-complex relations in TAP-MS PPI networks into the clustering process, by implementing a new restarting strategy during the process of random walk. Through experimentation on un-weighted and weighted TAP-MS data sets, we validated biological significance of our results by mapping them to manually curated complexes. Results showed that, by incorporating non-binary, co-membership information, significant improvement has been achieved in terms of both statistical measurements and biological relevance. Better accuracy demonstrates that the proposed method outperformed several state-of-the-art clustering algorithms for the detection of protein complexes in TAP-MS data.

  5. Computational identification of binding energy hot spots in protein-RNA complexes using an ensemble approach.

    Science.gov (United States)

    Pan, Yuliang; Wang, Zixiang; Zhan, Weihua; Deng, Lei

    2018-05-01

    Identifying RNA-binding residues, especially energetically favored hot spots, can provide valuable clues for understanding the mechanisms and functional importance of protein-RNA interactions. Yet, limited availability of experimentally recognized energy hot spots in protein-RNA crystal structures leads to the difficulties in developing empirical identification approaches. Computational prediction of RNA-binding hot spot residues is still in its infant stage. Here, we describe a computational method, PrabHot (Prediction of protein-RNA binding hot spots), that can effectively detect hot spot residues on protein-RNA binding interfaces using an ensemble of conceptually different machine learning classifiers. Residue interaction network features and new solvent exposure characteristics are combined together and selected for classification with the Boruta algorithm. In particular, two new reference datasets (benchmark and independent) have been generated containing 107 hot spots from 47 known protein-RNA complex structures. In 10-fold cross-validation on the training dataset, PrabHot achieves promising performances with an AUC score of 0.86 and a sensitivity of 0.78, which are significantly better than that of the pioneer RNA-binding hot spot prediction method HotSPRing. We also demonstrate the capability of our proposed method on the independent test dataset and gain a competitive advantage as a result. The PrabHot webserver is freely available at http://denglab.org/PrabHot/. leideng@csu.edu.cn. Supplementary data are available at Bioinformatics online.

  6. Large-scale proteomic identification of S100 proteins in breast cancer tissues

    International Nuclear Information System (INIS)

    Cancemi, Patrizia; Di Cara, Gianluca; Albanese, Nadia Ninfa; Costantini, Francesca; Marabeti, Maria Rita; Musso, Rosa; Lupo, Carmelo; Roz, Elena; Pucci-Minafra, Ida

    2010-01-01

    Attempts to reduce morbidity and mortality in breast cancer is based on efforts to identify novel biomarkers to support prognosis and therapeutic choices. The present study has focussed on S100 proteins as a potentially promising group of markers in cancer development and progression. One reason of interest in this family of proteins is because the majority of the S100 genes are clustered on a region of human chromosome 1q21 that is prone to genomic rearrangements. Moreover, there is increasing evidence that S100 proteins are often up-regulated in many cancers, including breast, and this is frequently associated with tumour progression. Samples of breast cancer tissues were obtained during surgical intervention, according to the bioethical recommendations, and cryo-preserved until used. Tissue extracts were submitted to proteomic preparations for 2D-IPG. Protein identification was performed by N-terminal sequencing and/or peptide mass finger printing. The majority of the detected S100 proteins were absent, or present at very low levels, in the non-tumoral tissues adjacent to the primary tumor. This finding strengthens the role of S100 proteins as putative biomarkers. The proteomic screening of 100 cryo-preserved breast cancer tissues showed that some proteins were ubiquitously expressed in almost all patients while others appeared more sporadic. Most, if not all, of the detected S100 members appeared reciprocally correlated. Finally, from the perspective of biomarkers establishment, a promising finding was the observation that patients which developed distant metastases after a three year follow-up showed a general tendency of higher S100 protein expression, compared to the disease-free group. This article reports for the first time the comparative proteomic screening of several S100 protein members among a large group of breast cancer patients. The results obtained strongly support the hypothesis that a significant deregulation of multiple S100 protein members is

  7. Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

    Directory of Open Access Journals (Sweden)

    Madlung Johannes

    2010-05-01

    Full Text Available Abstract Background Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i. Results The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set. All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase. Conclusions Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

  8. Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma

    DEFF Research Database (Denmark)

    Møller, Holger Jon; Peterslund, Niels Anker; Graversen, Jonas Heilskov

    2002-01-01

    enabled identification of a soluble plasma form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular domain (scavenger receptor cysteine-rich 1-9). A sandwich enzyme-linked immunosorbent assay was established and used to measure the s...... a level of sHbSR above the range of healthy persons. Patients with myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels (up to 67.3 mg/L). In conclusion, sHbSR is an abundant plasma protein potentially valuable in monitoring patients with infections and myelomonocytic leukemia....

  9. Bottom–up protein identifications from microliter quantities of individual human tear samples. Important steps towards clinical relevance.

    Directory of Open Access Journals (Sweden)

    Peter Raus

    2015-12-01

    With 375 confidently identified proteins in the healthy adult tear, the obtained results are comprehensive and in large agreement with previously published observations on pooled samples of multiple patients. We conclude that, to a limited extent, bottom–up tear protein identifications from individual patients may have clinical relevance.

  10. Visual Analysis of DNA Microarray Data for Accurate Molecular Identification of Non-albicans Candida Isolates from Patients with Candidemia Episodes

    OpenAIRE

    De Luca Ferrari, Michela; Ribeiro Resende, Mariângela; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Gonoi, Tohru; Mikami, Yuzuru; Tominaga, Kenichiro; Kamei, Katsuhiko; Zaninelli Schreiber, Angelica; Trabasso, Plinio; Moretti, Maria Luiza

    2013-01-01

    The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance.

  11. Identification and quantification of major bovine milk proteins by liquid chromatography.

    Science.gov (United States)

    Bordin, G; Cordeiro Raposo, F; de la Calle, B; Rodriguez, A R

    2001-08-31

    In the field of food quality, bovine milk products are of particular interest due to the social and economic importance of the dairy products market. However, the risk of fraudulent manipulation is high in this area, for instance, replacing milk powder by whey is very interesting from an economic point of view. Therefore, there is a need to have suitable analytical methods available for the determination of all milk components, which is currently not the case, especially for the main proteins. The detection of potential manipulations requires then a clear analytical characterisation of each type of bovine milk, what constitutes the goal of this work. The separation of the major milk proteinic components has been carried out by ion-pair reversed-phase HPLC with photodiode array detection, using a C4 column. The overall optimisation has been achieved using a statistical experimental design procedure. The identification of each protein was ascertained using retention times, peak area ratios and second derivative UV spectra. Quantification was based on calibration curves drawn using purified proteins. Major sources of uncertainty were identified and the full uncertainty budget was established. The procedure was initially developed using the skimmed milk powder certified reference material CRM 063R and then applied to various types of commercial milks as well as to raw milk. The method is able to separate and quantify the seven major proteins (K-casein, alphas2-casein, alphas1-casein, beta-casein, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A) in one run and also to provide precise determinations of the total protein concentration. These are important results towards the further development of a reference method for major proteins in milk. In addition, the use of a certified material reference is suggested in order to make comparisons of method performances possible.

  12. Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry.

    Science.gov (United States)

    Buhs, Sophia; Gerull, Helwe; Nollau, Peter

    2017-01-01

    Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.

  13. The Dictyostelium discoideum cellulose synthase: Structure/function analysis and identification of interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Richard L. Blanton

    2004-02-19

    OAK-B135 The major accomplishments of this project were: (1) the initial characterization of dcsA, the gene for the putative catalytic subunit of cellulose synthase in the cellular slime mold Dictyostelium discoideum; (2) the detection of a developmentally regulated event (unidentified, but perhaps a protein modification or association with a protein partner) that is required for cellulose synthase activity (i.e., the dcsA product is necessary, but not sufficient for cellulose synthesis); (3) the continued exploration of the developmental context of cellulose synthesis and DcsA; (4) the isolation of a GFP-DcsA-expressing strain (work in progress); and (5) the identification of Dictyostelium homologues for plant genes whose products play roles in cellulose biosynthesis. Although our progress was slow and many of our results negative, we did develop a number of promising avenues of investigation that can serve as the foundation for future projects.

  14. pMD-Membrane: A Method for Ligand Binding Site Identification in Membrane-Bound Proteins.

    Directory of Open Access Journals (Sweden)

    Priyanka Prakash

    2015-10-01

    Full Text Available Probe-based or mixed solvent molecular dynamics simulation is a useful approach for the identification and characterization of druggable sites in drug targets. However, thus far the method has been applied only to soluble proteins. A major reason for this is the potential effect of the probe molecules on membrane structure. We have developed a technique to overcome this limitation that entails modification of force field parameters to reduce a few pairwise non-bonded interactions between selected atoms of the probe molecules and bilayer lipids. We used the resulting technique, termed pMD-membrane, to identify allosteric ligand binding sites on the G12D and G13D oncogenic mutants of the K-Ras protein bound to a negatively charged lipid bilayer. In addition, we show that differences in probe occupancy can be used to quantify changes in the accessibility of druggable sites due to conformational changes induced by membrane binding or mutation.

  15. Accurate recapture identification for genetic mark–recapture studies with error-tolerant likelihood-based match calling and sample clustering

    Science.gov (United States)

    Sethi, Suresh; Linden, Daniel; Wenburg, John; Lewis, Cara; Lemons, Patrick R.; Fuller, Angela K.; Hare, Matthew P.

    2016-01-01

    Error-tolerant likelihood-based match calling presents a promising technique to accurately identify recapture events in genetic mark–recapture studies by combining probabilities of latent genotypes and probabilities of observed genotypes, which may contain genotyping errors. Combined with clustering algorithms to group samples into sets of recaptures based upon pairwise match calls, these tools can be used to reconstruct accurate capture histories for mark–recapture modelling. Here, we assess the performance of a recently introduced error-tolerant likelihood-based match-calling model and sample clustering algorithm for genetic mark–recapture studies. We assessed both biallelic (i.e. single nucleotide polymorphisms; SNP) and multiallelic (i.e. microsatellite; MSAT) markers using a combination of simulation analyses and case study data on Pacific walrus (Odobenus rosmarus divergens) and fishers (Pekania pennanti). A novel two-stage clustering approach is demonstrated for genetic mark–recapture applications. First, repeat captures within a sampling occasion are identified. Subsequently, recaptures across sampling occasions are identified. The likelihood-based matching protocol performed well in simulation trials, demonstrating utility for use in a wide range of genetic mark–recapture studies. Moderately sized SNP (64+) and MSAT (10–15) panels produced accurate match calls for recaptures and accurate non-match calls for samples from closely related individuals in the face of low to moderate genotyping error. Furthermore, matching performance remained stable or increased as the number of genetic markers increased, genotyping error notwithstanding.

  16. Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

    Science.gov (United States)

    Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

    2018-04-01

    A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

  17. Large scale identification and categorization of protein sequences using structured logistic regression

    DEFF Research Database (Denmark)

    Pedersen, Bjørn Panella; Ifrim, Georgiana; Liboriussen, Poul

    2014-01-01

    Abstract Background Structured Logistic Regression (SLR) is a newly developed machine learning tool first proposed in the context of text categorization. Current availability of extensive protein sequence databases calls for an automated method to reliably classify sequences and SLR seems well...... problem. Results Using SLR, we have built classifiers to identify and automatically categorize P-type ATPases into one of 11 pre-defined classes. The SLR-classifiers are compared to a Hidden Markov Model approach and shown to be highly accurate and scalable. Representing the bulk of currently known...... for further biochemical characterization and structural analysis....

  18. Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

    Science.gov (United States)

    Huang, Bill X.; Kim, Hee-Yong

    2013-01-01

    Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850

  19. Identification of Protein Pupylation Sites Using Bi-Profile Bayes Feature Extraction and Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Xiaowei Zhao

    2013-01-01

    Full Text Available Pupylation, one of the most important posttranslational modifications of proteins, typically takes place when prokaryotic ubiquitin-like protein (Pup is attached to specific lysine residues on a target protein. Identification of pupylation substrates and their corresponding sites will facilitate the understanding of the molecular mechanism of pupylation. Comparing with the labor-intensive and time-consuming experiment approaches, computational prediction of pupylation sites is much desirable for their convenience and fast speed. In this study, a new bioinformatics tool named EnsemblePup was developed that used an ensemble of support vector machine classifiers to predict pupylation sites. The highlight of EnsemblePup was to utilize the Bi-profile Bayes feature extraction as the encoding scheme. The performance of EnsemblePup was measured with a sensitivity of 79.49%, a specificity of 82.35%, an accuracy of 85.43%, and a Matthews correlation coefficient of 0.617 using the 5-fold cross validation on the training dataset. When compared with other existing methods on a benchmark dataset, the EnsemblePup provided better predictive performance, with a sensitivity of 80.00%, a specificity of 83.33%, an accuracy of 82.00%, and a Matthews correlation coefficient of 0.629. The experimental results suggested that EnsemblePup presented here might be useful to identify and annotate potential pupylation sites in proteins of interest. A web server for predicting pupylation sites was developed.

  20. Proteomic identification of early salicylate- and flg22-responsive redox-sensitive proteins in Arabidopsis

    KAUST Repository

    Liu, Peng

    2015-02-27

    Accumulation of reactive oxygen species (ROS) is one of the early defense responses against pathogen infection in plants. The mechanism about the initial and direct regulation of the defense signaling pathway by ROS remains elusive. Perturbation of cellular redox homeostasis by ROS is believed to alter functions of redox-sensitive proteins through their oxidative modifications. Here we report an OxiTRAQ-based proteomic study in identifying proteins whose cysteines underwent oxidative modifications in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional regulation, chromatin remodeling, RNA processing, post-translational modifications, and nucleocytoplasmic shuttling. The identification of the salicylate-/flg22-responsive redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding biological significance of their oxidative modifications during the plant defense response.

  1. Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

    Science.gov (United States)

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. A peptide affinity column for the identification of integrin alpha IIb-binding proteins.

    Science.gov (United States)

    Daxecker, Heide; Raab, Markus; Bernard, Elise; Devocelle, Marc; Treumann, Achim; Moran, Niamh

    2008-03-01

    To understand the regulation of integrin alpha(IIb)beta(3), a critical platelet adhesion molecule, we have developed a peptide affinity chromatography method using the known integrin regulatory motif, LAMWKVGFFKR. Using standard Fmoc chemistry, this peptide was synthesized onto a Toyopearl AF-Amino-650 M resin on a 6-aminohexanoic acid (Ahx) linker. Peptide density was controlled by acetylation of 83% of the Ahx amino groups. Four recombinant human proteins (CIB1, PP1, ICln and RN181), previously identified as binding to this integrin regulatory motif, were specifically retained by the column containing the integrin peptide but not by a column presenting an irrelevant peptide. Hemoglobin, creatine kinase, bovine serum albumin, fibrinogen and alpha-tubulin failed to bind under the chosen conditions. Immunodetection methods confirmed the binding of endogenous platelet proteins, including CIB1, PP1, ICln RN181, AUP-1 and beta3-integrin, from a detergent-free platelet lysate. Thus, we describe a reproducible method that facilitates the reliable extraction of specific integrin-binding proteins from complex biological matrices. This methodology may enable the sensitive and specific identification of proteins that interact with linear, membrane-proximal peptide motifs such as the integrin regulatory motif LAMWKVGFFKR.

  3. Identification of α(1,6)fucosylated proteins differentially expressed in human colorectal cancer

    International Nuclear Information System (INIS)

    Muinelo-Romay, Laura; Villar-Portela, Susana; Cuevas, Elisa; Gil-Martín, Emilio; Fernández-Briera, Almudena

    2011-01-01

    A universal hallmark of cancer cells is the change in their glycosylation phenotype. One of the most frequent alterations in the normal glycosylation pattern observed during carcinogenesis is the enhancement of α(1,6)linked fucose residues of glycoproteins, due to the up-regulation of the α(1,6)fucosyltransferase activity. Our previous results demonstrated the specific alteration of this enzyme activity and expression in colorectal cancer, suggesting its implication in tumour development and progression. In the current work we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in colorectal cancer. This strategy allowed the identification of a group of α(1,6)fucosylated proteins candidates to be involved in CRC malignancy. The majority of the identified proteins take part in cell signaling and interaction processes as well as in modulation of the immunological response. Likewise, we confirmed the increased expression of GRP94 in colorectal cancer tissue and the significant down-regulation of the IgGFcBP expression in tumour cells. All these results validate the importance of core-fucosylated proteins profile analysis to understand the mechanisms which promote cancer onset and progression and to discover new tumour markers or therapeutic targets

  4. Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis.

    Science.gov (United States)

    Hilton, Nicholas A; Sladewski, Thomas E; Perry, Jenna A; Pataki, Zemplen; Sinclair-Davis, Amy N; Muniz, Richard S; Tran, Holly L; Wurster, Jenna I; Seo, Jiwon; de Graffenried, Christopher L

    2018-05-21

    The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly-polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely-configured process in kinetoplastids. This article is protected by copyright. All rights reserved. © 2018 John Wiley & Sons Ltd.

  5. Biotic stress protein markers of Aquilaria sp. for gaharu species identification in Malaysia

    International Nuclear Information System (INIS)

    Azhar Mohamad; Abdul Rahim Harun

    2012-01-01

    Gaharu trees (Aquilaria) is in danger of extinction in the wild due to illegal logging. Its resin (Gaharu) is used for the production of highly valued incense throughout Asia. In Aquilaria sp. systemic induction of defense genes in response to mechanical wounding in nature is regulated by an 18-amino-acid peptide signal protein called systemin. This protein is produced in response to the natural stress at the vicinity of the wound and is also influenced by its genetic background. As the protein can be differentiated by its locality, the protein expressed is also found to be significantly different which, in turn, can be used for identification of this plant species. In this work, A. malaccensis and A. hirta were evaluated based on the targeted genes related to systemin. Targeted gene refers to specific sequence in genomic DNA. Sequence mining from public databases is part of the crucial process in getting the specific genes. The sequences will go through alignment step to identify conserved region prior to primer design. The primers were used in Polymerase Chain Reaction (PCR) techniques to amplify the conserved regions. It was found that both samples can be differentiated. This would be useful for plant breeders, trader and planter in ensuring authentic planting materials. This paper will describe the use of targeted genes primers as markers in identifying the Aquilaria species. (author)

  6. Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

    International Nuclear Information System (INIS)

    Jacob, Jaison; Louis, John M.; Nesheiwat, Issa; Torchia, Dennis A.

    2002-01-01

    Analysis of 2D [ 13 C, 1 H]-HSQC spectra of biosynthetic fractionally 13 C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional 13 C labeling yields aromatic rings in which some of the 13 C- 13 C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the δ-, ε- and ζ-carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the 13 C constant-time period in 2D [ 13 C, 1 H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic 13 C CSA and 13 C- 1 H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution 13 C constant-time spectra with good sensitivity

  7. Accelerated identification of proteins by mass spectrometry by employing covalent pre-gel staining with Uniblue A.

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    Marco A Mata-Gómez

    Full Text Available BACKGROUND: The identification of proteins by mass spectrometry is a standard method in biopharmaceutical quality control and biochemical research. Prior to identification by mass spectrometry, proteins are usually pre-separated by electrophoresis. However, current protein staining and de-staining protocols are tedious and time consuming, and therefore prolong the sample preparation time for mass spectrometry. METHODOLOGY AND PRINCIPAL FINDINGS: We developed a 1-minute covalent pre-gel staining protocol for proteins, which does not require de-staining before the mass spectrometry analysis. We investigated the electrophoretic properties of derivatized proteins and peptides and studied their behavior in mass spectrometry. Further, we elucidated the preferred reaction of proteins with Uniblue A and demonstrate the integration of the peptide derivatization into typical informatics tools. CONCLUSIONS AND SIGNIFICANCE: The Uniblue A staining method drastically speeds up the sample preparation for the mass spectrometry based identification of proteins. The application of this chemo-proteomic strategy will be advantageous for routine quality control of proteins and for time-critical tasks in protein analysis.

  8. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

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    Stabel Silvia

    2002-04-01

    Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

  9. Identification of similar regions of protein structures using integrated sequence and structure analysis tools

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    Heiland Randy

    2006-03-01

    Full Text Available Abstract Background Understanding protein function from its structure is a challenging problem. Sequence based approaches for finding homology have broad use for annotation of both structure and function. 3D structural information of protein domains and their interactions provide a complementary view to structure function relationships to sequence information. We have developed a web site http://www.sblest.org/ and an API of web services that enables users to submit protein structures and identify statistically significant neighbors and the underlying structural environments that make that match using a suite of sequence and structure analysis tools. To do this, we have integrated S-BLEST, PSI-BLAST and HMMer based superfamily predictions to give a unique integrated view to prediction of SCOP superfamilies, EC number, and GO term, as well as identification of the protein structural environments that are associated with that prediction. Additionally, we have extended UCSF Chimera and PyMOL to support our web services, so that users can characterize their own proteins of interest. Results Users are able to submit their own queries or use a structure already in the PDB. Currently the databases that a user can query include the popular structural datasets ASTRAL 40 v1.69, ASTRAL 95 v1.69, CLUSTER50, CLUSTER70 and CLUSTER90 and PDBSELECT25. The results can be downloaded directly from the site and include function prediction, analysis of the most conserved environments and automated annotation of query proteins. These results reflect both the hits found with PSI-BLAST, HMMer and with S-BLEST. We have evaluated how well annotation transfer can be performed on SCOP ID's, Gene Ontology (GO ID's and EC Numbers. The method is very efficient and totally automated, generally taking around fifteen minutes for a 400 residue protein. Conclusion With structural genomics initiatives determining structures with little, if any, functional characterization

  10. Identification of odorant binding proteins and chemosensory proteins in Microplitis mediator as well as functional characterization of chemosensory protein 3.

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    Yong Peng

    Full Text Available Odorant binding proteins (OBPs and chemosensory proteins (CSPs play important roles in transporting semiochemicals through the sensillar lymph to olfactory receptors in insect antennae. In the present study, twenty OBPs and three CSPs were identified from the antennal transcriptome of Microplitis mediator. Ten OBPs (MmedOBP11-20 and two CSPs (MmedCSP2-3 were newly identified. The expression patterns of these new genes in olfactory and non-olfactory tissues were investigated by real-time quantitative PCR (qPCR measurement. The results indicated that MmedOBP14, MmedOBP18, MmedCSP2 and MmedCSP3 were primarily expressed in antennae suggesting potential olfactory roles in M. mediator. However, other genes including MmedOBP11-13, 15-17, 19-20 appeared to be expressed at higher levels in body parts than in antennae. Focusing on the functional characterization of MmedCSP3, immunocytochemistry and fluorescent competitive binding assays were conducted indoors. It was found that MmedCSP3 was specifically located in the sensillum lymph of olfactory sensilla basiconca type 2. The recombinant MmedCSP3 could bind several types of host insects odors and plant volatiles. Interestingly, three sex pheromone components of Noctuidae insects, cis-11-hexadecenyl aldehyde (Z11-16: Ald, cis-11-hexadecanol (Z11-16: OH, and trans-11-tetradecenyl acetate (E11-14: Ac, showed high binding affinities (Ki = 17.24-18.77 μM. The MmedCSP3 may be involved in locating host insects. Our data provide a base for further investigating the physiological roles of OBPs and CSPs in M. mediator, and extend the function of MmedCSP3 in chemoreception of M. mediator.

  11. Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

    Science.gov (United States)

    Zhang, Cathy X. Y.; Brooks, Brian W.; Huang, Hongsheng; Pagotto, Franco

    2016-01-01

    ABSTRACT The Gram-positive bacterium Listeria monocytogenes causes a significant percentage of the fatalities among foodborne illnesses in humans. Surface proteins specifically expressed in a wide range of L. monocytogenes serotypes under selective enrichment culture conditions could serve as potential biomarkers for detection and isolation of this pathogen via antibody-based methods. Our study aimed to identify such biomarkers. Interrogation of the L. monocytogenes serotype 4b strain F2365 genome identified 130 putative or known surface proteins. The homologues of four surface proteins, LMOf2365_0578, LMOf2365_0581, LMOf2365_0639, and LMOf2365_2117, were assessed as biomarkers due to the presence of conserved regions among strains of L. monocytogenes which are variable among other Listeria species. Rabbit polyclonal antibodies against the four recombinant proteins revealed the expression of only LMOf2365_0639 on the surface of serotype 4b strain LI0521 cells despite PCR detection of mRNA transcripts for all four proteins in the organism. Three of 35 monoclonal antibodies (MAbs) to LMOf2365_0639, MAbs M3643, M3644, and M3651, specifically recognized 42 (91.3%) of 46 L. monocytogenes lineage I and II isolates grown in nonselective brain heart infusion medium. While M3644 and M3651 reacted with 14 to 15 (82.4 to 88.2%) of 17 L. monocytogenes lineage I and II isolates, M3643 reacted with 22 (91.7%) of 24 lineage I, II, and III isolates grown in selective enrichment media (UVM1, modified Fraser, Palcam, and UVM2 media). The three MAbs exhibited only weak reactivities (the optical densities at 414 nm were close to the cutoff value) to some other Listeria species grown in selective enrichment media. Collectively, the data indicate the potential of LMOf2365_0639 as a surface biomarker of L. monocytogenes, with the aid of specific MAbs, for pathogen detection, identification, and isolation in clinical, environmental, and food samples. IMPORTANCE L. monocytogenes is

  12. Identification of salivary mucin MUC7 binding proteins from Streptococcus gordonii

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    Thornton David J

    2009-08-01

    Full Text Available Abstract Background The salivary mucin MUC7 (previously known as MG2 can adhere to various strains of streptococci that are primary colonizers and predominant microorganisms of the oral cavity. Although there is a growing interest in interaction between oral pathogens and salivary mucins, studies reporting the specific binding sites on the bacteria are rather limited. Identification and characterization of the specific interacting proteins on the bacterial cell surface, termed adhesins, are crucial to further understand host-pathogen interactions. Results We demonstrate here, using purified MUC7 to overlay blots of SDS-extracts of Streptococcus gordonii cell surface proteins, 4 MUC7-binding bands, with apparent molecular masses of 62, 78, 84 and 133 kDa from the Streptococcus gordonii strain, PK488. Putative adhesins were identified by in-gel digestion and subsequent nanoLC-tandem mass spectrometry analysis of resultant peptides. The 62 kDa and 84 kDa bands were identified as elongation factor (EF Tu and EF-G respectively. The 78 kDa band was a hppA gene product; the 74 kDa oligopeptide-binding lipoprotein. The 133 kDa band contained two proteins; alpha enolase and DNA-directed RNA polymerase, beta' subunit. Some of these proteins, for example alpha enolase are expected to be intracellular, however, flow cytometric analysis confirmed its location on the bacterial surface. Conclusion Our data demonstrated that S. gordonii expressed a number of putative MUC7 recognizing proteins and these contribute to MUC7 mucin binding of this streptococcal strain.

  13. Identification of an exported heat shock protein 70 in Plasmodium falciparum

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    Grover Manish

    2013-01-01

    Full Text Available Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer’s clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

  14. Identification of a key structural element for protein folding within beta-hairpin turns.

    Science.gov (United States)

    Kim, Jaewon; Brych, Stephen R; Lee, Jihun; Logan, Timothy M; Blaber, Michael

    2003-05-09

    Specific residues in a polypeptide may be key contributors to the stability and foldability of the unique native structure. Identification and prediction of such residues is, therefore, an important area of investigation in solving the protein folding problem. Atypical main-chain conformations can help identify strains within a folded protein, and by inference, positions where unique amino acids may have a naturally high frequency of occurrence due to favorable contributions to stability and folding. Non-Gly residues located near the left-handed alpha-helical region (L-alpha) of the Ramachandran plot are a potential indicator of structural strain. Although many investigators have studied mutations at such positions, no consistent energetic or kinetic contributions to stability or folding have been elucidated. Here we report a study of the effects of Gly, Ala and Asn substitutions found within the L-alpha region at a characteristic position in defined beta-hairpin turns within human acidic fibroblast growth factor, and demonstrate consistent effects upon stability and folding kinetics. The thermodynamic and kinetic data are compared to available data for similar mutations in other proteins, with excellent agreement. The results have identified that Gly at the i+3 position within a subset of beta-hairpin turns is a key contributor towards increasing the rate of folding to the native state of the polypeptide while leaving the rate of unfolding largely unchanged.

  15. Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

    International Nuclear Information System (INIS)

    Shaw, Debra J.; Morse, Robert; Todd, Adrian G.; Eggleton, Paul; Lorson, Christian L.; Young, Philip J.

    2009-01-01

    The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

  16. Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, Debra J.; Morse, Robert; Todd, Adrian G. [Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom); Eggleton, Paul [Inflammation and Musculoskeletal Disease, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom); MRC Immunochemistry Unit, University of Oxford, Oxford OX1 3QU (United Kingdom); Lorson, Christian L. [Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, University of Missouri, Columbia, MO 65211 (United States); Young, Philip J., E-mail: philip.young@pms.ac.uk [Clinical Neurobiology, IBCS, Peninsula College of Medicine and Dentistry, Exeter EX1 2LU (United Kingdom)

    2009-12-25

    The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

  17. Identification of small peptides arising from hydrolysis of meat proteins in dry fermented sausages.

    Science.gov (United States)

    López, Constanza M; Bru, Elena; Vignolo, Graciela M; Fadda, Silvina G

    2015-06-01

    In this study, proteolysis and low molecular weight (LMW) peptides (<3kDa) from commercial Argentinean fermented sausages were characterized by applying a peptidomic approach. Protein profiles and peptides obtained by Tricine-SDS-PAGE and RP-HPLC-MS, respectively, allowed distinguishing two different types of fermented sausages, although no specific biomarkers relating to commercial brands or quality were recognized. From electrophoresis, α-actin, myoglobin, creatine kinase M-type and L-lactate dehydrogenase were degraded at different intensities. In addition, a partial characterization of fermented sausage peptidome through the identification of 36 peptides, in the range of 1000-2100 Da, arising from sarcoplasmic (28) and myofibrillar (8) proteins was achieved. These peptides had been originated from α-actin, myoglobin, and creatine kinase M-type, but also from the hydrolysis of other proteins not previously reported. Although muscle enzymes exerted a major role on peptidogenesis, microbial contribution cannot be excluded as it was postulated herein. This work represents a first peptidomic approach for fermented sausages, thereby providing a baseline to define key peptides acting as potential biomarkers. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. P185-M Protein Identification and Validation of Results in Workflows that Integrate over Various Instruments, Datasets, Search Engines

    Science.gov (United States)

    Hufnagel, P.; Glandorf, J.; Körting, G.; Jabs, W.; Schweiger-Hufnagel, U.; Hahner, S.; Lubeck, M.; Suckau, D.

    2007-01-01

    Analysis of complex proteomes often results in long protein lists, but falls short in measuring the validity of identification and quantification results on a greater number of proteins. Biological and technical replicates are mandatory, as is the combination of the MS data from various workflows (gels, 1D-LC, 2D-LC), instruments (TOF/TOF, trap, qTOF or FTMS), and search engines. We describe a database-driven study that combines two workflows, two mass spectrometers, and four search engines with protein identification following a decoy database strategy. The sample was a tryptically digested lysate (10,000 cells) of a human colorectal cancer cell line. Data from two LC-MALDI-TOF/TOF runs and a 2D-LC-ESI-trap run using capillary and nano-LC columns were submitted to the proteomics software platform ProteinScape. The combined MALDI data and the ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen), and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined maximum false-positive rate of 5%. ProteinScape combined the data to one LC-MALDI and one LC-ESI dataset. The initial separate searches from the two combined datasets generated eight independent peptide lists. These were compiled into an integrated protein list using the ProteinExtractor algorithm. An initial evaluation of the generated data led to the identification of approximately 1200 proteins. Result integration on a peptide level allowed discrimination of protein isoforms that would not have been possible with a mere combination of protein lists.

  19. Combining metal oxide affinity chromatography (MOAC and selective mass spectrometry for robust identification of in vivo protein phosphorylation sites

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    Weckwerth Wolfram

    2005-11-01

    Full Text Available Abstract Background Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Results Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC and selective ion trap mass spectrometry. The complete approach involves (i enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment, (iii identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. Conclusion A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to

  20. Metabolite signal identification in accurate mass metabolomics data with MZedDB, an interactive m/z annotation tool utilising predicted ionisation behaviour 'rules'

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    Snowdon Stuart

    2009-07-01

    Full Text Available Abstract Background Metabolomics experiments using Mass Spectrometry (MS technology measure the mass to charge ratio (m/z and intensity of ionised molecules in crude extracts of complex biological samples to generate high dimensional metabolite 'fingerprint' or metabolite 'profile' data. High resolution MS instruments perform routinely with a mass accuracy of Results Metabolite 'structures' harvested from publicly accessible databases were converted into a common format to generate a comprehensive archive in MZedDB. 'Rules' were derived from chemical information that allowed MZedDB to generate a list of adducts and neutral loss fragments putatively able to form for each structure and calculate, on the fly, the exact molecular weight of every potential ionisation product to provide targets for annotation searches based on accurate mass. We demonstrate that data matrices representing populations of ionisation products generated from different biological matrices contain a large proportion (sometimes > 50% of molecular isotopes, salt adducts and neutral loss fragments. Correlation analysis of ESI-MS data features confirmed the predicted relationships of m/z signals. An integrated isotope enumerator in MZedDB allowed verification of exact isotopic pattern distributions to corroborate experimental data. Conclusion We conclude that although ultra-high accurate mass instruments provide major insight into the chemical diversity of biological extracts, the facile annotation of a large proportion of signals is not possible by simple, automated query of current databases using computed molecular formulae. Parameterising MZedDB to take into account predicted ionisation behaviour and the biological source of any sample improves greatly both the frequency and accuracy of potential annotation 'hits' in ESI-MS data.

  1. Identification of a 251 gene expression signature that can accurately detect M. tuberculosis in patients with and without HIV co-infection.

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    Noor Dawany

    Full Text Available BACKGROUND: Co-infection with tuberculosis (TB is the leading cause of death in HIV-infected individuals. However, diagnosis of TB, especially in the presence of an HIV co-infection, can be limiting due to the high inaccuracy associated with the use of conventional diagnostic methods. Here we report a gene signature that can identify a tuberculosis infection in patients co-infected with HIV as well as in the absence of HIV. METHODS: We analyzed global gene expression data from peripheral blood mononuclear cell (PBMC samples of patients that were either mono-infected with HIV or co-infected with HIV/TB and used support vector machines to identify a gene signature that can distinguish between the two classes. We then validated our results using publically available gene expression data from patients mono-infected with TB. RESULTS: Our analysis successfully identified a 251-gene signature that accurately distinguishes patients co-infected with HIV/TB from those infected with HIV only, with an overall accuracy of 81.4% (sensitivity = 76.2%, specificity = 86.4%. Furthermore, we show that our 251-gene signature can also accurately distinguish patients with active TB in the absence of an HIV infection from both patients with a latent TB infection and healthy controls (88.9-94.7% accuracy; 69.2-90% sensitivity and 90.3-100% specificity. We also demonstrate that the expression levels of the 251-gene signature diminish as a correlate of the length of TB treatment. CONCLUSIONS: A 251-gene signature is described to (a detect TB in the presence or absence of an HIV co-infection, and (b assess response to treatment following anti-TB therapy.

  2. MAGGIE Component 1: Identification and Purification of Native and Recombinant Multiprotein Complexes and Modified Proteins from Pyrococcus furiosus

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W. [University of Georgia; W. W. Adams, Michael

    2014-01-07

    Virtualy all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes (PCs), the composition of which is largely unknown. Structural genomics efforts have demonstrated that less than 25% of the genes in a given prokaryotic genome will yield stable, soluble proteins when expressed using a one-ORF-at-a-time approach. We proposed that much of the remaining 75% of the genes encode proteins that are part of multiprotein complexes or are modified post-translationally, for example, with metals. The problem is that PCs and metalloproteins (MPs) cannot be accurately predicted on a genome-wide scale. The only solution to this dilemma is to experimentally determine PCs and MPs in biomass of a model organism and to develop analytical tools that can then be applied to the biomass of any other organism. In other words, organisms themselves must be analyzed to identify their PCs and MPs: “native proteomes” must be determined. This information can then be utilized to design multiple ORF expression systems to produce recombinant forms of PCs and MPs. Moreover, the information and utility of this approach can be enhanced by using a hyperthermophile, one that grows optimally at 100°C, as a model organism. By analyzing the native proteome at close to 100 °C below the optimum growth temperature, we will trap reversible and dynamic complexes, thereby enabling their identification, purification, and subsequent characterization. The model organism for the current study is Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100°C. It is grown up to 600-liter scale and kg quantities of biomass are available. In this project we identified native PCs and MPs using P. furiosus biomass (with MS/MS analyses to identify proteins by component 4). In addition, we provided samples of abundant native PCs and MPs for structural characterization (using SAXS by component 5). We also designed and evaluated generic bioinformatics and

  3. Complex mixture analysis of peptides using LC/LC-MS/MS and data-dependent protein identification

    International Nuclear Information System (INIS)

    Wasinger, V.; Corthals, G.

    2001-01-01

    The comprehensive identification of proteins within complex solutions by mass-spectrometry largely depends on the sensitivity, resolving power and sampling efficiency of the technology. An integrated orthogonal approach using Strong Cation Exchange-Reverse Phase-MS/MS (SCX-RP-MS/MS) was used to evaluate the data-dependent Collision Induced Dissociation (CID) of yeast peptides. Reverse phase gradient times of 4, 10. 30, 90, and 180 minutes allowed the identification of hundreds of proteins in a nearly automated fashion from nuclear, membrane, and cytosolic distributions. Many proteins from typically difficult to resolve regions of two-dimensional gels, such as >100kDa, > pI 9.0 and Codon Adaptation Index < 0.2, were also identified using this multi-dimensional separation technology. Few low mass proteins (<10kDa) were identified. The impact of scan-range and duty-cycle on CID of peptides will be discussed

  4. Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement

    DEFF Research Database (Denmark)

    Kublik, Anja; Deobald, Darja; Hartwig, Stefanie

    2016-01-01

    electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250–270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster...... of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi....

  5. Accurate identification of ALK positive lung carcinoma patients: novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry.

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    Esther Conde

    Full Text Available BACKGROUND: Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit and an automated ALK FISH scanning system (FDA-cleared in a series of non-small cell lung cancer tumor samples. METHODS: Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit, which was automatically captured and scored by using Bioview's automated scanning system. RESULTS: All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. CONCLUSIONS: The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.

  6. Identification of Secretory Proteins in Mycobacterium tuberculosis Using Pseudo Amino Acid Composition

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    Huan Yang

    2016-01-01

    Full Text Available Tuberculosis is killing millions of lives every year and on the blacklist of the most appalling public health problems. Recent findings suggest that secretory protein of Mycobacterium tuberculosis may serve the purpose of developing specific vaccines and drugs due to their antigenicity. Responding to global infectious disease, we focused on the identification of secretory proteins in Mycobacterium tuberculosis. A novel method called MycoSec was designed by incorporating g-gap dipeptide compositions into pseudo amino acid composition. Analysis of variance-based technique was applied in the process of feature selection and a total of 374 optimal features were obtained and used for constructing the final predicting model. In the jackknife test, MycoSec yielded a good performance with the area under the receiver operating characteristic curve of 0.93, demonstrating that the proposed system is powerful and robust. For user’s convenience, the web server MycoSec was established and an obliging manual on how to use it was provided for getting around any trouble unnecessary.

  7. Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

    Science.gov (United States)

    Lasalde, Clarivel; Rivera, Andrea V; León, Alfredo J; González-Feliciano, José A; Estrella, Luis A; Rodríguez-Cruz, Eva N; Correa, María E; Cajigas, Iván J; Bracho, Dina P; Vega, Irving E; Wilkinson, Miles F; González, Carlos I

    2014-02-01

    One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

  8. Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study▿†

    Science.gov (United States)

    Wang, He; Xiao, Meng; Kong, Fanrong; Chen, Sharon; Dou, Hong-Tao; Sorrell, Tania; Li, Ruo-Yu; Xu, Ying-Chun

    2011-01-01

    Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria. PMID:21389150

  9. Putative drug and vaccine target protein identification using comparative genomic analysis of KEGG annotated metabolic pathways of Mycoplasma hyopneumoniae.

    Science.gov (United States)

    Damte, Dereje; Suh, Joo-Won; Lee, Seung-Jin; Yohannes, Sileshi Belew; Hossain, Md Akil; Park, Seung-Chun

    2013-07-01

    In the present study, a computational comparative and subtractive genomic/proteomic analysis aimed at the identification of putative therapeutic target and vaccine candidate proteins from Kyoto Encyclopedia of Genes and Genomes (KEGG) annotated metabolic pathways of Mycoplasma hyopneumoniae was performed for drug design and vaccine production pipelines against M.hyopneumoniae. The employed comparative genomic and metabolic pathway analysis with a predefined computational systemic workflow extracted a total of 41 annotated metabolic pathways from KEGG among which five were unique to M. hyopneumoniae. A total of 234 proteins were identified to be involved in these metabolic pathways. Although 125 non homologous and predicted essential proteins were found from the total that could serve as potential drug targets and vaccine candidates, additional prioritizing parameters characterize 21 proteins as vaccine candidate while druggability of each of the identified proteins evaluated by the DrugBank database prioritized 42 proteins suitable for drug targets. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. Identification of the functional domains of the telomere protein Rap1 in Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ikumi Fujita

    Full Text Available The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1(+ have revealed that the long N-terminal region (1-456 a.a. [amino acids] of Rap1 (full length: 693 a.a. is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457-693 a.a. containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.

  11. Identification of glycan structure alterations on cell membrane proteins in desoxyepothilone B resistant leukemia cells.

    Science.gov (United States)

    Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H

    2011-11-01

    Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.

  12. Proteogenomic Analysis Greatly Expands the Identification of Proteins Related to Reproduction in the Apogamous Fern Dryopteris affinis ssp. affinis.

    Science.gov (United States)

    Grossmann, Jonas; Fernández, Helena; Chaubey, Pururawa M; Valdés, Ana E; Gagliardini, Valeria; Cañal, María J; Russo, Giancarlo; Grossniklaus, Ueli

    2017-01-01

    Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis . For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a "species-specific transcriptome database" (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non-model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models.

  13. Bayesian network model for identification of pathways by integrating protein interaction with genetic interaction data.

    Science.gov (United States)

    Fu, Changhe; Deng, Su; Jin, Guangxu; Wang, Xinxin; Yu, Zu-Guo

    2017-09-21

    Molecular interaction data at proteomic and genetic levels provide physical and functional insights into a molecular biosystem and are helpful for the construction of pathway structures complementarily. Despite advances in inferring biological pathways using genetic interaction data, there still exists weakness in developed models, such as, activity pathway networks (APN), when integrating the data from proteomic and genetic levels. It is necessary to develop new methods to infer pathway structure by both of interaction data. We utilized probabilistic graphical model to develop a new method that integrates genetic interaction and protein interaction data and infers exquisitely detailed pathway structure. We modeled the pathway network as Bayesian network and applied this model to infer pathways for the coherent subsets of the global genetic interaction profiles, and the available data set of endoplasmic reticulum genes. The protein interaction data were derived from the BioGRID database. Our method can accurately reconstruct known cellular pathway structures, including SWR complex, ER-Associated Degradation (ERAD) pathway, N-Glycan biosynthesis pathway, Elongator complex, Retromer complex, and Urmylation pathway. By comparing N-Glycan biosynthesis pathway and Urmylation pathway identified from our approach with that from APN, we found that our method is able to overcome its weakness (certain edges are inexplicable). According to underlying protein interaction network, we defined a simple scoring function that only adopts genetic interaction information to avoid the balance difficulty in the APN. Using the effective stochastic simulation algorithm, the performance of our proposed method is significantly high. We developed a new method based on Bayesian network to infer detailed pathway structures from interaction data at proteomic and genetic levels. The results indicate that the developed method performs better in predicting signaling pathways than previously

  14. On plate graphite supported sample processing for simultaneous lipid and protein identification by matrix assisted laser desorption ionization mass spectrometry.

    Science.gov (United States)

    Calvano, Cosima Damiana; van der Werf, Inez Dorothé; Sabbatini, Luigia; Palmisano, Francesco

    2015-05-01

    The simultaneous identification of lipids and proteins by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) after direct on-plate processing of micro-samples supported on colloidal graphite is demonstrated. Taking advantages of large surface area and thermal conductivity, graphite provided an ideal substrate for on-plate proteolysis and lipid extraction. Indeed proteins could be efficiently digested on-plate within 15 min, providing sequence coverages comparable to those obtained by conventional in-solution overnight digestion. Interestingly, detection of hydrophilic phosphorylated peptides could be easily achieved without any further enrichment step. Furthermore, lipids could be simultaneously extracted/identified without any additional treatment/processing step as demonstrated for model complex samples such as milk and egg. The present approach is simple, efficient, of large applicability and offers great promise for protein and lipid identification in very small samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Spectroscopic confirmation of the optical identification of X-ray sources used to determine accurate positions for the anomalous X-ray pulsars 1E 2259+58.6 and 4U 0142+61

    Science.gov (United States)

    van den Berg, M.; Verbunt, F.

    2001-03-01

    Optical spectra show that two proposed counterparts for X-ray sources detected near 1E 2259+58.6 are late G stars, and a proposed counterpart for a source near 4U 0142+61 is a dMe star. The X-ray luminosities are as expected for such stars. We thus confirm the optical identification of the three X-ray objects, and thereby the correctness of the accurate positions for 1E 2259+58.6 and 4U 0142+61 based on them. Based on observations made with the William Herschel Telescope operated on the island of La Palma by the Isaac Newton Group in the Spanish Observatorio del Roque de los Muchachos of the Instituto de Astrofisica de Canarias.

  16. SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kandaswamy, Krishna Kumar [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Graduate School for Computing in Medicine and Life Sciences, University of Luebeck, 23538 Luebeck (Germany); Pugalenthi, Ganesan [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Hartmann, Enno; Kalies, Kai-Uwe [Centre for Structural and Cell Biology in Medicine, Institute of Biology, University of Luebeck, 23538 Luebeck (Germany); Moeller, Steffen [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Suganthan, P.N. [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Martinetz, Thomas, E-mail: martinetz@inb.uni-luebeck.de [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany)

    2010-01-15

    Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

  17. SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

    International Nuclear Information System (INIS)

    Kandaswamy, Krishna Kumar; Pugalenthi, Ganesan; Hartmann, Enno; Kalies, Kai-Uwe; Moeller, Steffen; Suganthan, P.N.; Martinetz, Thomas

    2010-01-01

    Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

  18. Identification of membrane-associated proteins with pathogenic potential expressed by Corynebacterium pseudotuberculosis grown in animal serum.

    Science.gov (United States)

    Raynal, José Tadeu; Bastos, Bruno Lopes; Vilas-Boas, Priscilla Carolinne Bagano; Sousa, Thiago de Jesus; Costa-Silva, Marcos; de Sá, Maria da Conceição Aquino; Portela, Ricardo Wagner; Moura-Costa, Lília Ferreira; Azevedo, Vasco; Meyer, Roberto

    2018-01-25

    Previous works defining antigens that might be used as vaccine targets against Corynebacterium pseudotuberculosis, which is the causative agent of sheep and goat caseous lymphadenitis, have focused on secreted proteins produced in a chemically defined culture media. Considering that such antigens might not reflect the repertoire of proteins expressed during infection conditions, this experiment aimed to investigate the membrane-associated proteins with pathogenic potential expressed by C. pseudotuberculosis grown directly in animal serum. Its membrane-associated proteins have been extracted using an organic solvent enrichment methodology, followed by LC-MS/MS and bioinformatics analysis for protein identification and classification. The results revealed 22 membrane-associated proteins characterized as potentially pathogenic. An interaction network analysis indicated that the four potentially pathogenic proteins ciuA, fagA, OppA4 and OppCD were biologically connected within two distinct network pathways, which were both associated with the ABC Transporters KEGG pathway. These results suggest that C. pseudotuberculosis pathogenesis might be associated with the transport and uptake of nutrients; other seven identified potentially pathogenic membrane proteins also suggest that pathogenesis might involve events of bacterial resistance and adhesion. The proteins herein reported potentially reflect part of the protein repertoire expressed during real infection conditions and might be tested as vaccine antigens.

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

    Science.gov (United States)

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

    2013-12-19

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

  20. Protein-energy malnutrition in the rehabilitation setting: Evidence to improve identification.

    Science.gov (United States)

    Marshall, Skye

    2016-04-01

    Methods of identifying malnutrition in the rehabilitation setting require further examination so that patient outcomes may be improved. The purpose of this narrative review was to: (1) examine the defining characteristics of malnutrition, starvation, sarcopenia and cachexia; (2) review the validity of nutrition screening tools and nutrition assessment tools in the rehabilitation setting; and (3) determine the prevalence of malnutrition in the rehabilitation setting by geographical region and method of diagnosis. A narrative review was conducted drawing upon international literature. Starvation represents one form of malnutrition. Inadequate energy and protein intake are the critical factor in the aetiology of malnutrition, which is distinct from sarcopenia and cachexia. Eight nutrition screening tools and two nutrition assessment tools have been evaluated for criterion validity in the rehabilitation setting, and consideration must be given to the resources of the facility and the patient group in order to select the appropriate tool. The prevalence of malnutrition in the rehabilitation setting ranges from 14-65% worldwide with the highest prevalence reported in rural, European and Australian settings. Malnutrition is highly prevalent in the rehabilitation setting, and consideration must be given to the patient group when determining the most appropriate method of identification so that resources may be used efficaciously and the chance of misdiagnosis minimised. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

    Directory of Open Access Journals (Sweden)

    Xingui Tian

    2015-10-01

    Full Text Available Human adenovirus type 55 (HAdV55 is a newly identified re-emergent acute respiratory disease (ARD pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152, A55R2 (residues 179 to 187, A55R4 (residues 247 to 259 and A55R7 (residues 429 to 443, were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA and neutralization tests (NT. Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3 and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis.

  2. Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

    Science.gov (United States)

    Tian, Xingui; Ma, Qiang; Jiang, Zaixue; Huang, Junfeng; Liu, Qian; Lu, Xiaomei; Luo, Qingming; Zhou, Rong

    2015-01-01

    Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. PMID:26516903

  3. Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Robb, Victoria A.; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H.

    2003-06-11

    Meningiomas are common tumors of the central nervous system, however, the mechanisms under lying their pathogenesis are largely undefined. Two members of the Protein 4.1 super family, the neuro fibromatosis 2 (NF2) gene product (merlin/schwannomin) and Protein 4.1B have been implicated as meningioma tumor suppressors. In this report, we demonstrate that another Protein 4.1 family member, Protein 4.1R, also functions as a meningioma tumor suppressor. Based on the assignment of the Protein 4.1R gene to chromosome 1p32-36, a common region of deletion observed in meningiomas, we analyzed Protein 4.1R expression in meningioma cell lines and surgical tumor specimens. We observed loss of Protein 4.1R protein expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningioma as by immuno histo chemistry (IHC). Analysis of a subset of these sporadic meningiomas by fluorescent in situ hybridization (FISH) with a Protein 4.1R specific probe demonstrated 100 percent concordance with the IHC results. In support of a meningioma tumor suppressor function, over expression of Protein 4.1R resulted in suppression of IOMM-Lee and CH157MN cell proliferation. Similar to the Protein 4.1B and merlin meningioma tumor suppressors, Protein 4.1R localization in the membrane fraction increased significantly under conditions of growth arrest in vitro. Lastly, Protein 4.1R interacted with some known merlin/Protein 4.1B interactors such as CD44 and bII-spectrin, but did not associate with the Protein 4.1B interactors 14-3-3 and PRMT3 or the merlin binding proteins SCHIP-1 and HRS. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor important in the molecular pathogenesis of meningioma.

  4. Sequence Identification, Recombinant Production, and Analysis of the Self-Assembly of Egg Stalk Silk Proteins from Lacewing Chrysoperla carnea.

    Science.gov (United States)

    Neuenfeldt, Martin; Scheibel, Thomas

    2017-06-13

    Egg stalk silks of the common green lacewing Chrysoperla carnea likely comprise at least three different silk proteins. Based on the natural spinning process, it was hypothesized that these proteins self-assemble without shear stress, as adult lacewings do not use a spinneret. To examine this, the first sequence identification and determination of the gene expression profile of several silk proteins and various transcript variants thereof was conducted, and then the three major proteins were recombinantly produced in Escherichia coli encoded by their native complementary DNA (cDNA) sequences. Circular dichroism measurements indicated that the silk proteins in aqueous solutions had a mainly intrinsically disordered structure. The largest silk protein, which we named ChryC1, exhibited a lower critical solution temperature (LCST) behavior and self-assembled into fibers or film morphologies, depending on the conditions used. The second silk protein, ChryC2, self-assembled into nanofibrils and subsequently formed hydrogels. Circular dichroism and Fourier transform infrared spectroscopy confirmed conformational changes of both proteins into beta sheet rich structures upon assembly. ChryC3 did not self-assemble into any morphology under the tested conditions. Thereby, through this work, it could be shown that recombinant lacewing silk proteins can be produced and further used for studying the fiber formation of lacewing egg stalks.

  5. Identification of immunogenic proteins and evaluation of four recombinant proteins as potential vaccine antigens from Vibrio anguillarum in flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Xing, Jing; Xu, Hongsen; Wang, Yang; Tang, Xiaoqian; Sheng, Xiuzhen; Zhan, Wenbin

    2017-05-31

    Vibrio anguillarum is a severe bacterial pathogen that can infect a wide range of fish species. Identification of immunogenic proteins and development of vaccine are essential for disease prevention. In this study, immunogenic proteins were screened and identified from V. anguillarum, and then protective efficacy of the immunogenic proteins was evaluated. Immunogenic proteins in V. anguillarum whole cell were detected by Western blotting (WB) using immunized flounder (Paralichthys olivaceus) serum, and then identified by Mass spectrometry (MS). The recombinant proteins of four identified immunogenic proteins were produced and immunized to fish, and then percentages of surface membrane immunoglobulin-positive (sIg+) cells in peripheral blood lymphocytes (PBL), total antibodies, antibodies against V. anguillarum, antibodies against recombinant proteins and relative percent survival (RPS) were measured, respectively. The results showed that five immunogenic proteins, VAA, Groel, OmpU, PteF and SpK, were identified; their recombinant proteins, rOmpU, rGroel, rSpK and rVAA, could induce the proliferation of sIg+ cells in PBL and production of total antibodies, antibodies against V. anguillarum and antibodies against the recombinant proteins; their protection against V. anguillarum showed 64.86%, 72.97%, 21.62% and 78.38% RPS, respectively. The results revealed that the immunoproteomic technique using fish anti-V. anguillarum serum provided an efficient way to screen the immunogenic protein for vaccine antigen. Moreover, the rVAA, rGroel and rOmpU had potential to be vaccine candidates against V. anguillarum infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Computational Identification of Protein Pupylation Sites by Using Profile-Based Composition of k-Spaced Amino Acid Pairs.

    Directory of Open Access Journals (Sweden)

    Md Mehedi Hasan

    Full Text Available Prokaryotic proteins are regulated by pupylation, a type of post-translational modification that contributes to cellular function in bacterial organisms. In pupylation process, the prokaryotic ubiquitin-like protein (Pup tagging is functionally analogous to ubiquitination in order to tag target proteins for proteasomal degradation. To date, several experimental methods have been developed to identify pupylated proteins and their pupylation sites, but these experimental methods are generally laborious and costly. Therefore, computational methods that can accurately predict potential pupylation sites based on protein sequence information are highly desirable. In this paper, a novel predictor termed as pbPUP has been developed for accurate prediction of pupylation sites. In particular, a sophisticated sequence encoding scheme [i.e. the profile-based composition of k-spaced amino acid pairs (pbCKSAAP] is used to represent the sequence patterns and evolutionary information of the sequence fragments surrounding pupylation sites. Then, a Support Vector Machine (SVM classifier is trained using the pbCKSAAP encoding scheme. The final pbPUP predictor achieves an AUC value of 0.849 in 10-fold cross-validation tests and outperforms other existing predictors on a comprehensive independent test dataset. The proposed method is anticipated to be a helpful computational resource for the prediction of pupylation sites. The web server and curated datasets in this study are freely available at http://protein.cau.edu.cn/pbPUP/.

  7. Accurate prediction of subcellular location of apoptosis proteins combining Chou’s PseAAC and PsePSSM based on wavelet denoising

    Science.gov (United States)

    Chen, Cheng; Chen, Rui-Xin; Wang, Lei; Wang, Ming-Hui; Zhang, Yan

    2017-01-01

    Apoptosis proteins subcellular localization information are very important for understanding the mechanism of programmed cell death and the development of drugs. The prediction of subcellular localization of an apoptosis protein is still a challenging task because the prediction of apoptosis proteins subcellular localization can help to understand their function and the role of metabolic processes. In this paper, we propose a novel method for protein subcellular localization prediction. Firstly, the features of the protein sequence are extracted by combining Chou's pseudo amino acid composition (PseAAC) and pseudo-position specific scoring matrix (PsePSSM), then the feature information of the extracted is denoised by two-dimensional (2-D) wavelet denoising. Finally, the optimal feature vectors are input to the SVM classifier to predict subcellular location of apoptosis proteins. Quite promising predictions are obtained using the jackknife test on three widely used datasets and compared with other state-of-the-art methods. The results indicate that the method proposed in this paper can remarkably improve the prediction accuracy of apoptosis protein subcellular localization, which will be a supplementary tool for future proteomics research. PMID:29296195

  8. Binomial probability distribution model-based protein identification algorithm for tandem mass spectrometry utilizing peak intensity information.

    Science.gov (United States)

    Xiao, Chuan-Le; Chen, Xiao-Zhou; Du, Yang-Li; Sun, Xuesong; Zhang, Gong; He, Qing-Yu

    2013-01-04

    Mass spectrometry has become one of the most important technologies in proteomic analysis. Tandem mass spectrometry (LC-MS/MS) is a major tool for the analysis of peptide mixtures from protein samples. The key step of MS data processing is the identification of peptides from experimental spectra by searching public sequence databases. Although a number of algorithms to identify peptides from MS/MS data have been already proposed, e.g. Sequest, OMSSA, X!Tandem, Mascot, etc., they are mainly based on statistical models considering only peak-matches between experimental and theoretical spectra, but not peak intensity information. Moreover, different algorithms gave different results from the same MS data, implying their probable incompleteness and questionable reproducibility. We developed a novel peptide identification algorithm, ProVerB, based on a binomial probability distribution model of protein tandem mass spectrometry combined with a new scoring function, making full use of peak intensity information and, thus, enhancing the ability of identification. Compared with Mascot, Sequest, and SQID, ProVerB identified significantly more peptides from LC-MS/MS data sets than the current algorithms at 1% False Discovery Rate (FDR) and provided more confident peptide identifications. ProVerB is also compatible with various platforms and experimental data sets, showing its robustness and versatility. The open-source program ProVerB is available at http://bioinformatics.jnu.edu.cn/software/proverb/ .

  9. Identification of T1D susceptibility genes within the MHC region by combining protein interaction networks and SNP genotyping data

    DEFF Research Database (Denmark)

    Brorsson, C.; Hansen, Niclas Tue; Hansen, Kasper Lage

    2009-01-01

    genes. We have developed a novel method that combines single nucleotide polymorphism (SNP) genotyping data with protein-protein interaction (ppi) networks to identify disease-associated network modules enriched for proteins encoded from the MHC region. Approximately 2500 SNPs located in the 4 Mb MHC......To develop novel methods for identifying new genes that contribute to the risk of developing type 1 diabetes within the Major Histocompatibility Complex (MHC) region on chromosome 6, independently of the known linkage disequilibrium (LD) between human leucocyte antigen (HLA)-DRB1, -DQA1, -DQB1...... region were analysed in 1000 affected offspring trios generated by the Type 1 Diabetes Genetics Consortium (T1DGC). The most associated SNP in each gene was chosen and genes were mapped to ppi networks for identification of interaction partners. The association testing and resulting interacting protein...

  10. The Search Engine for Multi-Proteoform Complexes: An Online Tool for the Identification and Stoichiometry Determination of Protein Complexes.

    Science.gov (United States)

    Skinner, Owen S; Schachner, Luis F; Kelleher, Neil L

    2016-12-08

    Recent advances in top-down mass spectrometry using native electrospray now enable the analysis of intact protein complexes with relatively small sample amounts in an untargeted mode. Here, we describe how to characterize both homo- and heteropolymeric complexes with high molecular specificity using input data produced by tandem mass spectrometry of whole protein assemblies. The tool described is a "search engine for multi-proteoform complexes," (SEMPC) and is available for free online. The output is a list of candidate multi-proteoform complexes and scoring metrics, which are used to define a distinct set of one or more unique protein subunits, their overall stoichiometry in the intact complex, and their pre- and post-translational modifications. Thus, we present an approach for the identification and characterization of intact protein complexes from native mass spectrometry data. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  11. Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements.

    Directory of Open Access Journals (Sweden)

    Olga V Viktorovskaya

    2016-08-01

    Full Text Available There are currently no vaccines or antivirals available for dengue virus infection, which can cause dengue hemorrhagic fever and death. A better understanding of the host pathogen interaction is required to develop effective therapies to treat DENV. In particular, very little is known about how cellular RNA binding proteins interact with viral RNAs. RNAs within cells are not naked; rather they are coated with proteins that affect localization, stability, translation and (for viruses replication.Seventy-nine novel RNA binding proteins for dengue virus (DENV were identified by cross-linking proteins to dengue viral RNA during a live infection in human cells. These cellular proteins were specific and distinct from those previously identified for poliovirus, suggesting a specialized role for these factors in DENV amplification. Knockdown of these proteins demonstrated their function as viral host factors, with evidence for some factors acting early, while others late in infection. Their requirement by DENV for efficient amplification is likely specific, since protein knockdown did not impair the cell fitness for viral amplification of an unrelated virus. The protein abundances of these host factors were not significantly altered during DENV infection, suggesting their interaction with DENV RNA was due to specific recruitment mechanisms. However, at the global proteome level, DENV altered the abundances of proteins in particular classes, including transporter proteins, which were down regulated, and proteins in the ubiquitin proteasome pathway, which were up regulated.The method for identification of host factors described here is robust and broadly applicable to all RNA viruses, providing an avenue to determine the conserved or distinct mechanisms through which diverse viruses manage the viral RNA within cells. This study significantly increases the number of cellular factors known to interact with DENV and reveals how DENV modulates and usurps

  12. Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates.

    Directory of Open Access Journals (Sweden)

    Christy Catherine

    Full Text Available Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA.

  13. Identification and quantification of major maillard cross-links in human serum albumin and lens protein. Evidence for glucosepane as the dominant compound.

    Science.gov (United States)

    Biemel, Klaus M; Friedl, D Alexander; Lederer, Markus O

    2002-07-12

    Glycation reactions leading to protein modifications (advanced glycation end products) contribute to various pathologies associated with the general aging process and long term complications of diabetes. However, only few relevant compounds have so far been detected in vivo. We now report on the first unequivocal identification of the lysine-arginine cross-links glucosepane 5, DOGDIC 6, MODIC 7, and GODIC 8 in human material. For their accurate quantification by coupled liquid chromatography-electrospray ionization mass spectrometry, (13)C-labeled reference compounds were synthesized independently. Compounds 5-8 are formed via the alpha-dicarbonyl compounds N(6)-(2,3-dihydroxy-5,6-dioxohexyl)-l-lysinate (1a,b), 3-deoxyglucosone (), methylglyoxal (), and glyoxal (), respectively. The protein-bound dideoxyosone 1a,b seems to be of prime significance for cross-linking because it presumably is not detoxified by mammalian enzymes as readily as 2-4. Hence, the follow-up product glucosepane 5 was found to be the dominant compound. Up to 42.3 pmol of 5/mg of protein was identified in human serum albumin of diabetics; the level of 5 correlates markedly with the glycated hemoglobin HbA(1c). In the water-insoluble fraction of lens proteins from normoglycemics, concentration of 5 ranges between 132.3 and 241.7 pmol/mg. The advanced glycoxidation end product GODIC 8 is elevated significantly in brunescent lenses, indicating enhanced oxidative stress in this material. Compounds 5-8 thus appear predestined as markers for pathophysiological processes.

  14. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

    Directory of Open Access Journals (Sweden)

    Perera Rajika L

    2004-12-01

    Full Text Available Abstract Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44, kinases (EGFR-cytoplasmic domain, CDK2 and 4, proteases (MMP1, CASP2, signal transduction proteins (GRB2, RAF1, HRAS and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX. Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY, or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx and maltose binding protein (MBP were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases

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

    Science.gov (United States)

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

    2015-03-03

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

  16. Identification of in planta protein–protein interactions using IP-MS

    NARCIS (Netherlands)

    Jamge, Suraj; Angenent, Gerco; Bemer, Marian

    2018-01-01

    Gene regulation by transcription factors involves complex protein interaction networks, which include chromatin remodeling and modifying proteins as an integral part. Decoding these protein interactions is crucial for our understanding of chromatin-mediated gene regulation. Here, we describe a

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer

    International Nuclear Information System (INIS)

    McGoldrick, Christopher A; Jiang, Yu-Lin; Paromov, Victor; Brannon, Marianne; Krishnan, Koyamangalath; Stone, William L

    2014-01-01

    Esterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells. Cellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results. The major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 3.4.19.1) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells. These data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH

  19. Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates.

    Directory of Open Access Journals (Sweden)

    Carol B Fowler

    2010-12-01

    Full Text Available Proteomic studies of formalin-fixed paraffin-embedded (FFPE tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing.In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%. Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates.These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form suitable for proteomic analysis.

  20. Elevated Pressure Improves the Extraction and Identification of Proteins Recovered from Formalin-Fixed, Paraffin-Embedded Tissue Surrogates

    Science.gov (United States)

    Fowler, Carol B.; Chesnick, Ingrid E.; Moore, Cedric D.; O'Leary, Timothy J.; Mason, Jeffrey T.

    2010-01-01

    Background Proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues are frustrated by the inability to extract proteins from archival tissue in a form suitable for analysis by 2-D gel electrophoresis or mass spectrometry. This inability arises from the difficulty of reversing formaldehyde-induced protein adducts and cross-links within FFPE tissues. We previously reported the use of elevated hydrostatic pressure as a method for efficient protein recovery from a hen egg-white lysozyme tissue surrogate, a model system developed to study formalin fixation and histochemical processing. Principal Findings In this study, we demonstrate the utility of elevated hydrostatic pressure as a method for efficient protein recovery from FFPE mouse liver tissue and a complex multi-protein FFPE tissue surrogate comprised of hen egg-white lysozyme, bovine carbonic anhydrase, bovine ribonuclease A, bovine serum albumin, and equine myoglobin (55∶15∶15∶10∶5 wt%). Mass spectrometry of the FFPE tissue surrogates retrieved under elevated pressure showed that both the low and high-abundance proteins were identified with sequence coverage comparable to that of the surrogate mixture prior to formaldehyde treatment. In contrast, non-pressure-extracted tissue surrogate samples yielded few positive and many false peptide identifications. Studies with soluble formalin-treated bovine ribonuclease A demonstrated that pressure modestly inhibited the rate of reversal (hydrolysis) of formaldehyde-induced protein cross-links. Dynamic light scattering studies suggest that elevated hydrostatic pressure and heat facilitate the recovery of proteins free of formaldehyde adducts and cross-links by promoting protein unfolding and hydration with a concomitant reduction in the average size of the protein aggregates. Conclusions These studies demonstrate that elevated hydrostatic pressure treatment is a promising approach for improving the recovery of proteins from FFPE tissues in a form

  1. Identification of differentially expressed reproductive and metabolic proteins in the female abalone (Haliotis laevigata) gonad following artificial induction of spawning.

    Science.gov (United States)

    Mendoza-Porras, Omar; Botwright, Natasha A; Reverter, Antonio; Cook, Mathew T; Harris, James O; Wijffels, Gene; Colgrave, Michelle L

    2017-12-01

    Inefficient control of temperate abalone spawning prevents pair-wise breeding and production of abalone with highly marketable traits. Traditionally, abalone farmers have used a combination of UV irradiation and application of temperature gradients to the tank water to artificially induce spawning. Proteins are known to regulate crucial processes such as respiration, muscle contraction, feeding, growth and reproduction. Spawning as a pre-requisite of abalone reproduction is likely to be regulated, in part, by endogenous proteins. A first step in elucidating the mechanisms that regulate spawning is to identify which proteins are directly involved during spawning. The present study examined protein expression following traditional spawning induction in the Haliotis laevigata female. Gonads were collected from abalone in the following physiological states: (1) spawning; (2) post-spawning; and (3) failed-to-spawn. Differential protein abundance was initially assessed using two-dimensional difference in-gel electrophoresis coupled with mass spectrometry for protein identification. A number of reproductive proteins such as vitellogenin, vitelline envelope zona pellucida domain 29 and prohibitin, and metabolic proteins such as thioredoxin peroxidase, superoxide dismutase and heat shock proteins were identified. Differences in protein abundance levels between physiological states were further assessed using scheduled multiple reaction monitoring mass spectrometry. Positive associations were observed between the abundance of specific proteins, such as heat shock cognate 70 and peroxiredoxin 6, and the propensity or failure to spawn in abalone. These findings have contributed to better understand both the effects of oxidative and heat stress over abalone physiology and their influence on abalone spawning. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  2. Large scale identification and categorization of protein sequences using structured logistic regression.

    Directory of Open Access Journals (Sweden)

    Bjørn P Pedersen

    Full Text Available BACKGROUND: Structured Logistic Regression (SLR is a newly developed machine learning tool first proposed in the context of text categorization. Current availability of extensive protein sequence databases calls for an automated method to reliably classify sequences and SLR seems well-suited for this task. The classification of P-type ATPases, a large family of ATP-driven membrane pumps transporting essential cations, was selected as a test-case that would generate important biological information as well as provide a proof-of-concept for the application of SLR to a large scale bioinformatics problem. RESULTS: Using SLR, we have built classifiers to identify and automatically categorize P-type ATPases into one of 11 pre-defined classes. The SLR-classifiers are compared to a Hidden Markov Model approach and shown to be highly accurate and scalable. Representing the bulk of currently known sequences, we analysed 9.3 million sequences in the UniProtKB and attempted to classify a large number of P-type ATPases. To examine the distribution of pumps on organisms, we also applied SLR to 1,123 complete genomes from the Entrez genome database. Finally, we analysed the predicted membrane topology of the identified P-type ATPases. CONCLUSIONS: Using the SLR-based classification tool we are able to run a large scale study of P-type ATPases. This study provides proof-of-concept for the application of SLR to a bioinformatics problem and the analysis of P-type ATPases pinpoints new and interesting targets for further biochemical characterization and structural analysis.

  3. A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma.

    Science.gov (United States)

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

    2016-04-09

    Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes.

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

    Directory of Open Access Journals (Sweden)

    Delphine Vincent

    Full Text Available 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.

  5. Sequence protein identification by randomized sequence database and transcriptome mass spectrometry (SPIDER-TMS): from manual to automatic application of a 'de novo sequencing' approach.

    Science.gov (United States)

    Pascale, Raffaella; Grossi, Gerarda; Cruciani, Gabriele; Mecca, Giansalvatore; Santoro, Donatello; Sarli Calace, Renzo; Falabella, Patrizia; Bianco, Giuliana

    Sequence protein identification by a randomized sequence database and transcriptome mass spectrometry software package has been developed at the University of Basilicata in Potenza (Italy) and designed to facilitate the determination of the amino acid sequence of a peptide as well as an unequivocal identification of proteins in a high-throughput manner with enormous advantages of time, economical resource and expertise. The software package is a valid tool for the automation of a de novo sequencing approach, overcoming the main limits and a versatile platform useful in the proteomic field for an unequivocal identification of proteins, starting from tandem mass spectrometry data. The strength of this software is that it is a user-friendly and non-statistical approach, so protein identification can be considered unambiguous.

  6. Identification of polycystic ovary syndrome potential drug targets based on pathobiological similarity in the protein-protein interaction network

    OpenAIRE

    Huang, Hao; He, Yuehan; Li, Wan; Wei, Wenqing; Li, Yiran; Xie, Ruiqiang; Guo, Shanshan; Wang, Yahui; Jiang, Jing; Chen, Binbin; Lv, Junjie; Zhang, Nana; Chen, Lina; He, Weiming

    2016-01-01

    Polycystic ovary syndrome (PCOS) is one of the most common endocrinological disorders in reproductive aged women. PCOS and Type 2 Diabetes (T2D) are closely linked in multiple levels and possess high pathobiological similarity. Here, we put forward a new computational approach based on the pathobiological similarity to identify PCOS potential drug target modules (PPDT-Modules) and PCOS potential drug targets in the protein-protein interaction network (PPIN). From the systems level and biologi...

  7. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

    Science.gov (United States)

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu; Nielsen, Maria E O; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J; Andersen, Jens S; Yao, Gang

    2012-09-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

  8. Identification and comparison of macrophage-induced proteins and proteins induced under various stress conditions in Brucella abortus.

    OpenAIRE

    Rafie-Kolpin, M; Essenberg, R C; Wyckoff, J H

    1996-01-01

    Brucella abortus is a facultative intracellular pathogen of cattle and humans that is capable of survival inside macrophages. In order to understand how B. abortus copes with the conditions during intracellular growth in macrophages, the protein synthesis pattern of the bacteria grown inside bovine macrophages has been compared with that of bacteria grown in the cell culture medium by two-dimensional polyacrylamide gel electrophoresis. Approximately 24 new proteins that are not detected in th...

  9. Nitrate as a probe of cytochrome c surface: crystallographic identification of crucial "hot spots" for protein-protein recognition.

    Science.gov (United States)

    De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

    2014-06-01

    The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. BioJava-ModFinder: identification of protein modifications in 3D structures from the Protein Data Bank.

    Science.gov (United States)

    Gao, Jianjiong; Prlic, Andreas; Bi, Chunxiao; Bluhm, Wolfgang F; Dimitropoulos, Dimitris; Xu, Dong; Bourne, Philip E; Rose, Peter W

    2017-07-01

    We developed a new software tool, BioJava-ModFinder, for identifying protein modifications observed in 3D structures archived in the Protein Data Bank (PDB). Information on more than 400 types of protein modifications were collected and curated from annotations in PDB, RESID, and PSI-MOD. We divided these modifications into three categories: modified residues, attachment modifications, and cross-links. We have developed a systematic method to identify these modifications in 3D protein structures. We have integrated this package with the RCSB PDB web application and added protein modification annotations to the sequence diagram and structure display. By scanning all 3D structures in the PDB using BioJava-ModFinder, we identified more than 30 000 structures with protein modifications, which can be searched, browsed, and visualized on the RCSB PDB website. BioJava-ModFinder is available as open source (LGPL license) at ( https://github.com/biojava/biojava/tree/master/biojava-modfinder ). The RCSB PDB can be accessed at http://www.rcsb.org . pwrose@ucsd.edu. © The Author 2017. Published by Oxford University Press.

  11. Identification of cytosolic peroxisome proliferator binding protein as a member of the heat shock protein HSP70 family.

    Science.gov (United States)

    Alvares, K; Carrillo, A; Yuan, P M; Kawano, H; Morimoto, R I; Reddy, J K

    1990-01-01

    Clofibrate and many of its structural analogues induce proliferation of peroxisomes in the hepatic parenchymal cells of rodents and certain nonrodent species including primates. This induction is tissue specific, occurring mainly in the liver parenchymal cells and to a lesser extent in the kidney cortical epithelium. The induction of peroxisomes is associated with a predictable pleiotropic response, characterized by hepatomegaly, and increased activities and mRNA levels of certain peroxisomal enzymes. Using affinity chromatography, we had previously isolated a protein that binds to clofibric acid. We now show that this protein is homologous with the heat shock protein HSP70 family by analysis of amino acid sequences of isolated peptides from trypsin-treated clofibric acid binding protein and by cross-reactivity with a monoclonal antibody raised against the conserved region of the 70-kDa heat shock proteins. The clofibric acid-Sepharose column could bind HSP70 proteins isolated from various species, which could then be eluted with either clofibric acid or ATP. Conversely, when a rat liver cytosol containing multiple members of the HSP70 family was passed through an ATP-agarose column, and eluted with clofibric acid, only P72 (HSC70) was eluted. These results suggest that clofibric acid, a peroxisome proliferator, preferentially interacts with P72 at or near the ATP binding site. Images PMID:2371272

  12. Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

    Energy Technology Data Exchange (ETDEWEB)

    Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko [Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 (Japan); Umezawa, Kazuo, E-mail: umezawa@aichi-med-u.ac.jp [Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195 (Japan)

    2016-08-05

    15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. -- Highlights: •Fifteen-deoxyspergualin (DSG) is an immunosuppressive agent clinically used. •We have identified PCBP2, an RNA-binding protein, as a molecular target of DSG. •Alteration of PCBP2 activity may explain the immunosuppressive activity of DSG.

  13. ContaMiner and ContaBase: a webserver and database for early identification of unwantedly crystallized protein contaminants

    Science.gov (United States)

    Hungler, Arnaud; Momin, Afaque; Diederichs, Kay; Arold, Stefan, T.

    2016-01-01

    Solving the phase problem in protein X-ray crystallography relies heavily on the identity of the crystallized protein, especially when molecular replacement (MR) methods are used. Yet, it is not uncommon that a contaminant crystallizes instead of the protein of interest. Such contaminants may be proteins from the expression host organism, protein fusion tags or proteins added during the purification steps. Many contaminants co-purify easily, crystallize and give good diffraction data. Identification of contaminant crystals may take time, since the presence of the contaminant is unexpected and its identity unknown. A webserver (ContaMiner) and a contaminant database (ContaBase) have been established, to allow fast MR-based screening of crystallographic data against currently 62 known contaminants. The web-based ContaMiner (available at http://strube.cbrc.kaust.edu.sa/contaminer/) currently produces results in 5 min to 4 h. The program is also available in a github repository and can be installed locally. ContaMiner enables screening of novel crystals at synchrotron beamlines, and it would be valuable as a routine safety check for ‘crystallization and preliminary X-ray analysis’ publications. Thus, in addition to potentially saving X-ray crystallographers much time and effort, ContaMiner might considerably lower the risk of publishing erroneous data. PMID:27980519

  14. Identification of RNAIII-binding proteins in Staphylococcus aureus using tethered RNAs and streptavidin aptamers based pull-down assay.

    Science.gov (United States)

    Zhang, Xu; Zhu, Qing; Tian, Tian; Zhao, Changlong; Zang, Jianye; Xue, Ting; Sun, Baolin

    2015-05-15

    It has been widely recognized that small RNAs (sRNAs) play important roles in physiology and virulence control in bacteria. In Staphylococcus aureus, many sRNAs have been identified and some of them have been functionally studied. Since it is difficult to identify RNA-binding proteins (RBPs), very little has been known about the RBPs in S. aureus, especially those associated with sRNAs. Here we adopted a tRNA scaffold streptavidin aptamer based pull-down assay to identify RBPs in S. aureus. The tethered RNA was successfully captured by the streptavidin magnetic beads, and proteins binding to RNAIII were isolated and analyzed by mass spectrometry. We have identified 81 proteins, and expressed heterologously 9 of them in Escherichia coli. The binding ability of the recombinant proteins with RNAIII was further analyzed by electrophoresis mobility shift assay, and the result indicates that proteins CshA, RNase J2, Era, Hu, WalR, Pyk, and FtsZ can bind to RNAIII. This study suggests that some proteins can bind to RNA III in S. aureus, and may be involved in RNA III function. And tRSA based pull-down assay is an effective method to search for RBPs in bacteria, which should facilitate the identification and functional study of RBPs in diverse bacterial species.

  15. Proteomic identification of S-nitrosylated Golgi proteins: new insights into endothelial cell regulation by eNOS-derived NO.

    Directory of Open Access Journals (Sweden)

    Panjamaporn Sangwung

    Full Text Available Endothelial nitric oxide synthase (eNOS is primarily localized on the Golgi apparatus and plasma membrane caveolae in endothelial cells. Previously, we demonstrated that protein S-nitrosylation occurs preferentially where eNOS is localized. Thus, in endothelial cells, Golgi proteins are likely to be targets for S-nitrosylation. The aim of this study was to identify S-nitrosylated Golgi proteins and attribute their S-nitrosylation to eNOS-derived nitric oxide in endothelial cells.Golgi membranes were isolated from rat livers. S-nitrosylated Golgi proteins were determined by a modified biotin-switch assay coupled with mass spectrometry that allows the identification of the S-nitrosylated cysteine residue. The biotin switch assay followed by Western blot or immunoprecipitation using an S-nitrosocysteine antibody was also employed to validate S-nitrosylated proteins in endothelial cell lysates.Seventy-eight potential S-nitrosylated proteins and their target cysteine residues for S-nitrosylation were identified; 9 of them were Golgi-resident or Golgi/endoplasmic reticulum (ER-associated proteins. Among these 9 proteins, S-nitrosylation of EMMPRIN and Golgi phosphoprotein 3 (GOLPH3 was verified in endothelial cells. Furthermore, S-nitrosylation of these proteins was found at the basal levels and increased in response to eNOS stimulation by the calcium ionophore A23187. Immunofluorescence microscopy and immunoprecipitation showed that EMMPRIN and GOLPH3 are co-localized with eNOS at the Golgi apparatus in endothelial cells. S-nitrosylation of EMMPRIN was notably increased in the aorta of cirrhotic rats.Our data suggest that the selective S-nitrosylation of EMMPRIN and GOLPH3 at the Golgi apparatus in endothelial cells results from the physical proximity to eNOS-derived nitric oxide.

  16. Autoimmunity to Tropomyosin-Specific Peptides Induced by Mycobacterium leprae in Leprosy Patients: Identification of Mimicking Proteins.

    Science.gov (United States)

    Singh, Itu; Yadav, Asha Ram; Mohanty, Keshar Kunja; Katoch, Kiran; Sharma, Prashant; Pathak, Vinay Kumar; Bisht, Deepa; Gupta, Umesh D; Sengupta, Utpal

    2018-01-01

    It has been shown earlier that there is a rise in the levels of autoantibodies and T cell response to cytoskeletal proteins in leprosy. Our group recently demonstrated a rise in both T and B cell responses to keratin and myelin basic protein in all types of leprosy patients and their associations in type 1 reaction (T1R) group of leprosy. In this study, we investigated the association of levels of autoantibodies and lymphoproliferation against myosin in leprosy patients across the spectrum and tried to find out the mimicking proteins or epitopes between host protein and protein/s of Mycobacterium leprae . One hundred and sixty-nine leprosy patients and 55 healthy controls (HC) were enrolled in the present study. Levels of anti-myosin antibodies and T-cell responses against myosin were measured by ELISA and lymphoproliferation assay, respectively. Using 2-D gel electrophoresis, western blot and MALDI-TOF/TOF antibody-reactive spots were identified. Three-dimensional structure of mimicking proteins was modeled by online server. B cell epitopes of the proteins were predicted by BCPREDS server 1.0 followed by identification of mimicking epitopes. Mice of inbred BALB/c strain were hyperimmunized with M. leprae soluble antigen (MLSA) and splenocytes and lymph node cells of these animals were adoptively transferred to naïve mice. Highest level of anti-myosin antibodies was noted in sera of T1R leprosy patients. We observed significantly higher levels of lymphoproliferative response ( p  leprae . We found four mimicking epitopes between these sequences. These data suggest that these mimicking proteins tropomyosin and ATP-dependent Clp protease ATP-binding subunit of M. leprae or more precisely mimicking epitopes (four B cell epitopes) might be responsible for extensive tissue damage during type1 reaction in leprosy.

  17. Matrix-assisted laser desorption/ionization coupled with quadrupole/orthogonal acceleration time-of-flight mass spectrometry for protein discovery, identification, and structural analysis.

    Science.gov (United States)

    Baldwin, M A; Medzihradszky, K F; Lock, C M; Fisher, B; Settineri, T A; Burlingame, A L

    2001-04-15

    The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel

  18. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): Identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42

    International Nuclear Information System (INIS)

    Shinjo, K.; Koland, J.G.; Hart, M.J.; Narasimhan, V.; Cerione, R.A.; Johnson, D.I.; Evans, T.

    1990-01-01

    The authors have isolated cDNA clones from a human placental library that code for a low molecular weight GTP-binding protein originally designated G p (also called G25K). This identification is based on comparisons with the available peptide sequences for the purified human G p protein and the use of two highly specific anti-peptide antibodies. The predicted amino acid sequence of the protein is very similar to those of various members of the ras superfamily of low molecular weight GTP-binding proteins, including the N-, Ki-, and Ha-ras proteins (30-35% identical), the rho proteins and the rac proteins. The highest degree of sequence identity (80%) is found with the Saccharomyces cerevisiae cell division-cycle protein CDC42. The human placental gene, which they designate CDC42Hs, complements the cdc42-1 mutation in S. cerevisiae, which suggests that this GTP-binding protein is the human homolog of the yeast protein

  19. Aniline-induced nitrosative stress in rat spleen: Proteomic identification of nitrated proteins

    International Nuclear Information System (INIS)

    Fan Xiuzhen; Wang Jianling; Soman, Kizhake V.; Ansari, G.A.S.; Khan, M. Firoze

    2011-01-01

    Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6 kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity. - Highlights: → Proteomic approaches are used to identify nitrated proteins in the spleen. → Twenty five nitrated proteins were found only in the spleen of aniline-treated rats. → Aniline exposure led to increased iNOS mRNA and protein

  20. Identification of fibrinogen-binding proteins of Aspergillus fumigatus using proteomic approach.

    Science.gov (United States)

    Upadhyay, Santosh Kumar; Gautam, Poonam; Pandit, Hrishikesh; Singh, Yogendra; Basir, Seemi Farhat; Madan, Taruna

    2012-03-01

    Aspergillus fumigatus, the main etiological agent for various forms of human aspergillosis, gets access to the respiratory system of human host by inhalation of airborne conidia. These conidia possibly adhere to extracellular matrix (ECM) proteins. Among the ECM proteins involved in adherence, fibrinogen is thought to be crucial. Here, we studied whether A. fumigatus three-week culture filtrate (3wcf) proteins promote binding of A. fumigatus to ECM proteins and promote fungal growth. We observed that incubation of ECM with 3wcf proteins led to dose- and time-dependent increase in adherence of conidia to the ECM. In order to identify the catalogue of fibrinogen-binding A. fumigatus proteins, we carried out fibrinogen affinity blotting using two-dimensional gel electrophoresed 3wcf proteins. A total of 15 fibrinogen-binding protein spots corresponding to 7 unique proteins were identified in 3wcf using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF). Among these, 4 proteins, namely, beta-glucosidase, alpha-mannosidase, pectate lyase A and oryzin precursor were predicted to have cell wall or extracellular localization, whereas amidase family protein and two hypothetical proteins did not display the signal sequence. This study reports seven novel fibrinogen-binding proteins of A. fumigatus, some of which could be further explored for targeting the adhesion phenomenon as antifungal strategy.

  1. Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato

    NARCIS (Netherlands)

    Rep, Martijn; Dekker, Henk L.; Vossen, Jack H.; de Boer, Albert D.; Houterman, Petra M.; Speijer, Dave; Back, Jaap W.; de Koster, Chris G.; Cornelissen, Ben J. C.

    2002-01-01

    The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during

  2. Identification of arsenite-and arsenic diglutathione-binding proteins in human hepatocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Mizumura, Ayano; Watanabe, Takayuki [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Kobayashi, Yayoi [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Environmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan); Hirano, Seishiro [Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi, Inage, Chiba 263-8522 (Japan); Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)

    2010-01-15

    It is generally accepted that trivalent arsenicals are more toxic than the corresponding pentavalent arsenicals, since trivalent arsenicals bind the thiol groups of biomolecules, leading to a deterioration in cellular functions. In the present study, we prepared three different arsenic-bound sepharoses and investigated the binding of hepatic cytosolic proteins to pentavalent, trivalent, and glutathione-conjugated trivalent arsenicals. SDS-PAGE showed no proteins bound to pentavalent arsenic specifically. In contrast, we found a number of proteins that have specific and high affinity for trivalent arsenic. Two of those proteins were identified: protein disulfide isomerase-related protein 5 (PDSIRP5) and peroxiredoxin 1/enhancer protein (PRX1/EP). These proteins have vicinal cysteines, as previously reported. In contrast, one of the prominent proteins that did not bind to trivalent arsenic was identified as calreticulin precursor. Although there are 3 cysteines in calreticulin precursor, two of the cysteines are spaced more than 25 amino acids apart. Five synthetic peptides containing 2 vicinal cysteines were prepared to study whether they would inhibit the binding of PDSIRP5, PRX1/EP, and other arsenic-binding proteins to trivalent arsenicals. Only two of the five peptides effectively inhibited binding, suggesting that other amino acids besides the 2 vicinal cysteines may modulate the affinity of cysteine-rich proteins for trivalent arsenicals. We further investigated hepatic cytosolic proteins that bound specifically to glutathione-conjugated trivalent arsenic, which is the most abundant form of arsenical in bile fluid. Four proteins that bound specifically to glutathione-conjugated trivalent arsenic were identified; interestingly, these proteins were different from the trivalent arsenic-binding proteins. These results suggest that although glutathione-conjugation is an important process in the metabolism, excretion, and detoxification of arsenicals, glutathione

  3. Identification, characterization, and synthesis of peptide epitopes and a recombinant six-epitope protein for Trichomonas vaginalis serodiagnosis

    Directory of Open Access Journals (Sweden)

    Alderete JF

    2013-08-01

    Full Text Available J F Alderete, Calvin J NeaceSchool of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USAAbstract: There is a need for a rapid, accurate serodiagnostic test useful for both women and men infected by Trichomonas vaginalis, which causes the number one sexually transmitted infection (STI. Women and men exposed to T. vaginalis make serum antibody to fructose-1,6-bisphosphate aldolase (ALD, α-enolase (ENO, and glyceraldehyde-3-phosphate dehydrogenase (GAP. We identified, by epitope mapping, the common and distinct epitopes of each protein detected by the sera of women patients with trichomonosis and by the sera of men highly seropositive to the immunogenic protein α-actinin (positive control sera. We analyzed the amino acid sequences to determine the extent of identity of the epitopes of each protein with other proteins in the databanks. This approach identified epitopes unique to T. vaginalis, indicating these peptide-epitopes as possible targets for a serodiagnostic test. Individual or combinations of 15-mer peptide epitopes with low to no identity with other proteins were reactive with positive control sera from both women and men but were unreactive with negative control sera. These analyses permitted the synthesis of a recombinant His6 fusion protein of 111 amino acids with an Mr of ~13.4 kDa, which consisted of 15-mer peptides of two distinct epitopes each for ALD, ENO, and GAP. This recombinant protein was purified by affinity chromatography. This composite protein was detected by enzyme-linked immunosorbent assay (ELISA, dot blots, and immunoblots, using positive control sera from women and men. These data indicate that it is possible to identify epitopes and that either singly, in combination, or as a composite protein represent targets for a point-of-care serodiagnostic test for T. vaginalis.Keywords: diagnostics, point-of-care, targets, trichomonosis

  4. iTRAQ-Based Identification of Proteins Related to Muscle Growth in the Pacific Abalone, Haliotis discus hannai

    Directory of Open Access Journals (Sweden)

    Jianfang Huang

    2017-10-01

    Full Text Available The abalone Haliotis discus hannai is an important aquaculture species that is grown for human consumption. However, little is known of the genetic mechanisms governing muscle growth in this species, particularly with respect to proteomics. The isobaric tag for relative and absolute quantitation (iTRAQ method allows for sensitive and accurate protein quantification. Our study was the first to use iTRAQ-based quantitative proteomics to investigate muscle growth regulation in H. discus hannai. Among the 1904 proteins identified from six samples, 125 proteins were differentially expressed in large specimens of H. discus hannai as compared to small specimens. In the large specimens, 47 proteins were upregulated and 78 were downregulated. Many of the significant Kyoto Encyclopedia of Genes and Genomes (KEGG pathways, including these differentially expressed proteins, were closely related to muscle growth, including apoptosis, thyroid hormone signaling, regulation of the actin cytoskeleton, and viral myocarditis (p < 0.05. Our quantitative real-time polymerase chain reaction (qRT-PCR analyses suggested that the alterations in expression levels observed in the differentially expressed proteins were consistent with the alterations observed in the encoding mRNAs, indicating the repeatability of our proteomic approach. Our findings contribute to the knowledge of the molecular mechanisms of muscle growth in H. discus hannai.

  5. Structure-sequence based analysis for identification of conserved regions in proteins

    Science.gov (United States)

    Zemla, Adam T; Zhou, Carol E; Lam, Marisa W; Smith, Jason R; Pardes, Elizabeth

    2013-05-28

    Disclosed are computational methods, and associated hardware and software products for scoring conservation in a protein structure based on a computationally identified family or cluster of protein structures. A method of computationally identifying a family or cluster of protein structures in also disclosed herein.

  6. Identification of structural similarities between putative transmission proteins of Polymyxa and Spongospora transmitted bymoviruses and furoviruses.

    Science.gov (United States)

    Dessens, J T; Meyer, M

    1996-01-01

    Comparison of amino acid sequence and hydropathy profiles shows conserved, structural similarities between the capsid readthrough protein of potato mop top virus (transmitted by Spongospora subterranea) and furovirus and bymovirus proteins implicated in transmission by Polymyxa spp. This suggests that these proteins have a common ancestry and are involved in a common biological process: virus transmission by plasmodiophorid fungi.

  7. Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

    International Nuclear Information System (INIS)

    Witt, P.L.; Bownds, M.D.

    1987-01-01

    Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger

  8. Integrative Identification of Arabidopsis Mitochondrial Proteome and Its Function Exploitation through Protein Interaction Network

    Science.gov (United States)

    Cui, Jian; Liu, Jinghua; Li, Yuhua; Shi, Tieliu

    2011-01-01

    Mitochondria are major players on the production of energy, and host several key reactions involved in basic metabolism and biosynthesis of essential molecules. Currently, the majority of nucleus-encoded mitochondrial proteins are unknown even for model plant Arabidopsis. We reported a computational framework for predicting Arabidopsis mitochondrial proteins based on a probabilistic model, called Naive Bayesian Network, which integrates disparate genomic data generated from eight bioinformatics tools, multiple orthologous mappings, protein domain properties and co-expression patterns using 1,027 microarray profiles. Through this approach, we predicted 2,311 candidate mitochondrial proteins with 84.67% accuracy and 2.53% FPR performances. Together with those experimental confirmed proteins, 2,585 mitochondria proteins (named CoreMitoP) were identified, we explored those proteins with unknown functions based on protein-protein interaction network (PIN) and annotated novel functions for 26.65% CoreMitoP proteins. Moreover, we found newly predicted mitochondrial proteins embedded in particular subnetworks of the PIN, mainly functioning in response to diverse environmental stresses, like salt, draught, cold, and wound etc. Candidate mitochondrial proteins involved in those physiological acitivites provide useful targets for further investigation. Assigned functions also provide comprehensive information for Arabidopsis mitochondrial proteome. PMID:21297957

  9. Ribosomal subunit protein typing using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification and discrimination of Aspergillus species.

    Science.gov (United States)

    Nakamura, Sayaka; Sato, Hiroaki; Tanaka, Reiko; Kusuya, Yoko; Takahashi, Hiroki; Yaguchi, Takashi

    2017-04-26

    Accurate identification of Aspergillus species is a very important subject. Mass spectral fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is generally employed for the rapid identification of fungal isolates. However, the results are based on simple mass spectral pattern-matching, with no peak assignment and no taxonomic input. We propose here a ribosomal subunit protein (RSP) typing technique using MALDI-TOF MS for the identification and discrimination of Aspergillus species. The results are concluded to be phylogenetic in that they reflect the molecular evolution of housekeeping RSPs. The amino acid sequences of RSPs of genome-sequenced strains of Aspergillus species were first verified and compared to compile a reliable biomarker list for the identification of Aspergillus species. In this process, we revealed that many amino acid sequences of RSPs (about 10-60%, depending on strain) registered in the public protein databases needed to be corrected or newly added. The verified RSPs were allocated to RSP types based on their mass. Peak assignments of RSPs of each sample strain as observed by MALDI-TOF MS were then performed to set RSP type profiles, which were then further processed by means of cluster analysis. The resulting dendrogram based on RSP types showed a relatively good concordance with the tree based on β-tubulin gene sequences. RSP typing was able to further discriminate the strains belonging to Aspergillus section Fumigati. The RSP typing method could be applied to identify Aspergillus species, even for species within section Fumigati. The discrimination power of RSP typing appears to be comparable to conventional β-tubulin gene analysis. This method would therefore be suitable for species identification and discrimination at the strain to species level. Because RSP typing can characterize the strains within section Fumigati, this method has potential as a powerful and reliable tool in

  10. Identification of host cell proteins which interact with herpes simplex virus type 1 tegument protein pUL37.

    Science.gov (United States)

    Kelly, Barbara J; Diefenbach, Eve; Fraefel, Cornel; Diefenbach, Russell J

    2012-01-20

    The herpes simplex virus type 1 (HSV-1) structural tegument protein pUL37, which is conserved across the Herpesviridae family, is known to be essential for secondary envelopment during the egress of viral particles. To shed light on additional roles of pUL37 during viral replication a yeast two-hybrid screen of a human brain cDNA library was undertaken. This screen identified ten host cell proteins as potential pUL37 interactors. One of the interactors, serine threonine kinase TAOK3, was subsequently confirmed to interact with pUL37 using an in vitro pulldown assay. Such host cell/pUL37 interactions provide further insights into the multifunctional role of this herpesviral tegument protein. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Purification of a protein from serum of cattle with hepatic lipidosis, and identification of the protein as haptoglobin.

    Science.gov (United States)

    Yoshino, K; Katoh, N; Takahashi, K; Yuasa, A

    1992-06-01

    A protein that has 2 subunits with molecular weight of 35,000 and 23,000 was detected in serum of cattle with hepatic lipidosis (fatty liver). The protein was purified from serum obtained from a cow with fatty liver, and was identified as haptoglobin, which is known to have hemoglobin-binding capacity and to be an acute-phase protein. To assess the relevance of haptoglobin in fatty liver, cattle were classified in 3 groups (healthy control, haptoglobin-positive, and haptoglobin-negative); liver triglyceride content and several serum biochemical variables were evaluated for the 3 groups. Compared with the control and haptoglobin-negative cattle, haptoglobin-positive cattle had significantly (P less than 0.01) higher liver triglyceride content, serum bilirubin concentration, and aspartate transaminase activity. Serum haptoglobin concentration was high in slaughter cattle (27 of 40 cattle tested), particularly in cows (20/28).

  12. Finding the "bio" in biobased products: electrophoretic identification of wheat proteins in processed products.

    Science.gov (United States)

    Robertson, George H; Hurkman, William J; Cao, Trung K; Tanaka, Charlene K; Orts, William J

    2010-04-14

    Verification of the biocontent in biobased or "green" products identifies genuine products, exposes counterfeit copies, supports or refutes content claims, and ensures consumer confidence. When the biocontent includes protein, elemental nitrogen analysis is insufficient for verification since non-protein, but nitrogen-rich, content also may be present. However, the proteins can be extracted, separated by electrophoretic methods, and detected by UV absorption, protein stain, or immunoblotting. We utilized capillary zone electrophoresis (CZE) to separate proteins in a gliadin fraction that had been dissolved in aqueous ethanol (70%) and polyacrylamide gel electrophoresis (PAGE) to separate proteins in a gliadin-plus-glutenin fraction that had been dissolved in water containing both sodium dodecyl sulfate (SDS) and a reducing agent, dithiothreitol (DTT). We sought to verify the presence of these wheat grain proteins in wheat bread, a wheat flake cereal, wheat beer, and an enclosure for an antique automobile ignition coil reputed to contain wheat gluten. Proteins extracted from commercial wheat, corn, and soy flours served as standards, and proteins from heat-altered wheat served as process condition references. This approach successfully identified wheat proteins in these products especially if the process temperature did not exceed 120 degrees C. Above this temperature attenuation was nearly complete for proteins analyzed by CZE, but wheat-like patterns could still be recognized by one- and two-dimensional PAGE. Immunoblots reacted with grain-specific antibodies confirmed the identities of the cereal component especially when the protein pattern was greatly altered by thermal modification, specific protein adsorption, or protein digestion. In addition to verifying that wheat proteins are present, the complementary use of these methods can reveal whether whole wheat gluten or merely an alcohol-soluble fraction had been used in the specific product and indicate the

  13. Decreasing the amount of trypsin in in-gel digestion leads to diminished chemical noise and improved protein identifications.

    Science.gov (United States)

    Hu, Mo; Liu, Yanhua; Yu, Kaiwen; Liu, Xiaoyun

    2014-09-23

    Pre-fractionation by gel electrophoresis is often combined with liquid chromatography-mass spectrometry (LC-MS) for large-scale profiling of complex protein samples. An essential component of this widely applied proteomic platform is in-gel protein digestion. In nearly two decades of practicing this approach, an extremely high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. Here we report that trypsin autolysis products contribute to the bulk of chemical noise in in-gel digestion and remarkably we found evidence that the amount of trypsin can be slashed by an order of magnitude with comparable digestion performance. By revising perhaps the most critical element of this decade-old digestion protocol, the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered cost due to enzyme expenditure. More importantly, substantially reduced chemical noise (i.e., trypsin self-cleavage products) as a result of less enzyme usage translates into more protein identifications when limited amounts of samples are the interest of interrogation. In-gel digestion is one of the most widely used methods in proteomics. An exceedingly high level of trypsin has been utilized due to the consideration of slow enzyme diffusion into the gel matrix. This requirement has been faithfully kept in nearly two decades of practicing this approach. Here we report that trypsin concentration can be slashed by at least an order of magnitude while still providing comparable digestion performance. Thus the proteomics community relying on gel separation prior to LC-MS analysis will benefit instantly from much lowered enzyme cost. More importantly, substantially reduced chemical noise (i.e., trypsin autolysis products) due to less enzyme usage translates into ~30% more protein identifications when limited amounts of protein samples are analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Systematic identification of yeast proteins extracted into model wine during aging on the yeast lees.

    Science.gov (United States)

    Rowe, Jeffrey D; Harbertson, James F; Osborne, James P; Freitag, Michael; Lim, Juyun; Bakalinsky, Alan T

    2010-02-24

    Total protein and protein-associated mannan concentrations were measured, and individual proteins were identified during extraction into model wines over 9 months of aging on the yeast lees following completion of fermentations by seven wine strains of Saccharomyces cerevisiae. In aged wines, protein-associated mannan increased about 6-fold (+/-66%), while total protein only increased 2-fold (+/-20%), which resulted in a significantly greater protein-associated mannan/total protein ratio for three strains. A total of 219 proteins were identified among all wine samples taken over the entire time course. Of the 17 "long-lived" proteins detected in all 9 month samples, 13 were cell wall mannoproteins, and four were glycolytic enzymes. Most cytosolic proteins were not detected after 6 months. Native mannosylated yeast invertase was assayed for binding to wine tannin and was found to have a 10-fold lower affinity than nonglycosylated bovine serum albumin. Enrichment of mannoproteins in the aged model wines implies greater solution stability than other yeast proteins and the possibility that their contributions to wine quality may persist long after bottling.

  15. Identification of proteins sensitive to thermal stress in human neuroblastoma and glioma cell lines.

    Directory of Open Access Journals (Sweden)

    Guilian Xu

    Full Text Available Heat-shock is an acute insult to the mammalian proteome. The sudden elevation in temperature has far-reaching effects on protein metabolism, leads to a rapid inhibition of most protein synthesis, and the induction of protein chaperones. Using heat-shock in cells of neuronal (SH-SY5Y and glial (CCF-STTG1 lineage, in conjunction with detergent extraction and sedimentation followed by LC-MS/MS proteomic approaches, we sought to identify human proteins that lose solubility upon heat-shock. The two cell lines showed largely overlapping profiles of proteins detected by LC-MS/MS. We identified 58 proteins in detergent insoluble fractions as losing solubility in after heat shock; 10 were common between the 2 cell lines. A subset of the proteins identified by LC-MS/MS was validated by immunoblotting of similarly prepared fractions. Ultimately, we were able to definitively identify 3 proteins as putatively metastable neural proteins; FEN1, CDK1, and TDP-43. We also determined that after heat-shock these cells accumulate insoluble polyubiquitin chains largely linked via lysine 48 (K-48 residues. Collectively, this study identifies human neural proteins that lose solubility upon heat-shock. These proteins may represent components of the human proteome that are vulnerable to misfolding in settings of proteostasis stress.

  16. Identification of proteins whose synthesis in Saccharomyces cerevisiae is induced by DNA damage and heat shock

    International Nuclear Information System (INIS)

    Gailit, James

    1990-01-01

    Protein synthesis in Saccharomyces cerevisiae after exposure to ultraviolet light (UV) was examined by two-dimensional gel electrophoresis of pulse-labelled proteins. The synthesis of 12 distinct proteins was induced by treatment with UV doses of 10-200 J/m 2 . The induced proteins differed in minimum dose necessary for induction, maximum dose at which induction still occurred and constitutive level present in unirradiated cells. A chemical mutagen, 4-nitroquinoline-1-oxide, induced synthesis of the same proteins. Induction after UV treatment was observed in seven different yeast strains, including three mutants deficient in DNA repair. Synthesis of five of the proteins was also induced by brief heat shock treatment. These five may be members of a family of proteins whose synthesis is regulated by two different pathways responding to different types of stress. (author)

  17. [Identification and characterization of proteins from human bronchial secretion (author's transl)].

    Science.gov (United States)

    Laine, A; Hayem, A

    1976-03-01

    An analysis of bronchial mucus proteins was carried out by crossed immunoelectrophoresis. Before electrophoretic migration, sputum was treated with Ecteola-cellulose, which retains acid mucins. The proteins were then extracted by a phosphate/saline buffer pH 7.5. Crossed immunoelectrophoresis of the "bronchial extracts" was carried out with an anti-human serum: fifteen proteins were detected. Among them, IgA and protease inhibitiors play an important role in bronchial pathology. Bronchial extracts were also studied with immune serums against milk proteins, whole saliva and proteins of bronchial mucus. Bronchotransferrin, amylase and two esterases were characterized. Four other proteins were also detected with immune serums against bronchial mucus-proteins: their biological role is still unknown.

  18. Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen*

    Science.gov (United States)

    Sharma, Aditya K.; Arora, Divya; Singh, Lalit K.; Gangwal, Aakriti; Sajid, Andaleeb; Molle, Virginie; Singh, Yogendra; Nandicoori, Vinay Kumar

    2016-01-01

    Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events. PMID:27758870

  19. Identification of novel type 1 diabetes candidate genes by integrating genome-wide association data, protein-protein interactions, and human pancreatic islet gene expression

    DEFF Research Database (Denmark)

    Bergholdt, Regine; Brorsson, Caroline; Palleja, Albert

    2012-01-01

    Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated with dis......-cells. Our results provide novel insight to the mechanisms behind type 1 diabetes pathogenesis and, thus, may provide the basis for the design of novel treatment strategies.......Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated...... with disease, and they do not typically inform the broader context in which the disease genes operate. Here, we integrated type 1 diabetes GWAS data with protein-protein interactions to construct biological networks of relevance for disease. A total of 17 networks were identified. To prioritize...

  20. Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

    Institute of Scientific and Technical Information of China (English)

    Yi Feng; Zhong-Min Tian; Ming-Xi Wan; Zhao-Bin Zheng

    2007-01-01

    AIM: To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy.METHODS: Total proteins from human hepatocarcinomacell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite.Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)and database searching.RESULTS: We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin,endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein Ⅰ,peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION: Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

  1. Identification of multiply charged proteins and amino acid clusters by liquid nitrogen assisted spray ionization mass spectrometry.

    Science.gov (United States)

    Kumar Kailasa, Suresh; Hasan, Nazim; Wu, Hui-Fen

    2012-08-15

    The development of liquid nitrogen assisted spray ionization mass spectrometry (LNASI MS) for the analysis of multiply charged proteins (insulin, ubiquitin, cytochrome c, α-lactalbumin, myoglobin and BSA), peptides (glutathione, HW6, angiotensin-II and valinomycin) and amino acid (arginine) clusters is described. The charged droplets are formed by liquid nitrogen assisted sample spray through a stainless steel nebulizer and transported into mass analyzer for the identification of multiply charged protein ions. The effects of acids and modifier volumes for the efficient ionization of the above analytes in LNASI MS were carefully investigated. Multiply charged proteins and amino acid clusters were effectively identified by LNASI MS. The present approach can effectively detect the multiply charged states of cytochrome c at 400 nM. A comparison between LNASI and ESI, CSI, SSI and V-EASI methods on instrumental conditions, applied temperature and observed charge states for the multiply charged proteins, shows that the LNASI method produces the good quality spectra of amino acid clusters at ambient conditions without applied any electric field and heat. To date, we believe that the LNASI method is the most simple, low cost and provided an alternative paradigm for production of multiply charged ions by LNASI MS, just as ESI-like ions yet no need for applying any electrical field and it could be operated at low temperature for generation of highly charged protein/peptide ions. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Identification of Besnoitia besnoiti proteins that showed differences in abundance between tachyzoite and bradyzoite stages by difference gel electrophoresis.

    Science.gov (United States)

    Fernández-García, Aurora; Alvarez-García, Gema; Marugán-Hernández, Virginia; García-Lunar, Paula; Aguado-Martínez, Adriana; Risco-Castillo, Verónica; Ortega-Mora, Luis M

    2013-07-01

    Bovine besnoitiosis is a chronic and debilitating disease, caused by the apicomplexan parasite Besnoitia besnoiti. Infection of cattle by B. besnoiti is governed by the tachyzoite stage, which is related to acute infection, and the bradyzoite stage gathered into macroscopic cysts located in subcutaneous tissue in the skin, mucosal membranes and sclera conjunctiva and related to persistence and chronic infection. However, the entire life cycle of this parasite and the molecular mechanisms underlying tachyzoite-to-bradyzoite conversion remain unknown. In this context, a different antigenic pattern has been observed between tachyzoite and bradyzoite extracts. Thus, to identify stage-specific proteins, a difference gel electrophoresis (DIGE) approach was used on tachyzoite and bradyzoite extracts followed by mass spectrometry (MS) analysis. A total of 130 and 132 spots were differentially expressed in bradyzoites and tachyzoites, respectively (average ratio ± 1.5, Presult, 5 up-regulated bradyzoite proteins (GAPDH, ENO1, LDH, SOD and RNA polymerase) and 5 up-regulated tachyzoite proteins (ENO2; LDH; ATP synthase; HSP70 and PDI) were identified. The present results set the basis for the identification of new proteins as drug targets. Moreover, the role of these proteins in tachyzoite-to-bradyzoite conversion and the role of the host cell environment should be a subject of further research.

  3. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    Science.gov (United States)

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  4. Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

    International Nuclear Information System (INIS)

    Mariano, Andrea C.; Andrade, Maxuel O.; Santos, Anesia A.; Carolino, Sonia M.B.; Oliveira, Marli L.; Baracat-Pereira, Maria Cristina; Brommonshenkel, Sergio H.; Fontes, Elizabeth P.B.

    2004-01-01

    Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed

  5. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

    Directory of Open Access Journals (Sweden)

    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  6. Protein Tyrosine Nitration : Selectivity, Physicochemical and Biological Consequences, Denitration, and Proteomics Methods for the Identification of Tyrosine-Nitrated Proteins

    NARCIS (Netherlands)

    Abello, Nicolas; Kerstjens, Huib A. M.; Postma, Dirkje S.; Bischoff, Rainer

    Protein tyrosine nitration (PTN) is a post-translational modification occurring under the action of a nitrating agent. Tyrosine is modified in the 3-position of the phenolic ring through the addition of a nitro group (NO(2)). In the present article, we review the main nitration reactions and

  7. Nitrate as a probe of cytochrome c surface : crystallographic identification of crucial "hot spots" for protein-protein recognition

    NARCIS (Netherlands)

    De March, Matteo; Demitri, Nicola; De Zorzi, Rita; Casini, Angela; Gabbiani, Chiara; Guerri, Annalisa; Messori, Luigi; Geremia, Silvano

    The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive

  8. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein

    DEFF Research Database (Denmark)

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu

    2012-01-01

    previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense...

  9. Identification of tyrosine-phosphorylated proteins associated with metastasis and functional analysis of FER in human hepatocellular carcinoma cells

    International Nuclear Information System (INIS)

    Li, Haiyu; Ren, Zhenggang; Kang, Xiaonan; Zhang, Lan; Li, Xuefei; Wang, Yan; Xue, Tongchun; Shen, Yuefang; Liu, Yinkun

    2009-01-01

    Aberrant activity of tyrosine-phosphorylated proteins is commonly associated with HCC metastasis. Cell signaling events driven by these proteins are implicated in numerous processes that alter cancer cell behavior. Exploring the activities and signaling pathways of these proteins in HCC metastasis may help in identifying new candidate molecules for HCC-targeted therapy. Hep3B (a nonmetastatic HCC cell line) and MHCC97H (a highly metastatic HCC cell line) were used in this study, and the tyrosine-phosphorylated proteins expressed in these cell lines were profiled by a phosphoproteomics technique based on LC-MS/MS. Protein-protein interaction and functional clustering analyses were performed to determine the activities of the identified proteins and the signaling pathways closely related to HCC metastasis. In both cell lines, a total of 247 phosphotyrosine (pTyr) proteins containing 281 pTyr sites were identified without any stimulation. The involvement of almost 30% of these in liver or liver cancer has not been reported previously. Biological process clustering analysis indicated that pTyr proteins involved in cell motility, migration, protein autophosphorylation, cell-cell communication, and antiapoptosis functions were overexpressed during metastasis. Pathway clustering analysis revealed that signaling pathways such as those involved in EGFR signaling, cytokine- and chemokine-mediated signal transduction, and the PI3K and JAK-STAT cascades were significantly activated during HCC metastasis. Moreover, noncanonical regulation of the JNK cascade might also provide new targets for HCC metastasis. After comparing the pTyr proteins that were differentially expressed during HCC cell metastasis, we selected FER, a nonreceptor tyrosine kinase, and validated its role in terms of both expression and function. The data confirmed that FER might play a critical role in the invasion and metastasis of HCC. The identification of pTyr proteins and signaling pathways associated

  10. Proteome analysis of barley seeds: Identification of major proteins from two-dimensional gels (pl 4-7)

    DEFF Research Database (Denmark)

    Østergaard, O.; Finnie, Christine; Laugesen, S.

    2004-01-01

    inhibitors), and proteins related to desiccation and oxidative stress. Sixty-four of the identifications were made using expressed sequence tags (ESTs). Numerous spots in the 2-D gel pattern changed during germination (micromalting) and an intensely stained area which contained large amounts of the serpin......Germination of monocotyledonous plants involves activation and de novo synthesis of enzymes that degrade cell walls and starch and mobilize stored endosperm reserves for embryo growth. Two-dimensional (2-D) gel electrophoresis and mass spectrometry were applied to identify major water...

  11. Identification of multidrug resistance protein 1 (MRP1/ABCC1) as a molecular gate for cellular export of cobalamin

    DEFF Research Database (Denmark)

    Beedholm-Ebsen, Rasmus; van de Wetering, Koen; Hardlei, Tore

    2010-01-01

    transporters by cellular gene silencing showed a role in cellular Cbl efflux of the ATP-binding cassette (ABC)-drug transporter, ABCC1, alias multidrug resistance protein 1 (MRP1), which is present in the basolateral membrane of intestinal epithelium and in other cells. The ability of MRP1 to mediate ATP...... and kidney. In contrast, Cbl accumulates in the terminal part of the intestine of these mice, suggesting a functional malabsorption because of a lower epithelial basolateral Cbl efflux. The identification of this Cbl export mechanism now allows the delineation of a coherent pathway for Cbl trafficking from...

  12. Identification of Protein Complexes Using Weighted PageRank-Nibble Algorithm and Core-Attachment Structure.

    Science.gov (United States)

    Peng, Wei; Wang, Jianxin; Zhao, Bihai; Wang, Lusheng

    2015-01-01

    Protein complexes play a significant role in understanding the underlying mechanism of most cellular functions. Recently, many researchers have explored computational methods to identify protein complexes from protein-protein interaction (PPI) networks. One group of researchers focus on detecting local dense subgraphs which correspond to protein complexes by considering local neighbors. The drawback of this kind of approach is that the global information of the networks is ignored. Some methods such as Markov Clustering algorithm (MCL), PageRank-Nibble are proposed to find protein complexes based on random walk technique which can exploit the global structure of networks. However, these methods ignore the inherent core-attachment structure of protein complexes and treat adjacent node equally. In this paper, we design a weighted PageRank-Nibble algorithm which assigns each adjacent node with different probability, and propose a novel method named WPNCA to detect protein complex from PPI networks by using weighted PageRank-Nibble algorithm and core-attachment structure. Firstly, WPNCA partitions the PPI networks into multiple dense clusters by using weighted PageRank-Nibble algorithm. Then the cores of these clusters are detected and the rest of proteins in the clusters will be selected as attachments to form the final predicted protein complexes. The experiments on yeast data show that WPNCA outperforms the existing methods in terms of both accuracy and p-value. The software for WPNCA is available at "http://netlab.csu.edu.cn/bioinfomatics/weipeng/WPNCA/download.html".

  13. Identification of novel human damage response proteins targeted through yeast orthology.

    Directory of Open Access Journals (Sweden)

    J Peter Svensson

    Full Text Available Studies in Saccharomyces cerevisiae show that many proteins influence cellular survival upon exposure to DNA damaging agents. We hypothesized that human orthologs of these S. cerevisiae proteins would also be required for cellular survival after treatment with DNA damaging agents. For this purpose, human homologs of S. cerevisiae proteins were identified and mapped onto the human protein-protein interaction network. The resulting human network was highly modular and a series of selection rules were implemented to identify 45 candidates for human toxicity-modulating proteins. The corresponding transcripts were targeted by RNA interference in human cells. The cell lines with depleted target expression were challenged with three DNA damaging agents: the alkylating agents MMS and 4-NQO, and the oxidizing agent t-BuOOH. A comparison of the survival revealed that the majority (74% of proteins conferred either sensitivity or resistance. The identified human toxicity-modulating proteins represent a variety of biological functions: autophagy, chromatin modifications, RNA and protein metabolism, and telomere maintenance. Further studies revealed that MMS-induced autophagy increase the survival of cells treated with DNA damaging agents. In summary, we show that damage recovery proteins in humans can be identified through homology to S. cerevisiae and that many of the same pathways are represented among the toxicity modulators.

  14. Comparative proteomics analysis of oral cancer cell lines: identification of cancer associated proteins

    Science.gov (United States)

    2014-01-01

    Background A limiting factor in performing proteomics analysis on cancerous cells is the difficulty in obtaining sufficient amounts of starting material. Cell lines can be used as a simplified model system for studying changes that accompany tumorigenesis. This study used two-dimensional gel electrophoresis (2DE) to compare the whole cell proteome of oral cancer cell lines vs normal cells in an attempt to identify cancer associated proteins. Results Three primary cell cultures of normal cells with a limited lifespan without hTERT immortalization have been successfully established. 2DE was used to compare the whole cell proteome of these cells with that of three oral cancer cell lines. Twenty four protein spots were found to have changed in abundance. MALDI TOF/TOF was then used to determine the identity of these proteins. Identified proteins were classified into seven functional categories – structural proteins, enzymes, regulatory proteins, chaperones and others. IPA core analysis predicted that 18 proteins were related to cancer with involvements in hyperplasia, metastasis, invasion, growth and tumorigenesis. The mRNA expressions of two proteins – 14-3-3 protein sigma and Stress-induced-phosphoprotein 1 – were found to correlate with the corresponding proteins’ abundance. Conclusions The outcome of this analysis demonstrated that a comparative study of whole cell proteome of cancer versus normal cell lines can be used to identify cancer associated proteins. PMID:24422745

  15. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    Science.gov (United States)

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

    2016-09-02

    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

  16. Identification of karyopherin α1 and α7 interacting proteins in porcine tissue.

    Directory of Open Access Journals (Sweden)

    Ki-Eun Park

    Full Text Available Specialized trafficking systems in eukaryotic cells serve a critical role in partitioning intracellular proteins between the nucleus and cytoplasm. Cytoplasmic proteins (including chromatin remodeling enzymes and transcription factors must gain access to the nucleus to exert their functions to properly program fundamental cellular events ranging from cell cycle progression to gene transcription. Knowing that nuclear import mediated by members of the karyopherin α family of transport receptors plays a critical role in regulating development and differentiation, we wanted to determine the identity of proteins that are trafficked by this karyopherin α pathway. To this end, we performed a GST pull-down assay using porcine orthologs of karyopherin α1 (KPNA1 and karyopherin α7 (KPNA7 and prey protein derived from porcine fibroblast cells and used a liquid chromatography and tandem mass spectrometry (LC-MS/MS approach to determine the identity of KPNA1 and KPNA7 interacting proteins. Our screen revealed that the proteins that interact with KPNA1 and KPNA7 are generally nuclear proteins that possess nuclear localization signals. We further validated two candidate proteins from this screen and showed that they are able to be imported into the nucleus in vivo and also interact with members of the karyopherin α family of proteins in vitro. Our results also reveal the utility of using a GST pull-down approach coupled with LC-MS/MS to screen for protein interaction partners in a non-traditional model system.

  17. Identification and analysis of YELLOW protein family genes in the silkworm, Bombyx mori

    Directory of Open Access Journals (Sweden)

    Yi Yong-Zhu

    2006-08-01

    Full Text Available Abstract Background The major royal jelly proteins/yellow (MRJP/YELLOW family possesses several physiological and chemical functions in the development of Apis mellifera and Drosophila melanogaster. Each protein of the family has a conserved domain named MRJP. However, there is no report of MRJP/YELLOW family proteins in the Lepidoptera. Results Using the YELLOW protein sequence in Drosophila melanogaster to BLAST silkworm EST database, we found a gene family composed of seven members with a conserved MRJP domain each and named it YELLOW protein family of Bombyx mori. We completed the cDNA sequences with RACE method. The protein of each member possesses a MRJP domain and a putative cleavable signal peptide consisting of a hydrophobic sequence. In view of genetic evolution, the whole Bm YELLOW protein family composes a monophyletic group, which is distinctly separate from Drosophila melanogaster and Apis mellifera. We then showed the tissue expression profiles of Bm YELLOW protein family genes by RT-PCR. Conclusion A Bombyx mori YELLOW protein family is found to be composed of at least seven members. The low homogeneity and unique pattern of gene expression by each member among the family ensure us to prophesy that the members of Bm YELLOW protein family would play some important physiological functions in silkworm development.

  18. Identification of mammalian proteins cross-linked to DNA by ionizing radiation.

    Science.gov (United States)

    Barker, Sharon; Weinfeld, Michael; Zheng, Jing; Li, Liang; Murray, David

    2005-10-07

    Ionizing radiation (IR) is an important environmental risk factor for various cancers and also a major therapeutic agent for cancer treatment. Exposure of mammalian cells to IR induces several types of damage to DNA, including double- and single-strand breaks, base and sugar damage, as well as DNA-DNA and DNA-protein cross-links (DPCs). Little is known regarding the biological consequences of DPCs. Identifying the proteins that become cross-linked to DNA by IR would be an important first step in this regard. We have therefore undertaken a proteomics study to isolate and identify proteins involved in IR-induced DPCs. DPCs were induced in AA8 Chinese hamster ovary or GM00637 human fibroblast cells using 0-4 gray of gamma-rays under either aerated or hypoxic conditions. DPCs were isolated using a recently developed method, and proteins were identified by mass spectrometry. We identified 29 proteins as being cross-linked to DNA by IR under aerated and/or hypoxic conditions. The identified proteins include structural proteins, actin-associated proteins, transcription regulators, RNA-splicing components, stress-response proteins, cell cycle regulatory proteins, and GDP/GTP-binding proteins. The involvement of several proteins (actin, histone H2B, and others) in DPCs was confirmed by using Western blot analysis. The dose responsiveness of DPC induction was examined by staining one-dimensional SDS-polyacrylamide gels with SYPRO Tangerine followed by analysis using fluorescence imaging. Quantitation of the fluorescence signal indicated no significant difference in total yields of IR-induced DPCs generated under aerated or hypoxic conditions, although differences were observed for several individual protein bands.

  19. Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis.

    Science.gov (United States)

    Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan Mhai; Mondol, Sobuj; Ahmed, C M Sabbir

    2014-01-01

    Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim-sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein-protein interaction. In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to develop and discover more effective and specific

  20. Genome-Wide Identification and Analysis of Genes Encoding PHD-Finger Protein in Tomato

    International Nuclear Information System (INIS)

    Hayat, S.; Cheng, Z.; Chen, X.

    2016-01-01

    The PHD-finger proteins are conserved in eukaryotic organisms and are involved in a variety of important functions in different biological processes in plants. However, the function of PHD fingers are poorly known in tomato (Solanum lycopersicum L.). In current study, we identified 45 putative genes coding Phd finger protein in tomato distributed on 11 chromosomes except for chromosome 8. Some of the genes encode other conserved key domains besides Phd-finger. Phylogenetic analysis of these 45 proteins resulted in seven clusters. Most Phd finger proteins were predicted to PML body location. These PHD-finger genes displayed differential expression either in various organs, at different development stages and under stresses in tomato. Our study provides the first systematic analysis of PHD-finger genes and proteins in tomato. This preliminary study provides a very useful reference information for Phd-finger proteins in tomato. They will be helpful for cloning and functional study of tomato PHD-finger genes. (author)

  1. Identification of Common Epitopes on a Conserved Region of NSs Proteins Among Tospoviruses of Watermelon silver mottle virus Serogroup.

    Science.gov (United States)

    Chen, Tsung-Chi; Huang, Ching-Wen; Kuo, Yan-Wen; Liu, Fang-Lin; Yuan, Chao-Hsiu Hsuan; Hsu, Hei-Ti; Yeh, Shyi-Dong

    2006-12-01

    ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.

  2. Identification of StARD3 as a lutein-binding protein in the macula of the primate retina.

    Science.gov (United States)

    Li, Binxing; Vachali, Preejith; Frederick, Jeanne M; Bernstein, Paul S

    2011-04-05

    Lutein, zeaxanthin, and their metabolites are the xanthophyll carotenoids that form the macular pigment of the human retina. Epidemiological evidence suggests that high levels of these carotenoids in the diet, serum, and macula are associated with a decreased risk of age-related macular degeneration (AMD), and the AREDS2 study is prospectively testing this hypothesis. Understanding the biochemical mechanisms underlying the selective uptakes of lutein and zeaxanthin into the human macula may provide important insights into the physiology of the human macula in health and disease. GSTP1 is the macular zeaxanthin-binding protein, but the identity of the human macular lutein-binding protein has remained elusive. Prior identification of the silkworm lutein-binding protein (CBP) as a member of the steroidogenic acute regulatory domain (StARD) protein family and selective labeling of monkey photoreceptor inner segments with an anti-CBP antibody provided an important clue for identifying the primate retina lutein-binding protein. The homology of CBP with all 15 human StARD proteins was analyzed using database searches, Western blotting, and immunohistochemistry, and we here provide evidence to identify StARD3 (also known as MLN64) as a human retinal lutein-binding protein. Antibody to StARD3, N-62 StAR, localizes to all neurons of monkey macular retina and especially cone inner segments and axons, but does not colocalize with the Müller cell marker, glutamine synthetase. Further, recombinant StARD3 selectively binds lutein with high affinity (K(D) = 0.45 μM) when assessed by surface plasmon resonance (SPR) binding assays. Our results demonstrate previously unrecognized, specific interactions of StARD3 with lutein and provide novel avenues for exploring its roles in human macular physiology and disease.

  3. Identification of StARD3 as a Lutein-binding Protein in the Macula of the Primate Retina†

    Science.gov (United States)

    Li, Binxing; Vachali, Preejith; Frederick, Jeanne M.; Bernstein, Paul S.

    2011-01-01

    Lutein, zeaxanthin and their metabolites are the xanthophyll carotenoids that form the macular pigment of the human retina. Epidemiological evidence suggests that high levels of these carotenoids in the diet, serum and macula are associated with decreased risk of age-related macular degeneration (AMD), and the AREDS2 study is prospectively testing this hypothesis. Understanding the biochemical mechanisms underlying the selective uptakes of lutein and zeaxanthin into the human macula may provide important insights into the physiology of the human macula in health and disease. GSTP1 is the macular zeaxanthin-binding protein, but the identity of the human macular lutein-binding protein has remained elusive. Prior identification of the silkworm lutein-binding protein (CBP) as a member of the steroidogenic acute regulatory domain (StARD) protein family, and selective labeling of monkey photoreceptor inner segments by anti-CBP antibody provided an important clue toward identifying the primate retina lutein-binding protein. Homology of CBP to all 15 human StARD proteins was analyzed using database searches, western blotting and immunohistochemistry, and we here provide evidence to identify StARD3 (also known as MLN64) as a human retinal lutein-binding protein. Further, recombinant StARD3 selectively binds lutein with high affinity (KD = 0.45 micromolar) when assessed by surface plasmon resonance (SPR) binding assays. Our results demonstrate previously unrecognized, specific interactions of StARD3 with lutein and provide novel avenues to explore its roles in human macular physiology and disease. PMID:21322544

  4. Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

    Science.gov (United States)

    Rabbani, M A G; Ribaudo, Michael; Guo, Ju-Tao; Barik, Sailen

    2016-12-15

    A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-β. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3. The innate immunity of the host, typified by interferon (IFN), is a major antiviral defense. IFN inhibits virus growth by inducing a large number of IFN-stimulated gene (ISG) proteins, several of which have been shown to have specific antiviral functions. Parainfluenza virus type 3 (PIV3) is major pathogen of children, and no reliable vaccine or specific antiviral against it currently exists. In this article, we report several ISG proteins that strongly inhibit PIV3 growth, the use of which may allow a better antiviral regimen targeting PIV3. Copyright © 2016, American Society for Microbiology

  5. Identification and analysis of potential targets in Streptococcus sanguinis using computer aided protein data analysis

    Science.gov (United States)

    Chowdhury, Md Rabiul Hossain; Bhuiyan, Md IqbalKaiser; Saha, Ayan; Mosleh, Ivan MHAI; Mondol, Sobuj; Ahmed, C M Sabbir

    2014-01-01

    Purpose Streptococcus sanguinis is a Gram-positive, facultative aerobic bacterium that is a member of the viridans streptococcus group. It is found in human mouths in dental plaque, which accounts for both dental cavities and bacterial endocarditis, and which entails a mortality rate of 25%. Although a range of remedial mediators have been found to control this organism, the effectiveness of agents such as penicillin, amoxicillin, trimethoprim–sulfamethoxazole, and erythromycin, was observed. The emphasis of this investigation was on finding substitute and efficient remedial approaches for the total destruction of this bacterium. Materials and methods In this computational study, various databases and online software were used to ascertain some specific targets of S. sanguinis. Particularly, the Kyoto Encyclopedia of Genes and Genomes databases were applied to determine human nonhomologous proteins, as well as the metabolic pathways involved with those proteins. Different software such as Phyre2, CastP, DoGSiteScorer, the Protein Function Predictor server, and STRING were utilized to evaluate the probable active drug binding site with its known function and protein–protein interaction. Results In this study, among 218 essential proteins of this pathogenic bacterium, 81 nonhomologous proteins were accrued, and 15 proteins that are unique in several metabolic pathways of S. sanguinis were isolated through metabolic pathway analysis. Furthermore, four essentially membrane-bound unique proteins that are involved in distinct metabolic pathways were revealed by this research. Active sites and druggable pockets of these selected proteins were investigated with bioinformatic techniques. In addition, this study also mentions the activity of those proteins, as well as their interactions with the other proteins. Conclusion Our findings helped to identify the type of protein to be considered as an efficient drug target. This study will pave the way for researchers to

  6. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

    Science.gov (United States)

    Morimoto, Shimpei; Yahara, Koji

    2018-03-01

    Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

  7. Identification of Salt-Tolerant Sinorhizobium sp Strain BL3 Membrane Proteins Based on Proteomics

    DEFF Research Database (Denmark)

    Tanthanuch, Waraporn; Mohammed, Shabaz; Matthiesen, Rune

    2010-01-01

    functional categories, the two biggest of which were energy production and conversion, and proteins not in clusters of orthologous groups (COGs). In addition, a comparative analysis of membrane proteins between salt-stressed and non-stressed BL3 cells was conducted using a membrane enrichment method and off-line...... SCX fractionation coupled to nanoLC-MS/MS. These techniques would be useful for further comparative analysis of membrane proteins that function in the response to environmental stress....

  8. Imaging mass spectrometry in papillary thyroid carcinoma for the identification and validation of biomarker proteins.

    Science.gov (United States)

    Min, Kyueng-Whan; Bang, Joo-Young; Kim, Kwang Pyo; Kim, Wan-Seop; Lee, Sang Hwa; Shanta, Selina Rahman; Lee, Jeong Hwa; Hong, Ji Hye; Lim, So Dug; Yoo, Young-Bum; Na, Chan-Hyun

    2014-07-01

    Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-µm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2.

  9. A machine learning approach for the identification of odorant binding proteins from sequence-derived properties

    Directory of Open Access Journals (Sweden)

    Suganthan PN

    2007-09-01

    Full Text Available Abstract Background Odorant binding proteins (OBPs are believed to shuttle odorants from the environment to the underlying odorant receptors, for which they could potentially serve as odorant presenters. Although several sequence based search methods have been exploited for protein family prediction, less effort has been devoted to the prediction of OBPs from sequence data and this area is more challenging due to poor sequence identity between these proteins. Results In this paper, we propose a new algorithm that uses Regularized Least Squares Classifier (RLSC in conjunction with multiple physicochemical properties of amino acids to predict odorant-binding proteins. The algorithm was applied to the dataset derived from Pfam and GenDiS database and we obtained overall prediction accuracy of 97.7% (94.5% and 98.4% for positive and negative classes respectively. Conclusion Our study suggests that RLSC is potentially useful for predicting the odorant binding proteins from sequence-derived properties irrespective of sequence similarity. Our method predicts 92.8% of 56 odorant binding proteins non-homologous to any protein in the swissprot database and 97.1% of the 414 independent dataset proteins, suggesting the usefulness of RLSC method for facilitating the prediction of odorant binding proteins from sequence information.

  10. Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins

    DEFF Research Database (Denmark)

    Peltier, J B; Friso, G; Kalume, D E

    2000-01-01

    The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modificati......The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post......-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed...... sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess...

  11. Identification of immunogenic and virulence-associated Campylobacter jejuni proteins

    DEFF Research Database (Denmark)

    Nielsen, Lene Nørby; Luijkx, Thomas A.; Vegge, Christina Skovgaard

    2012-01-01

    With the aim of identifying proteins important for host interaction and virulence, we have screened an expression library of NCTC 11168 Campylobacter jejuni genes for highly immunogenic proteins. A commercial C. jejuni open reading frame (ORF) library consisting of more than 1,600 genes was trans......With the aim of identifying proteins important for host interaction and virulence, we have screened an expression library of NCTC 11168 Campylobacter jejuni genes for highly immunogenic proteins. A commercial C. jejuni open reading frame (ORF) library consisting of more than 1,600 genes...

  12. Identification of species- and tissue-specific proteins using proteomic strategy

    Science.gov (United States)

    Chernukha, I. M.; Vostrikova, N. L.; Kovalev, L. I.; Shishkin, S. S.; Kovaleva, M. A.; Manukhin, Y. S.

    2017-09-01

    Proteomic technologies have proven to be very effective for detecting biochemical changes in meat products, such as changes in tissue- and species-specific proteins. In the tissues of cattle, pig, horse and camel M. longissimus dorsi both tissue- and species specific proteins were detected using two dimensional electrophoresis. Species-specific isoforms of several muscle proteins were also identified. The identified and described proteins of cattle, pig, horse and camel skeletal muscles (including mass spectra of the tryptic peptides) were added to the national free access database “Muscle organ proteomics”. This research has enabled the development of new highly sensitive technologies for meat product quality control against food fraud.

  13. Proteomic identification of dysferlin-interacting protein complexes in human vascular endothelium

    International Nuclear Information System (INIS)

    Leung, Cleo; Utokaparch, Soraya; Sharma, Arpeeta; Yu, Carol; Abraham, Thomas; Borchers, Christoph; Bernatchez, Pascal

    2011-01-01

    Highlights: ► Bi-directional (inward and outward) movement of GFP-dysferlin in COS-7 cells. ► Dysferlin interacts with key signaling proteins for transcytosis in EC. ► Dysferlin mediates trafficking of vesicles carrying protein cargos in EC. -- Abstract: Dysferlin is a membrane-anchored protein known to facilitate membrane repair in skeletal muscles following mechanical injury. Mutations of dysferlin gene impair sarcolemma integrity, a hallmark of certain forms of muscular dystrophy in patients. Dysferlin contains seven calcium-dependent C2 binding domains, which are required to promote fusion of intracellular membrane vesicles. Emerging evidence reveal the unexpected expression of dysferlin in non-muscle, non-mechanically active tissues, such as endothelial cells, which cast doubts over the belief that ferlin proteins act exclusively as membrane repair proteins. We and others have shown that deficient trafficking of membrane bound proteins in dysferlin-deficient cells, suggesting that dysferlin might mediate trafficking of client proteins. Herein, we describe the intracellular trafficking and movement of GFP-dysferlin positive vesicles in unfixed reconstituted cells using live microscopy. By performing GST pull-down assays followed by mass spectrometry, we identified dysferlin binding protein complexes in human vascular endothelial cells. Together, our data further support the claims that dysferlin not only mediates membrane repair but also trafficking of client proteins, u