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Sample records for accurate protein identification

  1. Mass spectrometry based protein identification with accurate statistical significance assignment

    OpenAIRE

    Alves, Gelio; Yu, Yi-Kuo

    2014-01-01

    Motivation: Assigning statistical significance accurately has become increasingly important as meta data of many types, often assembled in hierarchies, are constructed and combined for further biological analyses. Statistical inaccuracy of meta data at any level may propagate to downstream analyses, undermining the validity of scientific conclusions thus drawn. From the perspective of mass spectrometry based proteomics, even though accurate statistics for peptide identification can now be ach...

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

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    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.

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

    Science.gov (United States)

    Ramkissoon, Kevin R; Miller, Jennifer K; 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.

  4. Towards an accurate bioimpedance identification

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    Sanchez, B.; Louarroudi, E.; Bragos, R.; Pintelon, R.

    2013-04-01

    This paper describes the local polynomial method (LPM) for estimating the time-invariant bioimpedance frequency response function (FRF) considering both the output-error (OE) and the errors-in-variables (EIV) identification framework and compare it with the traditional cross— and autocorrelation spectral analysis techniques. The bioimpedance FRF is measured with the multisine electrical impedance spectroscopy (EIS) technique. To show the overwhelming accuracy of the LPM approach, both the LPM and the classical cross— and autocorrelation spectral analysis technique are evaluated through the same experimental data coming from a nonsteady-state measurement of time-varying in vivo myocardial tissue. The estimated error sources at the measurement frequencies due to noise, σnZ, and the stochastic nonlinear distortions, σZNL, have been converted to Ω and plotted over the bioimpedance spectrum for each framework. Ultimately, the impedance spectra have been fitted to a Cole impedance model using both an unweighted and a weighted complex nonlinear least square (CNLS) algorithm. A table is provided with the relative standard errors on the estimated parameters to reveal the importance of which system identification frameworks should be used.

  5. Accurate in silico identification of species-specific acetylation sites by integrating protein sequence-derived and functional features

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    Li, Yuan; Wang, Mingjun; Wang, Huilin; Tan, Hao; Zhang, Ziding; Webb, Geoffrey I.; Song, Jiangning

    2014-07-01

    Lysine acetylation is a reversible post-translational modification, playing an important role in cytokine signaling, transcriptional regulation, and apoptosis. To fully understand acetylation mechanisms, identification of substrates and specific acetylation sites is crucial. Experimental identification is often time-consuming and expensive. Alternative bioinformatics methods are cost-effective and can be used in a high-throughput manner to generate relatively precise predictions. Here we develop a method termed as SSPKA for species-specific lysine acetylation prediction, using random forest classifiers that combine sequence-derived and functional features with two-step feature selection. Feature importance analysis indicates functional features, applied for lysine acetylation site prediction for the first time, significantly improve the predictive performance. We apply the SSPKA model to screen the entire human proteome and identify many high-confidence putative substrates that are not previously identified. The results along with the implemented Java tool, serve as useful resources to elucidate the mechanism of lysine acetylation and facilitate hypothesis-driven experimental design and validation.

  6. Accurate pose estimation for forensic identification

    Science.gov (United States)

    Merckx, Gert; Hermans, Jeroen; Vandermeulen, Dirk

    2010-04-01

    In forensic authentication, one aims to identify the perpetrator among a series of suspects or distractors. A fundamental problem in any recognition system that aims for identification of subjects in a natural scene is the lack of constrains on viewing and imaging conditions. In forensic applications, identification proves even more challenging, since most surveillance footage is of abysmal quality. In this context, robust methods for pose estimation are paramount. In this paper we will therefore present a new pose estimation strategy for very low quality footage. Our approach uses 3D-2D registration of a textured 3D face model with the surveillance image to obtain accurate far field pose alignment. Starting from an inaccurate initial estimate, the technique uses novel similarity measures based on the monogenic signal to guide a pose optimization process. We will illustrate the descriptive strength of the introduced similarity measures by using them directly as a recognition metric. Through validation, using both real and synthetic surveillance footage, our pose estimation method is shown to be accurate, and robust to lighting changes and image degradation.

  7. 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

    Fast and Accurate Identification of Cross-Linked Peptides for the structural analysis of large protein complexes and to elucidate interaction networks. Salman Tahir Jimi-Carlo Bukowski-Wills; Morten Rasmussen; Juri RappsilberWellcome Trust Centre for Cell Biology, Edinburgh , United Kingdom   Novel...

  8. 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 .

  9. Accurate Identification of Cancerlectins through Hybrid Machine Learning Technology

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    Jieru Zhang

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  11. PILER-CR: Fast and accurate identification of CRISPR repeats

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    Edgar Robert C

    2007-01-01

    Full Text Available Abstract Background Sequencing of prokaryotic genomes has recently revealed the presence of CRISPR elements: short, highly conserved repeats separated by unique sequences of similar length. The distinctive sequence signature of CRISPR repeats can be found using general-purpose repeat- or pattern-finding software tools. However, the output of such tools is not always ideal for studying these repeats, and significant effort is sometimes needed to build additional tools and perform manual analysis of the output. Results We present PILER-CR, a program specifically designed for the identification and analysis of CRISPR repeats. The program executes rapidly, completing a 5 Mb genome in around 5 seconds on a current desktop computer. We validate the algorithm by manual curation and by comparison with published surveys of these repeats, finding that PILER-CR has both high sensitivity and high specificity. We also present a catalogue of putative CRISPR repeats identified in a comprehensive analysis of 346 prokaryotic genomes. Conclusion PILER-CR is a useful tool for rapid identification and classification of CRISPR repeats. The software is donated to the public domain. Source code and a Linux binary are freely available at http://www.drive5.com/pilercr.

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

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    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.

  13. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics

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    Zheng-Wei Li

    2016-08-01

    Full Text Available Protein-protein interactions (PPIs occur at almost all levels of cell functions and play crucial roles in various cellular processes. Thus, identification of PPIs is critical for deciphering the molecular mechanisms and further providing insight into biological processes. Although a variety of high-throughput experimental techniques have been developed to identify PPIs, existing PPI pairs by experimental approaches only cover a small fraction of the whole PPI networks, and further, those approaches hold inherent disadvantages, such as being time-consuming, expensive, and having high false positive rate. Therefore, it is urgent and imperative to develop automatic in silico approaches to predict PPIs efficiently and accurately. In this article, we propose a novel mixture of physicochemical and evolutionary-based feature extraction method for predicting PPIs using our newly developed discriminative vector machine (DVM classifier. The improvements of the proposed method mainly consist in introducing an effective feature extraction method that can capture discriminative features from the evolutionary-based information and physicochemical characteristics, and then a powerful and robust DVM classifier is employed. To the best of our knowledge, it is the first time that DVM model is applied to the field of bioinformatics. When applying the proposed method to the Yeast and Helicobacter pylori (H. pylori datasets, we obtain excellent prediction accuracies of 94.35% and 90.61%, respectively. The computational results indicate that our method is effective and robust for predicting PPIs, and can be taken as a useful supplementary tool to the traditional experimental methods for future proteomics research.

  14. Highly Accurate Prediction of Protein-Protein Interactions via Incorporating Evolutionary Information and Physicochemical Characteristics.

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    Li, Zheng-Wei; You, Zhu-Hong; Chen, Xing; Gui, Jie; Nie, Ru

    2016-01-01

    Protein-protein interactions (PPIs) occur at almost all levels of cell functions and play crucial roles in various cellular processes. Thus, identification of PPIs is critical for deciphering the molecular mechanisms and further providing insight into biological processes. Although a variety of high-throughput experimental techniques have been developed to identify PPIs, existing PPI pairs by experimental approaches only cover a small fraction of the whole PPI networks, and further, those approaches hold inherent disadvantages, such as being time-consuming, expensive, and having high false positive rate. Therefore, it is urgent and imperative to develop automatic in silico approaches to predict PPIs efficiently and accurately. In this article, we propose a novel mixture of physicochemical and evolutionary-based feature extraction method for predicting PPIs using our newly developed discriminative vector machine (DVM) classifier. The improvements of the proposed method mainly consist in introducing an effective feature extraction method that can capture discriminative features from the evolutionary-based information and physicochemical characteristics, and then a powerful and robust DVM classifier is employed. To the best of our knowledge, it is the first time that DVM model is applied to the field of bioinformatics. When applying the proposed method to the Yeast and Helicobacter pylori (H. pylori) datasets, we obtain excellent prediction accuracies of 94.35% and 90.61%, respectively. The computational results indicate that our method is effective and robust for predicting PPIs, and can be taken as a useful supplementary tool to the traditional experimental methods for future proteomics research.

  15. A statistical method for assessing peptide identification confidence in accurate mass and time tag proteomics.

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    Stanley, Jeffrey R; Adkins, Joshua N; Slysz, Gordon W; Monroe, Matthew E; Purvine, Samuel O; Karpievitch, Yuliya V; Anderson, Gordon A; Smith, Richard D; Dabney, Alan R

    2011-08-15

    Current algorithms for quantifying peptide identification confidence in the accurate mass and time (AMT) tag approach assume that the AMT tags themselves have been correctly identified. However, there is uncertainty in the identification of AMT tags, because this is based on matching LC-MS/MS fragmentation spectra to peptide sequences. In this paper, we incorporate confidence measures for the AMT tag identifications into the calculation of probabilities for correct matches to an AMT tag database, resulting in a more accurate overall measure of identification confidence for the AMT tag approach. The method is referenced as Statistical Tools for AMT Tag Confidence (STAC). STAC additionally provides a uniqueness probability (UP) to help distinguish between multiple matches to an AMT tag and a method to calculate an overall false discovery rate (FDR). STAC is freely available for download, as both a command line and a Windows graphical application.

  16. Identification of Microorganisms by High Resolution Tandem Mass Spectrometry with Accurate Statistical Significance

    Science.gov (United States)

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

    2016-02-01

    Correct and rapid identification of microorganisms is the key to the success of many important applications in health and safety, including, but not limited to, infection treatment, food safety, and biodefense. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is challenging correct microbial identification because of the large number of choices present. To properly disentangle candidate 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 peptidome profiles of microbes to better separate them and by designing an analysis method that yields accurate statistical significance. Here, we present an analysis pipeline that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using MS/MS data of 81 samples, each composed of a single known microorganism, that the proposed pipeline can correctly identify microorganisms at least at the genus and species levels. We have also shown that the proposed pipeline computes accurate statistical significances, i.e., E-values for identified peptides and unified E-values for identified microorganisms. The proposed analysis pipeline has been implemented in MiCId, a freely available software for Microorganism Classification and Identification. MiCId is available for download at http://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html.

  17. A Statistical Method for Assessing Peptide Identification Confidence in Accurate Mass and Time Tag Proteomics

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    Stanley, Jeffrey R.; Adkins, Joshua N.; Slysz, Gordon W.; Monroe, Matthew E.; Purvine, Samuel O.; Karpievitch, Yuliya V.; Anderson, Gordon A.; Smith, Richard D.; Dabney, Alan R.

    2011-07-15

    High-throughput proteomics is rapidly evolving to require high mass measurement accuracy for a variety of different applications. Increased mass measurement accuracy in bottom-up proteomics specifically allows for an improved ability to distinguish and characterize detected MS features, which may in turn be identified by, e.g., matching to entries in a database for both precursor and fragmentation mass identification methods. Many tools exist with which to score the identification of peptides from LC-MS/MS measurements or to assess matches to an accurate mass and time (AMT) tag database, but these two calculations remain distinctly unrelated. Here we present a statistical method, Statistical Tools for AMT tag Confidence (STAC), which extends our previous work incorporating prior probabilities of correct sequence identification from LC-MS/MS, as well as the quality with which LC-MS features match AMT tags, to evaluate peptide identification confidence. Compared to existing tools, we are able to obtain significantly more high-confidence peptide identifications at a given false discovery rate and additionally assign confidence estimates to individual peptide identifications. Freely available software implementations of STAC are available in both command line and as a Windows graphical application.

  18. Identification and Quantification of Protein Glycosylation

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    Ziv Roth

    2012-01-01

    Full Text Available Glycosylation is one of the most abundant posttranslation modifications of proteins, and accumulating evidence indicate that the vast majority of proteins in eukaryotes are glycosylated. Glycosylation plays a role in protein folding, interaction, stability, and mobility, as well as in signal transduction. Thus, by regulating protein activity, glycosylation is involved in the normal functioning of the cell and in the development of diseases. Indeed, in the past few decades there has been a growing realization of the importance of protein glycosylation, as aberrant glycosylation has been implicated in metabolic, neurodegenerative, and neoplastic diseases. Thus, the identification and quantification of protein-borne oligosaccharides have become increasingly important both in the basic sciences of biochemistry and glycobiology and in the applicative sciences, particularly biomedicine and biotechnology. Here, we review the state-of-the-art methodologies for the identification and quantification of oligosaccharides, specifically N- and O-glycosylated proteins.

  19. What's in a Name? The Impact of Accurate Staphylococcus pseudintermedius Identification on Appropriate Antimicrobial Susceptibility Testing.

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    Limbago, Brandi M

    2016-03-01

    Bacteria in the Staphylococcus intermedius group, including Staphylococcus pseudintermedius, often encode mecA-mediated methicillin resistance. Reliable detection of this phenotype for proper treatment and infection control decisions requires that these coagulase-positive staphylococci are accurately identified and specifically that they are not misidentified as S. aureus. As correct species level bacterial identification becomes more commonplace in clinical laboratories, one can expect to see changes in guidance for antimicrobial susceptibility testing and interpretation. The study by Wu et al. in this issue (M. T. Wu, C.-A. D. Burnham, L. F. Westblade, J. Dien Bard, S. D. Lawhon, M. A. Wallace, T. Stanley, E. Burd, J. Hindler, R. M. Humphries, J Clin Microbiol 54:535-542, 2016, http://dx.doi.org/10.1128/JCM.02864-15) highlights the impact of robust identification of S. intermedius group organisms on the selection of appropriate antimicrobial susceptibility testing methods and interpretation.

  20. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

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    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  1. Post-Electrophoretic Identification of Oxidized Proteins

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    Conrad Craig

    2000-01-01

    Full Text Available 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.

  2. A novel PCR-based approach for accurate identification of Vibrio parahaemolyticus

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    Ruichao eLi

    2016-01-01

    Full Text Available A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the blaCARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Phylogenetic analysis showed that blaCARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this blaCARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh, toxR and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of blaCARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by blaCARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification.

  3. A Novel PCR-Based Approach for Accurate Identification of Vibrio parahaemolyticus.

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    Li, Ruichao; Chiou, Jiachi; Chan, Edward Wai-Chi; Chen, Sheng

    2016-01-01

    A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the bla CARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Homologous analysis showed that bla CARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this bla CARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of bla CARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by bla CARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification.

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

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    Guerry, Paul; Duong, Viet Dung; Herrmann, Torsten, E-mail: torsten.herrmann@ens-lyon.fr [Université de Lyon (UMR 5280 CNRS, Ecole Normale Supérieure de Lyon, Université Claude Bernard Lyon 1), Institut des Sciences Analytiques, Centre de RMN à très Hauts Champs (France)

    2015-08-15

    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.

  5. 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.

  6. 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.

  7. Identification of "Known Unknowns" Utilizing Accurate Mass Data and ChemSpider

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    Little, James L.; Williams, Antony J.; Pshenichnov, Alexey; Tkachenko, Valery

    2012-01-01

    In many cases, an unknown to an investigator is actually known in the chemical literature, a reference database, or an internet resource. We refer to these types of compounds as "known unknowns." ChemSpider is a very valuable internet database of known compounds useful in the identification of these types of compounds in commercial, environmental, forensic, and natural product samples. The database contains over 26 million entries from hundreds of data sources and is provided as a free resource to the community. Accurate mass mass spectrometry data is used to query the database by either elemental composition or a monoisotopic mass. Searching by elemental composition is the preferred approach. However, it is often difficult to determine a unique elemental composition for compounds with molecular weights greater than 600 Da. In these cases, searching by the monoisotopic mass is advantageous. In either case, the search results are refined by sorting the number of references associated with each compound in descending order. This raises the most useful candidates to the top of the list for further evaluation. These approaches were shown to be successful in identifying "known unknowns" noted in our laboratory and for compounds of interest to others.

  8. PlantLoc: an accurate web server for predicting plant protein subcellular localization by substantiality motif

    OpenAIRE

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

    2013-01-01

    Knowledge of subcellular localizations (SCLs) of plant proteins relates to their functions and aids in understanding the regulation of biological processes at the cellular level. We present PlantLoc, a highly accurate and fast webserver for predicting the multi-label SCLs of plant proteins. The PlantLoc server has two innovative characters: building localization motif libraries by a recursive method without alignment and Gene Ontology information; and establishing simple architecture for rapi...

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

    Directory of Open Access Journals (Sweden)

    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

  10. Analysis of inteins in the Candida parapsilosis complex for simple and accurate species identification.

    Science.gov (United States)

    Prandini, Tâmara Heloísa Rocha; Theodoro, Raquel Cordeiro; Bruder-Nascimento, Ariane C M O; Scheel, Christina M; Bagagli, Eduardo

    2013-09-01

    Inteins are coding sequences that are transcribed and translated with flanking sequences and then are excised by an autocatalytic process. There are two types of inteins in fungi, mini-inteins and full-length inteins, both of which present a splicing domain containing well-conserved amino acid sequences. Full-length inteins also present a homing endonuclease domain that makes the intein a mobile genetic element. These parasitic genetic elements are located in highly conserved genes and may allow for the differentiation of closely related species of the Candida parapsilosis (psilosis) complex. The correct identification of the three psilosis complex species C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis is very important in the clinical setting for improving antifungal therapy and patient care. In this work, we analyzed inteins that are present in the vacuolar ATPase gene VMA and in the threonyl-tRNA synthetase gene ThrRS in 85 strains of the Candida psilosis complex (46 C. parapsilosis, 17 C. metapsilosis, and 22 C. orthopsilosis). Here, we describe an accessible and accurate technique based on a single PCR that is able to differentiate the psilosis complex based on the VMA intein. Although the ThrRS intein does not distinguish the three species of the psilosis complex by PCR product size, it can differentiate them by sequencing and phylogenetic analysis. Furthermore, this intein is unusually present as both mini- and full-length forms in C. orthopsilosis. Additional population studies should be performed to address whether this represents a common intraspecific variability or the presence of subspecies within C. orthopsilosis.

  11. Accurate Target Identification Using Multi-look Fusion of Low Quality Target Signatures

    Science.gov (United States)

    2008-12-01

    qualité, ce qui pourrait avoir des conséquences importantes pour les applications pratiques. D’une part, l’apparition de technologies de capteurs et...identification performance and this is not adequate for many target identification applications . Furthermore, in order for the single-look procedure to...obtenues qu’avec un un seul capteur . Toutefois, force est de constater que le rendement de l’identification correcte d’objectifs par l’approche

  12. Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry

    OpenAIRE

    Brown, Kristy J; Marathi, Ramya; Fiorillo, Alyson A; Ciccimaro, Eugene F.; Sharma, Seema; Rowlands, David S.; Rayavarapu, Sree; Nagaraju, Kanneboyina; Eric P. Hoffman; Hathout, Yetrib

    2012-01-01

    Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based ...

  13. Automated protein subfamily identification and classification.

    Directory of Open Access Journals (Sweden)

    Duncan P Brown

    2007-08-01

    Full Text Available Function prediction by homology is widely used to provide preliminary functional annotations for genes for which experimental evidence of function is unavailable or limited. This approach has been shown to be prone to systematic error, including percolation of annotation errors through sequence databases. Phylogenomic analysis avoids these errors in function prediction but has been difficult to automate for high-throughput application. To address this limitation, we present a computationally efficient pipeline for phylogenomic classification of proteins. This pipeline uses the SCI-PHY (Subfamily Classification in Phylogenomics algorithm for automatic subfamily identification, followed by subfamily hidden Markov model (HMM construction. A simple and computationally efficient scoring scheme using family and subfamily HMMs enables classification of novel sequences to protein families and subfamilies. Sequences representing entirely novel subfamilies are differentiated from those that can be classified to subfamilies in the input training set using logistic regression. Subfamily HMM parameters are estimated using an information-sharing protocol, enabling subfamilies containing even a single sequence to benefit from conservation patterns defining the family as a whole or in related subfamilies. SCI-PHY subfamilies correspond closely to functional subtypes defined by experts and to conserved clades found by phylogenetic analysis. Extensive comparisons of subfamily and family HMM performances show that subfamily HMMs dramatically improve the separation between homologous and non-homologous proteins in sequence database searches. Subfamily HMMs also provide extremely high specificity of classification and can be used to predict entirely novel subtypes. The SCI-PHY Web server at http://phylogenomics.berkeley.edu/SCI-PHY/ allows users to upload a multiple sequence alignment for subfamily identification and subfamily HMM construction. Biologists wishing to

  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.

  15. 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

  16. Fast and accurate multivariate Gaussian modeling of protein families: predicting residue contacts and protein-interaction partners.

    Directory of Open Access Journals (Sweden)

    Carlo Baldassi

    Full Text Available In the course of evolution, proteins show a remarkable conservation of their three-dimensional structure and their biological function, leading to strong evolutionary constraints on the sequence variability between homologous proteins. Our method aims at extracting such constraints from rapidly accumulating sequence data, and thereby at inferring protein structure and function from sequence information alone. Recently, global statistical inference methods (e.g. direct-coupling analysis, sparse inverse covariance estimation have achieved a breakthrough towards this aim, and their predictions have been successfully implemented into tertiary and quaternary protein structure prediction methods. However, due to the discrete nature of the underlying variable (amino-acids, exact inference requires exponential time in the protein length, and efficient approximations are needed for practical applicability. Here we propose a very efficient multivariate Gaussian modeling approach as a variant of direct-coupling analysis: the discrete amino-acid variables are replaced by continuous Gaussian random variables. The resulting statistical inference problem is efficiently and exactly solvable. We show that the quality of inference is comparable or superior to the one achieved by mean-field approximations to inference with discrete variables, as done by direct-coupling analysis. This is true for (i the prediction of residue-residue contacts in proteins, and (ii the identification of protein-protein interaction partner in bacterial signal transduction. An implementation of our multivariate Gaussian approach is available at the website http://areeweb.polito.it/ricerca/cmp/code.

  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. Accurate refinement of docked protein complexes using evolutionary information and deep learning.

    Science.gov (United States)

    Akbal-Delibas, Bahar; Farhoodi, Roshanak; Pomplun, Marc; Haspel, Nurit

    2016-06-01

    One of the major challenges for protein docking methods is to accurately discriminate native-like structures from false positives. Docking methods are often inaccurate and the results have to be refined and re-ranked to obtain native-like complexes and remove outliers. In a previous work, we introduced AccuRefiner, a machine learning based tool for refining protein-protein complexes. Given a docked complex, the refinement tool produces a small set of refined versions of the input complex, with lower root-mean-square-deviation (RMSD) of atomic positions with respect to the native structure. The method employs a unique ranking tool that accurately predicts the RMSD of docked complexes with respect to the native structure. In this work, we use a deep learning network with a similar set of features and five layers. We show that a properly trained deep learning network can accurately predict the RMSD of a docked complex with 1.40 Å error margin on average, by approximating the complex relationship between a wide set of scoring function terms and the RMSD of a docked structure. The network was trained on 35000 unbound docking complexes generated by RosettaDock. We tested our method on 25 different putative docked complexes produced also by RosettaDock for five proteins that were not included in the training data. The results demonstrate that the high accuracy of the ranking tool enables AccuRefiner to consistently choose the refinement candidates with lower RMSD values compared to the coarsely docked input structures.

  19. Accurate Prediction of One-Dimensional Protein Structure Features Using SPINE-X.

    Science.gov (United States)

    Faraggi, Eshel; Kloczkowski, Andrzej

    2017-01-01

    Accurate prediction of protein secondary structure and other one-dimensional structure features is essential for accurate sequence alignment, three-dimensional structure modeling, and function prediction. SPINE-X is a software package to predict secondary structure as well as accessible surface area and dihedral angles ϕ and ψ. For secondary structure SPINE-X achieves an accuracy of between 81 and 84 % depending on the dataset and choice of tests. The Pearson correlation coefficient for accessible surface area prediction is 0.75 and the mean absolute error from the ϕ and ψ dihedral angles are 20(∘) and 33(∘), respectively. The source code and a Linux executables for SPINE-X are available from Research and Information Systems at http://mamiris.com .

  20. 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...... genome and allows identification, mapping, cloning and assistance in gene prediction of any protein for which minimal mass spectrometric information can be obtained. Several novel proteins from Arabidopsis thaliana and human have been discovered in this way....

  1. 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.

  2. Accurate De Novo Prediction of Protein Contact Map by Ultra-Deep Learning Model

    Science.gov (United States)

    Li, Zhen; Zhang, Renyu

    2017-01-01

    Motivation Protein contacts contain key information for the understanding of protein structure and function and thus, contact prediction from sequence is an important problem. Recently exciting progress has been made on this problem, but the predicted contacts for proteins without many sequence homologs is still of low quality and not very useful for de novo structure prediction. Method This paper presents a new deep learning method that predicts contacts by integrating both evolutionary coupling (EC) and sequence conservation information through an ultra-deep neural network formed by two deep residual neural networks. The first residual network conducts a series of 1-dimensional convolutional transformation of sequential features; the second residual network conducts a series of 2-dimensional convolutional transformation of pairwise information including output of the first residual network, EC information and pairwise potential. By using very deep residual networks, we can accurately model contact occurrence patterns and complex sequence-structure relationship and thus, obtain higher-quality contact prediction regardless of how many sequence homologs are available for proteins in question. Results Our method greatly outperforms existing methods and leads to much more accurate contact-assisted folding. Tested on 105 CASP11 targets, 76 past CAMEO hard targets, and 398 membrane proteins, the average top L long-range prediction accuracy obtained by our method, one representative EC method CCMpred and the CASP11 winner MetaPSICOV is 0.47, 0.21 and 0.30, respectively; the average top L/10 long-range accuracy of our method, CCMpred and MetaPSICOV is 0.77, 0.47 and 0.59, respectively. Ab initio folding using our predicted contacts as restraints but without any force fields can yield correct folds (i.e., TMscore>0.6) for 203 of the 579 test proteins, while that using MetaPSICOV- and CCMpred-predicted contacts can do so for only 79 and 62 of them, respectively. Our contact

  3. Identification of SUMO target proteins by quantitative proteomics

    DEFF Research Database (Denmark)

    Andersen, Jens S; Matic, Ivan; Vertegaal, Alfred C O

    2009-01-01

    The identification of target proteins for small ubiquitin-like modifiers (SUMOs) is a critical step towards a detailed understanding of the cellular functions of SUMOs. Substrate protein identification for SUMOs is hampered by the low abundance of SUMO targets, the finding that only a small fract...

  4. Unifying protein inference and peptide identification with feedback to update consistency between peptides.

    Science.gov (United States)

    Shi, Jinhong; Chen, Bolin; Wu, Fang-Xiang

    2013-01-01

    We first propose a new method to process peptide identification reports from databases search engines. Then via it we develop a method for unifying protein inference and peptide identification by adding a feedback from protein inference to peptide identification. The feedback information is a list of high-confidence proteins, which is used to update an adjacency matrix between peptides. The adjacency matrix is used in the regularization of peptide scores. Logistic regression (LR) is used to compute the probability of peptide identification with the regularized scores. Protein scores are then calculated with the LR probability of peptides. Instead of selecting the best peptide match for each MS/MS, we select multiple peptides. By testing on two datasets, the results have shown that the proposed method can robustly assign accurate probabilities to peptides, and have a higher discrimination power than PeptideProphet to distinguish correct and incorrect identified peptides. Additionally, not only can our method infer more true positive proteins but also infer less false positive proteins than ProteinProphet at the same false positive rate. The coverage of inferred proteins is also significantly increased due to the selection of multiple peptides for each MS/MS and the improvement of their scores by the feedback from the inferred proteins.

  5. Phage display library screening for identification of interacting protein partners.

    Science.gov (United States)

    Addepalli, Balasubrahmanyam; Rao, Suryadevara; Hunt, Arthur G

    2015-01-01

    Phage display is a versatile high-throughput screening method employed to understand and improve the chemical biology, be it production of human monoclonal antibodies or identification of interacting protein partners. A majority of cell proteins operate in a concerted fashion either by stable or transient interactions. Such interactions can be mediated by recognition of small amino acid sequence motifs on the protein surface. Phage display can play a crucial role in identification of such motifs. This report describes the use of phage display for the identification of high affinity sequence motifs that could be responsible for interactions with a target (bait) protein.

  6. HMM-FRAME: accurate protein domain classification for metagenomic sequences containing frameshift errors

    Directory of Open Access Journals (Sweden)

    Sun Yanni

    2011-05-01

    Full Text Available Abstract Background Protein domain classification is an important step in metagenomic annotation. The state-of-the-art method for protein domain classification is profile HMM-based alignment. However, the relatively high rates of insertions and deletions in homopolymer regions of pyrosequencing reads create frameshifts, causing conventional profile HMM alignment tools to generate alignments with marginal scores. This makes error-containing gene fragments unclassifiable with conventional tools. Thus, there is a need for an accurate domain classification tool that can detect and correct sequencing errors. Results We introduce HMM-FRAME, a protein domain classification tool based on an augmented Viterbi algorithm that can incorporate error models from different sequencing platforms. HMM-FRAME corrects sequencing errors and classifies putative gene fragments into domain families. It achieved high error detection sensitivity and specificity in a data set with annotated errors. We applied HMM-FRAME in Targeted Metagenomics and a published metagenomic data set. The results showed that our tool can correct frameshifts in error-containing sequences, generate much longer alignments with significantly smaller E-values, and classify more sequences into their native families. Conclusions HMM-FRAME provides a complementary protein domain classification tool to conventional profile HMM-based methods for data sets containing frameshifts. Its current implementation is best used for small-scale metagenomic data sets. The source code of HMM-FRAME can be downloaded at http://www.cse.msu.edu/~zhangy72/hmmframe/ and at https://sourceforge.net/projects/hmm-frame/.

  7. Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis.

    Science.gov (United States)

    Zhu, Lijuan; Wang, Zhikai; Wang, Wenwen; Wang, Chunli; Hua, Shasha; Su, Zeqi; Brako, Larry; Garcia-Barrio, Minerva; Ye, Mingliang; Wei, Xuan; Zou, Hanfa; Ding, Xia; Liu, Lifang; Liu, Xing; Yao, Xuebiao

    2015-11-06

    Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.

  8. 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.

  9. Poisonous or non-poisonous plants? DNA-based tools and applications for accurate identification.

    Science.gov (United States)

    Mezzasalma, Valerio; Ganopoulos, Ioannis; Galimberti, Andrea; Cornara, Laura; Ferri, Emanuele; Labra, Massimo

    2017-01-01

    Plant exposures are among the most frequently reported cases to poison control centres worldwide. This is a growing condition due to recent societal trends oriented towards the consumption of wild plants as food, cosmetics, or medicine. At least three general causes of plant poisoning can be identified: plant misidentification, introduction of new plant-based supplements and medicines with no controls about their safety, and the lack of regulation for the trading of herbal and phytochemical products. Moreover, an efficient screening for the occurrence of plants poisonous to humans is also desirable at the different stages of the food supply chain: from the raw material to the final transformed product. A rapid diagnosis of intoxication cases is necessary in order to provide the most reliable treatment. However, a precise taxonomic characterization of the ingested species is often challenging. In this review, we provide an overview of the emerging DNA-based tools and technologies to address the issue of poisonous plant identification. Specifically, classic DNA barcoding and its applications using High Resolution Melting (Bar-HRM) ensure high universality and rapid response respectively, whereas High Throughput Sequencing techniques (HTS) provide a complete characterization of plant residues in complex matrices. The pros and cons of each approach have been evaluated with the final aim of proposing a general user's guide to molecular identification directed to different stakeholder categories interested in the diagnostics of poisonous plants.

  10. Application of the antibiotic batumin for accurate and rapid identification of staphylococcal small colony variants

    Directory of Open Access Journals (Sweden)

    Churkina Larisa N

    2012-07-01

    Full Text Available Abstract Background Staphylococcus aureus is a major human pathogen causing significant morbidity and mortality. The S. aureus colonies in osteomyelitis, in patients with cystic fibrosis and patients with endoprosthesis rejection frequently have an atypical morphology, i.e. staphylococcal small-colony variants, which form a naturally occurring subpopulation of clinically important staphylococci. Identification of these small colony variants is difficult, because of the loss of typical phenotypic characteristics of these variants. We wanted to improve and simplify the diagnosis of staphylococcal infection using a diagnostic preparation, consisting of 5 μg batumin paper disks. Batumin possesses a unique selective activity against all studied Staphylococcus spp., whereas all other species tested thus far are batumin resistant. We assessed the efficacy of the batumin diagnostic preparation to identify staphylococcal small colony variants, isolated from osteomyelitis patients. Findings With the batumin diagnostic preparation, all 30 tested staphylococcal small-colony variants had a growth inhibition zone around the disk of minimum 25 mm, accordant with the inhibition zones of the parent strains, isolated from the same patients. Conclusions The batumin diagnostic preparation correctly identified the small-colony variants of S. aureus, S. haemolyticus and S. epidermidis as belonging to the genus Staphylococcus, which differ profoundly from parental strains and are difficult to identify with standard methods. Identification of staphylococcal small-colony variants with the batumin diagnostic preparation is technically simple and can facilitate practical laboratory work.

  11. 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.

  12. 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

  13. Innovative Flow Cytometry Allows Accurate Identification of Rare Circulating Cells Involved in Endothelial Dysfunction

    Science.gov (United States)

    Boraldi, Federica; Bartolomeo, Angelica; De Biasi, Sara; Orlando, Stefania; Costa, Sonia; Cossarizza, Andrea; Quaglino, Daniela

    2016-01-01

    Introduction Although rare, circulating endothelial and progenitor cells could be considered as markers of endothelial damage and repair potential, possibly predicting the severity of cardiovascular manifestations. A number of studies highlighted the role of these cells in age-related diseases, including those characterized by ectopic calcification. Nevertheless, their use in clinical practice is still controversial, mainly due to difficulties in finding reproducible and accurate methods for their determination. Methods Circulating mature cells (CMC, CD45-, CD34+, CD133-) and circulating progenitor cells (CPC, CD45dim, CD34bright, CD133+) were investigated by polychromatic high-speed flow cytometry to detect the expression of endothelial (CD309+) or osteogenic (BAP+) differentiation markers in healthy subjects and in patients affected by peripheral vascular manifestations associated with ectopic calcification. Results This study shows that: 1) polychromatic flow cytometry represents a valuable tool to accurately identify rare cells; 2) the balance of CD309+ on CMC/CD309+ on CPC is altered in patients affected by peripheral vascular manifestations, suggesting the occurrence of vascular damage and low repair potential; 3) the increase of circulating cells exhibiting a shift towards an osteoblast-like phenotype (BAP+) is observed in the presence of ectopic calcification. Conclusion Differences between healthy subjects and patients with ectopic calcification indicate that this approach may be useful to better evaluate endothelial dysfunction in a clinical context. PMID:27560136

  14. A transition radiation detector which features 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 Inst. of Physics and Engineering, Moscow (Russia 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 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 {times} l0{sup 2}. The single-wire, track-position resolution for the TRD is {approximately}230{mu}m.

  15. A transition radiation detector which features 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 Inst. of Physics and Engineering (Russian Federation)); Bennett, M.; Chikanian, A.; Kumar, S.; Mitchell, J.T.; Pope, K. (Yale Univ., New Haven, CT (United States))

    1993-04-01

    The authors 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 10[sup 2]. The single-wire, track-position resolution for the TRD is [approximately] [mu]m.

  16. 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.

  17. Fast and Accurate Discovery of Degenerate Linear Motifs in Protein Sequences

    Science.gov (United States)

    Levy, Emmanuel D.; Michnick, Stephen W.

    2014-01-01

    Linear motifs mediate a wide variety of cellular functions, which makes their characterization in protein sequences crucial to understanding cellular systems. However, the short length and degenerate nature of linear motifs make their discovery a difficult problem. Here, we introduce MotifHound, an algorithm particularly suited for the discovery of small and degenerate linear motifs. MotifHound performs an exact and exhaustive enumeration of all motifs present in proteins of interest, including all of their degenerate forms, and scores the overrepresentation of each motif based on its occurrence in proteins of interest relative to a background (e.g., proteome) using the hypergeometric distribution. To assess MotifHound, we benchmarked it together with state-of-the-art algorithms. The benchmark consists of 11,880 sets of proteins from S. cerevisiae; in each set, we artificially spiked-in one motif varying in terms of three key parameters, (i) number of occurrences, (ii) length and (iii) the number of degenerate or “wildcard” positions. The benchmark enabled the evaluation of the impact of these three properties on the performance of the different algorithms. The results showed that MotifHound and SLiMFinder were the most accurate in detecting degenerate linear motifs. Interestingly, MotifHound was 15 to 20 times faster at comparable accuracy and performed best in the discovery of highly degenerate motifs. We complemented the benchmark by an analysis of proteins experimentally shown to bind the FUS1 SH3 domain from S. cerevisiae. Using the full-length protein partners as sole information, MotifHound recapitulated most experimentally determined motifs binding to the FUS1 SH3 domain. Moreover, these motifs exhibited properties typical of SH3 binding peptides, e.g., high intrinsic disorder and evolutionary conservation, despite the fact that none of these properties were used as prior information. MotifHound is available (http://michnick.bcm.umontreal.ca or http

  18. Conformational energy range of ligands in protein crystal structures: The difficult quest for accurate understanding.

    Science.gov (United States)

    Peach, Megan L; Cachau, Raul E; Nicklaus, Marc C

    2017-02-24

    In this review, we address a fundamental question: What is the range of conformational energies seen in ligands in protein-ligand crystal structures? This value is important biophysically, for better understanding the protein-ligand binding process; and practically, for providing a parameter to be used in many computational drug design methods such as docking and pharmacophore searches. We synthesize a selection of previously reported conflicting results from computational studies of this issue and conclude that high ligand conformational energies really are present in some crystal structures. The main source of disagreement between different analyses appears to be due to divergent treatments of electrostatics and solvation. At the same time, however, for many ligands, a high conformational energy is in error, due to either crystal structure inaccuracies or incorrect determination of the reference state. Aside from simple chemistry mistakes, we argue that crystal structure error may mainly be because of the heuristic weighting of ligand stereochemical restraints relative to the fit of the structure to the electron density. This problem cannot be fixed with improvements to electron density fitting or with simple ligand geometry checks, though better metrics are needed for evaluating ligand and binding site chemistry in addition to geometry during structure refinement. The ultimate solution for accurately determining ligand conformational energies lies in ultrahigh-resolution crystal structures that can be refined without restraints.

  19. FAMSA: Fast and accurate multiple sequence alignment of huge protein families

    Science.gov (United States)

    Deorowicz, Sebastian; Debudaj-Grabysz, Agnieszka; Gudyś, Adam

    2016-01-01

    Rapid development of modern sequencing platforms has contributed to the unprecedented growth of protein families databases. The abundance of sets containing hundreds of thousands of sequences is a formidable challenge for multiple sequence alignment algorithms. The article introduces FAMSA, a new progressive algorithm designed for fast and accurate alignment of thousands of protein sequences. Its features include the utilization of the longest common subsequence measure for determining pairwise similarities, a novel method of evaluating gap costs, and a new iterative refinement scheme. What matters is that its implementation is highly optimized and parallelized to make the most of modern computer platforms. Thanks to the above, quality indicators, i.e. sum-of-pairs and total-column scores, show FAMSA to be superior to competing algorithms, such as Clustal Omega or MAFFT for datasets exceeding a few thousand sequences. Quality does not compromise on time or memory requirements, which are an order of magnitude lower than those in the existing solutions. For example, a family of 415519 sequences was analyzed in less than two hours and required no more than 8 GB of RAM. FAMSA is available for free at http://sun.aei.polsl.pl/REFRESH/famsa. PMID:27670777

  20. 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,…

  1. Identification of mitochondrial proteins of malaria parasite using analysis of variance.

    Science.gov (United States)

    Ding, Hui; Li, Dongmei

    2015-02-01

    As a parasitic protozoan, Plasmodium falciparum (P. falciparum) can cause malaria. The mitochondrial proteins of malaria parasite play important roles in the discovery of anti-malarial drug targets. Thus, accurate identification of mitochondrial proteins of malaria parasite is a key step for understanding their functions and finding potential drug targets. In this work, we developed a sequence-based method to identify the mitochondrial proteins of malaria parasite. At first, we extended adjoining dipeptide composition to g-gap dipeptide composition for discretely formulating the protein sequences. Subsequently, the analysis of variance (ANOVA) combined with incremental feature selection (IFS) was used to pick out the optimal features. Finally, the jackknife cross-validation was used to evaluate the performance of the proposed model. Evaluation results showed that the maximum accuracy of 97.1% could be achieved by using 101 optimal 5-gap dipeptides. The comparison with previous methods demonstrated that our method was accurate and efficient.

  2. A tri-stage cluster identification model for accurate analysis of seismic catalogs

    Directory of Open Access Journals (Sweden)

    S. J. Nanda

    2013-02-01

    Full Text Available In this paper we propose a tri-stage cluster identification model that is a combination of a simple single iteration distance algorithm and an iterative K-means algorithm. In this study of earthquake seismicity, the model considers event location, time and magnitude information from earthquake catalog data to efficiently classify events as either background or mainshock and aftershock sequences. Tests on a synthetic seismicity catalog demonstrate the efficiency of the proposed model in terms of accuracy percentage (94.81% for background and 89.46% for aftershocks. The close agreement between lambda and cumulative plots for the ideal synthetic catalog and that generated by the proposed model also supports the accuracy of the proposed technique. There is flexibility in the model design to allow for proper selection of location and magnitude ranges, depending upon the nature of the mainshocks present in the catalog. The effectiveness of the proposed model also is evaluated by the classification of events in three historic catalogs: California, Japan and Indonesia. As expected, for both synthetic and historic catalog analysis it is observed that the density of events classified as background is almost uniform throughout the region, whereas the density of aftershock events are higher near the mainshocks.

  3. Rapid and Accurate Identification of Animal Species in Natural Leather Goods by Liquid Chromatography/Mass Spectrometry.

    Science.gov (United States)

    Izuchi, Yukari; Takashima, Tsuneo; Hatano, Naoya

    2016-01-01

    The demand for leather goods has grown globally in recent years. Industry revenue is forecast to reach $91.2 billion by 2018. There is an ongoing labelling problem in the leather items market, in that it is currently impossible to identify the species that a given piece of leather is derived from. To address this issue, we developed a rapid and simple method for the specific identification of leather derived from cattle, horses, pigs, sheep, goats, and deer by analysing peptides produced by the trypsin-digestion of proteins contained in leather goods using liquid chromatography/mass spectrometry. We determined species-specific amino acid sequences by liquid chromatography/tandem mass spectrometry analysis using the Mascot software program and demonstrated that collagen α-1(I), collagen α-2(I), and collagen α-1(III) from the dermal layer of the skin are particularly useful in species identification.

  4. An improved Bathocuproine assay for accurate valence identification and quantification of copper bound by biomolecules.

    Science.gov (United States)

    Chen, Dinglong; Darabedian, Narek; Li, Zhiqiang; Kai, Tianhan; Jiang, Dianlu; Zhou, Feimeng

    2016-03-15

    Copper is an essential metal in all organisms. Reliably quantifying and identifying the copper content and oxidation state is crucial, since the information is essential to understanding protein structure and function. Chromophoric ligands, such as Bathocuproine (BC) and its water-soluble analog, Bathocuproinedisulfonic acid (BCS), preferentially bind Cu(I) over Cu(II), and therefore have been widely used as optical probes to determine the oxidation state of copper bound by biomolecules. However, the BCS assay is commonly misused, leading to erroneous conclusions regarding the role of copper in biological processes. By measuring the redox potential of Cu(II)-BCS2 and conducting UV-vis absorption measurements in the presence of oxidizable amino acids, the thermodynamic origin of the potential artifacts becomes evident. The BCS assay was improved by introducing a strong Cu(II) chelator EDTA prior to the addition of BCS to prevent interference that might arise from Cu(II) present in the sample. The strong Cu(II) chelator rids of all the potential errors inherent in the conventional BCS assay. Applications of the improved assay to peptides and protein containing oxidizable amino acid residues confirm that free Cu(II) no longer leads to artifacts, thereby resolving issues related to this persistently misused colorimetric assay of Cu(I) in biological systems.

  5. Evaluating de novo sequencing in proteomics: already an accurate alternative to database-driven peptide identification?

    Science.gov (United States)

    Muth, Thilo; Renard, Bernhard Y

    2017-03-21

    While peptide identifications in mass spectrometry (MS)-based shotgun proteomics are mostly obtained using database search methods, high-resolution spectrum data from modern MS instruments nowadays offer the prospect of improving the performance of computational de novo peptide sequencing. The major benefit of de novo sequencing is that it does not require a reference database to deduce full-length or partial tag-based peptide sequences directly from experimental tandem mass spectrometry spectra. Although various algorithms have been developed for automated de novo sequencing, the prediction accuracy of proposed solutions has been rarely evaluated in independent benchmarking studies. The main objective of this work is to provide a detailed evaluation on the performance of de novo sequencing algorithms on high-resolution data. For this purpose, we processed four experimental data sets acquired from different instrument types from collision-induced dissociation and higher energy collisional dissociation (HCD) fragmentation mode using the software packages Novor, PEAKS and PepNovo. Moreover, the accuracy of these algorithms is also tested on ground truth data based on simulated spectra generated from peak intensity prediction software. We found that Novor shows the overall best performance compared with PEAKS and PepNovo with respect to the accuracy of correct full peptide, tag-based and single-residue predictions. In addition, the same tool outpaced the commercial competitor PEAKS in terms of running time speedup by factors of around 12-17. Despite around 35% prediction accuracy for complete peptide sequences on HCD data sets, taken as a whole, the evaluated algorithms perform moderately on experimental data but show a significantly better performance on simulated data (up to 84% accuracy). Further, we describe the most frequently occurring de novo sequencing errors and evaluate the influence of missing fragment ion peaks and spectral noise on the accuracy. Finally

  6. 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

  7. Evaluating Peptide Mass Fingerprinting-based Protein Identification

    Institute of Scientific and Technical Information of China (English)

    Senthilkumar; Damodaran; Troy; D.; Wood; Priyadharsini; Nagarajan; Richard; A.; Rabin

    2007-01-01

    Identification of proteins by mass spectrometry (MS) is an essential step in pro- teomic studies and is typically accomplished by either peptide mass fingerprinting (PMF) or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when high- throughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.

  8. Fitmunk: improving protein structures by accurate, automatic modeling of side-chain conformations.

    Science.gov (United States)

    Porebski, Przemyslaw Jerzy; Cymborowski, Marcin; Pasenkiewicz-Gierula, Marta; Minor, Wladek

    2016-02-01

    Improvements in crystallographic hardware and software have allowed automated structure-solution pipelines to approach a near-`one-click' experience for the initial determination of macromolecular structures. However, in many cases the resulting initial model requires a laborious, iterative process of refinement and validation. A new method has been developed for the automatic modeling of side-chain conformations that takes advantage of rotamer-prediction methods in a crystallographic context. The algorithm, which is based on deterministic dead-end elimination (DEE) theory, uses new dense conformer libraries and a hybrid energy function derived from experimental data and prior information about rotamer frequencies to find the optimal conformation of each side chain. In contrast to existing methods, which incorporate the electron-density term into protein-modeling frameworks, the proposed algorithm is designed to take advantage of the highly discriminatory nature of electron-density maps. This method has been implemented in the program Fitmunk, which uses extensive conformational sampling. This improves the accuracy of the modeling and makes it a versatile tool for crystallographic model building, refinement and validation. Fitmunk was extensively tested on over 115 new structures, as well as a subset of 1100 structures from the PDB. It is demonstrated that the ability of Fitmunk to model more than 95% of side chains accurately is beneficial for improving the quality of crystallographic protein models, especially at medium and low resolutions. Fitmunk can be used for model validation of existing structures and as a tool to assess whether side chains are modeled optimally or could be better fitted into electron density. Fitmunk is available as a web service at http://kniahini.med.virginia.edu/fitmunk/server/ or at http://fitmunk.bitbucket.org/.

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

    DEFF Research Database (Denmark)

    Nordhoff, E; Krogsdam, A M; Jorgensen, 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...

  10. 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

  11. High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1.

    Directory of Open Access Journals (Sweden)

    Jennifer L Schehr

    Full Text Available Expression of programmed-death ligand 1 (PD-L1 in non-small cell lung cancer (NSCLC is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT.To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP technology.Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33-100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples.Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the

  12. High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1

    Science.gov (United States)

    Schultz, Zachery D.; Warrick, Jay W.; Guckenberger, David J.; Pezzi, Hannah M.; Sperger, Jamie M.; Heninger, Erika; Saeed, Anwaar; Leal, Ticiana; Mattox, Kara; Traynor, Anne M.; Campbell, Toby C.; Berry, Scott M.; Beebe, David J.; Lang, Joshua M.

    2016-01-01

    Background Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)). Methods To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology. Results Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33–100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples. Conclusions Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria

  13. 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...

  14. 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…

  15. 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...

  16. Simple Protein Complex Purification and Identification Method Suitable for High- throughput Mapping of Protein Interaction Networks

    Energy Technology Data Exchange (ETDEWEB)

    Markillie, Lye Meng; Lin, Chiann Tso; Adkins, Joshua N.; Auberry, Deanna L.; Hill, Eric A.; Hooker, Brian S.; Moore, Priscilla A.; Moore, Ronald J.; Shi, Liang; Wiley, H. S.; Kery, Vladimir

    2005-04-11

    Most of the current methods for purification and identification of protein complexes use endogenous expression of affinity tagged bait, tandem affinity tag purification of protein complexes followed by specific elution of complexes from beads, gel separation, in-gel digestion and mass spectrometric analysis of protein interactors. We propose a single affinity tag in vitro pulldown assay with denaturing elution, trypsin digestion in organic solvent and LC ESI MS/MS protein identification using SEQUEST analysis. Our method is simple, easy to scale up and automate thus suitable for high throughput mapping of protein interaction networks and functional proteomics.

  17. 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.

  18. 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)

    Poleksic, Aleksandar; Yao, Yuan; Tong, Hanghang; Meng, Patrick; Xie, Lei

    2016-01-01

    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

  19. BioID Identification of Lamin-Associated Proteins.

    Science.gov (United States)

    Mehus, Aaron A; Anderson, Ruthellen H; Roux, Kyle J

    2016-01-01

    A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The identification of proteins associated with lamins is likely to provide fundamental insight into these mechanisms. BioID (proximity-dependent biotin identification) is a unique and powerful method for identifying protein-protein and proximity-based interactions in living cells. BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID-fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry. BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. BioID is applicable to insoluble proteins such as lamins that are often refractory to study by other methods and can identify weak and/or transient interactions. We discuss the use of BioID to elucidate novel lamin-interacting proteins and its applications in a broad range of biological systems, and provide detailed protocols to guide new applications.

  20. FastRNABindR: Fast and Accurate Prediction of Protein-RNA Interface Residues.

    Directory of Open Access Journals (Sweden)

    Yasser El-Manzalawy

    Full Text Available A wide range of biological processes, including regulation of gene expression, protein synthesis, and replication and assembly of many viruses are mediated by RNA-protein interactions. However, experimental determination of the structures of protein-RNA complexes is expensive and technically challenging. Hence, a number of computational tools have been developed for predicting protein-RNA interfaces. Some of the state-of-the-art protein-RNA interface predictors rely on position-specific scoring matrix (PSSM-based encoding of the protein sequences. The computational efforts needed for generating PSSMs severely limits the practical utility of protein-RNA interface prediction servers. In this work, we experiment with two approaches, random sampling and sequence similarity reduction, for extracting a representative reference database of protein sequences from more than 50 million protein sequences in UniRef100. Our results suggest that random sampled databases produce better PSSM profiles (in terms of the number of hits used to generate the profile and the distance of the generated profile to the corresponding profile generated using the entire UniRef100 data as well as the accuracy of the machine learning classifier trained using these profiles. Based on our results, we developed FastRNABindR, an improved version of RNABindR for predicting protein-RNA interface residues using PSSM profiles generated using 1% of the UniRef100 sequences sampled uniformly at random. To the best of our knowledge, FastRNABindR is the only protein-RNA interface residue prediction online server that requires generation of PSSM profiles for query sequences and accepts hundreds of protein sequences per submission. Our approach for determining the optimal BLAST database for a protein-RNA interface residue classification task has the potential of substantially speeding up, and hence increasing the practical utility of, other amino acid sequence based predictors of protein-protein

  1. Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR.

    Directory of Open Access Journals (Sweden)

    Erin P Price

    Full Text Available Burkholderia ubonensis is an environmental bacterium belonging to the Burkholderia cepacia complex (Bcc, a group of genetically related organisms that are associated with opportunistic but generally nonfatal infections in healthy individuals. In contrast, the near-neighbour species Burkholderia pseudomallei causes melioidosis, a disease that can be fatal in up to 95% of cases if left untreated. B. ubonensis is frequently misidentified as B. pseudomallei from soil samples using selective culturing on Ashdown's medium, reflecting both the shared environmental niche and morphological similarities of these species. Additionally, B. ubonensis shows potential as an important biocontrol agent in B. pseudomallei-endemic regions as certain strains possess antagonistic properties towards B. pseudomallei. Current methods for characterising B. ubonensis are laborious, time-consuming and costly, and as such this bacterium remains poorly studied. The aim of our study was to develop a rapid and inexpensive real-time PCR-based assay specific for B. ubonensis. We demonstrate that a novel B. ubonensis-specific assay, Bu550, accurately differentiates B. ubonensis from B. pseudomallei and other species that grow on selective Ashdown's agar. We anticipate that Bu550 will catalyse research on B. ubonensis by enabling rapid identification of this organism from Ashdown's-positive colonies that are not B. pseudomallei.

  2. 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.

  3. Combining computer algorithms with experimental approaches permits the rapid and accurate identification of T cell epitopes from defined antigens.

    Science.gov (United States)

    Schirle, M; Weinschenk, T; Stevanović, S

    2001-11-01

    The identification of T cell epitopes from immunologically relevant antigens remains a critical step in the development of vaccines and methods for monitoring of T cell responses. This review presents an overview of strategies that employ computer algorithms for the selection of candidate peptides from defined proteins and subsequent verification of their in vivo relevance by experimental approaches. Several computer algorithms are currently being used for epitope prediction of various major histocompatibility complex (MHC) class I and II molecules, based either on the analysis of natural MHC ligands or on the binding properties of synthetic peptides. Moreover, the analysis of proteasomal digests of peptides and whole proteins has led to the development of algorithms for the prediction of proteasomal cleavages. In order to verify the generation of the predicted peptides during antigen processing in vivo as well as their immunogenic potential, several experimental approaches have been pursued in the recent past. Mass spectrometry-based bioanalytical approaches have been used specifically to detect predicted peptides among isolated natural ligands. Other strategies employ various methods for the stimulation of primary T cell responses against the predicted peptides and subsequent testing of the recognition pattern towards target cells that express the antigen.

  4. Accurate prediction of interfacial residues in two-domain proteins using evolutionary information: implications for three-dimensional modeling.

    Science.gov (United States)

    Bhaskara, Ramachandra M; Padhi, Amrita; Srinivasan, Narayanaswamy

    2014-07-01

    With the preponderance of multidomain proteins in eukaryotic genomes, it is essential to recognize the constituent domains and their functions. Often function involves communications across the domain interfaces, and the knowledge of the interacting sites is essential to our understanding of the structure-function relationship. Using evolutionary information extracted from homologous domains in at least two diverse domain architectures (single and multidomain), we predict the interface residues corresponding to domains from the two-domain proteins. We also use information from the three-dimensional structures of individual domains of two-domain proteins to train naïve Bayes classifier model to predict the interfacial residues. Our predictions are highly accurate (∼85%) and specific (∼95%) to the domain-domain interfaces. This method is specific to multidomain proteins which contain domains in at least more than one protein architectural context. Using predicted residues to constrain domain-domain interaction, rigid-body docking was able to provide us with accurate full-length protein structures with correct orientation of domains. We believe that these results can be of considerable interest toward rational protein and interaction design, apart from providing us with valuable information on the nature of interactions.

  5. Toward more accurate ancestral protein genotype-phenotype reconstructions with the use of species tree-aware gene trees.

    Science.gov (United States)

    Groussin, Mathieu; Hobbs, Joanne K; Szöllősi, Gergely J; Gribaldo, Simonetta; Arcus, Vickery L; Gouy, Manolo

    2015-01-01

    The resurrection of ancestral proteins provides direct insight into how natural selection has shaped proteins found in nature. By tracing substitutions along a gene phylogeny, ancestral proteins can be reconstructed in silico and subsequently synthesized in vitro. This elegant strategy reveals the complex mechanisms responsible for the evolution of protein functions and structures. However, to date, all protein resurrection studies have used simplistic approaches for ancestral sequence reconstruction (ASR), including the assumption that a single sequence alignment alone is sufficient to accurately reconstruct the history of the gene family. The impact of such shortcuts on conclusions about ancestral functions has not been investigated. Here, we show with simulations that utilizing information on species history using a model that accounts for the duplication, horizontal transfer, and loss (DTL) of genes statistically increases ASR accuracy. This underscores the importance of the tree topology in the inference of putative ancestors. We validate our in silico predictions using in vitro resurrection of the LeuB enzyme for the ancestor of the Firmicutes, a major and ancient bacterial phylum. With this particular protein, our experimental results demonstrate that information on the species phylogeny results in a biochemically more realistic and kinetically more stable ancestral protein. Additional resurrection experiments with different proteins are necessary to statistically quantify the impact of using species tree-aware gene trees on ancestral protein phenotypes. Nonetheless, our results suggest the need for incorporating both sequence and DTL information in future studies of protein resurrections to accurately define the genotype-phenotype space in which proteins diversify.

  6. Methods and Approaches to Mass Spectroscopy Based Protein Identification

    Science.gov (United States)

    This book chapter is a review of current mass spectrometers and the role in the field of proteomics. Various instruments are discussed and their strengths and weaknesses are highlighted. In addition, the methods of protein identification using a mass spectrometer are explained as well as data vali...

  7. Accurate design of co-assembling multi-component protein nanomaterials.

    Science.gov (United States)

    King, Neil P; Bale, Jacob B; Sheffler, William; McNamara, Dan E; Gonen, Shane; Gonen, Tamir; Yeates, Todd O; Baker, David

    2014-06-05

    The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.

  8. Protein corona composition does not accurately predict hematocompatibility of colloidal gold nanoparticles.

    Science.gov (United States)

    Dobrovolskaia, Marina A; Neun, Barry W; Man, Sonny; Ye, Xiaoying; Hansen, Matthew; Patri, Anil K; Crist, Rachael M; McNeil, Scott E

    2014-10-01

    Proteins bound to nanoparticle surfaces are known to affect particle clearance by influencing immune cell uptake and distribution to the organs of the mononuclear phagocytic system. The composition of the protein corona has been described for several types of nanomaterials, but the role of the corona in nanoparticle biocompatibility is not well established. In this study we investigate the role of nanoparticle surface properties (PEGylation) and incubation times on the protein coronas of colloidal gold nanoparticles. While neither incubation time nor PEG molecular weight affected the specific proteins in the protein corona, the total amount of protein binding was governed by the molecular weight of PEG coating. Furthermore, the composition of the protein corona did not correlate with nanoparticle hematocompatibility. Specialized hematological tests should be used to deduce nanoparticle hematotoxicity. From the clinical editor: It is overall unclear how the protein corona associated with colloidal gold nanoparticles may influence hematotoxicity. This study warns that PEGylation itself may be insufficient, because composition of the protein corona does not directly correlate with nanoparticle hematocompatibility. The authors suggest that specialized hematological tests must be used to deduce nanoparticle hematotoxicity.

  9. 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.

  10. VORFFIP-Driven Dock: V-D2OCK, a Fast and Accurate Protein Docking Strategy

    Science.gov (United States)

    Segura, Joan; Marín-López, Manuel Alejandro; Jones, Pamela F.; Oliva, Baldo; Fernandez-Fuentes, Narcis

    2015-01-01

    The experimental determination of the structure of protein complexes cannot keep pace with the generation of interactomic data, hence resulting in an ever-expanding gap. As the structural details of protein complexes are central to a full understanding of the function and dynamics of the cell machinery, alternative strategies are needed to circumvent the bottleneck in structure determination. Computational protein docking is a valid and valuable approach to model the structure of protein complexes. In this work, we describe a novel computational strategy to predict the structure of protein complexes based on data-driven docking: VORFFIP-driven dock (V-D2OCK). This new approach makes use of our newly described method to predict functional sites in protein structures, VORFFIP, to define the region to be sampled during docking and structural clustering to reduce the number of models to be examined by users. V-D2OCK has been benchmarked using a validated and diverse set of protein complexes and compared to a state-of-art docking method. The speed and accuracy compared to contemporary tools justifies the potential use of VD2OCK for high-throughput, genome-wide, protein docking. Finally, we have developed a web interface that allows users to browser and visualize V-D2OCK predictions from the convenience of their web-browsers. PMID:25763838

  11. VORFFIP-driven dock: V-D2OCK, a fast and accurate protein docking strategy.

    Directory of Open Access Journals (Sweden)

    Joan Segura

    Full Text Available The experimental determination of the structure of protein complexes cannot keep pace with the generation of interactomic data, hence resulting in an ever-expanding gap. As the structural details of protein complexes are central to a full understanding of the function and dynamics of the cell machinery, alternative strategies are needed to circumvent the bottleneck in structure determination. Computational protein docking is a valid and valuable approach to model the structure of protein complexes. In this work, we describe a novel computational strategy to predict the structure of protein complexes based on data-driven docking: VORFFIP-driven dock (V-D2OCK. This new approach makes use of our newly described method to predict functional sites in protein structures, VORFFIP, to define the region to be sampled during docking and structural clustering to reduce the number of models to be examined by users. V-D2OCK has been benchmarked using a validated and diverse set of protein complexes and compared to a state-of-art docking method. The speed and accuracy compared to contemporary tools justifies the potential use of VD2OCK for high-throughput, genome-wide, protein docking. Finally, we have developed a web interface that allows users to browser and visualize V-D2OCK predictions from the convenience of their web-browsers.

  12. Identification of chikungunya virus interacting proteins in mammalian cells

    Indian Academy of Sciences (India)

    Mandar S Paingankar; Vidya A Arankalle

    2014-06-01

    Identification and characterization of virus host interactions is an essential step for the development of novel antiviral strategies. Very few studies have been targeted towards identification of chikungunya virus (CHIKV) interacting host proteins. In current study, virus overlay protein binding assay (VOPBA) and matrix-assisted laser desorption/ionization time of flight analysis (MALDI TOF/TOF) were employed for the identification of CHIKV binding proteins in mammalian cells. HSP70 and actin were identified as virus binding proteins in HEK-293T and Vero-E6 cells, whereas STAT-2 was identified as an additional protein in Vero-E6 cells. Pre-incubation with anti-HSP70 antibody and miRNA silencing of HSP70 significantly reduced the CHIKV production in HEK-293T and Vero-E6 cells at early time points. These results suggest that CHIKV exploits the housekeeping molecules such as actin, HSP70 and STAT-2 to establish infection in the mammalian cells.

  13. Bioinformatics pipeline for functional identification and characterization of proteins

    Science.gov (United States)

    Skarzyńska, Agnieszka; Pawełkowicz, Magdalena; Krzywkowski, Tomasz; Świerkula, Katarzyna; PlÄ der, Wojciech; Przybecki, Zbigniew

    2015-09-01

    The new sequencing methods, called Next Generation Sequencing gives an opportunity to possess a vast amount of data in short time. This data requires structural and functional annotation. Functional identification and characterization of predicted proteins could be done by in silico approches, thanks to a numerous computational tools available nowadays. However, there is a need to confirm the results of proteins function prediction using different programs and comparing the results or confirm experimentally. Here we present a bioinformatics pipeline for structural and functional annotation of proteins.

  14. Protein identification using nano liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Negroni, Luc

    2007-01-01

    Tandem mass spectrometry is an efficient technique for the identification of peptides on the basis of their fragmentation pattern (MS/MS scan). It can generate individual spectra for each peptide, thereby creating a powerful tool for protein identification on the basis of peptide characterization. This important advance in automatic data acquisition has allowed an efficient association between liquid chromatography and tandem mass spectrometry, and the use of nanocolumns and nanoelectrospray ionization has dramatically increased the efficiency of this method. Now large sets of peptides can be identified at a femtomole level. At the end of the process, batch processing of the MS/MS spectra produces peptide lists that identify purified proteins or protein mixtures with high confidence.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Accurate proteome-wide protein quantification from high-resolution 15N mass spectra.

    Science.gov (United States)

    Khan, Zia; Amini, Sasan; Bloom, Joshua S; Ruse, Cristian; Caudy, Amy A; Kruglyak, Leonid; Singh, Mona; Perlman, David H; Tavazoie, Saeed

    2011-12-19

    In quantitative mass spectrometry-based proteomics, the metabolic incorporation of a single source of 15N-labeled nitrogen has many advantages over using stable isotope-labeled amino acids. However, the lack of a robust computational framework for analyzing the resulting spectra has impeded wide use of this approach. We have addressed this challenge by introducing a new computational methodology for analyzing 15N spectra in which quantification is integrated with identification. Application of this method to an Escherichia coli growth transition reveals significant improvement in quantification accuracy over previous methods.

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

    Science.gov (United States)

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background 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. Method 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. Results 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). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  1. 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.

  2. 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

  3. Accurate retention time determination of co-eluting proteins in analytical chromatography by means of spectral data.

    Science.gov (United States)

    Dismer, Florian; Hansen, Sigrid; Oelmeier, Stefan Alexander; Hubbuch, Jürgen

    2013-03-01

    Chromatography is the method of choice for the separation of proteins, at both analytical and preparative scale. Orthogonal purification strategies for industrial use can easily be implemented by combining different modes of adsorption. Nevertheless, with flexibility comes the freedom of choice and optimal conditions for consecutive steps need to be identified in a robust and reproducible fashion. One way to address this issue is the use of mathematical models that allow for an in silico process optimization. Although this has been shown to work, model parameter estimation for complex feedstocks becomes the bottleneck in process development. An integral part of parameter assessment is the accurate measurement of retention times in a series of isocratic or gradient elution experiments. As high-resolution analytics that can differentiate between proteins are often not readily available, pure protein is mandatory for parameter determination. In this work, we present an approach that has the potential to solve this problem. Based on the uniqueness of UV absorption spectra of proteins, we were able to accurately measure retention times in systems of up to four co-eluting compounds. The presented approach is calibration-free, meaning that prior knowledge of pure component absorption spectra is not required. Actually, pure protein spectra can be determined from co-eluting proteins as part of the methodology. The approach was tested for size-exclusion chromatograms of 38 mixtures of co-eluting proteins. Retention times were determined with an average error of 0.6 s (1.6% of average peak width), approximated and measured pure component spectra showed an average coefficient of correlation of 0.992.

  4. Physicochemical property distributions for accurate and rapid pairwise protein homology detection

    Directory of Open Access Journals (Sweden)

    Oehmen Christopher S

    2010-03-01

    Full Text Available Abstract Background The challenge of remote homology detection is that many evolutionarily related sequences have very little similarity at the amino acid level. Kernel-based discriminative methods, such as support vector machines (SVMs, that use vector representations of sequences derived from sequence properties have been shown to have superior accuracy when compared to traditional approaches for the task of remote homology detection. Results We introduce a new method for feature vector representation based on the physicochemical properties of the primary protein sequence. A distribution of physicochemical property scores are assembled from 4-mers of the sequence and normalized based on the null distribution of the property over all possible 4-mers. With this approach there is little computational cost associated with the transformation of the protein into feature space, and overall performance in terms of remote homology detection is comparable with current state-of-the-art methods. We demonstrate that the features can be used for the task of pairwise remote homology detection with improved accuracy versus sequence-based methods such as BLAST and other feature-based methods of similar computational cost. Conclusions A protein feature method based on physicochemical properties is a viable approach for extracting features in a computationally inexpensive manner while retaining the sensitivity of SVM protein homology detection. Furthermore, identifying features that can be used for generic pairwise homology detection in lieu of family-based homology detection is important for applications such as large database searches and comparative genomics.

  5. Identification of bacteriophage virion proteins by the ANOVA feature selection and analysis.

    Science.gov (United States)

    Ding, Hui; Feng, Peng-Mian; Chen, Wei; Lin, Hao

    2014-08-01

    The bacteriophage virion proteins play extremely important roles in the fate of host bacterial cells. Accurate identification of bacteriophage virion proteins is very important for understanding their functions and clarifying the lysis mechanism of bacterial cells. In this study, a new sequence-based method was developed to identify phage virion proteins. In the new method, the protein sequences were initially formulated by the g-gap dipeptide compositions. Subsequently, the analysis of variance (ANOVA) with incremental feature selection (IFS) was used to search for the optimal feature set. It was observed that, in jackknife cross-validation, the optimal feature set including 160 optimized features can produce the maximum accuracy of 85.02%. By performing feature analysis, we found that the correlation between two amino acids with one gap was more important than other correlations for phage virion protein prediction and that some of the 1-gap dipeptides were important and mainly contributed to the virion protein prediction. This analysis will provide novel insights into the function of phage virion proteins. On the basis of the proposed method, an online web-server, PVPred, was established and can be freely accessed from the website (http://lin.uestc.edu.cn/server/PVPred). We believe that the PVPred will become a powerful tool to study phage virion proteins and to guide the related experimental validations.

  6. HMMCAS: a web tool for the identification and domain annotations of Cas proteins.

    Science.gov (United States)

    Chai, Guoshi; Yu, Min; Jiang, Lixu; Duan, Yaocong; Huang, Jian

    2017-02-07

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immune systems are discovered in many bacteria and most archaea. These systems are encoded by cas (CRISPR-associated) operons that have an extremely diverse architecture. The most crucial step in the depiction of cas operons composition is the identification of cas genes or Cas proteins. With the continuous increase of the newly sequenced archaeal and bacterial genomes, the recognition of new Cas proteins is becoming possible, which not only provides candidates for novel genome editing tools but also helps to understand the prokaryotic immune system better. Here we describe HMMCAS, a web service for the detection of CRISPR-associated structural and functional domains in protein sequences. HMMCAS uses hmmscan similarity search algorithm in HMMER3.1 to provide a fast, interactive service based on a comprehensive collection of hidden Markov models of Cas protein family. It can accurately identify the Cas proteins including those fusion proteins, for example the Cas1-Cas4 fusion protein in Candidatus Chloracidobacterium thermophilum B (Cab. thermophilum B). HMMCAS can also find putative cas operon and determine which type it belongs to. HMMCAS is freely available at http://i.uestc.edu.cn/hmmcas.

  7. Using protein markers of embryo and seed storage proteins in identification of four pistachio cultivars

    Directory of Open Access Journals (Sweden)

    Ali Akbar Ehsanpour

    2010-12-01

    Full Text Available Identification of protein marker for Pistachio cultivars, as a valuable source of food is important. In this study, the protein patterns of embryo from four pistachio cultivars including Akbari, Ahmad Aghaei, Fandoghi and Kaleghouchi were analyzed using SDS-PAGE. The presence of protein bands about 90 and 45 killo dalton (kd in protein pattern of embryonic axes in cultivars Kaleghouchi and Akbari respectively and the absence of protein bands with approximate molecular weight 30 and 20 kd in protein pattern of cotyledons in cultivars Kaleghouchi and Akbari respectively can be used as protein markers for these pistachio cultivars. On the other hand, the maximum expression level of bands 45 kd in protein pattern of cotyledons could be indicative of a protein marker for cultivar Ahmad Aghaei.

  8. Identification of protein superfamily from structure- based sequence motif

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The structure-based sequence motif of the distant proteins in evolution, protein tyrosine phosphatases (PTP) Ⅰ and Ⅱ superfamilies, as an example, has been defined by the structural comparison, structure-based sequence alignment and analyses on substitution patterns of residues in common sequence conserved regions. And the phosphatases Ⅰ and Ⅱ can be correctly identified together by the structure-based PTP sequence motif from SWISS-PROT and TrEBML databases. The results show that the correct rates of identification are over 98%. This is the first time to identify PTP Ⅰ and Ⅱ together by this motif.

  9. Combining Evolutionary Information and an Iterative Sampling Strategy for Accurate Protein Structure Prediction.

    Directory of Open Access Journals (Sweden)

    Tatjana Braun

    2015-12-01

    Full Text Available Recent work has shown that the accuracy of ab initio structure prediction can be significantly improved by integrating evolutionary information in form of intra-protein residue-residue contacts. Following this seminal result, much effort is put into the improvement of contact predictions. However, there is also a substantial need to develop structure prediction protocols tailored to the type of restraints gained by contact predictions. Here, we present a structure prediction protocol that combines evolutionary information with the resolution-adapted structural recombination approach of Rosetta, called RASREC. Compared to the classic Rosetta ab initio protocol, RASREC achieves improved sampling, better convergence and higher robustness against incorrect distance restraints, making it the ideal sampling strategy for the stated problem. To demonstrate the accuracy of our protocol, we tested the approach on a diverse set of 28 globular proteins. Our method is able to converge for 26 out of the 28 targets and improves the average TM-score of the entire benchmark set from 0.55 to 0.72 when compared to the top ranked models obtained by the EVFold web server using identical contact predictions. Using a smaller benchmark, we furthermore show that the prediction accuracy of our method is only slightly reduced when the contact prediction accuracy is comparatively low. This observation is of special interest for protein sequences that only have a limited number of homologs.

  10. 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

  11. 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.

  12. Identification of AOSC-binding proteins in neurons

    Institute of Scientific and Technical Information of China (English)

    LIU Ming; NIE Qin; XIN Xianliang; GENG Meiyu

    2008-01-01

    Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer's Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

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

    OpenAIRE

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

    2016-01-01

    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 ...

  14. PETs: A Stable and Accurate Predictor of Protein-Protein Interacting Sites Based on Extremely-Randomized Trees.

    Science.gov (United States)

    Xia, Bin; Zhang, Hong; Li, Qianmu; Li, Tao

    2015-12-01

    Protein-protein interaction (PPI) plays crucial roles in the performance of various biological processes. A variety of methods are dedicated to identify whether proteins have interaction residues, but it is often more crucial to recognize each amino acid. In practical applications, the stability of a prediction model is as important as its accuracy. However, random sampling, which is widely used in previous prediction models, often brings large difference between each training model. In this paper, a Predictor of protein-protein interaction sites based on Extremely-randomized Trees (PETs) is proposed to improve the prediction accuracy while maintaining the prediction stability. In PETs, a cluster-based sampling strategy is proposed to ensure the model stability: first, the training dataset is divided into subsets using specific features; second, the subsets are clustered using K-means; and finally the samples are selected from each cluster. Using the proposed sampling strategy, samples which have different types of significant features could be selected independently from different clusters. The evaluation shows that PETs is able to achieve better accuracy while maintaining a good stability. The source code and toolkit are available at https://github.com/BinXia/PETs.

  15. 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.

  16. Identification of protein interacting partners using tandem affinity purification.

    Science.gov (United States)

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-02-25

    A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous

  17. HEASARC Astronomical Archive: GLIESE2MAS - Gliese Catalog Stars with Accurate Coordinates and 2MASS Cross-Identifications

    Data.gov (United States)

    National Aeronautics and Space Administration — This table contains precise epoch 2000 coordinates and cross-identifications to sources in the 2MASS Point Source Catalog for nearly all stars in the Gliese,...

  18. 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/.

  19. Amplified protein detection and identification through DNA-conjugated M13 bacteriophage.

    Science.gov (United States)

    Lee, Ju Hun; Domaille, Dylan W; Cha, Jennifer N

    2012-06-26

    Sensitive protein detection and accurate identification continues to be in great demand for disease screening in clinical and laboratory settings. For these diagnostics to be of clinical value, it is necessary to develop sensors that have high sensitivity but favorable cost-to-benefit ratios. However, many of these sensing platforms are thermally unstable or require significant materials synthesis, engineering, or fabrication. Recently, we demonstrated that naturally occurring M13 bacteriophage can serve as biological scaffolds for engineering protein diagnostics. These viruses have five copies of the pIII protein, which can bind specifically to target antigens, and thousands of pVIII coat proteins, which can be genetically or chemically modified to react with signal-producing materials, such as plasmon-shifting gold nanoparticles (Au NPs). In this report, we show that DNA-conjugated M13 bacteriophage can act as inexpensive protein sensors that can rapidly induce a color change in the presence of a target protein yet also offer the ability to identify the detected antigen in a separate step. Many copies of a specific DNA oligonucleotide were appended to each virus to create phage-DNA conjugates that can hybridize with DNA-conjugated gold nanoparticles. In the case of a colorimetric positive result, the identity of the antigen can also be easily determined by using a DNA microarray. This saves precious resources by establishing a rapid, quantitative method to first screen for the presence of antigen followed by a highly specific typing assay if necessary.

  20. Identification of Uropathogenic Escherichia coli Surface Proteins by Shotgun Proteomics

    Science.gov (United States)

    Walters, Matthew S.; Mobley, Harry L.T.

    2009-01-01

    Uropathogenic Escherichia coli (UPEC) cause the majority of uncomplicated urinary tract infections in humans. In the process of identifying candidate antigens for a vaccine, two methods for the identification of the UPEC surface proteome during growth in human urine were investigated. The first approach utilized a protease to ‘shave’ surface-exposed peptides from the bacterial cell surface and identify them by mass spectrometry. Although this approach has been successfully applied to a Gram-positive pathogen, the adaptation to Gram-negative UPEC resulted in cytoplasmic protein contamination. In a more direct approach, whole-cell bacteria were labeled with a biotin tag to indicate surface-exposed peptides and two-dimensional liquid chromatography-tandem mass spectrometry (2-DLC-MS/MS) was used to identify proteins isolated from the outer membrane. This method discovered 25 predicted outer membrane proteins expressed by UPEC while growing in human urine. Nine of the 25 predicted outer membrane proteins were part of iron transport systems or putative iron-regulated virulence proteins, indicating the importance of iron acquisition during growth in urine. One of the iron transport proteins identified, Hma, appears to be a promising vaccine candidate is being further investigated. The method described here presents a system to rapidly identify the outer membrane proteome of bacteria, which may prove valuable in vaccine development. PMID:19426766

  1. Identification of 24h Ixodes scapularis immunogenic tick saliva proteins.

    Science.gov (United States)

    Lewis, Lauren A; Radulović, Željko M; Kim, Tae K; Porter, Lindsay M; Mulenga, Albert

    2015-04-01

    Ixodes scapularis is arguably the most medically important tick species in the United States. This tick transmits 5 of the 14 human tick-borne disease (TBD) agents in the USA: Borrelia burgdorferi, Anaplasma phagocytophilum, B. miyamotoi, Babesia microti, and Powassan virus disease. Except for the Powassan virus disease, I. scapularis-vectored TBD agents require more than 24h post attachment to be transmitted. This study describes identification of 24h immunogenic I. scapularis tick saliva proteins, which could provide opportunities to develop strategies to stop tick feeding before transmission of the majority of pathogens. A 24h fed female I. scapularis phage display cDNA expression library was biopanned using rabbit antibodies to 24h fed I. scapularis female tick saliva proteins, subjected to next generation sequencing, de novo assembly, and bioinformatic analyses. A total of 182 contigs were assembled, of which ∼19% (35/182) are novel and did not show identity to any known proteins in GenBank. The remaining ∼81% (147/182) of contigs were provisionally identified based on matches in GenBank including ∼18% (27/147) that matched protein sequences previously annotated as hypothetical and putative tick saliva proteins. Others include proteases and protease inhibitors (∼3%, 5/147), transporters and/or ligand binding proteins (∼6%, 9/147), immunogenic tick saliva housekeeping enzyme-like (17%, 25/147), ribosomal protein-like (∼31%, 46/147), and those classified as miscellaneous (∼24%, 35/147). Notable among the miscellaneous class include antimicrobial peptides (microplusin and ricinusin), myosin-like proteins that have been previously found in tick saliva, and heat shock tick saliva protein. Data in this study provides the foundation for in-depth analysis of I. scapularis feeding during the first 24h, before the majority of TBD agents can be transmitted.

  2. Identification and phylogenetic analysis of Dictyostelium discoideum kinesin proteins

    Directory of Open Access Journals (Sweden)

    Glöckner Gernot

    2003-11-01

    Full Text Available Abstract Background Kinesins constitute a large superfamily of motor proteins in eukaryotic cells. They perform diverse tasks such as vesicle and organelle transport and chromosomal segregation in a microtubule- and ATP-dependent manner. In recent years, the genomes of a number of eukaryotic organisms have been completely sequenced. Subsequent studies revealed and classified the full set of members of the kinesin superfamily expressed by these organisms. For Dictyostelium discoideum, only five kinesin superfamily proteins (Kif's have already been reported. Results Here, we report the identification of thirteen kinesin genes exploiting the information from the raw shotgun reads of the Dictyostelium discoideum genome project. A phylogenetic tree of 390 kinesin motor domain sequences was built, grouping the Dictyostelium kinesins into nine subfamilies. According to known cellular functions or strong homologies to kinesins of other organisms, four of the Dictyostelium kinesins are involved in organelle transport, six are implicated in cell division processes, two are predicted to perform multiple functions, and one kinesin may be the founder of a new subclass. Conclusion This analysis of the Dictyostelium genome led to the identification of eight new kinesin motor proteins. According to an exhaustive phylogenetic comparison, Dictyostelium contains the same subset of kinesins that higher eukaryotes need to perform mitosis. Some of the kinesins are implicated in intracellular traffic and a small number have unpredictable functions.

  3. Rapid and accurate identification of Mycobacterium tuberculosis complex and common non-tuberculous mycobacteria by multiplex real-time PCR targeting different housekeeping genes.

    Science.gov (United States)

    Nasr Esfahani, Bahram; Rezaei Yazdi, Hadi; Moghim, Sharareh; Ghasemian Safaei, Hajieh; Zarkesh Esfahani, Hamid

    2012-11-01

    Rapid and accurate identification of mycobacteria isolates from primary culture is important due to timely and appropriate antibiotic therapy. Conventional methods for identification of Mycobacterium species based on biochemical tests needs several weeks and may remain inconclusive. In this study, a novel multiplex real-time PCR was developed for rapid identification of Mycobacterium genus, Mycobacterium tuberculosis complex (MTC) and the most common non-tuberculosis mycobacteria species including M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and the M. gordonae in three reaction tubes but under same PCR condition. Genetic targets for primer designing included the 16S rDNA gene, the dnaJ gene, the gyrB gene and internal transcribed spacer (ITS). Multiplex real-time PCR was setup with reference Mycobacterium strains and was subsequently tested with 66 clinical isolates. Results of multiplex real-time PCR were analyzed with melting curves and melting temperature (T (m)) of Mycobacterium genus, MTC, and each of non-tuberculosis Mycobacterium species were determined. Multiplex real-time PCR results were compared with amplification and sequencing of 16S-23S rDNA ITS for identification of Mycobacterium species. Sensitivity and specificity of designed primers were each 100 % for MTC, M. abscessus, M. fortuitum, M. avium complex, M. kansasii, and M. gordonae. Sensitivity and specificity of designed primer for genus Mycobacterium was 96 and 100 %, respectively. According to the obtained results, we conclude that this multiplex real-time PCR with melting curve analysis and these novel primers can be used for rapid and accurate identification of genus Mycobacterium, MTC, and the most common non-tuberculosis Mycobacterium species.

  4. Identification of anabolic steroids and derivatives using bioassay-guided fractionation,UHPLC/TOFMS analysis and accurate mass database searching

    NARCIS (Netherlands)

    Peters, R.J.B.; Rijk, J.C.W.; Bovee, T.F.H.; Nijrolder, A.W.J.M.; Lommen, A.; Nielen, M.W.F.

    2010-01-01

    Biological tests can be used to screen samples for large groups of compounds having a particular effect, but it is often difficult to identify a specific compound when a positive effect is observed. The identification of an unknown compound is a challenge for analytical chemistry in environmental an

  5. 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.

  6. SCPRED: Accurate prediction of protein structural class for sequences of twilight-zone similarity with predicting sequences

    Directory of Open Access Journals (Sweden)

    Chen Ke

    2008-05-01

    Full Text Available Abstract Background Protein structure prediction methods provide accurate results when a homologous protein is predicted, while poorer predictions are obtained in the absence of homologous templates. However, some protein chains that share twilight-zone pairwise identity can form similar folds and thus determining structural similarity without the sequence similarity would be desirable for the structure prediction. The folding type of a protein or its domain is defined as the structural class. Current structural class prediction methods that predict the four structural classes defined in SCOP provide up to 63% accuracy for the datasets in which sequence identity of any pair of sequences belongs to the twilight-zone. We propose SCPRED method that improves prediction accuracy for sequences that share twilight-zone pairwise similarity with sequences used for the prediction. Results SCPRED uses a support vector machine classifier that takes several custom-designed features as its input to predict the structural classes. Based on extensive design that considers over 2300 index-, composition- and physicochemical properties-based features along with features based on the predicted secondary structure and content, the classifier's input includes 8 features based on information extracted from the secondary structure predicted with PSI-PRED and one feature computed from the sequence. Tests performed with datasets of 1673 protein chains, in which any pair of sequences shares twilight-zone similarity, show that SCPRED obtains 80.3% accuracy when predicting the four SCOP-defined structural classes, which is superior when compared with over a dozen recent competing methods that are based on support vector machine, logistic regression, and ensemble of classifiers predictors. Conclusion The SCPRED can accurately find similar structures for sequences that share low identity with sequence used for the prediction. The high predictive accuracy achieved by SCPRED is

  7. Identification of a novel Rev-interacting cellular protein

    Directory of Open Access Journals (Sweden)

    Werner Thomas

    2005-04-01

    Full Text Available Abstract Background Human cell types respond differently to infection by human immunodeficiency virus (HIV. Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies. Results Yeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1. 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev. Conclusion These results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors.

  8. 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...... with spectra analyses using the SARAMIS database. Additionally, first results on subtyping of SEZ indicated that a more refined discrimination, for example for epidemiological surveys, may be possible...

  9. 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.

  10. Identification of proteins binding coding and non-coding human RNAs using protein microarrays

    Directory of Open Access Journals (Sweden)

    Siprashvili Zurab

    2012-11-01

    Full Text Available Abstract Background The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays. Results We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability. Conclusions Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions.

  11. Identification of divergent protein domains by combining HMM-HMM comparisons and co-occurrence detection.

    Directory of Open Access Journals (Sweden)

    Amel Ghouila

    Full Text Available Identification of protein domains is a key step for understanding protein function. Hidden Markov Models (HMMs have proved to be a powerful tool for this task. The Pfam database notably provides a large collection of HMMs which are widely used for the annotation of proteins in sequenced organisms. This is done via sequence/HMM comparisons. However, this approach may lack sensitivity when searching for domains in divergent species. Recently, methods for HMM/HMM comparisons have been proposed and proved to be more sensitive than sequence/HMM approaches in certain cases. However, these approaches are usually not used for protein domain discovery at a genome scale, and the benefit that could be expected from their utilization for this problem has not been investigated. Using proteins of P. falciparum and L. major as examples, we investigate the extent to which HMM/HMM comparisons can identify new domain occurrences not already identified by sequence/HMM approaches. We show that although HMM/HMM comparisons are much more sensitive than sequence/HMM comparisons, they are not sufficiently accurate to be used as a standalone complement of sequence/HMM approaches at the genome scale. Hence, we propose to use domain co-occurrence--the general domain tendency to preferentially appear along with some favorite domains in the proteins--to improve the accuracy of the approach. We show that the combination of HMM/HMM comparisons and co-occurrence domain detection boosts protein annotations. At an estimated False Discovery Rate of 5%, it revealed 901 and 1098 new domains in Plasmodium and Leishmania proteins, respectively. Manual inspection of part of these predictions shows that it contains several domain families that were missing in the two organisms. All new domain occurrences have been integrated in the EuPathDomains database, along with the GO annotations that can be deduced.

  12. Identification of divergent protein domains by combining HMM-HMM comparisons and co-occurrence detection.

    Science.gov (United States)

    Ghouila, Amel; Florent, Isabelle; Guerfali, Fatma Zahra; Terrapon, Nicolas; Laouini, Dhafer; Yahia, Sadok Ben; Gascuel, Olivier; Bréhélin, Laurent

    2014-01-01

    Identification of protein domains is a key step for understanding protein function. Hidden Markov Models (HMMs) have proved to be a powerful tool for this task. The Pfam database notably provides a large collection of HMMs which are widely used for the annotation of proteins in sequenced organisms. This is done via sequence/HMM comparisons. However, this approach may lack sensitivity when searching for domains in divergent species. Recently, methods for HMM/HMM comparisons have been proposed and proved to be more sensitive than sequence/HMM approaches in certain cases. However, these approaches are usually not used for protein domain discovery at a genome scale, and the benefit that could be expected from their utilization for this problem has not been investigated. Using proteins of P. falciparum and L. major as examples, we investigate the extent to which HMM/HMM comparisons can identify new domain occurrences not already identified by sequence/HMM approaches. We show that although HMM/HMM comparisons are much more sensitive than sequence/HMM comparisons, they are not sufficiently accurate to be used as a standalone complement of sequence/HMM approaches at the genome scale. Hence, we propose to use domain co-occurrence--the general domain tendency to preferentially appear along with some favorite domains in the proteins--to improve the accuracy of the approach. We show that the combination of HMM/HMM comparisons and co-occurrence domain detection boosts protein annotations. At an estimated False Discovery Rate of 5%, it revealed 901 and 1098 new domains in Plasmodium and Leishmania proteins, respectively. Manual inspection of part of these predictions shows that it contains several domain families that were missing in the two organisms. All new domain occurrences have been integrated in the EuPathDomains database, along with the GO annotations that can be deduced.

  13. 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).

  14. Simple and accurate determination of global tau(R) in proteins using (13)C or (15)N relaxation data.

    Science.gov (United States)

    Mispelter, J; Izadi-Pruneyre, N; Quiniou, E; Adjadj, E

    2000-03-01

    In the study of protein dynamics by (13)C or (15)N relaxation measurements different models from the Lipari-Szabo formalism are used in order to determine the motion parameters. The global rotational correlation time tau(R) of the molecule must be estimated prior to the analysis. In this Communication, the authors propose a new approach in determining an accurate value for tau(R) in order to realize the best fit of R(2) for the whole sequence of the protein, regardless of the different type of motions atoms may experience. The method first determines the highly structured regions of the sequence. For each corresponding site, the Lipari-Szabo parameters are calculated for R(1) and NOE, using an arbitrary value for tau(R). The chi(2) for R(2), summed over the selected sites, shows a clear minimum, as a function of tau(R). This minimum is used to better estimate a proper value for tau(R).

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

    Science.gov (United States)

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

    2016-10-01

    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 inflammatory disease. In this study, we demonstrated the preparation of DNA aptamer-conjugated peripheral blood mononuclear cells (Apt-PBMCs) that specifically capture human CRP. Live PBMCs functionalized with aptamers could detect different levels of human CRP by producing immune complexes with reporter antibody. The binding behavior of Apt-PBMCs toward highly concentrated CRP sites was also investigated. The immune responses of Apt-PBMCs were evaluated by measuring TNF-alpha secretion after stimulating the PBMCs with lipopolysaccharides. In summary, engineered Apt-PBMCs have potential applications as live cell based biosensors and for in vitro tracing of CRP secretion sites.

  16. Streptococcus dysgalactiae subsp. equisimilis Isolated From Infections in Dogs and Humans: Are Current Subspecies Identification Criteria accurate?

    Science.gov (United States)

    Ciszewski, Marcin; Zegarski, Kamil; Szewczyk, Eligia M

    2016-11-01

    Streptococcus dysgalactiae is a pyogenic species pathogenic both for humans and animals. Until recently, it has been considered an exclusive animal pathogen causing infections in wild as well as domestic animals. Currently, human infections are being reported with increasing frequency, and their clinical picture is often similar to the ones caused by Streptococcus pyogenes. Due to the fact that S. dysgalactiae is a heterogeneous species, it was divided into two subspecies: S. dysgalactiae subsp. equisimilis (SDSE) and S. dysgalactiae subsp. dysgalactiae (SDSD). The first differentiation criterion, described in 1996, was based on strain isolation source. Currently applied criteria, published in 1998, are based on hemolysis type and Lancefield group classification. In this study, we compared subspecies identification results for 36 strains isolated from clinical cases both in humans and animals. Species differentiation was based on two previously described criteria as well as MALDI-TOF and genetic analyses: RISA and 16S rRNA genes sequencing. Antimicrobial susceptibility profiles were also determined according to CLSI guidelines. The results presented in our study suggest that the subspecies differentiation criteria previously described in the above two literature positions seem to be inaccurate in analyzed group of strains, the hemolysis type on blood agar, and Lancefield classification should not be here longer considered as criteria in subspecies identification. The antimicrobial susceptibility tests indicate emerging of multiresistant human SDSE strains resistant also to vancomycin, linezolid and tigecycline, which might pose a substantial problem in treatment.

  17. Automatic Identification of Antibodies in the Protein Data Bank

    Institute of Scientific and Technical Information of China (English)

    LI Xun; WANG Renxiao

    2009-01-01

    An automatic method has been developed for identifying antibody entries in the protein data bank (PDB). Our method, called KIAb (Keyword-based Identification of Antibodies), parses PDB-format files to search for particular keywords relevant to antibodies, and makes judgment accordingly. Our method identified 780 entries as antibodies on the entire PDB. Among them, 767 entries were confirmed by manual inspection, indicating a high success rate of 98.3%. Our method recovered basically all of the entries compiled in the Summary of Antibody Crystal Structures (SACS) database. It also identified a number of entries missed by SACS. Our method thus provides a more com-plete mining of antibody entries in PDB with a very low false positive rate.

  18. Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems.

    Directory of Open Access Journals (Sweden)

    Ram Samudrala

    2009-04-01

    Full Text Available The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates--effector proteins--are not. We have used a novel computational approach to confidently identify new secreted effectors by integrating protein sequence-based features, including evolutionary measures such as the pattern of homologs in a range of other organisms, G+C content, amino acid composition, and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from the plant pathogen Pseudomonas syringae and validated on a set of effectors from the animal pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium after eliminating effectors with detectable sequence similarity. We show that this approach can predict known secreted effectors with high specificity and sensitivity. Furthermore, by considering a large set of effectors from multiple organisms, we computationally identify a common putative secretion signal in the N-terminal 20 residues of secreted effectors. This signal can be used to discriminate 46 out of 68 total known effectors from both organisms, suggesting that it is a real, shared signal applicable to many type III secreted effectors. We use the method to make novel predictions of secreted effectors in S. Typhimurium, some of which have been experimentally validated. We also apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis, identifying the majority of known secreted proteins in addition to providing a number of novel predictions. This approach provides a new way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal.

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

    Science.gov (United States)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

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

  20. Identification of hot-spot residues in protein-protein interactions by computational docking

    Directory of Open Access Journals (Sweden)

    Fernández-Recio Juan

    2008-10-01

    Full Text Available Abstract Background The study of protein-protein interactions is becoming increasingly important for biotechnological and therapeutic reasons. We can define two major areas therein: the structural prediction of protein-protein binding mode, and the identification of the relevant residues for the interaction (so called 'hot-spots'. These hot-spot residues have high interest since they are considered one of the possible ways of disrupting a protein-protein interaction. Unfortunately, large-scale experimental measurement of residue contribution to the binding energy, based on alanine-scanning experiments, is costly and thus data is fairly limited. Recent computational approaches for hot-spot prediction have been reported, but they usually require the structure of the complex. Results We have applied here normalized interface propensity (NIP values derived from rigid-body docking with electrostatics and desolvation scoring for the prediction of interaction hot-spots. This parameter identifies hot-spot residues on interacting proteins with predictive rates that are comparable to other existing methods (up to 80% positive predictive value, and the advantage of not requiring any prior structural knowledge of the complex. Conclusion The NIP values derived from rigid-body docking can reliably identify a number of hot-spot residues whose contribution to the interaction arises from electrostatics and desolvation effects. Our method can propose residues to guide experiments in complexes of biological or therapeutic interest, even in cases with no available 3D structure of the complex.

  1. Improving protein identification sensitivity by combining MS and MS/MS information for shotgun proteomics using LTQ-Orbitrap high mass accuracy data.

    Science.gov (United States)

    Lu, Bingwen; Motoyama, Akira; Ruse, Cristian; Venable, John; Yates, John R

    2008-03-15

    We investigated and compared three approaches for shotgun protein identification by combining MS and MS/MS information using LTQ-Orbitrap high mass accuracy data. In the first approach, we employed a unique mass identifier method where MS peaks matched to peptides predicted from proteins identified from an MS/MS database search are first subtracted before using the MS peaks as unique mass identifiers for protein identification. In the second method, we used an accurate mass and time tag method by building a potential mass and retention time database from previous MudPIT analyses. For the third method, we used a peptide mass fingerprinting-like approach in combination with a randomized database for protein identification. We show that we can improve protein identification sensitivity for low-abundance proteins by combining MS and MS/MS information. Furthermore, "one-hit wonders" from MS/MS database searching can be further substantiated by MS information and the approach improves the identification of low-abundance proteins. The advantages and disadvantages for the three approaches are then discussed.

  2. Accurate high-throughput identification of parallel G-quadruplex topology by a new tetraaryl-substituted imidazole.

    Science.gov (United States)

    Hu, Ming-Hao; Chen, Shuo-Bin; Wang, Yu-Qing; Zeng, You-Mei; Ou, Tian-Miao; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2016-09-15

    G-quadruplex nucleic acids are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. These structures exhibit extensive structural polymorphism and play a pivotal role in the control of a variety of cellular processes. To date, diverse approaches for high-throughput identification of G-quadruplex structures have been successfully developed, but high-throughput methods for further characterization of their topologies are still lacking. In this study, we report a new tetra-arylimidazole probe psIZCM-1, which was found to display significant and distinctive changes in both the absorption and the fluorescence spectra in the presence of parallel G-quadruplexes but show insignificant changes upon interactions with anti-parallel G-quadruplexes or other non-quadruplex oligonucleotides. In view of this dual-output feature, we used psIZCM-1 to identify the parallel G-quadruplexes from a large set of 314 oligonucleotides (including 300 G-quadruplex-forming oligonucleotides and 14 non-quadruplex oligonucleotides) via a microplate reader and accordingly established a high-throughput method for the characterization of parallel G-quadruplex topologies. The accuracy of this method was greater than 95%, which was much higher than that of the commercial probe NMM. To make the approach more practical, we further combined psIZCM-1 with another G-quadruplex probe IZCM-7 to realize the high-throughput classification of parallel, anti-parallel G-quadruplexes and non-quadruplex structures.

  3. Accurate Promoter and Enhancer Identification in 127 ENCODE and Roadmap Epigenomics Cell Types and Tissues by GenoSTAN

    Science.gov (United States)

    Zacher, Benedikt; Michel, Margaux; Schwalb, Björn; Cramer, Patrick; Tresch, Achim

    2017-01-01

    Accurate maps of promoters and enhancers are required for understanding transcriptional regulation. Promoters and enhancers are usually mapped by integration of chromatin assays charting histone modifications, DNA accessibility, and transcription factor binding. However, current algorithms are limited by unrealistic data distribution assumptions. Here we propose GenoSTAN (Genomic STate ANnotation), a hidden Markov model overcoming these limitations. We map promoters and enhancers for 127 cell types and tissues from the ENCODE and Roadmap Epigenomics projects, today’s largest compendium of chromatin assays. Extensive benchmarks demonstrate that GenoSTAN generally identifies promoters and enhancers with significantly higher accuracy than previous methods. Moreover, GenoSTAN-derived promoters and enhancers showed significantly higher enrichment of complex trait-associated genetic variants than current annotations. Altogether, GenoSTAN provides an easy-to-use tool to define promoters and enhancers in any system, and our annotation of human transcriptional cis-regulatory elements constitutes a rich resource for future research in biology and medicine. PMID:28056037

  4. Identification and validation of reference genes for accurate normalization of real-time quantitative PCR data in kiwifruit.

    Science.gov (United States)

    Ferradás, Yolanda; Rey, Laura; Martínez, Óscar; Rey, Manuel; González, Ma Victoria

    2016-05-01

    Identification and validation of reference genes are required for the normalization of qPCR data. We studied the expression stability produced by eight primer pairs amplifying four common genes used as references for normalization. Samples representing different tissues, organs and developmental stages in kiwifruit (Actinidia chinensis var. deliciosa (A. Chev.) A. Chev.) were used. A total of 117 kiwifruit samples were divided into five sample sets (mature leaves, axillary buds, stigmatic arms, fruit flesh and seeds). All samples were also analysed as a single set. The expression stability of the candidate primer pairs was tested using three algorithms (geNorm, NormFinder and BestKeeper). The minimum number of reference genes necessary for normalization was also determined. A unique primer pair was selected for amplifying the 18S rRNA gene. The primer pair selected for amplifying the ACTIN gene was different depending on the sample set. 18S 2 and ACT 2 were the candidate primer pairs selected for normalization in the three sample sets (mature leaves, fruit flesh and stigmatic arms). 18S 2 and ACT 3 were the primer pairs selected for normalization in axillary buds. No primer pair could be selected for use as the reference for the seed sample set. The analysis of all samples in a single set did not produce the selection of any stably expressing primer pair. Considering data previously reported in the literature, we validated the selected primer pairs amplifying the FLOWERING LOCUS T gene for use in the normalization of gene expression in kiwifruit.

  5. LC-MS/MS methods for absolute quantification and identification of proteins associated with chimeric plant oil bodies.

    Science.gov (United States)

    Capuano, Floriana; Bond, Nicholas J; Gatto, Laurent; Beaudoin, Frédéric; Napier, Johnathan A; Benvenuto, Eugenio; Lilley, Kathryn S; Baschieri, Selene

    2011-12-15

    Oil bodies (OBs) are plant cell organelles that consist of a lipid core surrounded by a phospholipid monolayer embedded with specialized proteins such as oleosins. Recombinant proteins expressed in plants can be targeted to OBs as fusions with oleosin. This expression strategy is attractive because OBs are easily enriched and purified from other cellular components, based on their unique physicochemical properties. For recombinant OBs to be a potential therapeutic agent in biomedical applications, it is necessary to comprehensively analyze and quantify both endogenous and heterologously expressed OB proteins. In this study, a mass spectrometry (MS)-based method was developed to accurately quantify an OB-targeted heterologously expressed fusion protein that has potential as a therapeutic agent. The effect of the chimeric oleosin expression upon the OB proteome in transgenic plants was also investigated, and the identification of new potential OB residents was pursued through a variety of liquid chromatography (LC)-MS/MS approaches. The results showed that the accumulation of the fusion protein on OBs was low. Moreover, no significant differences in the accumulation of OB proteins were revealed between transgenic and wild-type seeds. The identification of five new putative components of OB proteome was also reported.

  6. Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems

    Energy Technology Data Exchange (ETDEWEB)

    Samudrala, Ram; Heffron, Fred; McDermott, Jason E.

    2009-04-24

    The type III secretion system is an essential component for virulence in many Gram-negative bacteria. Though components of the secretion system apparatus are conserved, its substrates, effector proteins, are not. We have used a machine learning approach to identify new secreted effectors. The method integrates evolutionary measures, such as the pattern of homologs in a range of other organisms, and sequence-based features, such as G+C content, amino acid composition and the N-terminal 30 residues of the protein sequence. The method was trained on known effectors from Salmonella typhimurium and validated on a corresponding set of effectors from Pseudomonas syringae, after eliminating effectors with detectable sequence similarity. The method was able to identify all of the known effectors in P. syringae with a specificity of 84% and sensitivity of 82%. The reciprocal validation, training on P. syringae and validating on S. typhimurium, gave similar results with a specificity of 86% when the sensitivity level was 87%. These results show that type III effectors in disparate organisms share common features. We found that maximal performance is attained by including an N-terminal sequence of only 30 residues, which agrees with previous studies indicating that this region contains the secretion signal. We then used the method to define the most important residues in this putative secretion signal. Finally, we present novel predictions of secreted effectors in S. typhimurium, some of which have been experimentally validated, and apply the method to predict secreted effectors in the genetically intractable human pathogen Chlamydia trachomatis. This approach is a novel and effective way to identify secreted effectors in a broad range of pathogenic bacteria for further experimental characterization and provides insight into the nature of the type III secretion signal.

  7. Mass spectrometric identification of proteins and characterization of their post-translational modifications in proteome analysis

    DEFF Research Database (Denmark)

    Roepstorff, P; Larsen, Martin Røssel

    2001-01-01

    dominant strategies for identification of proteins from gels based on peptide mass spectrometric fingerprinting and partial sequencing by mass spectrometry are described. After identification of the proteins the next challenge in proteome analysis is characterization of their post-translational...... modifications. The general problems associated with characterization of these directly from gel separated proteins are described and the current state of art for the determination of phosphorylation, glycosylation and proteolytic processing is illustrated....

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

    OpenAIRE

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

    2011-01-01

    Dynamic range compression (DRC) by hexapeptide libraries increases MS/MS-based identification of lower-abundance proteins in complex mixtures. However, two unanswered questions impede fully realizing DRC’s potential in shotgun proteomics. First, does DRC enhance identification of post-translationally modified proteins? Second, can DRC be incorporated into a workflow enabling relative protein abundance profiling? We sought to answer both questions analyzing human whole saliva. Addressing quest...

  9. In silico identification of essential proteins in Corynebacterium pseudotuberculosis based on protein-protein interaction networks

    DEFF Research Database (Denmark)

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

    2016-01-01

    and decreased production of meat, wool, and milk. Current diagnosis or treatment protocols are not fully effective and, thus, require further research of Cp pathogenesis. RESULTS: Here, we mapped known protein-protein interactions (PPI) from various species to nine Cp strains to reconstruct parts...

  10. Identification of a novel resi-dent centrosomal protein

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    One human autoimmune serum was identified to react withcentrosomes by immunofluorescence. We applied the affinity purification of membrane-bound antibody technique and demonstrated that the antibodies present in this antiserum reacted with a 31/29 ku centrosomal antigen. Immunofluorescence showed that this antigen is located at centrosome in a cell-cycle independent manner, and thereby it belongs to the family of centrosomal residents. We then uti- lized this autoimmune serum and antibodies against centrin and gamma-tubulin to investigate changes of centrosome cycle kinetics during premature chromosome condensation (PCC) artificially induced in V79-8 cells. We show here that centrosomal proteins continue to express when cells are syn-chronized at G1/S boundary and S phase by Hydroxyurea (HU). During this time, the addition of caffeine causes cells with unreplicated genome to go into mitosis, and induces the separation of the replicated centrosomes. These results sug-gest that the coordination of DNA synthesis and centrosome replication in the normal cell cycle can be uncoupled. Cells ensure that centrosome duplicates once, and only once dur-ing each DNA synthesis cycle through the tight and subtle coordination of cell cycle engine molecules, and thereby the assembly of bipolar spindle and the accurate transmission of genetic information.

  11. 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.

  12. Identification and localization of the structural proteins of anguillid herpesvirus 1

    Directory of Open Access Journals (Sweden)

    van Beurden Steven J

    2011-10-01

    Full Text Available Abstract Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1 causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3. Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus.

  13. Identification of peptide and protein doping related drug compounds confiscated in Denmark between 2007-2013

    DEFF Research Database (Denmark)

    Hartvig, Rune Andersen; Holm, Niels Bjerre; Dalsgaard, Petur Weihe

    2014-01-01

    We present an overview of protein and peptide compounds confiscated in Denmark from late 2007 till late 2013 together with a description of a newly developed HRAM-LC-MS method used for identification. As examples of identification, we present data for the peptides AOD-9604, [D-Ala2, Gln8, Ala15, ...

  14. PSSP-RFE: accurate prediction of protein structural class by recursive feature extraction from PSI-BLAST profile, physical-chemical property and functional annotations.

    Directory of Open Access Journals (Sweden)

    Liqi Li

    Full Text Available Protein structure prediction is critical to functional annotation of the massively accumulated biological sequences, which prompts an imperative need for the development of high-throughput technologies. As a first and key step in protein structure prediction, protein structural class prediction becomes an increasingly challenging task. Amongst most homological-based approaches, the accuracies of protein structural class prediction are sufficiently high for high similarity datasets, but still far from being satisfactory for low similarity datasets, i.e., below 40% in pairwise sequence similarity. Therefore, we present a novel method for accurate and reliable protein structural class prediction for both high and low similarity datasets. This method is based on Support Vector Machine (SVM in conjunction with integrated features from position-specific score matrix (PSSM, PROFEAT and Gene Ontology (GO. A feature selection approach, SVM-RFE, is also used to rank the integrated feature vectors through recursively removing the feature with the lowest ranking score. The definitive top features selected by SVM-RFE are input into the SVM engines to predict the structural class of a query protein. To validate our method, jackknife tests were applied to seven widely used benchmark datasets, reaching overall accuracies between 84.61% and 99.79%, which are significantly higher than those achieved by state-of-the-art tools. These results suggest that our method could serve as an accurate and cost-effective alternative to existing methods in protein structural classification, especially for low similarity datasets.

  15. PSSP-RFE: accurate prediction of protein structural class by recursive feature extraction from PSI-BLAST profile, physical-chemical property and functional annotations.

    Science.gov (United States)

    Li, Liqi; Cui, Xiang; Yu, Sanjiu; Zhang, Yuan; Luo, Zhong; Yang, Hua; Zhou, Yue; Zheng, Xiaoqi

    2014-01-01

    Protein structure prediction is critical to functional annotation of the massively accumulated biological sequences, which prompts an imperative need for the development of high-throughput technologies. As a first and key step in protein structure prediction, protein structural class prediction becomes an increasingly challenging task. Amongst most homological-based approaches, the accuracies of protein structural class prediction are sufficiently high for high similarity datasets, but still far from being satisfactory for low similarity datasets, i.e., below 40% in pairwise sequence similarity. Therefore, we present a novel method for accurate and reliable protein structural class prediction for both high and low similarity datasets. This method is based on Support Vector Machine (SVM) in conjunction with integrated features from position-specific score matrix (PSSM), PROFEAT and Gene Ontology (GO). A feature selection approach, SVM-RFE, is also used to rank the integrated feature vectors through recursively removing the feature with the lowest ranking score. The definitive top features selected by SVM-RFE are input into the SVM engines to predict the structural class of a query protein. To validate our method, jackknife tests were applied to seven widely used benchmark datasets, reaching overall accuracies between 84.61% and 99.79%, which are significantly higher than those achieved by state-of-the-art tools. These results suggest that our method could serve as an accurate and cost-effective alternative to existing methods in protein structural classification, especially for low similarity datasets.

  16. Identification of Novel Perfluoroalkyl Ether Carboxylic Acids (PFECAs) and Sulfonic Acids (PFESAs) in Natural Waters Using Accurate Mass Time-of-Flight Mass Spectrometry (TOFMS).

    Science.gov (United States)

    Strynar, Mark; Dagnino, Sonia; McMahen, Rebecca; Liang, Shuang; Lindstrom, Andrew; Andersen, Erik; McMillan, Larry; Thurman, Michael; Ferrer, Imma; Ball, Carol

    2015-10-06

    Recent scientific scrutiny and concerns over exposure, toxicity, and risk have led to international regulatory efforts resulting in the reduction or elimination of certain perfluorinated compounds from various products and waste streams. Some manufacturers have started producing shorter chain per- and polyfluorinated compounds to try to reduce the potential for bioaccumulation in humans and wildlife. Some of these new compounds contain central ether oxygens or other minor modifications of traditional perfluorinated structures. At present, there has been very limited information published on these "replacement chemistries" in the peer-reviewed literature. In this study we used a time-of-flight mass spectrometry detector (LC-ESI-TOFMS) to identify fluorinated compounds in natural waters collected from locations with historical perfluorinated compound contamination. Our workflow for discovery of chemicals included sequential sampling of surface water for identification of potential sources, nontargeted TOFMS analysis, molecular feature extraction (MFE) of samples, and evaluation of features unique to the sample with source inputs. Specifically, compounds were tentatively identified by (1) accurate mass determination of parent and/or related adducts and fragments from in-source collision-induced dissociation (CID), (2) in-depth evaluation of in-source adducts formed during analysis, and (3) confirmation with authentic standards when available. We observed groups of compounds in homologous series that differed by multiples of CF2 (m/z 49.9968) or CF2O (m/z 65.9917). Compounds in each series were chromatographically separated and had comparable fragments and adducts produced during analysis. We detected 12 novel perfluoroalkyl ether carboxylic and sulfonic acids in surface water in North Carolina, USA using this approach. A key piece of evidence was the discovery of accurate mass in-source n-mer formation (H(+) and Na(+)) differing by m/z 21.9819, corresponding to the

  17. Identification of Bile Duct Paucity in Alagille Syndrome: Using CK7 and EMA Immunohistochemistry as a Reliable Panel for Accurate Diagnosis.

    Science.gov (United States)

    Herman, Haley K; Abramowsky, Carlos R; Caltharp, Shelley; Metry, Diana; Cundiff, Caitlin A; Romero, Rene; Gillespie, Scott E; Shehata, Bahig M

    2016-01-01

    Bile duct paucity is the absence or marked reduction in the number of interlobular bile ducts (ILBD) within portal tracts. Its syndromic variant, Alagille syndrome (ALGS), is a multisystem disorder with effects on the liver, cardiovascular system, skeleton, face, and eyes. It is inherited as an autosomal dominant trait due to defects in NOTCH signaling pathway. ALGS is characterized by vanishing ILBD with subsequent chronic obstructive cholestasis in approximately 89% of cases. Cholestasis stimulates formation of new bile ductules through a process of neoductular reaction, making it difficult to evaluate the presence or absence of ILBD. Therefore, finding a method to differentiate clearly between ILBD and the ductular proliferation is essential for accurate diagnosis. A database search identified 28 patients with confirmed diagnosis of ALGS between 1992 and 2014. Additionally, 7 controls were used. A panel of two immunostains, cytokeratin 7 (CK7) and epithelial membrane antigen (EMA), was performed. CK7 highlighted the bile duct epithelium of ILBD and ductular proliferation, while EMA stained only the brush border of ILBD. In our ALGS group, the ratio of EMA-positive ILBD to identified portal tracts was 12.6% (range, 0%-41%). However, this same ratio was 95.0% (range, 90%-100%) among control cases (P EMA, to differentiate ILBD from ductular proliferation in patients with cholestasis. With this panel, identification of bile duct paucity can be achieved. Additional studies, including molecular confirmation and clinical correlation, would provide a definitive diagnosis of ALGS.

  18. microTSS: accurate microRNA transcription start site identification reveals a significant number of divergent pri-miRNAs.

    Science.gov (United States)

    Georgakilas, Georgios; Vlachos, Ioannis S; Paraskevopoulou, Maria D; Yang, Peter; Zhang, Yuhong; Economides, Aris N; Hatzigeorgiou, Artemis G

    2014-12-10

    A large fraction of microRNAs (miRNAs) are derived from intergenic non-coding loci and the identification of their promoters remains 'elusive'. Here, we present microTSS, a machine-learning algorithm that provides highly accurate, single-nucleotide resolution predictions for intergenic miRNA transcription start sites (TSSs). MicroTSS integrates high-resolution RNA-sequencing data with active transcription marks derived from chromatin immunoprecipitation and DNase-sequencing to enable the characterization of tissue-specific promoters. MicroTSS is validated with a specifically designed Drosha-null/conditional-null mouse model, generated using the conditional by inversion (COIN) methodology. Analyses of global run-on sequencing data revealed numerous pri-miRNAs in human and mouse either originating from divergent transcription at promoters of active genes or partially overlapping with annotated long non-coding RNAs. MicroTSS is readily applicable to any cell or tissue samples and constitutes the missing part towards integrating the regulation of miRNA transcription into the modelling of tissue-specific regulatory networks.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. 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.

  1. 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...

  2. 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...

  3. Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches

    NARCIS (Netherlands)

    Kooij, V.; Venkatraman, V.; Kirk, J.A.; Ubaida-Mohien, C.; Graham, D.R.; Faber, M.J.; Eyk, J.E. Van

    2014-01-01

    PURPOSE: The identification of protein isoforms in complex biological samples is challenging. We, therefore, used an MS approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. EXPER

  4. Identification of fibrin clot-bound plasma proteins.

    Directory of Open Access Journals (Sweden)

    Simone Talens

    Full Text Available Several proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl(2 and aprotinin to citrated platelet-poor plasma and unbound proteins were washed away with Tris-buffered saline. Non-covalently bound proteins were extracted, separated with 2D gel electrophoresis and visualized with Sypro Ruby. Excised protein spots were analyzed with mass spectrometry. The identity of the proteins was verified by checking the mass of the protein, and, if necessary, by Western blot analysis. Next to established fibrin-binding proteins we identified several novel fibrin clot-bound plasma proteins, including α(2-macroglobulin, carboxypeptidase N, α(1-antitrypsin, haptoglobin, serum amyloid P, and the apolipoproteins A-I, E, J, and A-IV. The latter six proteins are associated with high-density lipoprotein particles. In addition we showed that high-density lipoprotein associated proteins were also present in fibrinogen preparations purified from plasma. Most plasma proteins in a fibrin clot can be classified into three groups according to either blood coagulation, protease inhibition or high-density lipoprotein metabolism. The presence of high-density lipoprotein in clots might point to a role in hemostasis.

  5. Gel Electrophoresis of Proteins for the Identification of Crop Varieties

    Institute of Scientific and Technical Information of China (English)

    LAN Hai-yan; LI Li-hui

    2002-01-01

    With the development of the international trade and agricultural science and technology, especially after the execution of the rules on protection of new plant varieties, considerable emphasis has been placed on variety identification. Many evidences have suggested that gel electrophoresis have great influence on this area. This paper reviewed study status of various gel electrophoresis, including development of the methods, comparison of these techniques, influence factors, practical applications, achievements obtained and aspects in the future study. With the wider range on protection of new plant varieties in China, electrophoresis will play a more important role in variety identification.

  6. Identification of proteins associated with amyloidosis by polarity index method.

    Science.gov (United States)

    Polanco, Carlos; Samaniego, José Lino; Uversky, Vladimir N; Castañón-González, Jorge Alberto; Buhse, Thomas; Leopold-Sordo, Marili; Madero-Arteaga, Alejandro; Morales-Reyes, Alicia; Tavera-Sierra, Lourdes; González-Bernal, Jesus A; Arias-Estrada, Miguel

    2015-01-01

    There is a natural protein form, insoluble and resistant to proteolysis, adopted by many proteins independently of their amino acid sequences via specific misfolding-aggregation process. This dynamic process occurs in parallel with or as an alternative to physiologic folding, generating toxic protein aggregates that are deposited and accumulated in various organs and tissues. These proteinaceous deposits typically represent bundles of β-sheet-enriched fibrillar species known as the amyloid fibrils that are responsible for serious pathological conditions, including but not limited to neurodegenerative diseases, grouped under the term amyloidoses. The proteins that might adopt this fibrillar conformation are some globular proteins and natively unfolded (or intrinsically disordered) proteins. Our work shows that intrinsically disordered and intrinsically ordered proteins can be reliably identified, discriminated, and differentiated by analyzing their polarity profiles generated using a computational tool known as the polarity index method (Polanco & Samaniego, 2009; Polanco et al., 2012; 2013; 2013a; 2014; 2014a; 2014b; 2014c; 2014d). We also show that proteins expressed in neurons can be differentiated from proteins in these two groups based on their polarity profiles, and also that this computational tool can be used to identify proteins associated with amyloidoses. The efficiency of the proposed method is high (i.e. 70%) as evidenced by the analysis of peptides and proteins in the APD2 database (2012), AVPpred database (2013), and CPPsite database (2013), the set of selective antibacterial peptides from del Rio et al. (2001), the sets of natively unfolded and natively folded proteins from Oldfield et al. (2005), the set of human revised proteins expressed in neurons, and non-human revised proteins expressed in neurons, from the Uniprot database (2014), and also the set of amyloidogenic proteins from the AmyPDB database (2014).

  7. Identification of a Protein that Purifies with the Scrapie Prion

    Science.gov (United States)

    Bolton, David C.; McKinley, Michael P.; Prusiner, Stanley B.

    1982-12-01

    Purification of prions from scrapie-infected hamster brain yielded a protein that was not found in a similar fraction from uninfected brain. The protein migrated with an apparent molecular size of 27,000 to 30,000 daltons in sodium dodecyl sulfate polyacrylamide gels. The resistance of this protein to digestion by proteinase K distinguished it from proteins of similar molecular weight found in normal hamster brain. Initial results suggest that the amount of this protein correlates with the titer of the agent.

  8. Identification of protein-protein interactions by standard gal4p-based yeast two-hybrid screening.

    Science.gov (United States)

    Wagemans, Jeroen; Lavigne, Rob

    2015-01-01

    Yeast two-hybrid (Y2H) screening permits identification of completely new protein interaction partners for a protein of interest, in addition to confirming binary protein-protein interactions. After discussing the general advantages and drawbacks of Y2H and existing alternatives, this chapter provides a detailed protocol for traditional Gal4p-based Y2H library screens in Saccharomyces cerevisiae AH109. This includes bait transformation, bait auto-activation testing, prey library transformation, Y2H evaluation, and subsequent identification of the prey plasmids. Moreover, a one-on-one mating protocol to confirm interactions between suspected partners is given. Finally, a quantitative α-galactosidase assay protocol to compare interaction strengths is provided.

  9. Toward accurate prediction of pKa values for internal protein residues: the importance of conformational relaxation and desolvation energy.

    Science.gov (United States)

    Wallace, Jason A; Wang, Yuhang; Shi, Chuanyin; Pastoor, Kevin J; Nguyen, Bao-Linh; Xia, Kai; Shen, Jana K

    2011-12-01

    Proton uptake or release controls many important biological processes, such as energy transduction, virus replication, and catalysis. Accurate pK(a) prediction informs about proton pathways, thereby revealing detailed acid-base mechanisms. Physics-based methods in the framework of molecular dynamics simulations not only offer pK(a) predictions but also inform about the physical origins of pK(a) shifts and provide details of ionization-induced conformational relaxation and large-scale transitions. One such method is the recently developed continuous constant pH molecular dynamics (CPHMD) method, which has been shown to be an accurate and robust pK(a) prediction tool for naturally occurring titratable residues. To further examine the accuracy and limitations of CPHMD, we blindly predicted the pK(a) values for 87 titratable residues introduced in various hydrophobic regions of staphylococcal nuclease and variants. The predictions gave a root-mean-square deviation of 1.69 pK units from experiment, and there were only two pK(a)'s with errors greater than 3.5 pK units. Analysis of the conformational fluctuation of titrating side-chains in the context of the errors of calculated pK(a) values indicate that explicit treatment of conformational flexibility and the associated dielectric relaxation gives CPHMD a distinct advantage. Analysis of the sources of errors suggests that more accurate pK(a) predictions can be obtained for the most deeply buried residues by improving the accuracy in calculating desolvation energies. Furthermore, it is found that the generalized Born implicit-solvent model underlying the current CPHMD implementation slightly distorts the local conformational environment such that the inclusion of an explicit-solvent representation may offer improvement of accuracy.

  10. Proteins of human milk. I. Identification of major components

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, N.G.; Powers, M.T.; Tollaksen, S.L.

    1982-04-01

    Traditionally, human milk proteins are identified largely by reference to bovine milk. Hence, to identify the major proteins in human milk, we subjected human and bovine milk, in parallel, to high-resolution two-dimensional electrophoresis. Isoelectric precipitation at pH 4.6 was our criterion for distinguishing whey proteins from those of the casein complex. The ..cap alpha..- and..beta..-caseins were identified on the basis of relative abundance, relative molecular mass, and relative isoelectric points. No protein disappeared from ISO-DALT patterns of human milk after rennin treatment, and no new protein comparable to bovine para K-casein appeared in the BASO-DALT patterns; this suggests that K-casein is absent from human milk. The proteins identified in human milk patterns include the ..cap alpha.. and ..beta.. casein families, lactalbumin, albumin, transferrin, IgA, and lactoferrin. Numerous additional proteins seen in patterns for human milk remain to be identified.

  11. Identification of Topological Network Modules in Perturbed Protein Interaction Networks

    Science.gov (United States)

    Sardiu, Mihaela E.; Gilmore, Joshua M.; Groppe, Brad; Florens, Laurence; Washburn, Michael P.

    2017-01-01

    Biological networks consist of functional modules, however detecting and characterizing such modules in networks remains challenging. Perturbing networks is one strategy for identifying modules. Here we used an advanced mathematical approach named topological data analysis (TDA) to interrogate two perturbed networks. In one, we disrupted the S. cerevisiae INO80 protein interaction network by isolating complexes after protein complex components were deleted from the genome. In the second, we reanalyzed previously published data demonstrating the disruption of the human Sin3 network with a histone deacetylase inhibitor. Here we show that disrupted networks contained topological network modules (TNMs) with shared properties that mapped onto distinct locations in networks. We define TMNs as proteins that occupy close network positions depending on their coordinates in a topological space. TNMs provide new insight into networks by capturing proteins from different categories including proteins within a complex, proteins with shared biological functions, and proteins disrupted across networks. PMID:28272416

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

    Science.gov (United States)

    Venner, Eric; Lisewski, Andreas Martin; Erdin, Serkan; Ward, R Matthew; Amin, Shivas R; Lichtarge, Olivier

    2010-12-13

    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.

  13. 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.

  14. Identification of Posttranslational Modification-Dependent Protein Interactions Using Yeast Surface Displayed Human Proteome Libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology.

  15. Phytochip: development of a DNA-microarray for rapid and accurate identification of Pseudo-nitzschia spp and other harmful algal species.

    Science.gov (United States)

    Noyer, Charlotte; Abot, Anne; Trouilh, Lidwine; Leberre, Véronique Anton; Dreanno, Catherine

    2015-05-01

    Detection of harmful algal blooms has become a challenging concern because of the direct impacts on public health and economy. The identification of toxic dinoflagellates and diatoms in monitoring programs requires an extensive taxonomic expertise and is time consuming. Advances in molecular biology have allowed the development of new approaches, more rapid, accurate and cost-effective for detecting these microorganisms. In this context, we developed a new DNA microarray (called, Phytochip) for the simultaneous detection of multiple HAB species with a particular emphasis on Pseudo-nitzschia species. Oligonucleotide probes were designed along the rRNA operon. After DNA extraction, the target rDNA genes were amplified and labeled using an asymmetric PCR; then, the amplicons were hybridized to the oligonucleotide probes present on the chips. The total assay from seawater sampling to data acquisition can be performed within a working day. Specificity and sensitivity were assessed by using monoclonal cultures, mixtures of species and field samples spiked with a known amount of cultured cells. The Phytochip with its 81 validated oligonucleotide probes was able to detect 12 species of Pseudo-nitzschia and 11 species of dinoflagellates among which were 3 species of Karenia and 3 species of Alexandrium. The Phytochip was applied to environmental samples already characterized by light microscopy and cloned into DNA libraries. The hybridizations on the Phytochip were in good agreement with the sequences retrieved from the clone libraries and the microscopic observations. The Phytochip enables a reliable multiplex detection of phytoplankton and can assist a water quality monitoring program as well as more general ecological research.

  16. 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.

  17. 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.

  18. 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.

  19. Identification of Differentially Expressed Serum Proteins in Infectious Purpura Fulminans

    Directory of Open Access Journals (Sweden)

    Ting He

    2014-01-01

    Full Text Available Purpura fulminans (PF is a life-threatening hemorrhagic condition. Because of the rarity and randomness of the disease, no improvement in treatment has been made for a long time. In this study, we assessed the serum proteome response to PF by comparing serum proteins between healthy controls and PF patient. Liquid chromatography with tandem mass spectrometry (LC-MS/MS approach was used after depleting 6 abundant proteins of serum. In total, 262 proteins were confidently identified with 2 unique peptides, and 38 proteins were identified significantly up- (≥2 or downregulated (≤0.5 based on spectral counting ratios (SpCPF/N. In the 38 proteins with significant abundance changes, 11 proteins were previously known to be associated with burn or sepsis response, but 27 potentially novel proteins may be specifically associated with PF process. Two differentially expressed proteins, alpha-1-antitrypsin (SERPINA1 and alpha-2 antiplasmin (SERPINF2, were validated by Western blot. This is the first study where PF patient and healthy controls are compared in a proteomic study to elucidate proteins involved in the response to PF. This study provides an initial basis for future studies of PF, and the differentially expressed proteins might provide new therapeutic targets to decrease the mortality of PF.

  20. Identification of host proteins, Spata3 and Dkk2, interacting with Toxoplasma gondii micronemal protein MIC3.

    Science.gov (United States)

    Wang, Yifan; Fang, Rui; Yuan, Yuan; Pan, Ming; Hu, Min; Zhou, Yanqin; Shen, Bang; Zhao, Junlong

    2016-07-01

    As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii.

  1. A nested mixture model for protein identification using mass spectrometry

    CERN Document Server

    Li, Qunhua; Stephens, Matthew; 10.1214/09-AOAS316

    2010-01-01

    Mass spectrometry provides a high-throughput way to identify proteins in biological samples. In a typical experiment, proteins in a sample are first broken into their constituent peptides. The resulting mixture of peptides is then subjected to mass spectrometry, which generates thousands of spectra, each characteristic of its generating peptide. Here we consider the problem of inferring, from these spectra, which proteins and peptides are present in the sample. We develop a statistical approach to the problem, based on a nested mixture model. In contrast to commonly used two-stage approaches, this model provides a one-stage solution that simultaneously identifies which proteins are present, and which peptides are correctly identified. In this way our model incorporates the evidence feedback between proteins and their constituent peptides. Using simulated data and a yeast data set, we compare and contrast our method with existing widely used approaches (PeptideProphet/ProteinProphet) and with a recently publis...

  2. 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.

  3. Identification of new centrosome proteins by autoimmune patient sera

    Institute of Scientific and Technical Information of China (English)

    XIA Liang; LI Yan; YANG Dong; WANG LiMin; HE Fang; ZHOU ChunYuan; LI YongZhe; ZENG ChangQing; He DaCheng

    2007-01-01

    Compared to other subcellular organelles, centrosome proteome can hardly be studied, due to the difficulties in separation and purification of centrosome. Auto-antisera from 6 autoimmune patients, which recognized centrosome specifically in immunofluorescence, were used to identify the corresponding centrosomal proteins. The sera were first tested by Western blot on whole cell lysate, and all bound antibodies were then eluted from each single band in Western blot membrane to assure which antibody was responsible for the centrosome specific immunofluorescence staining. The corresponding proteins were obtained by immunoprecipitation and identified by mass spectrometry. Six centrosomal proteins, including 2 known centrosomal proteins and 4 proteins with unknown localization or reportedly non-centrosomal localization, were identified. These proteins apparently involve in cell cycle regulation, signal transduction pathways, molecular chaperons, and metabolism enzymes, which may reflect the expected functional diversity of centrosome.

  4. Identification of an epitope of SARS-coronavirus nucleocapsid protein

    Institute of Scientific and Technical Information of China (English)

    YING LIN; JIN WANG; HONG XIA WANG; HUA LIANG JIANG; JIAN HUA SHEN; YOU HUA XIE; YUAN WANG; GANG PEI; BEI FEN SHEN; JIA RUI WU; BING SUN; XU SHEN; RUI FU YANG; YI XUE LI; YONG YONG JI; YOU YU HE; MUDE SHI; WEI LU; TIE LIU SHI

    2003-01-01

    The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a majorvirion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV werepredicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodieswere isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-inducedpolyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, itwas confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratorysyndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified andN protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.

  5. 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 NBE

  6. A highly accurate protein structural class prediction approach using auto cross covariance transformation and recursive feature elimination.

    Science.gov (United States)

    Li, Xiaowei; Liu, Taigang; Tao, Peiying; Wang, Chunhua; Chen, Lanming

    2015-12-01

    Structural class characterizes the overall folding type of a protein or its domain. Many methods have been proposed to improve the prediction accuracy of protein structural class in recent years, but it is still a challenge for the low-similarity sequences. In this study, we introduce a feature extraction technique based on auto cross covariance (ACC) transformation of position-specific score matrix (PSSM) to represent a protein sequence. Then support vector machine-recursive feature elimination (SVM-RFE) is adopted to select top K features according to their importance and these features are input to a support vector machine (SVM) to conduct the prediction. Performance evaluation of the proposed method is performed using the jackknife test on three low-similarity datasets, i.e., D640, 1189 and 25PDB. By means of this method, the overall accuracies of 97.2%, 96.2%, and 93.3% are achieved on these three datasets, which are higher than those of most existing methods. This suggests that the proposed method could serve as a very cost-effective tool for predicting protein structural class especially for low-similarity datasets.

  7. Identification of proteins in fluid collected from nerve regeneration chambers

    Directory of Open Access Journals (Sweden)

    Ye Yilin

    2014-01-01

    Full Text Available We examined whether there are novel neurotrophic factors (NTFs in nerve regeneration conditioned fluid (NRCF. Nerve regeneration chamber models were established in the sciatic nerves of 25 New Zealand rabbits, and NRCF was extracted from the chambers l week postoperatively. Proteins in NRCF were separated by native polyacrylamide gel electrophoresis (PAGE, and Western blot and ELISA were used to identify the proteins. A novel NTF was identified in a protein fraction corresponding to 220 kDa.

  8. Proteomics of Soil and Sediment: Protein Identification by De Novo Sequencing of Mass Spectra Complements Traditional Database Searching

    Science.gov (United States)

    Miller, S.; Rizzo, A. I.; Waldbauer, J.

    2015-12-01

    Proteomics has the potential to elucidate the metabolic pathways and taxa responsible for in situ biogeochemical transformations. However, low rates of protein identification from high resolution mass spectra have been a barrier to the development of proteomics in complex environmental samples. Much of the difficulty lies in the computational challenge of linking mass spectra to their corresponding proteins. Traditional database search methods for matching peptide sequences to mass spectra are often inadequate due to the complexity of environmental proteomes and the large database search space, as we demonstrate with soil and sediment proteomes generated via a range of extraction methods. One alternative to traditional database searching is de novo sequencing, which identifies peptide sequences without the need for a database. BLAST can then be used to match de novo sequences to similar genetic sequences. Assigning confidence to putative identifications has been one hurdle for the implementation of de novo sequencing. We found that accurate de novo sequences can be screened by quality score and length. Screening criteria are verified by comparing the results of de novo sequencing and traditional database searching for well-characterized proteomes from simple biological systems. The BLAST hits of screened sequences are interrogated for taxonomic and functional information. We applied de novo sequencing to organic topsoil and marine sediment proteomes. Peak-rich proteomes, which can result from various extraction techniques, yield thousands of high-confidence protein identifications, an improvement over previous proteomic studies of soil and sediment. User-friendly software tools for de novo metaproteomics analysis have been developed. This "De Novo Analysis" Pipeline is also a faster method of data analysis than constructing a tailored sequence database for traditional database searching.

  9. Cross-Species Genome-Wide Identification of Evolutionary Conserved MicroProteins

    Science.gov (United States)

    Straub, Daniel

    2017-01-01

    MicroProteins are small single-domain proteins that act by engaging their targets into different, sometimes nonproductive protein complexes. In order to identify novel microProteins in any sequenced genome of interest, we have developed miPFinder, a program that identifies and classifies potential microProteins. In the past years, several microProteins have been discovered in plants where they are mainly involved in the regulation of development by fine-tuning transcription factor activities. The miPFinder algorithm identifies all up to date known plant microProteins and extends the microProtein concept beyond transcription factors to other protein families. Here, we reveal potential microProtein candidates in several plant and animal reference genomes. A large number of these microProteins are species-specific while others evolved early and are evolutionary highly conserved. Most known microProtein genes originated from large ancestral genes by gene duplication, mutation and subsequent degradation. Gene ontology analysis shows that putative microProtein ancestors are often located in the nucleus, and involved in DNA binding and formation of protein complexes. Additionally, microProtein candidates act in plant transcriptional regulation, signal transduction and anatomical structure development. MiPFinder is freely available to find microProteins in any genome and will aid in the identification of novel microProteins in plants and animals. PMID:28338802

  10. Identification of highly active flocculant proteins in bovine blood.

    Science.gov (United States)

    Piazza, George J; Nuñez, Alberto; Garcia, Rafael A

    2012-03-01

    Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits α and β) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, γ-globulin, α-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, α-globulin, and β-globulin were not flocculants. On a mass basis, hemoglobin, γ-globulin, α-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity.

  11. Identification of fibrin clot-bound plasma proteins

    NARCIS (Netherlands)

    S. Talens (Simone); F.W.G. Leebeek (Frank); J.A.A. Demmers (Jeroen); D.C. Rijken (Dingeman)

    2012-01-01

    textabstractSeveral proteins are known to bind to a fibrin network and to change clot properties or function. In this study we aimed to get an overview of fibrin clot-bound plasma proteins. A plasma clot was formed by adding thrombin, CaCl2 and aprotinin to citrated platelet-poor plasma and unbound

  12. Identification, cloning, and purification of protein antigens of Treponema pallidum.

    Science.gov (United States)

    Stamm, L V; Dallas, W S; Ray, P H; Bassford, P J

    1988-01-01

    Difficulties in culturing the bacterium Treponema pallidum have greatly hindered syphilis research. In recent years, several laboratories have begun applying recombinant DNA technology to the study of this organism. Recent work is summarized concerning the expression of T. pallidum DNA in Escherichia coli. A number of E. coli clones expressing treponemal protein antigens have been identified. In one instance, a recombinant protein was purified to homogeneity and shown to be identical to a highly immunogenic, native T. pallidum membrane protein of molecular weight 39,000, which was designated the basic membrane protein (BMP) of this organism. In addition, recent experiments are described that were designed to identify cell-surface proteins that would serve as the primary focus of our cloning efforts. Results obtained with use of several different approaches strongly suggest that the outer membrane of T. pallidum is an antigenically inert structure largely devoid of protein. However, a class of low-molecular-weight protein antigens have been identified that are actively secreted into the extracellular medium. Attempts currently are being made to clone these secreted proteins and investigate their roles in the pathogenesis and immunobiology of syphilis.

  13. Identification of proteins interacting with Arabidopsis ACD11

    DEFF Research Database (Denmark)

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

    2009-01-01

    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...

  14. Identification of new centrosome proteins by autoimmune patient sera

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Compared to other subcellular organelles, centrosome proteome can hardly be studied, due to the dif- ficulties in separation and purification of centrosome. Auto-antisera from 6 autoimmune patients, which recognized centrosome specifically in immunofluorescence, were used to identify the corresponding centrosomal proteins. The sera were first tested by Western blot on whole cell lysate, and all bound antibodies were then eluted from each single band in Western blot membrane to assure which antibody was responsible for the centrosome specific immunofluorescence staining. The corresponding pro- teins were obtained by immunoprecipitation and identified by mass spectrometry. Six centrosomal proteins, including 2 known centrosomal proteins and 4 proteins with unknown localization or report- edly non-centrosomal localization, were identified. These proteins apparently involve in cell cycle regulation, signal transduction pathways, molecular chaperons, and metabolism enzymes, which may reflect the expected functional diversity of centrosome.

  15. Identification of SNARE proteins in fish-Tilapia Oreochromis niloticus

    Institute of Scientific and Technical Information of China (English)

    HUANG Xiaohang; LAM Patrick P L; LIN Xuezheng; LIU Chenlin; BIAN Ji; GAISANO Herbert

    2007-01-01

    SNARE proteins are a group of membrane-associated proteins involved in exocytosis, secretion and membrane trafficking events in eukaryotic cells. Research on SNARE protein biology has become a more attractive field in recent years, which is applied to marine biology specifically to the fish Tilapia (Oreochromis niloticus). Plasma membrane fractions of different tissues of Tilapia, including brain, liver-pancreas, intestine, skin and muscle, were extracted, and immuno-decorated with isoform-specific antibodies to the SNARE families and associated proteins. The presence of Syntaxins -1A, 2 and 3, SNAP-23 and SNAP-25, VAMP-2, Munc-18-1 and Munc-13 in the brain was identified, which were differentially distributed in the other organ tissues of the fish Tilapia. The distinct distribution of SNARE and associated proteins will serve as the basis for further investigation into their special secretory function in these tissues of the fish.

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

    Science.gov (United States)

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

    2015-08-01

    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 NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 μg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs.

  17. 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.

  18. Backbone building from quadrilaterals: a fast and accurate algorithm for protein backbone reconstruction from alpha carbon coordinates.

    Science.gov (United States)

    Gront, Dominik; Kmiecik, Sebastian; Kolinski, Andrzej

    2007-07-15

    In this contribution, we present an algorithm for protein backbone reconstruction that comprises very high computational efficiency with high accuracy. Reconstruction of the main chain atomic coordinates from the alpha carbon trace is a common task in protein modeling, including de novo structure prediction, comparative modeling, and processing experimental data. The method employed in this work follows the main idea of some earlier approaches to the problem. The details and careful design of the present approach are new and lead to the algorithm that outperforms all commonly used earlier applications. BBQ (Backbone Building from Quadrilaterals) program has been extensively tested both on native structures as well as on near-native decoy models and compared with the different available existing methods. Obtained results provide a comprehensive benchmark of existing tools and evaluate their applicability to a large scale modeling using a reduced representation of protein conformational space. The BBQ package is available for downloading from our website at http://biocomp.chem.uw.edu.pl/services/BBQ/. This webpage also provides a user manual that describes BBQ functions in detail.

  19. Analytical approaches for the characterization and identification of olive (Olea europaea) oil proteins.

    Science.gov (United States)

    Esteve, Clara; D'Amato, Alfonsina; Marina, María Luisa; García, María Concepción; Righetti, Pier Giorgio

    2013-10-30

    Proteins in olive oil have been scarcely investigated probably due to the difficulty of working with such a lipidic matrix and the dramatically low abundance of proteins in this biological material. Additionally, this scarce information has generated contradictory results, thus requiring further investigations. This work treats this subject from a comprehensive point of view and proposes the use of different analytical approaches to delve into the characterization and identification of proteins in olive oil. Different extraction methodologies, including capture via combinational hexapeptide ligand libraries (CPLLs), were tried. A sequence of methodologies, starting with off-gel isoelectric focusing (IEF) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or high-performance liquid chromatography (HPLC) using an ultraperformance liquid chromatography (UPLC) column, was applied to profile proteins from olive seed, pulp, and oil. Besides this, and for the first time, a tentative identification of oil proteins by mass spectrometry has been attempted.

  20. On-tissue protein identification and imaging by MALDI-ion mobility mass spectrometry.

    Science.gov (United States)

    Stauber, Jonathan; MacAleese, Luke; Franck, Julien; Claude, Emmanuelle; Snel, Marten; Kaletas, Basak Kükrer; Wiel, Ingrid M V D; Wisztorski, Maxence; Fournier, Isabelle; Heeren, Ron M A

    2010-03-01

    MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for the detection and localization of drugs, proteins, and lipids on-tissue. Nevertheless, this approach can only perform identification of low mass molecules as lipids, pharmaceuticals, and peptides. In this article, a combination of approaches for the detection and imaging of proteins and their identification directly on-tissue is described after tryptic digestion. Enzymatic digestion protocols for different kinds of tissues--formalin fixed paraffin embedded (FFPE) and frozen tissues--are combined with MALDI-ion mobility mass spectrometry (IM-MS). This combination enables localization and identification of proteins via their related digested peptides. In a number of cases, ion mobility separates isobaric ions that cannot be identified by conventional MALDI time-of-flight (TOF) mass spectrometry. The amount of detected peaks per measurement increases (versus conventional MALDI-TOF), which enables mass and time selected ion images and the identification of separated ions. These experiments demonstrate the feasibility of direct proteins identification by ion-mobility-TOF IMS from tissue. The tissue digestion combined with MALDI-IM-TOF-IMS approach allows a proteomics "bottom-up" strategy with different kinds of tissue samples, especially FFPE tissues conserved for a long time in hospital sample banks. The combination of IM with IMS marks the development of IMS approaches as real proteomic tools, which brings new perspectives to biological studies.

  1. A Graph-Centric Approach for Metagenome-Guided Peptide and Protein Identification in Metaproteomics.

    Science.gov (United States)

    Tang, Haixu; Li, Sujun; Ye, Yuzhen

    2016-12-01

    Metaproteomic studies adopt the common bottom-up proteomics approach to investigate the protein composition and the dynamics of protein expression in microbial communities. When matched metagenomic and/or metatranscriptomic data of the microbial communities are available, metaproteomic data analyses often employ a metagenome-guided approach, in which complete or fragmental protein-coding genes are first directly predicted from metagenomic (and/or metatranscriptomic) sequences or from their assemblies, and the resulting protein sequences are then used as the reference database for peptide/protein identification from MS/MS spectra. This approach is often limited because protein coding genes predicted from metagenomes are incomplete and fragmental. In this paper, we present a graph-centric approach to improving metagenome-guided peptide and protein identification in metaproteomics. Our method exploits the de Bruijn graph structure reported by metagenome assembly algorithms to generate a comprehensive database of protein sequences encoded in the community. We tested our method using several public metaproteomic datasets with matched metagenomic and metatranscriptomic sequencing data acquired from complex microbial communities in a biological wastewater treatment plant. The results showed that many more peptides and proteins can be identified when assembly graphs were utilized, improving the characterization of the proteins expressed in the microbial communities. The additional proteins we identified contribute to the characterization of important pathways such as those involved in degradation of chemical hazards. Our tools are released as open-source software on github at https://github.com/COL-IU/Graph2Pro.

  2. Identification and Characterization of Proteins Associated with Plant Tolerance to Heat Stress

    Institute of Scientific and Technical Information of China (English)

    Bingru Huang; Chenping Xu

    2008-01-01

    Heat stress is a major abiotic stress limiting plant growth and productivity in many areas of the world. Understanding mechanisms of plant adaptation to heat stress would facilitate the development of heat-tolerant cultivars for improving productivity in warm climatic regions. Protein metabolism involving protein synthesis and degradation is one of the most sensitive processes to heat stress. Changes in the level and expression pattern of some proteins may play an important role in plant adaptation to heat stress. The identification of stress-responsive proteins and pathways has been facilitated by an increasing number of tools and resources, including two-dimensional electrophoresis and mass spectrometry, and the rapidly expanding nucleotide and amino acid sequence databases. Heat stress may induce or enhance protein expression or cause protein degradation. The induction of heat-responsive proteins, particularly heat shock proteins (HSPs), plays a key role in plant tolerance to heat stress. Protein degradation involving various proteases is also important in regulating plant responses to heat stress. This review provides an overview of recent research on proteomic profiling for the identification of heat-responsive proteins associated with heat tolerance, heat induction and characteristics of HSPs, and protein degradation in relation to plant responses to heat stress.

  3. Identification of novel amelogenin-binding proteins by proteomics analysis.

    Directory of Open Access Journals (Sweden)

    Takao Fukuda

    Full Text Available Emdogain (enamel matrix derivative, EMD is well recognized in periodontology. It is used in periodontal surgery to regenerate cementum, periodontal ligament, and alveolar bone. However, the precise molecular mechanisms underlying periodontal regeneration are still unclear. In this study, we investigated the proteins bound to amelogenin, which are suggested to play a pivotal role in promoting periodontal tissue regeneration. To identify new molecules that interact with amelogenin and are involved in osteoblast activation, we employed coupling affinity chromatography with proteomic analysis in fractionated SaOS-2 osteoblastic cell lysate. In SaOS-2 cells, many of the amelogenin-interacting proteins in the cytoplasm were mainly cytoskeletal proteins and several chaperone molecules of heat shock protein 70 (HSP70 family. On the other hand, the proteomic profiles of amelogenin-interacting proteins in the membrane fraction of the cell extracts were quite different from those of the cytosolic-fraction. They were mainly endoplasmic reticulum (ER-associated proteins, with lesser quantities of mitochondrial proteins and nucleoprotein. Among the identified amelogenin-interacting proteins, we validated the biological interaction of amelogenin with glucose-regulated protein 78 (Grp78/Bip, which was identified in both cytosolic and membrane-enriched fractions. Confocal co-localization experiment strongly suggested that Grp78/Bip could be an amelogenin receptor candidate. Further biological evaluations were examined by Grp78/Bip knockdown analysis with and without amelogenin. Within the limits of the present study, the interaction of amelogenin with Grp78/Bip contributed to cell proliferation, rather than correlate with the osteogenic differentiation in SaOS-2 cells. Although the biological significance of other interactions are not yet explored, these findings suggest that the differential effects of amelogenin-derived osteoblast activation could be of

  4. Proteomic identification of proteins in exosomes of patients with atherosclerosis

    Institute of Scientific and Technical Information of China (English)

    JIANG Mei; QUAN Jing; ZHANG Heng; DING Qian-qian; XIANG Meng; MENG Dan; SUN Ning; CHEN Si-feng

    2016-01-01

    AIM:Atherosclerosis primarily involved systemic arteries .Luminal surface , a monolayer of endothelial cells , of artery directly exposes to blood and is susceptible to active substances in the blood .Exosomes contain significantly amount of proteins and RNAs .Ex-osomes can be good and bad for cells , depending on their component .Thus, exosomes may contribute to atherosclerosis by affecting endothelial cells .This study analyzed the relationship of exosome proteins and atherosclerosis .METHODS: Fifty-six patients and healthy subjects were recruited and divided into two comparisons:healthy subjects vs atherosclerosis ( HS vs AS) , and hypertension vs hypertension plus atherosclerosis ( HT vs HT+AS) .Serum exosomes were decoded by protein mass spectrometry .The protein profile and function were analyzed by gene ontology ( GO) .RESULTS:It was found that five child terms repeatedly appeared in “response to stimulus” and “immune system process” of BP of the two categories ( HS vs AS and AS vs HT+AS):“positive regulation of innate immune response”,“immune response-activating signal transduction”,”activation of innate immune response”,“innate immune re-sponse-activating signal transduction” and “innate immune response activating cell surface receptor signaling pathway ”.Two child terms repeatedly showed in “binding” of MF of the two categories:“antigen binding” and “enzyme binding”.Two proteins, PSMA6 and PSMA7, were repeatedly shown in the two categories .CONCLUSION:GO analysis was utilized for structure hierarchy “tree” to illustrate these proteins involved in various terms in BP , CC and MF.The PPI analysis supplied proteins which may play potentially im-portant roles in AS process .Innate immune system and blood coagulation pathway contribute to AS formation .The proteins, PSMA6, PSMA7 and Annexin A2, may can be the new target proteins for prevention and treatment of AS .

  5. Large-format imaging plate and weissenberg camera for accurate protein crystallographic data collection using synchrotron radiation.

    Science.gov (United States)

    Sakabe, K; Sasaki, K; Watanabe, N; Suzuki, M; Wang, Z G; Miyahara, J; Sakabe, N

    1997-05-01

    Off-line and on-line protein data-collection systems using an imaging plate as a detector are described and their components reported. The off-line scanner IPR4080 was developed for a large-format imaging plate ;BASIII' of dimensions 400 x 400 mm and 400 x 800 mm. The characteristics of this scanner are a dynamic range of 10(5) photons pixel(-1), low background noise and high sensitivity. A means of reducing electronic noise and a method for finding the origin of the noise are discussed in detail. A dedicated screenless Weissenberg camera matching IPR4080 with synchrotron radiation was developed and installed on beamline BL6B at the Photon Factory. This camera can attach one or two sheets of 400 x 800 mm large-format imaging plate inside the film cassette by evacuation. The positional reproducibility of the imaging plate on the cassette is so good that the data can be processed by batch job. Data of 93% completeness up to 1.6 A resolution were collected on a single axis rotation and the value of R(merge) becomes 4% from a tetragonal lysozyme crystal using a set of two imaging-plate sheets. Comparing two types of imaging plates, the signal-to-noise ratio of the ST-VIP-type imaging plate is 25% better than that of the BASIII-type imaging plate for protein data collection using 1.0 and 0.7 A X-rays. A new on-line protein data-collection system with imaging plates is specially designed to use synchrotron radiation X-rays at maximum efficiency.

  6. A new strategy for protein interface identification using manifold learning method.

    Science.gov (United States)

    Wang, Bing; Huang, De-Shuang; Jiang, Changjun

    2014-06-01

    Protein interactions play vital roles in biological processes. The study for protein interface will allow people to elucidate the mechanism of protein interaction. However, a large portion of protein interface data is incorrectly collected in current studies. In this paper, a novel strategy of dataset reconstruction using manifold learning method has been proposed for dealing with the noises in the interaction interface data whose definition is based on the residue distances among the different chains within protein complexes. Three support vector machine-based predictors are constructed using different protein features to identify the functional sites involved in the formation of protein interface. The experimental results achieved in this work demonstrate that our strategy can remove noises, and therefore improve the ability for identification of protein interfaces with 77.8% accuracy.

  7. Proteomic identification of S-nitrosylated proteins in Arabidopsis

    DEFF Research Database (Denmark)

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

    2005-01-01

    purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic......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...

  8. 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

    Glycoproteins constitute a large fraction of the proteome. The fundamental role of protein glycosylation in cellular development, growth, and differentiation, tissue development, and in host-pathogen interactions is by now widely accepted. Proteome-wide characterization of glycoproteins...

  9. Identification of novel cyclic nucleotide binding proteins in Trypanosoma cruzi.

    Science.gov (United States)

    Jäger, Adriana V; De Gaudenzi, Javier G; Mild, Jesica G; Mc Cormack, Bárbara; Pantano, Sergio; Altschuler, Daniel L; Edreira, Martin M

    2014-12-01

    Cyclic AMP has been implicated as second messenger in a wide range of cellular processes. In the protozoan parasite Trypanosoma cruzi, cAMP is involved in the development of the parasite's life cycle. While cAMP effectors have been widely studied in other eukaryotic cells, little is known about cAMP's mechanism of action in T. cruzi. To date, only a cAMP-dependent protein kinase A (PKA) has been cloned and characterised in this parasite; however experimental evidence indicates the existence of cAMP-dependent, PKA-independent events. In order to identify new cAMP binding proteins as potential cAMP effectors, we carried out in silico studies using the predicted T. cruzi proteome. Using a combination of search methods 27 proteins with putative cNMP binding domains (CBDs) were identified. Phylogenetic analysis of the CBDs presented a homogeneous distribution, with sequences segregated into two main branches: one containing kinases-like proteins and the other gathering hypothetical proteins with different function or no other known. Comparative modelling of the strongest candidates provides support for the hypothesis that these proteins may give rise to structurally viable cyclic nucleotide binding domains. Pull-down and nucleotide displacement assays strongly suggest that TcCLB.508523.80 could bind cAMP and eventually be a new putative PKA-independent cAMP effector in T. cruzi.

  10. 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.

  11. Identification of a conserved interface between PUF and CPEB proteins.

    Science.gov (United States)

    Campbell, Zachary T; Menichelli, Elena; Friend, Kyle; Wu, Joann; Kimble, Judith; Williamson, James R; Wickens, Marvin

    2012-05-25

    Members of the PUF (Pumilio and FBF) and CPEB (cytoplasmic polyadenylation element-binding) protein families collaborate to regulate mRNA expression throughout eukaryotes. Here, we focus on the physical interactions between members of these two families, concentrating on Caenorhabditis elegans FBF-2 and CPB-1. To localize the site of interaction on FBF-2, we identified conserved amino acids within C. elegans PUF proteins. Deletion of an extended loop containing several conserved residues abolished binding to CPB-1. We analyzed alanine substitutions at 13 individual amino acids in FBF-2, each identified via its conservation. Multiple single point mutations disrupted binding to CPB-1 but not to RNA. Position Tyr-479 was particularly critical as multiple substitutions to other amino acids at this position did not restore binding. The complex of FBF-2 and CPB-1 repressed translation of an mRNA containing an FBF binding element. Repression required both proteins and was disrupted by FBF-2 alleles that failed to bind CPB-1 or RNA. The equivalent loop in human PUM2 is required for binding to human CPEB3 in vitro, although the primary sequences of the human and C. elegans PUF proteins have diverged in that region. Our findings define a key region in PUF/CPEB interactions and imply a conserved platform through which PUF proteins interact with their protein partners.

  12. Identification of cancer protein biomarkers using proteomic techniques

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-03-10

    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.

  13. Identification of cancer protein biomarkers using proteomic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Mor, Gil G. (Cheshire, CT); Ward, David C. (Las Vegas, NV); Bray-Ward, Patricia (Las Vegas, NV)

    2010-02-23

    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.

  14. 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.

  15. 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 too

  16. Non-targeted identification of prions and amyloid-forming proteins from yeast and mammalian cells.

    Science.gov (United States)

    Kryndushkin, Dmitry; Pripuzova, Natalia; Burnett, Barrington G; Shewmaker, Frank

    2013-09-20

    The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin.

  17. Identification of Actin-Binding Proteins from Maize Pollen

    Energy Technology Data Exchange (ETDEWEB)

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

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

    Directory of Open Access Journals (Sweden)

    Svetlana Lockwood

    Full Text Available BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

  19. 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

  20. Identification of novel Drosophila centromere-associated proteins.

    Science.gov (United States)

    Barth, Teresa K; Schade, Georg O M; Schmidt, Andreas; Vetter, Irene; Wirth, Marc; Heun, Patrick; Thomae, Andreas W; Imhof, Axel

    2014-10-01

    Centromeres are chromosomal regions crucial for correct chromosome segregation during mitosis and meiosis. They are epigenetically defined by centromeric proteins such as the centromere-specific histone H3-variant centromere protein A (CENP-A). In humans, 16 additional proteins have been described to be constitutively associated with centromeres throughout the cell cycle, known as the constitutive centromere-associated network (CCAN). In contrast, only one additional constitutive centromeric protein is known in Drosophila melanogaster (D.mel), the conserved CCAN member CENP-C. To gain further insights into D.mel centromere composition and biology, we analyzed affinity-purified chromatin prepared from D.mel cell lines expressing green fluorescent protein tagged histone three variants by MS. In addition to already-known centromeric proteins, we identified novel factors that were repeatedly enriched in affinity purification-MS experiments. We analyzed the cellular localization of selected candidates by immunocytochemistry and confirmed localization to the centromere and other genomic regions for ten factors. Furthermore, RNA interference mediated depletion of CG2051, CG14480, and hyperplastic discs, three of our strongest candidates, leads to elevated mitotic defects. Knockdowns of these candidates neither impair the localization of several known kinetochore proteins nor CENP-A(CID) loading, suggesting their involvement in alternative pathways that contribute to proper centromere function. In summary, we provide a comprehensive analysis of the proteomic composition of Drosophila centromeres. All MS data have been deposited in the ProteomeXchange with identifier PXD000758 (http://proteomecentral.proteomexchange.org/dataset/PXD000758).

  1. Identification of Disulfide Bonds in Protein Proteolytic Degradation Products Using de Novo-Protein Unique Sequence Tags Approach

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yufeng; Tolic, Nikola; Purvine, Samuel O.; Smith, Richard D.

    2010-08-01

    Disulfide bonds are a form of posttranslational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, and specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags) we unambiguously correlated the spectra to specific database proteins. Examination of the UStags’ prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to ~10 kDa.

  2. 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.

  3. Identification of giant Mimivirus protein functions using RNA interference

    Directory of Open Access Journals (Sweden)

    Haitham eSobhy

    2015-04-01

    Full Text Available Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans. We knocked down Mimivirus genes using short interfering RNA (siRNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as 3 genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate 4 proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases.

  4. Proteome identification of proteins interacting with histone methyltransferase SET8

    Institute of Scientific and Technical Information of China (English)

    Yi Qin; Huafang Ouyang; Jing Liu; Youhua Xie

    2013-01-01

    SET8 (also known as PR-Set7/9,SETD8,KMT5A),a member of the SET domain containing methyltransferase family,which specifically catalyzes mono-methylation of K20 on histone H4 (H4K20me1),has been implicated in multiple biological processes,such as gene transcriptional regulation,cell cycle control,genomic integrity maintenance and development.In this study,we used GST-SET8 fusion protein as bait to search for SET8 interaction partners to elucidate physiological functions of SET8.In combination with mass spectrometry,we identified 40 proteins that potentially interact with SET8.DDX21,a nucleolar protein,was further confirmed to associate with SET8.Furthermore,we discovered a novel function of SET8 in the regulation of rRNA transcription.

  5. Identification of Chlamydia trachomatis outer membrane complex proteins by differential proteomics.

    Science.gov (United States)

    Liu, Xiaoyun; Afrane, Mary; Clemmer, David E; Zhong, Guangming; Nelson, David E

    2010-06-01

    The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses of Chlamydia trachomatis serovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.

  6. 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 S10-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.

  7. 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.

  8. Identification of Dominant Immunogenic Bacteria and Bacterial Proteins in Periodontitis

    DEFF Research Database (Denmark)

    Agerbæk, Mette Rylev; Haubek, Dorte; Birkelund, Svend

    Marginal periodontitis is considered an infectious disease that triggers host inflammatory responses resulting in destruction of the periodontium. A complex biofilm of bacteria is associated with periodontitis. Some species have been identified as putative pathogens such as Porphyromonas gingivalis...... (P.g) and Actinobacillus actinomycetemcomitans (A.a), but the identity of dominate immunogens of these bacteria is poorly elucidated. The aim of the study was to identify dominant immunogenic proteins of P.g and A.a in patients suffering from chronic and aggressive periodontitis by proteomic analysis...... will be able to identify immunodominant proteins and potentially important virulence factors of putative periodontal pathogens....

  9. Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

    Science.gov (United States)

    Brgles, Marija; Kurtović, Tihana; Kovačič, Lidija; Križaj, Igor; Barut, Miloš; Lang Balija, Maja; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far-ammodytoxins (Atxs)-are contributing to the venom's toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

  10. Extraction and identification of membrane proteins from black widow spider eggs.

    Science.gov (United States)

    Fu, Si-Ling; Li, Jiang-Lin; Chen, Jia; Wang, Qiu-Ting; Li, Jian-Jun; Wang, Xian-Chun

    2015-07-18

    The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development.

  11. Identification of serum protein biomarkers for utrophin based DMD therapy

    Science.gov (United States)

    Guiraud, Simon; Edwards, Benjamin; Squire, Sarah E.; Babbs, Arran; Shah, Nandini; Berg, Adam; Chen, Huijia; Davies, Kay E.

    2017-01-01

    Despite promising therapeutic avenues, there is currently no effective treatment for Duchenne muscular dystrophy (DMD), a lethal monogenic disorder caused by the loss of the large cytoskeletal protein, dystrophin. A highly promising approach to therapy, applicable to all DMD patients irrespective to their genetic defect, is to modulate utrophin, a functional paralogue of dystrophin, able to compensate for the primary defects of DMD restoring sarcolemmal stability. One of the major difficulties in assessing the effectiveness of therapeutic strategies is to define appropriate outcome measures. In the present study, we utilised an aptamer based proteomics approach to profile 1,310 proteins in plasma of wild-type, mdx and Fiona (mdx overexpressing utrophin) mice. Comparison of the C57 and mdx sera revealed 83 proteins with statistically significant >2 fold changes in dystrophic serum abundance. A large majority of previously described biomarkers (ANP32B, THBS4, CAMK2A/B/D, CYCS, CAPNI) were normalised towards wild-type levels in Fiona animals. This work also identified potential mdx markers specific to increased utrophin (DUS3, TPI1) and highlights novel mdx biomarkers (GITR, MYBPC1, HSP60, SIRT2, SMAD3, CNTN1). We define a panel of putative protein mdx biomarkers to evaluate utrophin based strategies which may help to accelerate their translation to the clinic. PMID:28252048

  12. Screening and identification of proteins interacting with nucleostemin

    Institute of Scientific and Technical Information of China (English)

    Hai-Xia Yang; Geng-Lin Jin; Ling Meng; Jian-Zhi Zhang; Wen-Bin Liu; Cheng-Chao Shou

    2005-01-01

    AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS.METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and β-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing.To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively.Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially.RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A.CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

  13. A novel protein complex identification algorithm based on Connected Affinity Clique Extension (CACE).

    Science.gov (United States)

    Li, Peng; He, Tingting; Hu, Xiaohua; Zhao, Junmin; Shen, Xianjun; Zhang, Ming; Wang, Yan

    2014-06-01

    A novel algorithm based on Connected Affinity Clique Extension (CACE) for mining overlapping functional modules in protein interaction network is proposed in this paper. In this approach, the value of protein connected affinity which is inferred from protein complexes is interpreted as the reliability and possibility of interaction. The protein interaction network is constructed as a weighted graph, and the weight is dependent on the connected affinity coefficient. The experimental results of our CACE in two test data sets show that the CACE can detect the functional modules much more effectively and accurately when compared with other state-of-art algorithms CPM and IPC-MCE.

  14. Identification of Antigenic Proteins of the Nosocomial Pathogen Klebsiella pneumoniae

    Science.gov (United States)

    Hoppe, Sebastian; Bier, Frank F.; von Nickisch-Rosenegk, Markus

    2014-01-01

    The continuous expansion of nosocomial infections around the globe has become a precarious situation. Key challenges include mounting dissemination of multiple resistances to antibiotics, the easy transmission and the growing mortality rates of hospital-acquired bacterial diseases. Thus, new ways to rapidly detect these infections are vital. Consequently, researchers around the globe pursue innovative approaches for point-of-care devices. In many cases the specific interaction of an antigen and a corresponding antibody is pivotal. However, the knowledge about suitable antigens is lacking. The aim of this study was to identify novel antigens as specific diagnostic markers. Additionally, these proteins might be aptly used for the generation of vaccines to improve current treatment options. Hence, a cDNA-based expression library was constructed and screened via microarrays to detect novel antigens of Klebsiella pneumoniae, a prominent agent of nosocomial infections well-known for its extensive antibiotics resistance, especially by extended-spectrum beta-lactamases (ESBL). After screening 1536 clones, 14 previously unknown immunogenic proteins were identified. Subsequently, each protein was expressed in full-length and its immunodominant character examined by ELISA and microarray analyses. Consequently, six proteins were selected for epitope mapping and three thereof possessed linear epitopes. After specificity analysis, homology survey and 3d structural modelling, one epitope sequence GAVVALSTTFA of KPN_00363, an ion channel protein, was identified harboring specificity for K. pneumoniae. The remaining epitopes showed ambiguous results regarding the specificity for K. pneumoniae. The approach adopted herein has been successfully utilized to discover novel antigens of Campylobacter jejuni and Salmonella enterica antigens before. Now, we have transferred this knowledge to the key nosocomial agent, K. pneumoniae. By identifying several novel antigens and their linear

  15. Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Hung T. [BioMaPS Institute for Quantitative Biology, Rutgers University, Piscataway, New Jersey 08854 (United States); Pabit, Suzette A.; Meisburger, Steve P.; Pollack, Lois [School of Applied and Engineering Physics, Cornell University, Ithaca, New York 14853 (United States); Case, David A., E-mail: case@biomaps.rutgers.edu [BioMaPS Institute for Quantitative Biology, Rutgers University, Piscataway, New Jersey 08854 (United States); Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey 08854 (United States)

    2014-12-14

    A new method is introduced to compute X-ray solution scattering profiles from atomic models of macromolecules. The three-dimensional version of the Reference Interaction Site Model (RISM) from liquid-state statistical mechanics is employed to compute the solvent distribution around the solute, including both water and ions. X-ray scattering profiles are computed from this distribution together with the solute geometry. We describe an efficient procedure for performing this calculation employing a Lebedev grid for the angular averaging. The intensity profiles (which involve no adjustable parameters) match experiment and molecular dynamics simulations up to wide angle for two proteins (lysozyme and myoglobin) in water, as well as the small-angle profiles for a dozen biomolecules taken from the BioIsis.net database. The RISM model is especially well-suited for studies of nucleic acids in salt solution. Use of fiber-diffraction models for the structure of duplex DNA in solution yields close agreement with the observed scattering profiles in both the small and wide angle scattering (SAXS and WAXS) regimes. In addition, computed profiles of anomalous SAXS signals (for Rb{sup +} and Sr{sup 2+}) emphasize the ionic contribution to scattering and are in reasonable agreement with experiment. In cases where an absolute calibration of the experimental data at q = 0 is available, one can extract a count of the excess number of waters and ions; computed values depend on the closure that is assumed in the solution of the Ornstein–Zernike equations, with results from the Kovalenko–Hirata closure being closest to experiment for the cases studied here.

  16. 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...

  17. THE IDENTIFICATION AND CHARACTERIZATION OF AN IGE-INDUCING PROTEIN IN METARHIZIUM ANISOPLIAE EXTRACT

    Science.gov (United States)

    The Identification and Characterization of an IgE-Inducing Protein in Metarhizium anisopliae ExtractMarsha D.W. Ward1, Lisa B. Copeland1, Maura J. Donahue2, and Jody A. Shoemaker31ORD, NHEERL, US EPA, RTP, NC; 2Oak Ridge Institute for Science and Education, Cincinnati...

  18. 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

  19. Accurate flexible fitting of high-resolution protein structures to small-angle x-ray scattering data using a coarse-grained model with implicit hydration shell.

    Science.gov (United States)

    Zheng, Wenjun; Tekpinar, Mustafa

    2011-12-21

    Small-angle x-ray scattering (SAXS) is a powerful technique widely used to explore conformational states and transitions of biomolecular assemblies in solution. For accurate model reconstruction from SAXS data, one promising approach is to flexibly fit a known high-resolution protein structure to low-resolution SAXS data by computer simulations. This is a highly challenging task due to low information content in SAXS data. To meet this challenge, we have developed what we believe to be a novel method based on a coarse-grained (one-bead-per-residue) protein representation and a modified form of the elastic network model that allows large-scale conformational changes while maintaining pseudobonds and secondary structures. Our method optimizes a pseudoenergy that combines the modified elastic-network model energy with a SAXS-fitting score and a collision energy that penalizes steric collisions. Our method uses what we consider a new implicit hydration shell model that accounts for the contribution of hydration shell to SAXS data accurately without explicitly adding waters to the system. We have rigorously validated our method using five test cases with simulated SAXS data and three test cases with experimental SAXS data. Our method has successfully generated high-quality structural models with root mean-squared deviation of 1 ∼ 3 Å from the target structures.

  20. 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.

  1. Identification and characterization of immunogenic proteins of Mycoplasma genitalium

    DEFF Research Database (Denmark)

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

    2006-01-01

    . 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.......0383) and they had significantly higher antibody titers. By use of the rMgPa ELISA, this study further substantiates the importance of M. genitalium as a cause of male urethritis....

  2. Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis.

    Science.gov (United States)

    De Palma, Antonella; Roveri, Antonella; Zaccarin, Mattia; Benazzi, Louise; Daminelli, Simone; Pantano, Giorgia; Buttarello, Mauro; Ursini, Fulvio; Gion, Massimo; Mauri, Pier Luigi

    2010-08-13

    Since red blood cells (RBCs) lack nuclei and organelles, cell membrane is their main load-bearing component and, according to a dynamic interaction with the cytoskeleton compartment, plays a pivotal role in their functioning. Even if erythrocyte membranes are available in large quantities, the low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional 2D gel-based proteomic approaches. So, in order to increase the efficiency of RBC membrane proteome identification, here we took advantage of a simple and reproducible membrane sub-fractionation method coupled to Multidimensional Protein Identification Technology (MudPIT). In addition, the adoption of a stringent RBC filtration strategy from the whole blood, permitted to remove exhaustively contaminants, such as platelets and white blood cells, and to identify a total of 275 proteins in the three RBC membrane fractions collected and analysed. Finally, by means of software for the elaboration of the great quantity of data obtained and programs for statistical analysis and protein classification, it was possible to determine the validity of the entire system workflow and to assign the proper sub-cellular localization and function for the greatest number of the identified proteins.

  3. 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.

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

    Science.gov (United States)

    Connor, Daniel O; Zantow, Jonas; Hust, Michael; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2016-01-01

    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.

  5. Identification of Thylakoid Membrane Protein Complexes by Using a BN-Chip/MS Approach

    Institute of Scientific and Technical Information of China (English)

    Longquan Fan; Yinghong Pan

    2012-01-01

    Thylakoid membrane protein complexes of wheat (Triticum aestivum Linn.)play crucial roles in growth and crop production.Knowledge of the composition and structure of protein complexes,as well as protein interactions,will result in a much deeper understanding of metabolic pathways and cellular processes than protein identities alone,especially if the complexes can be separated in the native forms.Whereas the analysis of membrane protein complexes is a significant challenge due to their hydrophobic properties and relatively low abundance.A rapid and efficient method of identifying membrane protein complexes will greatly facilitate the investigation of agriculture.The present work developed an BN-Chip/MS approach for exhaustive separation and identification of protein complexes,by combining using blue-native polyacrylamide gel electrophoresis (BN-PAGE) and chip-based high-performance liquid chromatography quadruple time-of-flight tandem mass spectrometry (HPLC-Chip/ESI-QT-OF-MS,Chip/MS).By using this approach,seventy-five nonredundant proteins of wheat thylakoid membrane complexes were identified from digested 13 bands of BN-gel.When the protocol of BN separation was not used,only 37 nonredundant proteins had been identified and among of them 9 proteins were uniquely identi? ed.This BN-Chip/MS approach is rapid and efficient for identifying protein complexes in wheat thylakoid membranes,and also providing reliable foundations for further functional research of wheat chloroplast and for identifying protein complexes of other species.

  6. Advances in identification and validation of protein targets of natural products without chemical modification.

    Science.gov (United States)

    Chang, J; Kim, Y; Kwon, H J

    2016-05-04

    Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates.

  7. 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

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-02-01

    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 /sup 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 /sup 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.

  11. Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography.

    Directory of Open Access Journals (Sweden)

    Kevin Wang

    Full Text Available Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC, rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.

  12. Identification of Novel O-Linked Glycosylated Toxoplasma Proteins by Vicia villosa Lectin Chromatography.

    Science.gov (United States)

    Wang, Kevin; Peng, Eric D; Huang, Amy S; Xia, Dong; Vermont, Sarah J; Lentini, Gaelle; Lebrun, Maryse; Wastling, Jonathan M; Bradley, Peter J

    2016-01-01

    Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins.

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

    Directory of Open Access Journals (Sweden)

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

    Directory of Open Access Journals (Sweden)

    Xiuchun Ge

    Full Text Available BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Identification of bitter peptides in whey protein hydrolysate.

    Science.gov (United States)

    Liu, Xiaowei; Jiang, Deshou; Peterson, Devin G

    2014-06-25

    Bitterness of whey protein hydrolysates (WPH) can negatively affect product quality and limit utilization in food and pharmaceutical applications. Four main bitter peptides were identified in a commercial WPH by means of sensory-guided fractionation techniques that included ultrafiltration and offline two-dimensional reverse phase chromatography. LC-TOF-MS/MS analysis revealed the amino acid sequences of the bitter peptides were YGLF, IPAVF, LLF, and YPFPGPIPN that originated from α-lactalbumin, β-lactoglobulin, serum albumin, and β-casein, respectively. Quantitative LC-MS/MS analysis reported the concentrations of YGLF, IPAVF, LLF, and YPFPGPIPN to be 0.66, 0.58, 1.33, and 2.64 g/kg powder, respectively. Taste recombination analysis of an aqueous model consisting of all four peptides was reported to explain 88% of the bitterness intensity of the 10% WPH solution.

  17. Identification of host proteins associated with HIV-1 preintegration complexes isolated from infected CD4+ cells.

    Science.gov (United States)

    Raghavendra, Nidhanapati K; Shkriabai, Nikolozi; Graham, Robert Lj; Hess, Sonja; Kvaratskhelia, Mamuka; Wu, Li

    2010-08-11

    An integrated HIV-1 genomic DNA leads to an infected cell becoming either an active or a latent virus-producing cell. Upon appropriate activation, a latently infected cell can result in production of progeny viruses that spread the infection to uninfected cells. The host proteins influence several steps of HIV-1 infection including formation of the preintegration complex (PIC), a key nucleoprotein intermediate essential for integration of reverse transcribed viral DNA into the chromosome. Much effort has gone into the identification of host proteins contributing to the assembly of functional PICs. Experimental approaches included the use of yeast two-hybrid system, co-immunoprecipitation, affinity tagged HIV-1 viral proteins and in vitro reconstitution of salt-stripped PIC activity. Several host proteins identified using these approaches have been shown to affect HIV-1 replication in cells and influence catalytic activities of recombinant IN in vitro. However, the comprehensive identification and characterization of host proteins associated with HIV-1 PICs of infected cells have been hindered in part by the technical limitation in acquiring sufficient amount of catalytically active PICs. To efficiently identify additional host factors associated with PICs in infected cells, we have developed the following novel approach. The catalytically active PICs from HIV-1-infected CD4+ cells were isolated using biotinylated target DNA, and the proteins selectively co-purifying with PICs have been analyzed by mass spectrometry. This technology enabled us to reveal at least 19 host proteins that are associated with HIV-1 PICs, of which 18 proteins have not been described previously with respect to HIV-1 integration. Physiological functions of the identified proteins range from chromatin organization to protein transport. A detailed characterization of these host proteins could provide new insights into the mechanism of HIV-1 integration and uncover new antiviral targets to

  18. Identification of Protein Palmitoylation Inhibitors from a Scaffold Ranking Library

    Science.gov (United States)

    Hamel, Laura D.; Lenhart, Brian J.; Mitchell, David A.; Santos, Radleigh G.; Giulianotti, Marc A.; Deschenes, Robert J.

    2016-01-01

    The addition of palmitoyl moieties to proteins regulates their membrane targeting, subcellular localization, and stability. Dysregulation of the enzymes which catalyzed the palmitoyl addition and/or the substrates of these enzymes have been linked to cancer, cardiovascular, and neurological disorders, implying these enzymes and substrates are valid targets for pharmaceutical intervention. However, current chemical modulators of zDHHC PAT enzymes lack specificity and affinity, underscoring the need for screening campaigns to identify new specific, high affinity modulators. This report describes a mixture based screening approach to identify inhibitors of Erf2 activity. Erf2 is the Saccharomyces cerevisiae PAT responsible for catalyzing the palmitoylation of Ras2, an ortholog of the human Ras oncogene proteins. A chemical library developed by the Torrey Pines Institute for Molecular Studies consists of more than 30 million compounds designed around 68 molecular scaffolds that are systematically arranged into positional scanning and scaffold ranking formats. We have used this approach to identify and characterize several scaffold backbones and R-groups that reduce or eliminate the activity of Erf2 in vitro. Here, we present the analysis of one of the scaffold backbones, bis-cyclic piperazine. We identified compounds that inhibited Erf2 auto-palmitoylation activity using a fluorescence-based, coupled assay in a high throughput screening (HTS) format and validated the hits utilizing an orthogonal gel-based assay. Finally, we examined the effects of the compounds on cell growth in a yeast cell-based assay. Based on our results, we have identified specific, high affinity palmitoyl transferase inhibitors that will serve as a foundation for future compound design. PMID:27009891

  19. Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine.

    Science.gov (United States)

    Yang, Yanling; Wang, Lin; Yin, Jigang; Wang, Xinglong; Cheng, Shipeng; Lang, Xulong; Wang, Xiuran; Qu, Hailong; Sun, Chunhui; Wang, Jinglong; Zhang, Rui

    2011-10-01

    In order to screen immunogenic candidate antigens for the development of a brucellosis subunit vaccine, an immunoproteomic assay was used to identify immunogenic proteins from Brucella melitensis 16 M soluble proteins. In this study, a total of 56 immunodominant proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem mass spectrometry (LC-MS/MS). Two proteins of interest, riboflavin synthase alpha chain (RS-α) and Loraine synthase (LS-2), which are both involved in riboflavin synthesis, were detected by two-dimensional immunoblots using antisera obtained from Brucella-infected human and goats. LS-2, however, is an already well-known vaccine candidate. Therefore, we focussed our studies on the novel vaccine candidate RS-α. B. melitensis RS-α and LS-2 were then expressed in Escherichia coli as fusion proteins with His tag. The humoral and cellular immune responses to the recombinant (r)RS-α was characterized. In response to in vitro stimulation by rRS-α, splenocytes from mice vaccinated with rRS-α were able to produce γ-interferon (IFN-γ) and interleukin (IL)-2 but not interleukin (IL)-4 and interleukin (IL)-10. Furthermore, rRS-α or rLS-2-vaccinated mice were partially protected against B. melitensis infection. Our results suggested that we have developed a high-throughout, accurate, rapid and highly efficient method for the identification of candidate antigens by a combination of immunoproteomics with immunisation and bacterial challenge and rRs-α could be a useful candidate for the development of subunit vaccines against B. melitensis.

  20. 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...... of the proteins in the fluorescent spots excised directly from unstained 2DE gels provides sequence information that might be sufficient to design degenerate primers for gene cloning. Identified fluorescent proteins are in agreement with the coral species determined by visual examination of the samples....... The presented methodology is a viable alternative to direct gene cloning for the discovery of novel fluorescent proteins and will be further validated on other samples collected during the Galathea 3 expedition....

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

    Science.gov (United States)

    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.

  2. 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.

  3. A novel spectral library workflow to enhance protein identifications.

    Science.gov (United States)

    Li, Haomin; Zong, Nobel C; Liang, Xiangbo; Kim, Allen K; Choi, Jeong Ho; Deng, Ning; Zelaya, Ivette; Lam, Maggie; Duan, Huilong; Ping, Peipei

    2013-04-09

    The innovations in mass spectrometry-based investigations in proteome biology enable systematic characterization of molecular details in pathophysiological phenotypes. However, the process of delineating large-scale raw proteomic datasets into a biological context requires high-throughput data acquisition and processing. A spectral library search engine makes use of previously annotated experimental spectra as references for subsequent spectral analyses. This workflow delivers many advantages, including elevated analytical efficiency and specificity as well as reduced demands in computational capacity. In this study, we created a spectral matching engine to address challenges commonly associated with a library search workflow. Particularly, an improved sliding dot product algorithm, that is robust to systematic drifts of mass measurement in spectra, is introduced. Furthermore, a noise management protocol distinguishes spectra correlation attributed from noise and peptide fragments. It enables elevated separation between target spectral matches and false matches, thereby suppressing the possibility of propagating inaccurate peptide annotations from library spectra to query spectra. Moreover, preservation of original spectra also accommodates user contributions to further enhance the quality of the library. Collectively, this search engine supports reproducible data analyses using curated references, thereby broadening the accessibility of proteomics resources to biomedical investigators. This article is part of a Special Issue entitled: From protein structures to clinical applications.

  4. Normalization with genes encoding ribosomal proteins but not GAPDH provides an accurate quantification of gene expressions in neuronal differentiation of PC12 cells

    Directory of Open Access Journals (Sweden)

    Lim Qing-En

    2010-01-01

    Full Text Available Abstract Background Gene regulation at transcript level can provide a good indication of the complex signaling mechanisms underlying physiological and pathological processes. Transcriptomic methods such as microarray and quantitative real-time PCR require stable reference genes for accurate normalization of gene expression. Some but not all studies have shown that housekeeping genes (HGKs, β-actin (ACTB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, which are routinely used for normalization, may vary significantly depending on the cell/tissue type and experimental conditions. It is currently unclear if these genes are stably expressed in cells undergoing drastic morphological changes during neuronal differentiation. Recent meta-analysis of microarray datasets showed that some but not all of the ribosomal protein genes are stably expressed. To test the hypothesis that some ribosomal protein genes can serve as reference genes for neuronal differentiation, a genome-wide analysis was performed and putative reference genes were identified based on stability of expressions. The stabilities of these potential reference genes were then analyzed by reverse transcription quantitative real-time PCR in six differentiation conditions. Results Twenty stably expressed genes, including thirteen ribosomal protein genes, were selected from microarray analysis of the gene expression profiles of GDNF and NGF induced differentiation of PC12 cells. The expression levels of these candidate genes as well as ACTB and GAPDH were further analyzed by reverse transcription quantitative real-time PCR in PC12 cells differentiated with a variety of stimuli including NGF, GDNF, Forskolin, KCl and ROCK inhibitor, Y27632. The performances of these candidate genes as stable reference genes were evaluated with two independent statistical approaches, geNorm and NormFinder. Conclusions The ribosomal protein genes, RPL19 and RPL29, were identified as suitable reference genes

  5. Development of BIATECH-54 standard mixtures for assessment of protein identification and relative expression.

    Science.gov (United States)

    Kolker, Eugene; Hogan, Jason M; Higdon, Roger; Kolker, Natali; Landorf, Elizabeth; Yakunin, Alexander F; Collart, Frank R; van Belle, Gerald

    2007-10-01

    Mixtures of known proteins have been very useful in the assessment and validation of methods for high-throughput (HTP) MS (MS/MS) proteomics experiments. However, these test mixtures have generally consisted of few proteins at near equal concentration or of a single protein at varied concentrations. Such mixtures are too simple to effectively assess the validity of error rates for protein identification and differential expression in HTP MS/MS studies. This work aimed at overcoming these limitations and simulating studies of complex biological samples. We introduced a pair of 54-protein standard mixtures of variable concentrations with up to a 1000-fold dynamic range in concentration and up to ten-fold expression ratios with additional negative controls (infinite expression ratios). These test mixtures comprised 16 off-the-shelf Sigma-Aldrich proteins and 38 Shewanella oneidensis proteins produced in-house. The standard proteins were systematically distributed into three main concentration groups (high, medium, and low) and then the concentrations were varied differently for each mixture within the groups to generate different expression ratios. The mixtures were analyzed with both low mass accuracy LCQ and high mass accuracy FT-LTQ instruments. In addition, these 54 standard proteins closely follow the molecular weight distributions of both bacterial and human proteomes. As a result, these new standard mixtures allow for a much more realistic assessment of approaches for protein identification and label-free differential expression than previous mixtures. Finally, methodology and experimental design developed in this work can be readily applied in future to development of more complex standard mixtures for HTP proteomics studies.

  6. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrate

    DEFF Research Database (Denmark)

    Grønborg, Mads; Kristiansen, Troels Zakarias; Stensballe, Allan;

    2002-01-01

    proteins from untreated cells or those treated with calyculin A, a serine/threonine phosphatase inhibitor. Mass spectrometry-based analysis of bands from one-dimensional gels that were specifically observed in calyculin A-treated samples resulted in identification of several known serine...

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

    Directory of Open Access Journals (Sweden)

    Margaret Rohrbaugh

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

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

    Science.gov (United States)

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

    2016-12-01

    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.

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

    Science.gov (United States)

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

    2007-09-04

    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 alpha-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 alpha-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 alpha-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.

  10. Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening.

    Science.gov (United States)

    Han, Jin-Hee; Li, Jian; Wang, Bo; Lee, Seong-Kyun; Nyunt, Myat Htut; Na, Sunghun; Park, Jeong-Hyun; Han, Eun-Taek

    2015-08-01

    Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

  11. Protein feature based identification of cell cycle regulated proteins in yeast

    DEFF Research Database (Denmark)

    de Lichtenberg, Ulrik; Jensen, Thomas Skøt; Jensen, Lars Juhl;

    2003-01-01

    DNA microarrays have been used extensively to identify cell cycle regulated genes in yeast; however, the overlap in the genes identified is surprisingly small. We show that certain protein features can be used to distinguish cell cycle regulated genes from other genes with high confidence (features...... include protein phosphorylation, glycosylation, subcellular location and instability/degradation). We demonstrate that co-expressed, periodic genes encode proteins which share combinations of features, and provide an overview of the proteome dynamics during the cycle. A large set of novel putative cell...... cycle regulated proteins were identified, many of which have no known function....

  12. 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.

  13. Fast and accurate method for identifying high-quality protein-interaction modules by clique merging and its application to yeast.

    Science.gov (United States)

    Zhang, Chi; Liu, Song; Zhou, Yaoqi

    2006-04-01

    Molecular networks in cells are organized into functional modules, where genes in the same module interact densely with each other and participate in the same biological process. Thus, identification of modules from molecular networks is an important step toward a better understanding of how cells function through the molecular networks. Here, we propose a simple, automatic method, called MC(2), to identify functional modules by enumerating and merging cliques in the protein-interaction data from large-scale experiments. Application of MC(2) to the S. cerevisiae protein-interaction data produces 84 modules, whose sizes range from 4 to 69 genes. The majority of the discovered modules are significantly enriched with a highly specific process term (at least 4 levels below root) and a specific cellular component in Gene Ontology (GO) tree. The average fraction of genes with the most enriched GO term for all modules is 82% for specific biological processes and 78% for specific cellular components. In addition, the predicted modules are enriched with coexpressed proteins. These modules are found to be useful for annotating unknown genes and uncovering novel functions of known genes. MC(2) is efficient, and takes only about 5 min to identify modules from the current yeast gene interaction network with a typical PC (Intel Xeon 2.5 GHz CPU and 512 MB memory). The CPU time of MC(2) is affordable (12 h) even when the number of interactions is increased by a factor of 10. MC(2) and its results are publicly available on http://theory.med.buffalo.edu/MC2.

  14. 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.

  15. Identification of phosphorylation sites in the nucleocapsid protein (N protein) of SARS-coronavirus

    Science.gov (United States)

    Lin, Liang; Shao, Jianmin; Sun, Maomao; Liu, Jinxiu; Xu, Gongjin; Zhang, Xumin; Xu, Ningzhi; Wang, Rong; Liu, Siqi

    2007-12-01

    After decoding the genome of SARS-coronavirus (SARS-CoV), next challenge is to understand how this virus causes the illness at molecular bases. Of the viral structural proteins, the N protein plays a pivot role in assembly process of viral particles as well as viral replication and transcription. The SARS-CoV N proteins expressed in the eukaryotes, such as yeast and HEK293 cells, appeared in the multiple spots on two-dimensional electrophoresis (2DE), whereas the proteins expressed in E. coli showed a single 2DE spotE These 2DE spots were further examined by Western blot and MALDI-TOF/TOF MS, and identified as the N proteins with differently apparent pI values and similar molecular mass of 50 kDa. In the light of the observations and other evidences, a hypothesis was postulated that the SARS-CoV N protein could be phosphorylated in eukaryotes. To locate the plausible regions of phosphorylation in the N protein, two truncated N proteins were generated in E. coli and treated with PKC[alpha]. The two truncated N proteins after incubation of PKC[alpha] exhibited the differently electrophoretic behaviors on 2DE, suggesting that the region of 1-256 aa in the N protein was the possible target for PKC[alpha] phosphorylation. Moreover, the SARS-CoV N protein expressed in yeast were partially digested with trypsin and carefully analyzed by MALDI-TOF/TOF MS. In contrast to the completely tryptic digestion, these partially digested fragments generated two new peptide mass signals with neutral loss, and MS/MS analysis revealed two phosphorylated peptides located at the "dense serine" island in the N protein with amino acid sequences, GFYAEGSRGGSQASSRSSSR and GNSGNSTPGSSRGNSPARMASGGGK. With the PKC[alpha] phosphorylation treatment and the partially tryptic digestion, the N protein expressed in E. coli released the same peptides as observed in yeast cells. Thus, this investigation provided the preliminary data to determine the phosphorylation sites in the SARS-CoV N protein, and

  16. Identification of a novel hypocholesterolemic protein, major royal jelly protein 1, derived from royal jelly.

    Directory of Open Access Journals (Sweden)

    Yuri Kashima

    Full Text Available Royal jelly (RJ intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1, MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1 mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats.

  17. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae

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    Krehenbrink Martin

    2008-01-01

    Full Text Available Abstract Background Proteins secreted by bacteria play an important role in infection of eukaryotic hosts. Rhizobia infect the roots of leguminous plants and establish a mutually beneficial symbiosis. Proteins secreted during the infection process by some rhizobial strains can influence infection and modify the plant defence signalling pathways. The aim of this study was to systematically analyse protein secretion in the recently sequenced strain Rhizobium leguminosarum bv. viciae 3841. Results Similarity searches using defined protein secretion systems from other Gram-negative bacteria as query sequences revealed that R. l. bv. viciae 3841 has ten putative protein secretion systems. These are the general export pathway (GEP, a twin-arginine translocase (TAT secretion system, four separate Type I systems, one putative Type IV system and three Type V autotransporters. Mutations in genes encoding each of these (except the GEP were generated, but only mutations affecting the PrsDE (Type I and TAT systems were observed to affect the growth phenotype and the profile of proteins in the culture supernatant. Bioinformatic analysis and mass fingerprinting of tryptic fragments of culture supernatant proteins identified 14 putative Type I substrates, 12 of which are secreted via the PrsDE, secretion system. The TAT mutant was defective for the symbiosis, forming nodules incapable of nitrogen fixation. Conclusion None of the R. l. bv. viciae 3841 protein secretion systems putatively involved in the secretion of proteins to the extracellular space (Type I, Type IV, Type V is required for establishing the symbiosis with legumes. The PrsDE (Type I system was shown to be the major route of protein secretion in non-symbiotic cells and to secrete proteins of widely varied size and predicted function. This is in contrast to many Type I systems from other bacteria, which typically secrete specific substrates encoded by genes often localised in close proximity to

  18. Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function.

    Directory of Open Access Journals (Sweden)

    Maura Brioschi

    Full Text Available Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF. The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14 and non-failing human hearts (n = 13 were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS, the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01. We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK, whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF.

  19. Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function.

    Science.gov (United States)

    Brioschi, Maura; Polvani, Gianluca; Fratto, Pasquale; Parolari, Alessandro; Agostoni, Piergiuseppe; Tremoli, Elena; Banfi, Cristina

    2012-01-01

    Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14) and non-failing human hearts (n = 13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF.

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

    Institute of Scientific and Technical Information of China (English)

    Beuy Joob; Viroj Wiwanitkit

    2014-01-01

    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.

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

    Institute of Scientific and Technical Information of China (English)

    Beuy; Joob; Viroj; Wiwanitkit

    2014-01-01

    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 draggability within envelope glycoprotein GP2 from Ebola virus was done.According to this assessment,7 active pockets with varied draggability could be identified.

  2. Bioinformatics-Based Identification of Chemosensory Proteins in African Malaria Mosquito, Anopheles gambiae

    Institute of Scientific and Technical Information of China (English)

    Zhengxi Li; Zuorui Shen; Jingjiang Zhou; Lin Field

    2003-01-01

    Chemosensory proteins (CSPs) are identifiable by four spatially conserved Cysteine residues in their primary structure or by two disulfide bridges in their tertiary structure according to the previously identified olfactory specific-D related proteins. A genomics- and bioinformatics-based approach is taken in the present study to identify the putative CSPs in the malaria-carrying mosquito, Anopheles gambiae. The results show that five out of the nine annotated candidates are the most possible Anopheles CSPs of A. gambiae. This study lays the foundation for further functional identification of Anopheles CSPs, though all of these candidates need additional experimental verification.

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

    Energy Technology Data Exchange (ETDEWEB)

    Agarwal, Pratul K.

    2015-11-24

    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.

  4. 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.

  5. 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.

  6. Development of a Rapid and Accurate Identification Method for Citrobacter Species Isolated from Pork Products Using a Matrix-Assisted Laser-Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

    Science.gov (United States)

    Kwak, Hye-Lim; Han, Sun-Kyung; Park, Sunghoon; Park, Si Hong; Shim, Jae-Yong; Oh, Mihwa; Ricke, Steven C; Kim, Hae-Yeong

    2015-09-01

    Previous detection methods for Citrobacter are considered time consuming and laborious. In this study, we have developed a rapid and accurate detection method for Citrobacter species in pork products, using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). A total of 35 Citrobacter strains were isolated from 30 pork products and identified by both MALDI-TOF MS and 16S rRNA gene sequencing approaches. All isolates were identified to the species level by the MALDI-TOF MS, while 16S rRNA gene sequencing results could not discriminate them clearly. These results confirmed that MALDI-TOF MS is a more accurate and rapid detection method for the identification of Citrobacter species.

  7. Identification of proteins that interact with murine cytomegalovirus early protein M112-113 in brain

    Institute of Scientific and Technical Information of China (English)

    WANG Hui; LIU Xing-lou; SHU Sai-nan; HUANG Yong-jian; FANG Feng

    2011-01-01

    Background Murine cytomegalovirus (MCMV) early protein M112-113 is involved in viral DNA replication and believed to play a crucial role in the viral pathogenesis.To investigate the biological function of M112-113 protein in the pathogenesis of the brain disorders caused by cytomegalovirus (CMV),a screening for proteins interacting with M112-113 was performed by a yeast two-hybrid system.Methods Bait plasmid pGBKT7-M112-113 was constructed and transformed into AH109 yeast.After confirmation of the expression of MCMV M112-113 in yeast,the bait yeast was mated with a prey yeast containing mouse brain cDNA library plasmid to screen the proteins interacting with M112-113.Interactions between M112-113 and the obtained proteins were verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.Results Two proteins interacting with M112-113 were identified,including metastasis-associated 1 (MTA1) and zinc finger,CCHC domain containing 18 (ZCCHC18).M112-113 protein could interact with MTA1 or ZCCHC18 in yeast and mammalian cells.Conclusion The interactions of M112-113 with MTA1 or ZCCHC18 may be related to the pathogenesis of MCMV-associated disease in central nervous system.

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

    Directory of Open Access Journals (Sweden)

    Wenhan Zhu

    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. Identification of obscure yet conserved actin-associated proteins in Giardia lamblia.

    Science.gov (United States)

    Paredez, Alexander R; Nayeri, Arash; Xu, Jennifer W; Krtková, Jana; Cande, W Zacheus

    2014-06-01

    Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton.

  10. 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

  11. Rapid and accurate identification of species belonging to the Candida parapsilosis complex by real-time PCR and melting curve analysis.

    Science.gov (United States)

    Hays, Constantin; Duhamel, Chantal; Cattoir, Vincent; Bonhomme, Julie

    2011-04-01

    Candida parapsilosis is the second most frequent Candida species isolated from blood cultures. Since 2005, C. parapsilosis has been divided into three distinct species based on genetic traits: Candida parapsilosis, Candida metapsilosis and Candida orthopsilosis. The aim of this study was to develop a rapid real-time PCR assay able to distinguish these closely related species via a melting curve analysis. This identification method was optimized by using reference strains and well-characterized clinical isolates of Candida species. A single set of consensus primers was designed to amplify a 184 bp portion of the SADH gene in order to identify species based on the unique melt profile resulting from DNA sequence variations from each species of the complex. PCR products were detected with SYBR Green fluorescent dye and identification was established by melting curve analysis. For validation of the technique, a total of 116 clinical isolates, phenotypically identified as C. parapsilosis, were tested by real-time PCR and results were further compared with PCR-RFLP patterns of the SADH gene, used as the reference method. The melting curve analysis of amplified DNA could differentiate between C. parapsilosis (83.5 °C), C. metapsilosis (82.9 °C) and C. orthopsilosis (82.1 °C), with a sensitivity and specificity comparable to those of the reference method. One hundred and fourteen C. parapsilosis and two C. orthopsilosis isolates were identified among the clinical isolates. This method provides a simple, rapid and reliable identification of species belonging to the C. parapsilosis complex. This novel approach could be helpful for clinical and epidemiological investigations.

  12. Application of an Accurate and Validated Method for Identification and Quantification of Acrylamide in Bread, Biscuits and Other Bakery Products Using GC-MS/MS System

    OpenAIRE

    Negoiță,Mioara; Culețu,Alina

    2016-01-01

    A gas chromatography tandem mass spectrometry has been developed and validated for the separation, detection, identification and quantification of acrylamide in bread, biscuits and similar products. The method showed good precision with values lower than 6%. A good sensitivity was achieved for bread with 2.41 and 7.23 µg kg-1 limit of detection (LOD) and limit of quantification (LOQ), respectively, while for biscuits, LOD and LOQ were 4.63 and 13.89 µg kg-1, respectively. Accuracy obtained th...

  13. 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.

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

    Science.gov (United States)

    Chen, Jing; Canales, Lorena; Neal, Rachel E

    2011-07-07

    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.

  15. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    Science.gov (United States)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  16. Classification and Identification of Plant Fibrous Material with Different Species Using near Infrared Technique—A New Way to Approach Determining Biomass Properties Accurately within Different Species

    Science.gov (United States)

    Jiang, Wei; Zhou, Chengfeng; Han, Guangting; Via, Brian; Swain, Tammy; Fan, Zhaofei; Liu, Shaoyang

    2017-01-01

    Plant fibrous material is a good resource in textile and other industries. Normally, several kinds of plant fibrous materials used in one process are needed to be identified and characterized in advance. It is easy to identify them when they are in raw condition. However, most of the materials are semi products which are ground, rotted or pre-hydrolyzed. To classify these samples which include different species with high accuracy is a big challenge. In this research, both qualitative and quantitative analysis methods were chosen to classify six different species of samples, including softwood, hardwood, bast, and aquatic plant. Soft Independent Modeling of Class Analogy (SIMCA) and partial least squares (PLS) were used. The algorithm to classify different species of samples using PLS was created independently in this research. Results found that the six species can be successfully classified using SIMCA and PLS methods, and these two methods show similar results. The identification rates of kenaf, ramie and pine are 100%, and the identification rates of lotus, eucalyptus and tallow are higher than 94%. It is also found that spectra loadings can help pick up best wavenumber ranges for constructing the NIR model. Inter material distance can show how close between two species. Scores graph is helpful to choose the principal components numbers during the model construction. PMID:28105037

  17. Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

    Science.gov (United States)

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-03-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome

  18. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins

    Science.gov (United States)

    Tomar, Anil Kumar; Sooch, Balwinder Singh; Raj, Isha; Singh, Sarman; Singh, Tej P.; Yadav, Savita

    2011-01-01

    Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma. PMID:22182811

  19. Isolation and Identification of Concanavalin A Binding Glycoproteins from Human Seminal Plasma: A Step Towards Identification of Male Infertility Marker Proteins

    Directory of Open Access Journals (Sweden)

    Anil Kumar Tomar

    2011-01-01

    Full Text Available Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma.

  20. YahO protein as a calibrant for top-down proteomic identification of Shiga toxin using MALDI-TOF-TOF-MS/MS and post-source decay

    Science.gov (United States)

    Matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF-TOF) mass spectrometry is increasingly utilized for rapid top-down proteomic identification of proteins. This identification may involve analysis of either a pure protein or a protein mixture. For analysis of a pure protein...

  1. Proteomics meets blood banking: identification of protein targets for the improvement of platelet quality.

    Science.gov (United States)

    Schubert, Peter; Devine, Dana V

    2010-01-01

    Proteomics has brought new perspectives to the fields of hematology and transfusion medicine in the last decade. The steady improvement of proteomic technology is propelling novel discoveries of molecular mechanisms by studying protein expression, post-translational modifications and protein interactions. This review article focuses on the application of proteomics to the identification of molecular mechanisms leading to the deterioration of blood platelets during storage - a critical aspect in the provision of platelet transfusion products. Several proteomic approaches have been employed to analyse changes in the platelet protein profile during storage and the obtained data now need to be translated into platelet biochemistry in order to connect the results to platelet function. Targeted biochemical applications then allow the identification of points for intervention in signal transduction pathways. Once validated and placed in a transfusion context, these data will provide further understanding of the underlying molecular mechanisms leading to platelet storage lesion. Future aspects of proteomics in blood banking will aim to make use of protein markers identified for platelet storage lesion development to monitor proteome changes when alterations such as the use of additive solutions or pathogen reduction strategies are put in place in order to improve platelet quality for patients.

  2. Identification of proteins associated with Mycobacterium tuberculosis virulence pathway by their polar profile.

    Science.gov (United States)

    Polanco, Carlos; Castañón-González, Jorge Alberto; Mancilla, Raul; Buhse, Thomas; Samaniego, José Lino; Gimbel, Arturo

    2015-01-01

    With almost one third of the world population infected, tuberculosis is one of the most devastating diseases worldwide and it is a major threat to any healthcare system. With the mathematical-computational method named "Polarity Index Method", already published by this group, we identified, with high accuracy (70%), proteins related to Mycobacterium tuberculosis bacteria virulence pathway from the Tuberculist Database. The test considered the totality of proteins cataloged in the main domains: fungi, bacteria, and viruses from three databases: Antimicrobial Peptide Database (APD2), Tuberculist Database, Uniprot Database, and four antigens of Mycobacterium tuberculosis: PstS-1, 38-kDa, 19-kDa, and H37Rv ORF. The method described was calibrated with each database to achieve the same performance, showing a high percentage of coincidence in the identification of proteins associated with Mycobacterium tuberculosis bacteria virulence pathway located in the Tuberculist Database, and identifying a polar pattern regardless of the group studied. This method has already been used in the identification of diverse groups of proteins and peptides, showing that it is an effective discriminant. Its metric considers only one physico-chemical property, i.e. polarity.

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

    Directory of Open Access Journals (Sweden)

    Tokuko Iwamori

    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.

  4. Identification and Characterization of RBM44 as a Novel Intercellular Bridge Protein

    Science.gov (United States)

    Iwamori, Tokuko; Lin, Yi-Nan; Ma, Lang; Iwamori, Naoki; Matzuk, Martin M.

    2011-01-01

    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. PMID:21364893

  5. 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.

  6. 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.

  7. Identification of an immunogenic protein of Giardia lamblia using monoclonal antibodies generated from infected mice

    Directory of Open Access Journals (Sweden)

    Jael Quintero

    2013-08-01

    Full Text Available The humoral immune response plays an important role in the clearance of Giardia lamblia. However, our knowledge about the specific antigens of G. lamblia that induce a protective immune response is limited. The purpose of this study was to identify and characterise the immunogenic proteins of G. lamblia in a mouse model. We generated monoclonal antibodies (moAbs specific to G. lamblia (1B10, 2C9.D11, 3C10.E5, 3D10, 5G8.B5, 5F4, 4C7, 3C5 and 3C6 by fusing splenocytes derived from infected mice. Most of these moAbs recognised a band of ± 71 kDa (5G8 protein and this protein was also recognised by serum from the infected mice. We found that the moAbs recognised conformational epitopes of the 5G8 protein and that this antigen is expressed on the cell surface and inside trophozoites. Additionally, antibodies specific to the 5G8 protein induced strong agglutination (> 70-90% of trophozoites. We have thus identified a highly immunogenic antigen of G. lamblia that is recognised by the immune system of infected mice. In summary, this study describes the identification and partial characterisation of an immunogenic protein of G. lamblia. Additionally, we generated a panel of moAbs specific for this protein that will be useful for the biochemical and immunological characterisation of this immunologically interesting Giardia molecule.

  8. Identification and characterization of a microneme protein (NcMIC6) in Neospora caninum.

    Science.gov (United States)

    Li, Wensheng; Liu, Jing; Wang, Jing; Fu, Yong; Nan, Huizhu; Liu, Qun

    2015-08-01

    Neospora caninum, an Apicomplexa parasite, is the causative agent of neosporosis. As described for other members of Apicomplexa, microneme proteins (MICs) play a key role in attachment and invasion of host cells by N. caninum. Herein we identified N. caninum microneme protein 6 (NcMIC6) that is orthologous to Toxoplasma gondii microneme protein 6 (TgMIC6). The open reading frame of the NcMIC6 gene is 984 bp and encodes a 327 amino acid peptide. Sequence analysis showed that NcMIC6 included a signal peptide, a transmembrane region, three epidermal growth factor-like (EGF) domains, and two low complexity regions. Antibodies raised against recombinant NcMIC6 recognized an approximately 35-kDa native MIC6 protein in Western blots of N. caninum tachyzoites. Immunofluorescence analysis showed that NcMIC6 had a polar labeling pattern, which was consistent with localization of micronemes in the apical region. Pulse invasion assays showed that NcMIC6 translocated from the apical tip to the posterior end of the parasites. Secretion assays demonstrated that NcMIC6 was released into the supernatants. Importantly, it was clearly revealed by co-immunoprecipitation that NcMIC6 formed a complex with other two soluble microneme proteins (NcMIC1 and NcMIC4). In conclusion, identification and characterization of the novel microneme protein NcMIC6 may contribute to understanding how this protein functions during the parasite motility and host cell invasion.

  9. 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.

  10. Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae.

    Science.gov (United States)

    Hammerschmidt, S; Bethe, G; Remane, P H; Chhatwal, G S

    1999-04-01

    Lactoferrin (Lf), an iron-sequestering glycoprotein, predominates in mucosal secretions, where the level of free extracellular iron (10(-18) M) is not sufficient for bacterial growth. This represents a mechanism of resistance to bacterial infections by prevention of colonization of the host by pathogens. In this study we were able to show that Streptococcus pneumoniae specifically recognizes and binds the iron carrier protein human Lf (hLf). Pretreatment of pneumococci with proteases reduced hLf binding significantly, indicating that the hLf receptor is proteinaceous. Binding assays performed with 63 clinical isolates belonging to different serotypes showed that 88% of the tested isolates interacted with hLf. Scatchard analysis showed the existence of two hLf-binding proteins with dissociation constants of 5.7 x 10(-8) and 2.74 x 10(-7) M. The receptors were purified by affinity chromatography, and internal sequence analysis revealed that one of the S. pneumoniae proteins was homologous to pneumococcal surface protein A (PspA). The function of PspA as an hLf-binding protein was confirmed by the ability of purified PspA to bind hLf and to competitively inhibit hLf binding to pneumococci. S. pneumoniae may use the hLf-PspA interaction to overcome the iron limitation at mucosal surfaces, and this might represent a potential virulence mechanism.

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

    DEFF Research Database (Denmark)

    Lund, Christian Have

    GALACTURONOSYLTRANSFERASE1 (GAUT1) and GAUT7 has beesn identified and is essential for pectin biosynthesis. Interestingly, GAUT1 has been shown to be proteolytic processed from its transmembrane anchor domain and its catalytic domain is retained by GAUT7, thus ensuring biosynthesis of HG in the Golgi apparatus. Many...... methods exist in identifying protein-protein interaction (PPI) but many of these are developed for other organisms than plants and are most applicable for PPI detection in other organelles than the Golgi apparatus where pectin biosynthesis occurs. In this work, different PPI detection methods are examined...... for their ability to detect PPI inside the Golgi lumen. The first method tested was the commercially available splitubiquitin system from Dualsystems Biotech AG. This was applied to test binary interactions between proteins involved in HG and Rhamnogalacturonan I (RG-I) biosynthesis (see Manuscript II...

  12. 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.

  13. Enrichment of Functional Redox Reactive Proteins and Identification by Mass Spectrometry Results in Several Terminal Fe(III)-reducing Candidate Proteins in Shewanella oneidensis MR-1.

    Energy Technology Data Exchange (ETDEWEB)

    Elias, Dwayne A.; Yang, Feng; Mottaz, Heather M.; Beliaev, Alex S.; Lipton, Mary S.

    2007-02-01

    Identification of the proteins directly involved in microbial metal-reduction is important to understanding the biochemistry involved in heavy metal reduction/immobilization and the ultimate cleanup of DOE contaminated sites. Although previous strategies for the identification of these proteins have traditionally required laborious protein purification/characterization of metal-reducing capability, activity is often lost before the final purification step, thus creating a significant knowledge gap. In the current study, subcellular fractions of S. oneidensis MR-1 were enriched for Fe(III)-NTA reducing proteins in a single step using several orthogonal column matrices. The protein content of eluted fractions that demonstrated activity were determined by ultra high pressure liquid chromatography coupled with tandem mass spectrometry (LCMS/ MS). A comparison of the proteins identified from active fractions in all separations produced 30 proteins that may act as the terminal electron-accepting protein for Fe(III)-reduction. These include MtrA, MtrB, MtrC and OmcA as well as a number of other proteins not previously associated with Fe(III)-reduction. This is the first report of such an approach where the laborious procedures for protein purification are not required for identification of metal-reducing proteins. Such work provides the basis for a similar approach with other cultured organisms as well as analysis of sediment and groundwater samples from biostimulation efforts at contaminated sites.

  14. Novel small protein identification and quantitative proteomic analysis in Pseudomonas putida KT-­2440

    DEFF Research Database (Denmark)

    Yang, Xiaochen

    .This thesis investigated an industrial bacterium, Pseudomonas putida KT-2440, in two aspects. First, the research focused on discovering novel small proteins (s-proteins) in the bacterium. With large-scale approaches for gene identification, groups of novel s-proteins were identified and validated from...... the genome, transcriptome and proteome of the bacterium. The application of new research approach, ribosome profiling, enabled us to analysis novel open reading frames (ORFs) from different standpoint. Second, by quantitative proteomic approach, the differential expressions of genes were analyzed at proteome...... level under different environmental conditions. The results yield insights intothe adaptation of P. putida KT-2440 in different environments.Based on bioinformatic, proteomic and transcriptomic approaches, global gene expression was analyzed on both transcriptional and translational levels. Our research...

  15. Hidden Markov Models Incorporating Fuzzy Measures and Integrals for Protein Sequence Identification and Alignment

    Institute of Scientific and Technical Information of China (English)

    Niranjan P.Bidargaddi; Madlhu Chetty; Joarder Kamruzzaman

    2008-01-01

    Profile hidden Markov models (HMMs) based on classical HMMs have been widely applied for protein sequence identification. The formulation of the forward and backward variables in profile HMMs is made under statistical independence assumption of the probability theory. We propose a fuzzy profile HMM to overcome the limitations of that assumption and to achieve an improved alignment for protein sequences belonging to a given family. The proposed model fuzzifies the forward and backward variables by incorporating Sugeno fuzzy measures and Choquet integrals, thus further extends the generalized HMM. Based on the fuzzified forwardand backward variables, we propose a fuzzy Baum-Welch parameter estimation al-gorithm for profiles. The strong correlations and the sequence preference involved in the protein structures make this fuzzy architecture based model as a suitable candidate for building profiles of a given family, since the fuzzy set can handle uncertainties better than classical methods.

  16. A method for in vivo identification of bacterial small RNA-binding proteins.

    Science.gov (United States)

    Osborne, Jonathan; Djapgne, Louise; Tran, Bao Quoc; Goo, Young Ah; Oglesby-Sherrouse, Amanda G

    2014-12-01

    Small bacterial regulatory RNAs (sRNAs) have gained immense appreciation over the last decade for their roles in mediating posttranscriptional gene regulation of numerous physiological processes. Several proteins contribute to sRNA stability and regulation, most notably the Hfq RNA-binding protein. However, not all sRNAs rely on Hfq for their stability. It is therefore likely that other proteins contribute to the stability and function of certain bacterial sRNAs. Here, we describe a methodology for identifying in vivo-binding proteins of sRNAs, developed using the iron-responsive PrrF and PrrH sRNAs of Pseudomonas aeruginosa. RNA was isolated from iron-depleted cultures, which were irradiated to cross-link nucleoprotein complexes. Subsequently, PrrF- and PrrH-protein complexes were enriched using cDNA "bait", and enriched RNA-protein complexes were analyzed by tandem mass spectrometry to identify PrrF and PrrH associated proteins. This method identified Hfq as a potential PrrF- and PrrH-binding protein. Interestingly, Hfq was identified more often in samples probed with the PrrF cDNA "bait" as compared to the PrrH cDNA "bait", suggesting Hfq has a stronger binding affinity for the PrrF sRNAs in vivo. Hfq binding to the PrrF and PrrH sRNAs was validated by electrophoretic mobility shift assays with purified Hfq protein from P. aeruginosa. As such, this study demonstrates that in vivo cross-linking coupled with sequence-specific affinity chromatography and tandem mass spectrometry (SSAC-MS/MS) is an effective methodology for unbiased identification of bacterial sRNA-binding proteins.

  17. Accurate Chromosome Segregation at First Meiotic Division Requires AGO4, a Protein Involved in RNA-Dependent DNA Methylation in Arabidopsis thaliana.

    Science.gov (United States)

    Oliver, Cecilia; Santos, Juan Luis; Pradillo, Mónica

    2016-10-01

    The RNA-directed DNA methylation (RdDM) pathway is important for the transcriptional repression of transposable elements and for heterochromatin formation. Small RNAs are key players in this process by regulating both DNA and histone methylation. Taking into account that methylation underlies gene silencing and that there are genes with meiosis-specific expression profiles, we have wondered whether genes involved in RdDM could play a role during this specialized cell division. To address this issue, we have characterized meiosis progression in pollen mother cells from Arabidopsis thaliana mutant plants defective for several proteins related to RdDM. The most relevant results were obtained for ago4-1 In this mutant, meiocytes display a slight reduction in chiasma frequency, alterations in chromatin conformation around centromeric regions, lagging chromosomes at anaphase I, and defects in spindle organization. These abnormalities lead to the formation of polyads instead of tetrads at the end of meiosis, and might be responsible for the fertility defects observed in this mutant. Findings reported here highlight an involvement of AGO4 during meiosis by ensuring accurate chromosome segregation at anaphase I.

  18. Identification of potential molecular associations between chikungunya virus non-structural protein 2 and human host proteins.

    Science.gov (United States)

    Rana, J; Gulati, S; Rajasekharan, S; Gupta, A; Chaudhary, V; Gupta, S

    2017-01-01

    Chikungunya virus (CHIKV) non-structural protein 2 (nsP2) is considered to be the master regulator of viral RNA replication and host responses generated during viral infection. This protein has two main functional domains: an N-terminal domain which exhibits NTPase, RNA triphosphatase and helicase activities and a C-terminal protease domain. Understanding how CHIKV nsP2 interacts with its host proteins is essential for elucidating all the required processes for viral replication and pathogenesis along with the identification of potential targets for antiviral therapy. In current study yeast two-hybrid (Y2H) screening of a human fetal brain cDNA library was performed using nsP2 protein as bait. The analysis identified seven host proteins (CCDC130, CPNE6, POLR2C, MAPK9, EIF4A2, EEF1A1 and EIF3I) as putative interactors of CHIKV nsP2 which were selected for further analysis based on their roles in host cellular machinery. The gene ontology analysis indicates that these proteins are mainly involved in apoptosis, transcription and translational mechanism of host cell. Domain mapping of nsP2 revealed that these associations are not random connections but instead they have functional significance. Further studies to identify the amino acid residues and their chemical interactions that may help in opening new possibilities for preventing these interactions, thus reducing chances of chikungunya infection were performed. This study expands the understanding of CHIKV-host interactions and is important for rational approaches of discovering new antiviral agents.

  19. Identification of maturation and protein synthesis related proteins from porcine oocytes during in vitro maturation

    Directory of Open Access Journals (Sweden)

    Seo Kang

    2011-06-01

    Full Text Available Abstract Background In vitro maturation (IVM of mammalian oocytes is divided into the GV (germinal vesicle stage, MI (metaphase I stage and MII (metaphase II stage stages, and only fully mature oocytes have acquired the ability to be fertilized and initiate zygotic development. These observations have been mostly based on morphological evaluations, but the molecular events governing these processes are not fully understood. The aim of the present study was to better understand the processes involved in the molecular regulation of IVM using 2-DE analysis followed by mass spectrometry to identify proteins that are differentially expressed during oocyte IVM. Result A total of 16 up-regulated and 12 down-regulated proteins were identified. To investigate the IVM process, we specifically focused on the proteins that were up-regulated during the MII stage when compared with the GV stage, which included PRDX 2, GST, SPSY, myomegalin, PED4D, PRKAB 1, and DTNA. These up-regulated proteins were functionally involved in redox regulation and the cAMP-dependent pathway, which are essential for the intracellular signaling involved in oocyte maturation. Interestingly, the PDE4D and its partner, myomegalin, during the MII stage was consistently confirmed up-regulation by western blot analyses. Conclusion These results could be used to better understand some aspects of the molecular mechanisms underlying porcine oocyte maturation. This study identified some regulatory proteins that may have important roles in the molecular events involved in porcine oocyte maturation, particularly with respect to the regulation of oocyte meiotic resumption, MII arrest and oocyte activation. In addition, this study may have beneficial applications not only to basic science with respect to the improvement of oocyte culture conditions but also to mammalian reproductive biotechnology with potential implications.

  20. Accurate identification of UDP-glucuronosyltransferase 1A1 (UGT1A1) inhibitors using UGT1A1-overexpressing HeLa cells.

    Science.gov (United States)

    Sun, Hua; Zhou, Xiaotong; Wu, Baojian

    2015-01-01

    1. UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an irreplaceable role in detoxification of bilirubin and many drugs (e.g., SN-38). Here we aimed to explore the potential of UGT1A1-overexpressing HeLa cells (or HeLa1A1 cells) as a tool to accurately identify UGT1A1 inhibitors. 2. Determination of glucuronidation rates (β-estradiol and SN-38 as the substrates) was performed using HeLa1A1 cells and uridine diphosphoglucuronic acid (UDPGA)-supplemented cDNA expressed UGT1A1 enzyme (or microsomes). The inhibitory effects (IC50 values) of 20 structurally diverse compounds on the UGT1A1 activity were determined using HeLa1A1 cells and microsomal incubations. 3. In HeLa1A1 cells, the IC50 values for inhibition of β-estradiol glucuronidation by the tested compounds ranged from 0.33 to 94.6 µM. In the microsomal incubations, the IC50 values ranged from 0.47 to 155 µM. It was found that the IC50 values of all test compounds derived from the cells were well consistent with those from the microsomes (deviated by less than two-fold). Further, the IC50 values from the cells were strongly correlated with those from microsomes (r = 0.944, p HeLa cells were an appropriate tool to accurately depict the inhibition profiles of chemicals against UGT1A1.

  1. Identification of a 251 gene expression signature that can accurately detect M. tuberculosis in patients with and without HIV co-infection.

    Directory of Open Access Journals (Sweden)

    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. 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.

  3. 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.

  4. 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.

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

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    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.

  6. Closed-loop spontaneous baroreflex transfer function is inappropriate for system identification of neural arc but partly accurate for peripheral arc: predictability analysis.

    Science.gov (United States)

    Kamiya, Atsunori; Kawada, Toru; Shimizu, Shuji; Sugimachi, Masaru

    2011-04-01

    Although the dynamic characteristics of the baroreflex system have been described by baroreflex transfer functions obtained from open-loop analysis, the predictability of time-series output dynamics from input signals, which should confirm the accuracy of system identification, remains to be elucidated. Moreover, despite theoretical concerns over closed-loop system identification, the accuracy and the predictability of the closed-loop spontaneous baroreflex transfer function have not been evaluated compared with the open-loop transfer function. Using urethane and α-chloralose anaesthetized, vagotomized and aortic-denervated rabbits (n = 10), we identified open-loop baroreflex transfer functions by recording renal sympathetic nerve activity (SNA) while varying the vascularly isolated intracarotid sinus pressure (CSP) according to a binary random (white-noise) sequence (operating pressure ± 20 mmHg), and using a simplified equation to calculate closed-loop-spontaneous baroreflex transfer function while matching CSP with systemic arterial pressure (AP). Our results showed that the open-loop baroreflex transfer functions for the neural and peripheral arcs predicted the time-series SNA and AP outputs from measured CSP and SNA inputs, with r2 of 0.8 ± 0.1 and 0.8 ± 0.1, respectively. In contrast, the closed-loop-spontaneous baroreflex transfer function for the neural arc was markedly different from the open-loop transfer function (enhanced gain increase and a phase lead), and did not predict the time-series SNA dynamics (r2; 0.1 ± 0.1). However, the closed-loop-spontaneous baroreflex transfer function of the peripheral arc partially matched the open-loop transfer function in gain and phase functions, and had limited but reasonable predictability of the time-series AP dynamics (r2, 0.7 ± 0.1). A numerical simulation suggested that a noise predominantly in the neural arc under resting conditions might be a possible mechanism responsible for our findings. Furthermore

  7. Polyproline II structure in proteins: identification by chiroptical spectroscopies, stability, and functions.

    Science.gov (United States)

    Bochicchio, Brigida; Tamburro, Antonio Mario

    2002-11-01

    In the last years polyproline II (PPII) structure has been demonstrated to be essential to biological activities such as signal transduction, transcription, cell motility, and immune response. The polyproline left-handed helical structure was nearly unknown until now and often confused with unordered, disordered, irregular, unstructured, extended, or random coil conformations because it is neither alpha-helical nor beta-turn nor beta-sheet, i.e., a classical structure. In spite of the regularity of the PPII structure and, more precisely, its well-defined dihedral angle values, a typical feature of PPII structure is the absence of any intramolecular hydrogen bonds that renders the PPII structure indistinguishable from an irregular backbone structure by (1)H-NMR spectroscopy. The only way to unambiguously reveal PPII structure in solution is to use spectroscopies based on optical activity, such as circular dichroism (CD), vibrational circular dichroism (VCD), and Raman optical activity (ROA). Herein we focus on the identification of PPII structure by CD, widely considered to be the most reliable methodology. Then we report on VCD and ROA spectroscopies as tools in the identification of PPII structure. A third section is dedicated to the analysis of the stabilization of PPII conformation in aqueous solution. Finally, the significance of PPII in self-assembly processes, in elasticity of elastomeric proteins, and in proteins-(peptides) proteins molecular recognition processes are considered.

  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. 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.

  10. 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.

  11. 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

  12. Accurate X-ray position of the Anomalous X-ray Pulsar XTE J1810-197 and identification of its likely IR counterpart

    CERN Document Server

    Israel, G L; Mangano, V; Testa, V; Perna, R; Hummel, W; Mignani, R P; Ageorges, N; Curto, G L; Marco, O; Angelini, L; Campana, S; Covino, S; Marconi, G; Mereghetti, S; Stella, L

    2004-01-01

    We report the accurate sub-arcsec X-ray position of the new Anomalous X-ray Pulsar (AXP) XTE J1810-197, derived with a Chndra-HRC Target of Opportunity observation carried out in November 2003. We also report the discovery of a likely IR counterpart based on a VLT (IR band) Target of Opportunity observation carried out in October 2003. Our proposed counterpart is the only IR source (Ks=20.8) in the X-ray error circle. Its IR colors as well as the X-ray/IR flux ratio, are consistent with those of the counterparts of all other AXPs (at variance with field star colors). Deep Gunn-i band images obtained at the 3.6m ESO telescope detected no sources down to a limiting magnitude of 24.3. Moreover, we find that the pulsed fraction and count rates of XTE J1810-197 remained nearly unchanged since the previous Chandra and XMM-Newton observations (2003 August 27th and September 8th, respectively). We briefly discuss the implications of these results. In particular, we note that the transient (or at least highly variable...

  13. 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.

  14. 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.

  15. Identification of new hematopoietic cell subsets with a polyclonal antibody library specific for neglected proteins.

    Directory of Open Access Journals (Sweden)

    Monica Moro

    Full Text Available The identification of new markers, the expression of which defines new phenotipically and functionally distinct cell subsets, is a main objective in cell biology. We have addressed the issue of identifying new cell specific markers with a reverse proteomic approach whereby approximately 1700 human open reading frames encoding proteins predicted to be transmembrane or secreted have been selected in silico for being poorly known, cloned and expressed in bacteria. These proteins have been purified and used to immunize mice with the aim of obtaining polyclonal antisera mostly specific for linear epitopes. Such a library, made of about 1600 different polyclonal antisera, has been obtained and screened by flow cytometry on cord blood derived CD34+CD45dim cells and on peripheral blood derived mature lymphocytes (PBLs. We identified three new proteins expressed by fractions of CD34+CD45dim cells and eight new proteins expressed by fractions of PBLs. Remarkably, we identified proteins the presence of which had not been demonstrated previously by transcriptomic analysis. From the functional point of view, looking at new proteins expressed on CD34+CD45dim cells, we identified one cell surface protein (MOSC-1 the expression of which on a minority of CD34+ progenitors marks those CD34+CD45dim cells that will go toward monocyte/granulocyte differentiation. In conclusion, we show a new way of looking at the membranome by assessing expression of generally neglected proteins with a library of polyclonal antisera, and in so doing we have identified new potential subsets of hematopoietic progenitors and of mature PBLs.

  16. Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification.

    Science.gov (United States)

    Wright, Erik S; Yilmaz, L Safak; Corcoran, Andrew M; Ökten, Hatice E; Noguera, Daniel R

    2014-08-01

    Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).

  17. Identification of sites of ubiquitination in proteins: a fourier transform ion cyclotron resonance mass spectrometry approach.

    Science.gov (United States)

    Cooper, Helen J; Heath, John K; Jaffray, Ellis; Hay, Ronald T; Lam, Tukiet T; Marshall, Alan G

    2004-12-01

    Structural elucidation of posttranslationally modified peptides and proteins is of key importance in the understanding of an array of biological processes. Ubiquitination is a reversible modification that regulates many cellular functions. Consequences of ubiquitination depend on whether a single ubiquitin or polyubiquitin chain is added to the tagged protein. The lysine residue through which the polyubiquitin chain is formed is also critical for biological activity. Robust methods are therefore required to identify sites of ubiquitination modification, both in the target protein and in ubiquitin. Here, we demonstrate the suitability of Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, in conjunction with activated ion electron capture dissociation (AI ECD) or infrared multiphoton dissociation (IRMPD), for the analysis of ubiquitinated proteins. Polyubiquitinated substrate protein GST-Ubc5 was generated in vitro. Tryptic digests of polyubiquitinated species contain modified peptides in which the ubiquitin C-terminal Gly-Gly residues are retained on the modified lysine residues. Direct infusion microelectrospray FT-ICR of the digest and comparison with an in silico digest enables identification of modified peptides and therefore sites of ubiquitination. Fifteen sites of ubiquitination were identified in GST-Ubc5 and four sites in ubiquitin. Assignments were confirmed by AI ECD or IRMPD. The Gly-Gly modification is stable and both tandem mass spectrometric techniques are suitable, providing extensive sequence coverage and retention of the modification on backbone fragments.

  18. Ambient ionization-accurate mass spectrometry (AMI-AMS) for the identification of nonvisible set-off in food-contact materials.

    Science.gov (United States)

    Bentayeb, Karim; Ackerman, Luke K; Begley, Timothy H

    2012-02-29

    Set-off is the unintentional transfer of substances used in printing from the external printed surface of food packaging to the inner, food-contact surface. Ambient ionization-accurate mass spectrometry (AMI-AMS) detected and identified compounds from print set-off not visible to the human eye. AMI mass spectra from inner and outer surfaces of printed and nonprinted food packaging were compared to detect and identify nonvisible set-off components. A protocol to identify unknowns was developed using a custom open-source database of printing inks and food-packaging compounds. The protocol matched print-related food-contact surface ions with the molecular formulas of common ions, isotopes, and fragments of compounds from the database. AMI-AMS was able to detect print set-off and identify seven different compounds. Set-off on the packaging samples was confirmed using gas chromatographic-mass spectrometric (GC-MS) analysis of single-sided solvent extracts. N-Ethyl-2(and 4)-methylbenzenesulfonamide, 2,4-diphenyl-4-methyl-1(and 2)-pentene, and 2,4,7,9-tetramethyl-5-decyne-4,7-diol were present on the food-contact layer at concentrations from 0.21 to 2.7 ± 1.6 μg dm⁻², corresponding to nearly milligram per kilogram concentrations in the packaged food. Other minor set-off compounds were detected only by AMI-AMS, a fast, simple, and thorough technique to detect and identify set-off in food packaging.

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

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    Cancemi Patrizia

    2010-09-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusions 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

  20. 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.

  1. 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.

  2. A comparative analysis of computational approaches and algorithms for protein subcomplex identification.

    Science.gov (United States)

    Zaki, Nazar; Mora, Antonio

    2014-01-01

    High-throughput AP-MS methods have allowed the identification of many protein complexes. However, most post-processing methods of this type of data have been focused on detection of protein complexes and not its subcomplexes. Here, we review the results of some existing methods that may allow subcomplex detection and propose alternative methods in order to detect subcomplexes from AP-MS data. We assessed and drew comparisons between the use of overlapping clustering methods, methods based in the core-attachment model and our own prediction strategy (TRIBAL). The hypothesis behind TRIBAL is that subcomplex-building information may be concealed in the multiple edges generated by an interaction repeated in different contexts in raw data. The CACHET method offered the best results when the evaluation of the predicted subcomplexes was carried out using both the hypergeometric and geometric scores. TRIBAL offered the best performance when using a strict meet-min score.

  3. The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity.

    Directory of Open Access Journals (Sweden)

    Junfeng Han

    Full Text Available Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D. Thus, it is important to understand how glucagon receptor (GCGR activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO cells, and GCGR complexes were isolated by affinity purification (AP. Complexes were then analyzed by mass spectrometry (MS, and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon

  4. Computational identification of protein methylation sites through bi-profile Bayes feature extraction.

    Directory of Open Access Journals (Sweden)

    Jianlin Shao

    Full Text Available Protein methylation is one type of reversible post-translational modifications (PTMs, which plays vital roles in many cellular processes such as transcription activity, DNA repair. Experimental identification of methylation sites on proteins without prior knowledge is costly and time-consuming. In silico prediction of methylation sites might not only provide researches with information on the candidate sites for further determination, but also facilitate to perform downstream characterizations and site-specific investigations. In the present study, a novel approach based on Bi-profile Bayes feature extraction combined with support vector machines (SVMs was employed to develop the model for Prediction of Protein Methylation Sites (BPB-PPMS from primary sequence. Methylation can occur at many residues including arginine, lysine, histidine, glutamine, and proline. For the present, BPB-PPMS is only designed to predict the methylation status for lysine and arginine residues on polypeptides due to the absence of enough experimentally verified data to build and train prediction models for other residues. The performance of BPB-PPMS is measured with a sensitivity of 74.71%, a specificity of 94.32% and an accuracy of 87.98% for arginine as well as a sensitivity of 70.05%, a specificity of 77.08% and an accuracy of 75.51% for lysine in 5-fold cross validation experiments. Results obtained from cross-validation experiments and test on independent data sets suggest that BPB-PPMS presented here might facilitate the identification and annotation of protein methylation. Besides, BPB-PPMS can be extended to build predictors for other types of PTM sites with ease. For public access, BPB-PPMS is available at http://www.bioinfo.bio.cuhk.edu.hk/bpbppms.

  5. Identification of novel DNA repair proteins via primary sequence, secondary structure, and homology

    Directory of Open Access Journals (Sweden)

    Akutsu Tatsuya

    2009-01-01

    functionally unconfirmed proteins that are highly likely to be involved in the repair process. A new web service, INTREPED, has been made available for the immediate search and annotation of DNA repair proteins in newly sequenced genomes. Conclusion Despite complexity due to a multitude of repair pathways, combinations of sequence, structure, and homology with Support Vector Machines offer good methods in addition to existing homology searches for DNA repair protein identification and functional annotation. Most importantly, this study has uncovered relationships between the size of a genome and a genome's available repair repetoire, and offers a number of new predictions as well as a prediction service, both which reduce the search time and cost for novel repair genes and proteins.

  6. Isolation and Identification of Alicyclobacillus with High Dipicolinic Acid and Heat Resistant Proteins from Mango Juice

    Directory of Open Access Journals (Sweden)

    Hamid Reza Akhbariyoon

    2016-10-01

    Full Text Available Background and Objectives: Microbial spoilage of juices and industries related with Alicyclobacillus are considerable international issues. This spore-forming bacterium causes changes in juices odor and taste. The isolation and identification of Alicyclobacillus contamination in juice producing and packaging industries has an essential role in the prevention and control of this type of spoilage bacterium in HACCP (Hazard analysis and critical control points manner.Materials and Methods: A thermo-acidophilic, non-pathogenic and sporeforming bacterium was isolated from mango juice. Preliminary identification of the isolates was based on morphological, biochemical and physiological properties. Identification at species level was made by PCR amplification. The influence of temperature in the range of 25-65°C in the growth of bacterium and in the range of 80-120°C in spore-resistant and heat resistant proteins was investigated and compared with other spore producing bacteria.Results and Conclusion: Phylogenetic analysis of the 16S rRNA gene sequencing indicated that the isolated strain constituted a distinct lineage in the Alicyclobacillus cluster and submitted to NCBI with access number Alicyclobacillus HRM-5 KM983424.1. The spores resisted 110°C for 3 h, and produced 28% dipicolinic acid more comparable to Bacillus licheniformis. Also they could produce 0.69 mg heat resistance protein after 1.5 h treatment in 100°C. The results showed that this strain could have biotechnological applications.Conflict of interests: The authors declare no conflict of interest.

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

    KAUST Repository

    Liu, Pei

    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.

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

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    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.

  9. Identification and evaluation of reference genes for accurate gene expression normalization of fresh and frozen-thawed spermatozoa of water buffalo (Bubalus bubalis).

    Science.gov (United States)

    Ashish, Shende; Bhure, S K; Harikrishna, Pillai; Ramteke, S S; Muhammed Kutty, V H; Shruthi, N; Ravi Kumar, G V P P S; Manish, Mahawar; Ghosh, S K; Mihir, Sarkar

    2017-04-01

    The quantitative real time PCR (qRT-PCR) has become an important tool for gene-expression analysis for a selected number of genes in life science. Although large dynamic range, sensitivity and reproducibility of qRT-PCR is good, the reliability majorly depend on the selection of proper reference genes (RGs) employed for normalization. Although, RGs expression has been reported to vary considerably within same cell type with different experimental treatments. No systematic study has been conducted to identify and evaluate the appropriate RGs in spermatozoa of domestic animals. Therefore, this study was conducted to analyze suitable stable RGs in fresh and frozen-thawed spermatozoa. We have assessed 13 candidate RGs (BACT, RPS18s, RPS15A, ATP5F1, HMBS, ATP2B4, RPL13, EEF2, TBP, EIF2B2, MDH1, B2M and GLUT5) of different functions and pathways using five algorithms. Regardless of the approach, the ranking of the most and the least candidate RGs remained almost same. The comprehensive ranking by RefFinder showed GLUT5, ATP2B4 and B2M, MDH1 as the top two stable and least stable RGs, respectively. The expression levels of four heat shock proteins (HSP) were employed as a target gene to evaluate RGs efficiency for normalization. The results demonstrated an exponential difference in expression levels of the four HSP genes upon normalization of the data with the most stable and the least stable RGs. Our study, provides a convenient RGs for normalization of gene-expression of key metabolic pathways effected during freezing and thawing of spermatozoa of buffalo and other closely related bovines.

  10. 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.

  11. Identification and characterization of an Eimeria-conserved protein in Eimeria tenella.

    Science.gov (United States)

    Dong, Hui; Wang, Yange; Han, Hongyu; Li, Ting; Zhao, Qiping; Zhu, Shunhai; Li, Liujia; Wu, Youling; Huang, Bing

    2014-02-01

    The precocious lines of Eimeria spp. have unique phenotypes. However, the genetic basis of the precocious phenotype is still poorly understood. The identification of Eimeria genes controlling the precocious phenotype is of immense importance in the fight against coccidiosis. In the present study, a novel gene of Eimeria maxima was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). Homologous genes were also found in Eimeria tenella and Eimeria acervulina. Alignment of the amino acid sequences from E. tenella, E. maxima, and E. acervulina showed 80-86 % identity, demonstrating a conserved protein in different Eimeria spp. This gene, designated Eimeria-conserved protein (ECP), contained 235 amino acids with a predicted molecular mass of 25.4 kDa and had 100 % identity with one annotated protein from E. maxima (Emax_0517). Real-time PCR and Western blot analysis revealed that the expression of ECP at mRNA and protein level in E. tenella is developmentally regulated. Messenger RNA levels from the ECP gene were higher in sporozoites than in other developmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites). Expression of ECP protein was detected in unsporulated oocysts, increased in abundance in sporulated oocysts, and was most prominent in sporozoites. Thereafter, the level of the ECP protein decreased, and no ECP-specific protein was detected in second-generation merozoites. Immunostaining with anti-rECP indicated that ECP is highly concentrated in both refractile bodies (RB) of free sporozoites, but is located at the apical end of the sporozoites after invasion of DF-1 cells. The specific staining of the ECP protein becomes more intense in trophozoites and immature first-generation schizonts, but decreases in mature first-generation schizonts. Inhibition of the function of ECP using specific antibodies reduced the ability of E. tenella sporozoites to invade host cells. Compared with the

  12. 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.

  13. 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

  14. Identification and characterization of a cleavage site in the proteolysis of orf virus 086 protein

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    Xiaoping eWang

    2016-04-01

    Full Text Available The ORF virus (ORFV is among the parapoxvirus genus of the poxviridae family, but little is known about the proteolytic pathways of ORFV encoding proteins. By contrast, the proteolysis mechanism of the vaccinia virus has been extensively explored. Vaccinia virus core protein P4a undergoes a proteolytic process that takes place at a conserved cleavage site Ala-Gly-X (where X is any amino acid and participates in virus assembly. Bioinformatics analysis revealed that an ORFV encoding protein, ORFV086, has a similar structure to the Vaccinia virus P4a core protein. In this study, we focus on the kinetic analysis and proteolysis mechanism of ORFV086. We found, via kinetic analysis, that ORFV086 is a late gene that starts to express at 8 hours post infection at mRNA level and 12 to 24 hours post infection at the protein level. The ORFV086 precursor and a 21kDa fragment can be observed in mature ORFV virions. The same bands were detected at only 3 hours post infection, suggesting that both the ORFV086 precursor and the 21kDa fragment are viral structural proteins. ORFV086 was cleaved from 12 to 24 hours post infection. The cleavage took place at different sites,resulting in seven bands with differing molecular weights. Sequence alignment revealed that five putative cleavage sites were predicted at C-terminal and internal regions of ORFV086. To investigate whether those cleavage sites are involved in proteolytic processing, full length and several deletion mutant ORFV086 recombinant proteins were expressed and probed. The GGS site that produced a 21kDa cleavage fragment was confirmed by identification of N/C-terminal FLAG epitope recombinant proteins, site-directed mutagenesis and Pulse-chase analysis. Interestingly, chase results demonstrated that, at late times, ORFV086 is partially cleaved. Taken together, we concluded that GGS is a cleavage site in ORFV086 and produces a 21kDa fragment post infection. Both ORFV086 precursor and the 21kDa fragment

  15. Rapid identification of novel immunodominant proteins and characterization of a specific linear epitope of Campylobacter jejuni.

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    Sebastian Hoppe

    Full Text Available Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium's pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is

  16. Identification of an osteopontin-like protein in fish associated with mineral formation.

    Science.gov (United States)

    Fonseca, Vera G; Laizé, Vincent; Valente, Marta S; Cancela, M Leonor

    2007-09-01

    Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor-binding RGD motif, a negatively charged domain and numerous post-translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin-like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin-like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates.

  17. 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.

  18. Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

    Science.gov (United States)

    Hoppe, Sebastian; Bier, Frank F.; Nickisch-Rosenegk, Markus v.

    2013-01-01

    Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify

  19. Identification of pancreatic cancer invasion-related proteins by proteomic analysis

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    Clynes Martin

    2009-02-01

    Full Text Available Abstract Background Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion and Clone #8 (low invasion using proteomic profiling of an in vitro model of pancreatic cancer. Materials and methods Using 2D-DIGE followed by MALDI-TOF MS, two clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with high and low invasive capacities were incubated on matrigel 24 hours prior to analysis to stimulate cell-ECM contact and mimic in vivo interaction with the basement membrane. Results Sixty proteins were identified as being differentially expressed (> 1.2 fold change and p ≤ 0.05 between Clone #3 and Clone #8. Proteins found to have higher abundance levels in the highly invasive Clone #3 compared to the low invasive Clone #8 include members of the chaperone activity proteins and cytoskeleton constituents whereas metabolism-associated and catalytic proteins had lower abundance levels. Differential protein expression levels of ALDH1A1, VIM, STIP1 and KRT18 and GAPDH were confirmed by immunoblot. Using RNAi technology, STIP1 knockdown significantly reduced invasion and proliferation of the highly invasive Clone #3. Knockdown of another target, VIM by siRNA in Clone #3 cells also resulted in decreased invasion abilities of Clone #3. Elevated expression of STIP1 was observed in pancreatic tumour tissue compared to normal pancreas, whereas ALDH1A1 stained at lower levels in pancreatic tumours, as detected by immunohistochemistry. Conclusion Identification of targets which play a role in the highly invasive phenotype of pancreatic cancer may help to understand the biological behaviour, the rapid progression of this cancer and may be of importance in the development of new therapeutic strategies for pancreatic cancer.

  20. Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages.

    Science.gov (United States)

    Simpson, David J; Sacher, Jessica C; Szymanski, Christine M

    2016-01-11

    Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages.

  1. Identification of ICIS-1, a new protein involved in cilia stability.

    Science.gov (United States)

    Ponsard, Cecile; Skowron-Zwarg, Marie; Seltzer, Virginie; Perret, Eric; Gallinger, Julia; Fisch, Cathy; Dupuis-Williams, Pascale; Caruso, Nathalie; Middendorp, Sandrine; Tournier, Frederic

    2007-01-01

    Cilia are specialized organelles that exert critical functions in numerous organisms, including that of cell motility, fluid transport and protozoan locomotion. Ciliary architecture and function strictly depend on basal body formation, migration and axoneme elongation. Numerous ultrastructural studies have been undertaken in different species to elucidate the process of ciliogenesis. Recent analyses have led to identification of genes specifically expressed in ciliated organisms, but most proteins involved in ciliogenesis remain uncharacterized. Using human nasal epithelial cells capable of ciliary differentiation in vitro, differential display was carried out to identify new proteins associated with ciliogenesis. We isolated a new gene, ICIS-1 (Involved in CIlia Stability-1), upregulated during mucociliary differentiation. This gene is localized within the TGF-beta1 promoter and is ubiquitously expressed in human tissues. Functional analyses of gene expression inhibition by RNA interference in Paramecium tetraurelia indicated that the ICIS-1 homologue interfered with cilia stability or formation. These findings demonstrate that ICIS-1 is a new protein associated with ciliated cells and potentially related to cilia stability.

  2. Identification of Novel Milk Protein Gene Variants in Sahiwal Cattle Breed of Pakistan

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    Shahlla N. Mir

    2013-02-01

    Full Text Available This novel study was aimed at identification of new genetic variants in Sahiwal cattle breed of Pakistan and determined the effects of these variants on milk yield. Five major milk protein genes in Sahiwal cattle were analyzed and two single nucleotide polymorphisms identified through bi-directional sequencing. These include A to T in exon XI at position 11462 of the alpha s1 casein gene; resulting in a Glutamic Acid (GAA to Aspartic acid (GAU substitution at position 84 of alpha s1 casein protein and T to C change at position 8491 of the exon VII in beta-casein gene resulting in a Valine to Alanine substitution at position 197 of beta casein protein. Amplification Refractory Mutation System (ARMS and SNaPshot genotyping protocols were optimized for genotyping new genetic variants. The genotypes in both the alpha-s1 casein and beta casein genes were found associated with milk yield but their influence was not statistically significant. However, the least square means of milk yield for TT genotypes of alpha s1 casein and of beta casein genes were higher compared to other genotypes.

  3. Identification of archaeal proteins that affect the exosome function in vitro

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    Palhano Fernando L

    2010-05-01

    Full Text Available Abstract Background The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

  4. Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages

    Science.gov (United States)

    Simpson, David J.; Sacher, Jessica C.; Szymanski, Christine M.

    2016-01-01

    Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs). These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages. PMID:26761028

  5. Development of an Assay for the Identification of Receptor Binding Proteins from Bacteriophages

    Directory of Open Access Journals (Sweden)

    David J. Simpson

    2016-01-01

    Full Text Available Recently, a large number of new technologies have been developed that exploit the unique properties of bacteriophage receptor binding proteins (RBPs. These include their use in diagnostic applications that selectively capture bacteria and as therapeutics that reduce bacterial colonization in vivo. RBPs exhibit comparable, and in many cases superior, stability, receptor specificity, and affinity to other carbohydrate binding proteins such as antibodies or lectins. In order to further exploit the use of RBPs, we have developed an assay for discovering RBPs using phage genome expression libraries and protein screens to identify binding partners that recognize the host bacterium. When phage P22 was screened using this assay, Gp9 was the only RBP discovered, confirming previous predictions that this is the sole RBP encoded by this phage. We then examined the Escherichia coli O157:H7 typing phage 1 in our assay and identified a previously undescribed RBP. This general approach has the potential to assist in the identification of RBPs from other bacteriophages.

  6. Optimising methods for the preservation, capture and identification of ubiquitin chains and ubiquitylated proteins by immunoblotting

    Science.gov (United States)

    Emmerich, Christoph H.; Cohen, Philip

    2015-01-01

    Immunoblotting is a powerful technique for the semi-quantitative analysis of ubiquitylation events, and remains the most commonly used method to study this process due to its high specificity, speed, sensitivity and relatively low cost. However, the ubiquitylation of proteins is complex and, when the analysis is performed in an inappropriate manner, it can lead to the misinterpretation of results and to erroneous conclusions being reached. Here we discuss the advantages and disadvantages of the methods currently in use to analyse ubiquitin chains and protein ubiquitylation, and describe the procedures that we have found to be most useful for optimising the quality and reliability of the data that we have generated. We also highlight commonly encountered problems and the pitfalls inherent in some of these methods. Finally, we introduce a set of recommendations to help researchers obtain high quality data, especially those new to the field of ubiquitin signalling. The specific topics addressed in this article include sample preparation, the separation, detection and identification of particular ubiquitin chains by immunoblotting, and the analysis of ubiquitin chain topology through the combined use of ubiquitin-binding proteins and ubiquitin linkage-specific deubiquitylases. PMID:26325464

  7. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display

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    Meyer Torsten

    2012-06-01

    Full Text Available Abstract Background Solely in Europoe, Salmonella Typhimurium causes more than 100,000 infections per year. Improved detection of livestock colonised with S. Typhimurium is necessary to prevent foodborne diseases. Currently, commercially available ELISA assays are based on a mixture of O-antigens (LPS or total cell lysate of Salmonella and are hampered by cross-reaction. The identification of novel immunogenic proteins would be useful to develop ELISA based diagnostic assays with a higher specificity. Results A phage display library of the entire Salmonella Typhimurium genome was constructed and 47 immunogenic oligopeptides were identified using a pool of convalescent sera from pigs infected with Salmonella Typhimurium. The corresponding complete genes of seven of the identified oligopeptids were cloned. Five of them were produced in E. coli. The immunogenic character of these antigens was validated with sera from pigs infeced with S. Tyhimurium and control sera from non-infected animals. Finally, human antibody fragments (scFv against these five antigens were selected using antibody phage display and characterised. Conclusion In this work, we identified novel immunogenic proteins of Salmonella Typhimurium and generated antibody fragments against these antigens completely based on phage display. Five immunogenic proteins were validated using a panel of positive and negative sera for prospective applications in diagnostics of Salmonela Typhimurium.

  8. 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 (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.

  9. The role of proteomics in toxicology: identification of biomarkers of toxicity by protein expression analysis.

    Science.gov (United States)

    Kennedy, Sandy

    2002-01-01

    Proteomics, i.e. the high throughput separation, display and identification of proteins, has the potential to be a powerful tool in drug development. It could increase the predictability of early drug development and identify non-invasive biomarkers of toxicity or efficacy. This review provides an introduction to modern proteomics, with particular reference to applications in toxicology. A literature search was carried out to identify studies in two broad classes: screening/predictive toxicology, and mechanistic toxicology. The strengths and limitations of current methods and the likely impact of techniques in drug development are also considered. Proteomics can increase the speed and sensitivity of toxicological screening by identifying protein markers of toxicity. Proteomics studies have already provided insights into the mechanisms of action of a wide range of substances, from metals to peroxisome proliferators. Current limitations involving speed of throughput are being overcome by increasing automation and the development of new techniques. The isotope-coded affinity tag (ICAT) method appears particularly promising. The application of proteomics to drug development has given rise to the new field of pharmacoproteomics. New associations between proteins and toxicopathological effects are constantly being identified, and major progress is on the horizon as we move into the post-genomic era.

  10. 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

  11. Dihedral-Based Segment Identification and Classification of Biopolymers I: Proteins

    Science.gov (United States)

    2013-01-01

    A new structure classification scheme for biopolymers is introduced, which is solely based on main-chain dihedral angles. It is shown that by dividing a biopolymer into segments containing two central residues, a local classification can be performed. The method is referred to as DISICL, short for Dihedral-based Segment Identification and Classification. Compared to other popular secondary structure classification programs, DISICL is more detailed as it offers 18 distinct structural classes, which may be simplified into a classification in terms of seven more general classes. It was designed with an eye to analyzing subtle structural changes as observed in molecular dynamics simulations of biomolecular systems. Here, the DISICL algorithm is used to classify two databases of protein structures, jointly containing more than 10 million segments. The data is compared to two alternative approaches in terms of the amount of classified residues, average occurrence and length of structural elements, and pair wise matches of the classifications by the different programs. In an accompanying paper (Nagy, G.; Oostenbrink, C. Dihedral-based segment identification and classification of biopolymers II: Polynucleotides. J. Chem. Inf. Model. 2013, DOI: 10.1021/ci400542n), the analysis of polynucleotides is described and applied. Overall, DISICL represents a potentially useful tool to analyze biopolymer structures at a high level of detail. PMID:24364820

  12. Dihedral-based segment identification and classification of biopolymers I: proteins.

    Science.gov (United States)

    Nagy, Gabor; Oostenbrink, Chris

    2014-01-27

    A new structure classification scheme for biopolymers is introduced, which is solely based on main-chain dihedral angles. It is shown that by dividing a biopolymer into segments containing two central residues, a local classification can be performed. The method is referred to as DISICL, short for Dihedral-based Segment Identification and Classification. Compared to other popular secondary structure classification programs, DISICL is more detailed as it offers 18 distinct structural classes, which may be simplified into a classification in terms of seven more general classes. It was designed with an eye to analyzing subtle structural changes as observed in molecular dynamics simulations of biomolecular systems. Here, the DISICL algorithm is used to classify two databases of protein structures, jointly containing more than 10 million segments. The data is compared to two alternative approaches in terms of the amount of classified residues, average occurrence and length of structural elements, and pair wise matches of the classifications by the different programs. In an accompanying paper (Nagy, G.; Oostenbrink, C. Dihedral-based segment identification and classification of biopolymers II: Polynucleotides. J. Chem. Inf. Model. 2013, DOI: 10.1021/ci400542n), the analysis of polynucleotides is described and applied. Overall, DISICL represents a potentially useful tool to analyze biopolymer structures at a high level of detail.

  13. Abstract 5324: Pan-cancer identification of mutated pathways and protein complexes

    DEFF Research Database (Denmark)

    Leiserson, Mark D.; Vandin, Fabio; Wu, Hsin-Ta

    2014-01-01

    that are mutually exclusive across the tumor cohort. There are numerous examples of mutually exclusive mutations between interacting proteins; e.g. BRAF and KRAS in colorectal cancer. Dendrix++ generalizes this idea to find larger groups of mutually exclusive mutations.We applied HotNet2 and Dendrix++ to whole......Large-scale cancer sequencing efforts such as The Cancer Genome Atlas (TCGA) and others have shown that tumors exhibit extensive mutational heterogeneity with relatively few genes mutated at significant frequency and many genes mutated in only a small number of individuals. This long tail...... phenomenon complicates the identification of driver mutations by their observed frequency. The long tail is explained in part by the fact that driver mutations target genes in signaling and regulatory pathways, and these pathways may be perturbed by different mutations in different tumors.We developed two...

  14. Two dimensional SDS PAGE analysis of epididymal tissue proteins of normal and castrated bulls (Bos taurus) and identification of an androgen dependant protein

    Institute of Scientific and Technical Information of China (English)

    Gurunathgouda patil; V Girish Kumar; SG Ramachandra; AJ Rao; S Nandi

    2012-01-01

    A study was undertaken with an objective of two dimensionalSDS-PAGE analysis of protein profile in tissues of all caput, corpus and cauda of epididymis in both castrated and normal bulls and identification of androgen dependent protein/s in the epididymal tissues of bull(Bos taurus). Two dimensionalSDS-PAGE analysis for protein spots of different molecular weight(MW) with pI3.5 to7.35 and pI >7.35 to9.3 between the three regions in the normal and castrated bull as well as between normal and castrated bull of a particular region revealed significant differences. Similarly, comparison between the three regions of the normal and castrated bull epididymis for the number of protein spots irrespective of theMW in pI range of3.5 to7.35 and pI range >7.35 to 9.3 revealed significant differences.The number of protein spots found to be significantly higher in caput, corpus and cauda epididymis of the normal bull when compared to similar region of the castrated bull epididymis.Most of the proteins, which are secreted, are havingMW between 20 and85 kDa.Six proteins, which are known to be highly dependent on androgens, are also of acidic in nature except for one protein havingbasic pH.A protein spot(pI:6.55-6.85, andMW: <20 kDa) which appeared at the same site in all the three regions of the epididymis in the normal bull but was absent at the same site in all the three regions of the castrated bull was subjected for identification of the protein byMALDI-MS.The results revealed that this protein is an interferon-stimulated protein and it could beISG15/UCRP.

  15. Methylated-antibody affinity purification to improve proteomic identification of plant RNA polymerase Pol V complex and the interacting proteins

    Science.gov (United States)

    Qin, Guochen; Ma, Jun; Chen, Xiaomei; Chu, Zhaoqing; She, Yi-Min

    2017-01-01

    Affinity purification followed by enzymatic digestion and mass spectrometry has been widely utilized for the sensitive detection of interacting proteins and protein complexes in various organisms. In plants, the method is technically challenging due to the low abundance proteins, non-specific binding and difficulties of eluting interacting proteins from antibody beads. In this report, we describe a strategy to modify antibodies by reductive methylation of lysines without affecting their binding properties, followed by on-bead digestion of bound proteins with endoproteinase Lys-C. By this method, the antibody remains intact and does not interfere with the downstream identification of interacting proteins. Non-specific binding proteins were excluded using 14N/15N-metabolic labeling of wild-type and the transgenic plant counterparts. The method was employed to identify 12 co-immunoprecipitated protein subunits in Pol V complex and to discover 17 potential interacting protein targets in Arabidopsis. Our results demonstrated that the modification of antibodies by reductive dimethylation can improve the reliability and sensitivity of identifying low-abundance proteins through on-bead digestion and mass spectrometry. We also show that coupling this technique with chemical crosslinking enables in-depth characterization of endogenous protein complexes and the protein-protein interaction networks including mapping the surface topology and post-translational modifications of interacting proteins. PMID:28224978

  16. Iterative Non-m/z-sharing Rule for Confident and Sensitive Protein Identification of Non-shotgun Proteomics

    Institute of Scientific and Technical Information of China (English)

    YUN Dong; HE Fuchu; LU Haojie; WANG Haijian; ZHANG Yang; CHENG Gang; JIN Hong; YU Yanbao; XU Yawei; YANG Pengyuan

    2009-01-01

    Selecting reasonable matches from the database search results is crucial to mass spectrometry-based proteomics identification. However, the current score-based filter solution and decoy database methods are not effective enough to prevent all false positive and false negative selections. In this study, a systematic search strategy named iterative non-m/z-sharing (INMZS) analysis was proposed to address the problem. In the strategy, all search results were screened based on the share status of corresponding matched m/z, only the proteins that matched with exclusive m/z information were reserved for the confident matches. The researchers did further iterative search to improve the identification of minor components in a spot. Finally, identifications were confirmed by decoy database evaluation for the final phase of protein identification. Simulation and application tests of INMZS were implemented on a large human liver data set and standard protein cocktails. The result shows that INMZS plus decoy database evaluation is efficient in ensuring the confidence and sensitivity of 2-DE or similar non-shotgun based proteome identification.

  17. Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome

    Science.gov (United States)

    Leppert, Tami; Anex, Deon S.; Hilmer, Jonathan K.; Matsunami, Nori; Baird, Lisa; Stevens, Jeffery; Parsawar, Krishna; Durbin-Johnson, Blythe P.; Rocke, David M.; Nelson, Chad; Fairbanks, Daniel J.; Wilson, Andrew S.; Rice, Robert H.; Woodward, Scott R.; Bothner, Brian; Hart, Bradley R.; Leppert, Mark

    2016-01-01

    Human identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts. PMID:27603779

  18. Identification of small molecule binding sites within proteins using phage display technology.

    Energy Technology Data Exchange (ETDEWEB)

    Rodi, D. J.; Agoston, G. E.; Manon, R.; Lapcevich, R.; Green, S. J.; Makowski, L.; Biosciences Division; EntreMed Inc.; Florida State Univ.

    2001-11-01

    Affinity selection of peptides displayed on phage particles was used as the basis for mapping molecular contacts between small molecule ligands and their protein targets. Analysis of the crystal structures of complexes between proteins and small molecule ligands revealed that virtually all ligands of molecular weight 300 Da or greater have a continuous binding epitope of 5 residues or more. This observation led to the development of a technique for binding site identification which involves statistical analysis of an affinity-selected set of peptides obtained by screening of libraries of random, phage-displayed peptides against small molecules attached to solid surfaces. A random sample of the selected peptides is sequenced and used as input for a similarity scanning program which calculates cumulative similarity scores along the length of the putative receptor. Regions of the protein sequence exhibiting the highest similarity with the selected peptides proved to have a high probability of being involved in ligand binding. This technique has been employed successfully to map the contact residues in multiple known targets of the anticancer drugs paclitaxel (Taxol), docetaxel (Taxotere) and 2-methoxyestradiol and the glycosaminoglycan hyaluronan, and to identify a novel paclitaxel receptor [1]. These data corroborate the observation that the binding properties of peptides displayed on the surface of phage particles can mimic the binding properties of peptides in naturally occurring proteins. It follows directly that structural context is relatively unimportant for determining the binding properties of these disordered peptides. This technique represents a novel, rapid, high resolution method for identifying potential ligand binding sites in the absence of three-dimensional information and has the potential to greatly enhance the speed of development of novel small molecule pharmaceuticals.

  19. Platform for identification of Salmonella serovar differentiating bacterial proteins by top-down mass spectrometry: S. Typhimurium vs S. Heidelberg.

    Science.gov (United States)

    McFarland, Melinda A; Andrzejewski, Denis; Musser, Steven M; Callahan, John H

    2014-07-15

    Intact protein expression profiling has proven to be a powerful tool for bacterial subspecies differentiation. To facilitate typing, epidemiology, and trace-back of Salmonella contamination in the food supply, a minimum of serovar level differentiation is required. Subsequent identification and validation of marker proteins is integral to rapid screening development and to determining which proteins are subject to environmental pressure. Bacterial sequencing efforts have expanded the number of sequenced genomes available for single-nucleotide polymorphism (SNP) analyses, but annotation is often missing, start site errors are not uncommon, and the likelihood of expression is not known. In this work we show that the combination of intact protein expression profiles and top-down liquid chromatography-mass spectrometry (LC-MS/MS) facilitates the identification of proteins that result from expressed serovar specific nonsynonymous SNPs. Combinations of these marker proteins can be used in assays for rapid differentiation of bacteria. LC-MS generated intact protein expression profiles establish which bacterial protein masses differ across samples and can be determined without prior knowledge of the sample. Subsequent top-down LC-MS/MS is used to identify expressed proteins and their post-translational modifications (PTM), identify serovar specific markers, and validate genomic predicted orthologues as expressed biomarkers.

  20. Cy5 maleimide labelling for sensitive detection of free thiols in native protein extracts: identification of seed proteins targeted by barley thioredoxin h isoforms.

    Science.gov (United States)

    Maeda, Kenji; Finnie, Christine; Svensson, Birte

    2004-03-01

    Barley thioredoxin h isoforms HvTrxh1 and HvTrxh2 differ in temporal and spatial distribution and in kinetic properties. Target proteins of HvTrxh1 and HvTrxh2 were identified in mature seeds and in seeds after 72 h of germination. Improvement of the established method for identification of thioredoxin-targeted proteins based on two-dimensional electrophoresis and fluorescence labelling of thiol groups was achieved by application of a highly sensitive Cy5 maleimide dye and large-format two-dimensional gels, resulting in a 10-fold increase in the observed number of labelled protein spots. The technique also provided information about accessible thiol groups in the proteins identified in the barley seed proteome. In total, 16 different putative target proteins were identified from 26 spots using tryptic in-gel digestion, matrix-assisted laser-desorption ionization-time-of-flight MS and database search. HvTrxh1 and HvTrxh2 were shown to have similar target specificity. Barley alpha-amylase/subtilisin inhibitor, previously demonstrated to be reduced by both HvTrxh1 and HvTrxh2, was among the identified target proteins, confirming the suitability of the method. Several alpha-amylase/trypsin inhibitors, some of which are already known as target proteins of thioredoxin h, and cyclophilin known as a target protein of m-type thioredoxin were also identified. Lipid transfer protein, embryospecific protein, three chitinase isoenzymes, a single-domain glyoxalase-like protein and superoxide dismutase were novel identifications of putative target proteins, suggesting new physiological roles of thioredoxin h in barley seeds.

  1. Top-down proteomic identification of protein biomarkers of food-borne pathogens using MALDI-TOF-TOF-MS/MS

    Science.gov (United States)

    This chapter describes a step-by-step protocol and discussion of top-down proteomic identification of protein biomarkers of food-borne pathogens using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF-TOF-MS/MS) and web-based software developed in the Pro...

  2. 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

    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...

  3. Entamoeba invadens: Identification of a SERCA protein and effect of SERCA inhibitors on encystation.

    Science.gov (United States)

    Martínez-Higuera, Aarón; Herrera-Martínez, Mayra; Chávez-Munguía, Bibiana; Valle-Solís, Martha; Muñiz-Lino, Marcos A; Cázares-Apátiga, Javier; Rodríguez, Mario A

    2015-12-01

    Calcium has an important role on signaling of different cellular processes, including growth and differentiation. Signaling by calcium also has an essential function in pathogenesis and differentiation of the protozoan parasites Entamoeba histolytica and Entamoeba invadens. However, the proteins of these parasites that regulate the cytoplasmic concentration of this ion are poorly studied. In eukaryotic cells, the calcium-ATPase of the SERCA type plays an important role in calcium homeostasis by catalyzing the active efflux of calcium from cytoplasm to endoplasmic reticulum. Here, we reported the identification of SERCA of E. invadens (EiSERCA). This protein contains a putative sequence for endoplasmic reticulum retention and all domains involved in calcium transport identified in mammalian SERCA. By immunofluorescence assays, an antibody against SERCA of E. histolytica detected EiSERCA in a vesicular network in the cytoplasm of E. invadens trophozoites, co-localizing with calreticulin. Interestingly, EiSERCA was redistributed close to plasma membrane during encystation, suggesting that this pump could participate in regulate the calcium concentration during this process. In addition, thapsigargin and cyclopiazonic acid, both specific inhibitors of SERCA, affected the number and structure of cysts, supporting the hypothesis that calcium flux mediated by SERCA has an important role in the life cycle of Entamoeba.

  4. Identification of QTL Affecting Protein and Amino Acid Contents in Rice

    Institute of Scientific and Technical Information of China (English)

    ZHONG Ming; WANG Ling-qiang; YuAN De-jun; LUO Li-jun; Xu Cai-guo; HE Yu-qing

    2011-01-01

    The phenotypes of protein and amino acid contents were measured in an F9 recombinant inbred line population derived from a cross between Zhenshan 97B and Delong 208.A total of 48 and 64 QTLs were identified in 2004 and 2005,respectively.The contribution of each QTL to the phenotypic variation ranged from 4.0% to 43.7%.Most QTLs co-localized,forming 29 QTL clusters on the chromosomes with three major ones detected in both years,which were mapped on chromosomes 1,7 and 9,respectively.The two QTL clusters for amino acid content,qAa1 and qAa7,influenced almost all the traits with the allele from Zhenshan 97B,and the third QTL cluster for amino acid content,qAa9,increased the lysine content with the allele from Delong 208.A wide coincidence was found between the QTL detected under this study and the loci involved in amino acid metabolism pathways in nitrogen assimilation and transport,or protein biosynthesis.The results would facilitate the identification of candidate genes and could be used in marker-assisted selection for the favorable allele in rice quality improvement.

  5. Identification of compound-protein interactions through the analysis of gene ontology, KEGG enrichment for proteins and molecular fragments of compounds.

    Science.gov (United States)

    Chen, Lei; Zhang, Yu-Hang; Zheng, Mingyue; Huang, Tao; Cai, Yu-Dong

    2016-12-01

    Compound-protein interactions play important roles in every cell via the recognition and regulation of specific functional proteins. The correct identification of compound-protein interactions can lead to a good comprehension of this complicated system and provide useful input for the investigation of various attributes of compounds and proteins. In this study, we attempted to understand this system by extracting properties from both proteins and compounds, in which proteins were represented by gene ontology and KEGG pathway enrichment scores and compounds were represented by molecular fragments. Advanced feature selection methods, including minimum redundancy maximum relevance, incremental feature selection, and the basic machine learning algorithm random forest, were used to analyze these properties and extract core factors for the determination of actual compound-protein interactions. Compound-protein interactions reported in The Binding Databases were used as positive samples. To improve the reliability of the results, the analytic procedure was executed five times using different negative samples. Simultaneously, five optimal prediction methods based on a random forest and yielding maximum MCCs of approximately 77.55 % were constructed and may be useful tools for the prediction of compound-protein interactions. This work provides new clues to understanding the system of compound-protein interactions by analyzing extracted core features. Our results indicate that compound-protein interactions are related to biological processes involving immune, developmental and hormone-associated pathways.

  6. Accurate prediction of protein structural classes by incorporating predicted secondary structure information into the general form of Chou's pseudo amino acid composition.

    Science.gov (United States)

    Kong, Liang; Zhang, Lichao; Lv, Jinfeng

    2014-03-07

    Extracting good representation from protein sequence is fundamental for protein structural classes prediction tasks. In this paper, we propose a novel and powerful method to predict protein structural classes based on the predicted secondary structure information. At the feature extraction stage, a 13-dimensional feature vector is extracted to characterize general contents and spatial arrangements of the secondary structural elements of a given protein sequence. Specially, four segment-level features are designed to elevate discriminative ability for proteins from the α/β and α+β classes. After the features are extracted, a multi-class non-linear support vector machine classifier is used to implement protein structural classes prediction. We report extensive experiments comparing the proposed method to the state-of-the-art in protein structural classes prediction on three widely used low-similarity benchmark datasets: FC699, 1189 and 640. Our method achieves competitive performance on prediction accuracies, especially for the overall prediction accuracies which have exceeded the best reported results on all of the three datasets.

  7. Genes optimized by evolution for accurate and fast translation encode in Archaea and Bacteria a broad and characteristic spectrum of protein functions

    Directory of Open Access Journals (Sweden)

    Merkl Rainer

    2010-11-01

    Full Text Available Abstract Background In many microbial genomes, a strong preference for a small number of codons can be observed in genes whose products are needed by the cell in large quantities. This codon usage bias (CUB improves translational accuracy and speed and is one of several factors optimizing cell growth. Whereas CUB and the overrepresentation of individual proteins have been studied in detail, it is still unclear which high-level metabolic categories are subject to translational optimization in different habitats. Results In a systematic study of 388 microbial species, we have identified for each genome a specific subset of genes characterized by a marked CUB, which we named the effectome. As expected, gene products related to protein synthesis are abundant in both archaeal and bacterial effectomes. In addition, enzymes contributing to energy production and gene products involved in protein folding and stabilization are overrepresented. The comparison of genomes from eleven habitats shows that the environment has only a minor effect on the composition of the effectomes. As a paradigmatic example, we detailed the effectome content of 37 bacterial genomes that are most likely exposed to strongest selective pressure towards translational optimization. These effectomes accommodate a broad range of protein functions like enzymes related to glycolysis/gluconeogenesis and the TCA cycle, ATP synthases, aminoacyl-tRNA synthetases, chaperones, proteases that degrade misfolded proteins, protectants against oxidative damage, as well as cold shock and outer membrane proteins. Conclusions We made clear that effectomes consist of specific subsets of the proteome being involved in several cellular functions. As expected, some functions are related to cell growth and affect speed and quality of protein synthesis. Additionally, the effectomes contain enzymes of central metabolic pathways and cellular functions sustaining microbial life under stress situations. These

  8. 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.

  9. 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).

  10. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

    Science.gov (United States)

    Suzuki, Takashi; Brown, Judy J.; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

  11. WISCOD: A Statistical Web-Enabled Tool for the Identification of Significant Protein Coding Regions

    Directory of Open Access Journals (Sweden)

    Mireia Vilardell

    2014-01-01

    Full Text Available Classically, gene prediction programs are based on detecting signals such as boundary sites (splice sites, starts, and stops and coding regions in the DNA sequence in order to build potential exons and join them into a gene structure. Although nowadays it is possible to improve their performance with additional information from related species or/and cDNA databases, further improvement at any step could help to obtain better predictions. Here, we present WISCOD, a web-enabled tool for the identification of significant protein coding regions, a novel software tool that tackles the exon prediction problem in eukaryotic genomes. WISCOD has the capacity to detect real exons from large lists of potential exons, and it provides an easy way to use global P value called expected probability of being a false exon (EPFE that is useful for ranking potential exons in a probabilistic framework, without additional computational costs. The advantage of our approach is that it significantly increases the specificity and sensitivity (both between 80% and 90% in comparison to other ab initio methods (where they are in the range of 70–75%. WISCOD is written in JAVA and R and is available to download and to run in a local mode on Linux and Windows platforms.

  12. 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.

  13. A Novel IgM-capture enzyme-linked immunosorbent assay using recombinant Vag8 fusion protein for the accurate and early diagnosis of Bordetella pertussis infection.

    Science.gov (United States)

    Otsuka, Nao; Gotoh, Kensei; Nishimura, Naoko; Ozaki, Takao; Nakamura, Yukitsugu; Haga, Kiyohito; Yamazaki, Makoto; Gondaira, Fumio; Okada, Kenji; Miyaji, Yusuke; Toyoizumi-Ajisaka, Hiromi; Shibayama, Keigo; Arakawa, Yoshichika; Kamachi, Kazunari

    2016-05-01

    An ELISA that measures anti-PT IgG antibody has been used widely for the serodiagnosis of pertussis; however, the IgG-based ELISA is inadequate for patients during the acute phase of the disease because of the slow response of anti-PT IgG antibodies. To solve this problem, we developed a novel IgM-capture ELISA that measures serum anti-Bordetella pertussis Vag8 IgM levels for the accurate and early diagnosis of pertussis. First, we confirmed that Vag8 was highly expressed in all B. pertussis isolates tested (n = 30), but little or none in other Bordetella species, and that DTaP vaccines did not induce anti-Vag8 IgG antibodies in mice (i.e. the antibody level could be unaffected by the vaccination). To determine the immune response to Vag8 in B. pertussis infection, anti-Vag8 IgM levels were compared between 38 patients (acute phase of pertussis) and 29 healthy individuals using the anti-Vag8 IgM-capture ELISA. The results revealed that the anti-Vag8 IgM levels were significantly higher in the patients compared with the healthy individuals (P < 0.001). ROC analysis also showed that the anti-Vag8 IgM-capture ELISA has higher diagnostic accuracy (AUC, 0.92) than a commercial anti-PT IgG ELISA kit. Moreover, it was shown that anti-Vag8 IgM antibodies were induced earlier than anti-PT IgG antibodies on sequential patients' sera. These data indicate that our novel anti-Vag8 IgM-capture ELISA is a potentially useful tool for making the accurate and early diagnosis of B. pertussis infection.

  14. Reliable Identification of Cross-Linked Products in Protein Interaction Studies by 13C-Labeled p-Benzoylphenylalanine

    Science.gov (United States)

    Pettelkau, Jens; Ihling, Christian H.; Frohberg, Petra; van Werven, Lars; Jahn, Olaf; Sinz, Andrea

    2014-09-01

    We describe the use of the 13C-labeled artificial amino acid p-benzoyl-L-phenylalanine (Bpa) to improve the reliability of cross-linked product identification. Our strategy is exemplified for two protein-peptide complexes. These studies indicate that in many cases the identification of a cross-link without additional stable isotope labeling would result in an ambiguous assignment of cross-linked products. The use of a 13C-labeled photoreactive amino acid is considered to be preferred over the use of deuterated cross-linkers as retention time shifts in reversed phase chromatography can be ruled out. The observation of characteristic fragment ions additionally increases the reliability of cross-linked product assignment. Bpa possesses a broad reactivity towards different amino acids and the derived distance information allows mapping of spatially close amino acids and thus provides more solid structural information of proteins and protein complexes compared to the longer deuterated amine-reactive cross-linkers, which are commonly used for protein 3D-structure analysis and protein-protein interaction studies.

  15. Data Self-Recalibration and Mixture Mass Fingerprint Searching (DASER-MMF) to enhance protein identification within complex mixtures.

    Science.gov (United States)

    Danell, Ryan M; Ouvry-Patat, Severine A; Scarlett, Cameron O; Speir, J Paul; Borchers, Christoph H

    2008-12-01

    A novel algorithm based on Data Self-Recalibration and a subsequent Mixture Mass Fingerprint search (DASER-MMF) has been developed to improve the performance of protein identification from online 1D and 2D-LC-MS/MS experiments conducted on high-resolution mass spectrometers. Recalibration of 40% to 75% of the MS spectra in a human serum dataset is demonstrated with average errors of 0.3 +/- 0.3 ppm, regardless of the original calibration quality. With simple protein mixtures, the MMF search identifies new proteins not found in the MS/MS based search and increases the sequence coverage for identified proteins by six times. The high mass accuracy allows proteins to be identified with as little as three peptide mass hits. When applied to very complex samples, the MMF search shows less dramatic performance improvements. However, refinements such as additional discriminating factors utilized within the search space provide significant gains in protein identification ability and indicate that further enhancements are possible in this realm.

  16. Another turn of the screw in shaving Gram-positive bacteria: Optimization of proteomics surface protein identification in Streptococcus pneumoniae.

    Science.gov (United States)

    Olaya-Abril, Alfonso; Gómez-Gascón, Lidia; Jiménez-Munguía, Irene; Obando, Ignacio; Rodríguez-Ortega, Manuel J

    2012-06-27

    Bacterial surface proteins are of outmost importance as they play critical roles in the interaction between cells and their environment. In addition, they can be targets of either vaccines or antibodies. Proteomic analysis through "shaving" live cells with proteases has become a successful approach for a fast and reliable identification of surface proteins. However, this protocol has not been able to reach the goal of excluding cytoplasmic contamination, as cell lysis is an inherent process during culture and experimental manipulation. In this work, we carried out the optimization of the "shaving" strategy for the Gram-positive human pathogen Streptococcus pneumoniae, a bacterium highly susceptible to autolysis, and set up the conditions for maximizing the identification of surface proteins containing sorting or exporting signals, and for minimizing cytoplasmic contamination. We also demonstrate that cell lysis is an inherent process during culture and experimental manipulation, and that a low level of lysis is enough to contaminate a "surfome" preparation with peptides derived from cytoplasmic proteins. When the optimized conditions were applied to several clinical isolates, we found the majority of the proteins described to induce protection against pneumococcal infection. In addition, we found other proteins whose protection capacity has not been yet tested. In addition, we show the utility of this approach for providing antigens that can be used in serological tests for the diagnosis of pneumococcal disease.

  17. Identification of a substrate for Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus by a novel method for substrate identification.

    Science.gov (United States)

    Udo, H; Lam, C K; Mori, S; Inouye, M; Inouye, S

    2000-10-01

    Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases.

  18. 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/ .

  19. Identification of the gene encoding Brain Cell Membrane Protein 1 (BCMP1, a putative four-transmembrane protein distantly related to the Peripheral Myelin Protein 22 / Epithelial Membrane Proteins and the Claudins

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    Christophe Daniel

    2001-07-01

    Full Text Available Abstract Background A partial cDNA clone from dog thyroid presenting a very significant similarity with an uncharacterized mouse EST sequence was isolated fortuitously. We report here the identification of the complete mRNA and of the gene, the product of which was termed "brain cell membrane protein 1" (BCMP1. Results The 4 kb-long mRNA sequence exhibited an open-reading frame of only 543 b followed by a 3.2 kb-long 3' untranslated region containing several AUUUA instability motifs. Analysis of the encoded protein sequence identified the presence of four putative transmembrane domains. Similarity searches in protein domain databases identified partial sequence conservations with peripheral myelin protein 22 (PMP22/ epithelial membrane proteins (EMPs and Claudins, defining the encoded protein as representative of the existence of a novel subclass in this protein family. Northern-blot analysis of the expression of the corresponding mRNA in adult dog tissues revealed the presence of a huge amount of the 4 kb transcript in the brain. An EGFP-BCMP1 fusion protein expressed in transfected COS-7 cells exhibited a membranous localization as expected. The sequences encoding BCMP1 were assigned to chromosome X in dog, man and rat using radiation hybrid panels and were partly localized in the currently available human genome sequence. Conclusions We have identified the existence in several mammalian species of a gene encoding a putative four-transmembrane protein, BCMP1, wich defines a novel subclass in this family of proteins. In dog at least, the corresponding mRNA is highly present in brain cells. The chromosomal localization of the gene in man makes of it a likely candidate gene for X-linked mental retardation.

  20. Improvement of inhibitor identification for heat shock protein 90α by utilizing a red-shifted fluorescence polarization probe.

    Science.gov (United States)

    Qian, Jie; Holskin, Beverly P; Theroff, Jay; Underiner, Ted; Meyer, Sheryl L; Angeles, Thelma S

    2012-08-01

    Heat shock protein-90 (HSP90) is an ATP-dependent molecular chaperone with intrinsic ATPase activity. HSP90 is required for the stability and function of client proteins, many of which are involved in oncogenesis. Thus, identification of HSP90 inhibitors would potentially lead to the discovery of cancer therapeutics. Here, we present a high-throughput screening campaign utilizing two geldanamycin (GM)-labeled probes in a fluorescence polarization (FP) assay. For the primary screen, a previously reported green BODIPY-labeled GM (GM-BODIPY) was used to evaluate a library collection of about 400,000 compounds. From this screen, 3058 compounds showed >30% inhibition. To distinguish true positives from compound interference, a confirmatory screen was deemed necessary. Accordingly, a red-shifted FP binding assay was developed using GM labeled with red BODIPY. This tool enabled reliable identification of promising HSP90α inhibitors.

  1. In silicio identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae.

    Science.gov (United States)

    Caro, L H; Tettelin, H; Vossen, J H; Ram, A F; van den Ende, H; Klis, F M

    1997-12-01

    Use of the Von Heijne algorithm allowed the identification of 686 open reading frames (ORFs) in the genome of Saccharomyces cerevisiae that encode proteins with a potential N-terminal signal sequence for entering the secretory pathway. On further analysis, 51 of these proteins contain a potential glycosyl-phosphatidylinositol (GPI)-attachment signal. Seven additional ORFs were found to belong to this group. Upon examination of the possible GPI-attachment sites, it was found that in yeast the most probable amino acids for GPI-attachment as asparagine and glycine. In yeast, GPI-proteins are found at the cell surface, either attached to the plasma-membrane or as an intrinsic part of the cell wall. It was noted that plasma-membrane GPI-proteins possess a dibasic residue motif just before their predicted GPI-attachment site. Based on this, and on homologies between proteins, families of plasma-membrane and cell wall proteins were assigned, revealing 20 potential plasma-membrane and 38 potential cell wall proteins. For members of three plasma-membrane protein families, a function has been described. On the other hand, most of the cell wall proteins seem to be structural components of the wall, responsive to different growth conditions. The GPI-attachment site of yeast slightly differs from mammalian cells. This might be of use in the development of anti-fungal drugs.

  2. Identification of Giardia lamblia DHHC proteins and the role of protein S-palmitoylation in the encystation process.

    Science.gov (United States)

    Merino, María C; Zamponi, Nahuel; Vranych, Cecilia V; Touz, María C; Rópolo, Andrea S

    2014-07-01

    Protein S-palmitoylation, a hydrophobic post-translational modification, is performed by protein acyltransferases that have a common DHHC Cys-rich domain (DHHC proteins), and provides a regulatory switch for protein membrane association. In this work, we analyzed the presence of DHHC proteins in the protozoa parasite Giardia lamblia and the function of the reversible S-palmitoylation of proteins during parasite differentiation into cyst. Two specific events were observed: encysting cells displayed a larger amount of palmitoylated proteins, and parasites treated with palmitoylation inhibitors produced a reduced number of mature cysts. With bioinformatics tools, we found nine DHHC proteins, potential protein acyltransferases, in the Giardia proteome. These proteins displayed a conserved structure when compared to different organisms and are distributed in different monophyletic clades. Although all Giardia DHHC proteins were found to be present in trophozoites and encysting cells, these proteins showed a different intracellular localization in trophozoites and seemed to be differently involved in the encystation process when they were overexpressed. dhhc transgenic parasites showed a different pattern of cyst wall protein expression and yielded different amounts of mature cysts when they were induced to encyst. Our findings disclosed some important issues regarding the role of DHHC proteins and palmitoylation during Giardia encystation.

  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. Impact of injection solvent composition on protein identification in column-switching chip-liquid chromatography/mass spectrometry.

    Science.gov (United States)

    Houbart, V; Cobraiville, G; Nys, G; Merville, M-P; Fillet, M

    2016-05-06

    In shotgun proteomics, the gold standard technique is reversed-phase liquid chromatography coupled to mass spectrometry. Many researches have been carried out to study the effects on identification performances of chromatographic parameters such as the stationary phase and column dimensions, mobile phase composition and flow rate, as well as the gradient slope and length. However, little attention is usually paid to the injection solvent composition. In this study, we investigated the effect of the injection solvent on protein identification parameters (number of distinct peptides, amino acid coverage and MS/MS search score) as well as sensitivity. Tryptic peptides from six different proteins, covering a wide range of physicochemical properties, were employed as training set. Design of experiments was employed as a tool to highlight the factors related to the composition of the injection solvent that significantly influenced the obtained results. Optimal results for the training set were applied to analysis of more complex samples. The experiments pointed out optimising the composition of the injection solvent had a strong beneficial effect on all the considered responses. On the basis of these results, an approach to determine optimal conditions was proposed to maximise the protein identification performances and detection sensitivity.

  5. 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.

  6. The small subunit of the mammalian mitochondrial ribosome. Identification of the full complement of ribosomal proteins present.

    Science.gov (United States)

    Cavdar Koc, E; Burkhart, W; Blackburn, K; Moseley, A; Spremulli, L L

    2001-06-01

    Identification of all the protein components of the small subunit (28 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 28 S subunits followed by analysis of the resultant peptides by liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptide sequence information was used to search the human EST data bases and complete coding sequences of the proteins were assembled. The human mitochondrial ribosome has 29 distinct proteins in the small subunit. Fourteen of this group of proteins are homologs of the Escherichia coli 30 S ribosomal proteins S2, S5, S6, S7, S9, S10, S11, S12, S14, S15, S16, S17, S18, and S21. All of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. Surprisingly, three variants of ribosomal protein S18 are found in the mammalian and D. melanogaster mitochondrial ribosomes while C. elegans has two S18 homologs. The S18 homologs tend to be more closely related to chloroplast S18s than to prokaryotic S18s. No mitochondrial homologs to prokaryotic ribosomal proteins S1, S3, S4, S8, S13, S19, and S20 could be found in the peptides obtained from the whole 28 S subunit digests or by analysis of the available data bases. The remaining 15 proteins present in mammalian mitochondrial 28 S subunits (MRP-S22 through MRP-S36) are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of these proteins have a clear homolog in D. melanogaster while all but three can be found in the genome of C. elegans. Five of the mitochondrial specific ribosomal proteins have homologs in S. cerevisiae.

  7. Identification and evaluation of potential forensic marker proteins in vaginal fluid by liquid chromatography/mass spectrometry.

    Science.gov (United States)

    Igoh, Akihisa; Doi, Yusuke; Sakurada, Koichi

    2015-09-01

    Vaginal fluid is one of the most common body fluids found at crime scenes. Discriminating vaginal fluid from other body fluids is important in forensic science; however, few potential protein markers have been reported to date. Proteomic methods for identifying protein markers have gained attention, although few reports have applied this technology to forensic protein markers. Therefore, to identify characteristic vaginal proteins, we examined various body fluids (nasal secretions, saliva, urine, semen, vaginal fluids, and sweat) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry and peptide mass fingerprinting. We identified three components (average molecular mass values 17,237 ± 2, 18,063 ± 2, and 15,075 ± 1) detectable only in vaginal samples: two human small proline-rich protein 3 (SPRR3) isoforms and a human fatty acid-binding protein 5 (FABP5) with an acetylated (+42) N-terminal region lacking the initiator methionine residue (-131). Using ELISA, these yielded markedly high average values in vaginal fluids. The mass spectra of these proteins were not detected in infant saliva but were detected in the vaginal fluid throughout the menstrual cycle. The results of forensic analysis (detection limit, mixed body fluid samples, casework samples, and blind samples) suggest that these proteins are potential forensic markers. In conclusion, high SPRR3 and FABP5 expression levels, which may be used as potential markers for vaginal fluid identification in forensic science, were detected in vaginal fluids from healthy adults.

  8. Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog.

    Science.gov (United States)

    Martin, Dale D O; Vilas, Gonzalo L; Prescher, Jennifer A; Rajaiah, Gurram; Falck, John R; Bertozzi, Carolyn R; Berthiaume, Luc G

    2008-03-01

    Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1-3 months). Consequently, we developed a nonradioactive detection methodology in which a bio-orthogonal azidomyristate analog is specifically incorporated co- or post-translationally into proteins at N-terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million-fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase-cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post-translationally myristoylatable proteins (PKC epsilon, CD-IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post-translational myristoylation in apoptotic Jurkat T cells. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death.

  9. SHARP: genome-scale identification of gene-protein-reaction associations in cyanobacteria.

    Science.gov (United States)

    Krishnakumar, S; Durai, Dilip A; Wangikar, Pramod P; Viswanathan, Ganesh A

    2013-11-01

    Genome scale metabolic model provides an overview of an organism's metabolic capability. These genome-specific metabolic reconstructions are based on identification of gene to protein to reaction (GPR) associations and, in turn, on homology with annotated genes from other organisms. Cyanobacteria are photosynthetic prokaryotes which have diverged appreciably from their nonphotosynthetic counterparts. They also show significant evolutionary divergence from plants, which are well studied for their photosynthetic apparatus. We argue that context-specific sequence and domain similarity can add to the repertoire of the GPR associations and significantly expand our view of the metabolic capability of cyanobacteria. We took an approach that combines the results of context-specific sequence-to-sequence similarity search with those of sequence-to-profile searches. We employ PSI-BLAST for the former, and CDD, Pfam, and COG for the latter. An optimization algorithm was devised to arrive at a weighting scheme to combine the different evidences with KEGG-annotated GPRs as training data. We present the algorithm in the form of software "Systematic, Homology-based Automated Re-annotation for Prokaryotes (SHARP)." We predicted 3,781 new GPR associations for the 10 prokaryotes considered of which eight are cyanobacteria species. These new GPR associations fall in several metabolic pathways and were used to annotate 7,718 gaps in the metabolic network. These new annotations led to discovery of several pathways that may be active and thereby providing new directions for metabolic engineering of these species for production of useful products. Metabolic model developed on such a reconstructed network is likely to give better phenotypic predictions.

  10. Identification and analysis of the acetylated status of poplar proteins reveals analogous N-terminal protein processing mechanisms with other eukaryotes.

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    Chang-Cai Liu

    Full Text Available BACKGROUND: The N-terminal protein processing mechanism (NPM including N-terminal Met excision (NME and N-terminal acetylation (N(α-acetylation represents a common protein co-translational process of some eukaryotes. However, this NPM occurred in woody plants yet remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: To reveal the NPM in poplar, we investigated the N(α-acetylation status of poplar proteins during dormancy by combining tandem mass spectrometry with TiO2 enrichment of acetylated peptides. We identified 58 N-terminally acetylated (N(α-acetylated proteins. Most proteins (47, >81% are subjected to N(α-acetylation following the N-terminal removal of Met, indicating that N(α-acetylation and NME represent a common NPM of poplar proteins. Furthermore, we confirm that poplar shares the analogous NME and N(α-acetylation (NPM to other eukaryotes according to analysis of N-terminal features of these acetylated proteins combined with genome-wide identification of the involving methionine aminopeptidases (MAPs and N-terminal acetyltransferase (Nat enzymes in poplar. The N(α-acetylated reactions and the involving enzymes of these poplar proteins are also identified based on those of yeast and human, as well as the subcellular location information of these poplar proteins. CONCLUSIONS/SIGNIFICANCE: This study represents the first extensive investigation of N(α-acetylation events in woody plants, the results of which will provide useful resources for future unraveling the regulatory mechanisms of N(α-acetylation of proteins in poplar.

  11. Bovine and rodent tamm-horsfall protein (THP) genes: cloning, structural analysis, and promoter identification.

    Science.gov (United States)

    Yu, H; Papa, F; Sukhatme, V P

    1994-01-01

    We have isolated bovine and rodent cDNA and genomic clones encoding the kidney-specific Tamm-Horsfall protein (THP). In both species the gene contains 11 exons, the first of which is noncoding. Exon/intron junctions were analyzed and all were shown to follow the AG/GT rule. A kidney-specific DNase I hypersensitive site was mapped onto a rodent genomic fragment for which the sequence is highly conserved in three species (rat, cow, and human) over a stretch of 350 base pairs. Primer extension and RNase protection analysis identified a transcription start site at the 3' end of this conserved region. A TATA box is located at 32 nucleotides upstream of the start site in the bovine gene and 34 nucleotides upstream in the rodent gene. An inverted CCAAT motif occurs at 65 and 66 nucleotides upstream of the start site in the bovine and rodent genes, respectively. Other highly conserved regions were noted in this 350 bp region and these are likely to be binding sites for transcription factors. A functional assay based on an in vitro transcription system confirmed that the conserved region is an RNA Pol II promoter. The in vitro system accurately initiated transcription from the in vivo start site and was highly sensitive to inhibition by alpha-amanitin at a concentration of 2.5 micrograms/ml. These studies set the stage for the further definition of cis-acting sequences and trans-factors regulating expression of the THP gene, a model for kidney-specific gene expression.

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

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    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.

  13. A cell-based method for screening RNA-protein interactions: identification of constitutive transport element-interacting proteins.

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    Robert L Nakamura

    Full Text Available We have developed a mammalian cell-based screening platform to identify proteins that assemble into RNA-protein complexes. Based on Tat-mediated activation of the HIV LTR, proteins that interact with an RNA target elicit expression of a GFP reporter and are captured by fluorescence activated cell sorting. This "Tat-hybrid" screening platform was used to identify proteins that interact with the Mason Pfizer monkey virus (MPMV constitutive transport element (CTE, a structured RNA hairpin that mediates the transport of unspliced viral mRNAs from the nucleus to the cytoplasm. Several hnRNP-like proteins, including hnRNP A1, were identified and shown to interact with the CTE with selectivity in the reporter system comparable to Tap, a known CTE-binding protein. In vitro gel shift and pull-down assays showed that hnRNP A1 is able to form a complex with the CTE and Tap and that the RGG domain of hnRNP A1 mediates binding to Tap. These results suggest that hnRNP-like proteins may be part of larger export-competent RNA-protein complexes and that the RGG domains of these proteins play an important role in directing these binding events. The results also demonstrate the utility of the screening platform for identifying and characterizing new components of RNA-protein complexes.

  14. 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

    Full Text Available 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

  15. Accurate measurement of {sup 3}J{sub HNHα} couplings in small or disordered proteins from WATERGATE-optimized TROSY spectra

    Energy Technology Data Exchange (ETDEWEB)

    Roche, Julien; Ying, Jinfa; Bax, Ad, E-mail: bax@nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)

    2016-01-15

    Provided that care is taken in adjusting the WATERGATE element of a {sup 1}H–{sup 15}N TROSY-HSQC experiment, such that neither the water magnetization nor the {sup 1}H{sup α} protons are inverted by its final 180° pulse, {sup 3}J{sub HNHα} couplings can be measured directly from splittings in the {sup 1}H dimension of the spectrum. With band-selective {sup 1}H decoupling, very high {sup 15}N resolution can be achieved. A complete set of {sup 3}J{sub HNHα} values, ranging from 3.4 to 10.1 Hz was measured for the 56-residue third domain of IgG-binding protein G (GB3). Using the H–N–C{sup α}–H{sup α} dihedral angles extracted from a RDC-refined structure of GB3, {sup 3}J{sub HNHα} values predicted by a previously parameterized Karplus equation agree to within a root-mean-square deviation (rmsd) of 0.37 Hz with the experimental data. Values measured for the Alzheimer’s implicated Aβ{sup 1−40} peptide fit to within an rmsd of 0.45 Hz to random coil {sup 3}J{sub HNHα} values.

  16. Identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1.

    Directory of Open Access Journals (Sweden)

    Paolo Guazzi

    Full Text Available Loss-of-function mutations of the KRIT1 gene (CCM1 have been associated with the Cerebral Cavernous Malformation (CCM disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signaling networks. In previous work, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.

  17. 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.

  18. 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.

  19. Structure based design towards the identification of novel binding sites and inhibitors for the chikungunya virus envelope proteins.

    Science.gov (United States)

    Rashad, Adel A; Keller, Paul A

    2013-07-01

    Chikungunya virus is an emerging arbovirus that is widespread in tropical regions and is spreading quickly to temperate climates with recent epidemics in Africa, Asia, Europe and the Americas. It is having an increasingly major impact on humans with potentially life-threatening and debilitating arthritis. Thus far, neither vaccines nor medications are available to treat or control the virus and therefore, the development of medicinal chemistry is a vital and immediate issue that needs to be addressed. The viral envelope proteins play a major role during infection through mediation of binding and fusion with the infected cell surfaces. The possible binding target sites of the chikungunya virus envelope proteins have not previously been investigated; we describe here for the first time the identification of novel sites for potential binding on the chikungunya glycoprotein complexes and the identification of possible antagonists for these sites through virtual screening using two successive docking scores; FRED docking for fast precise screening, with the top hits then subjected to a ranking scoring using the AUTODOCK algorithm. Both the immature and the mature forms of the chikungunya envelope proteins were included in the study to increase the probability of finding positive and reliable hits. Some small molecules have been identified as good in silico chikungunya virus envelope proteins inhibitors and these could be good templates for drug design targeting this virus.

  20. Isolation of two strong poly (U binding proteins from moderate halophile Halomonas eurihalina and their identification as cold shock proteins.

    Directory of Open Access Journals (Sweden)

    Usha Kumari Garapati

    Full Text Available Cold shock proteins (Csp are known to be expressed in response to sudden decrease in temperature. They are thought to be involved in a number of cellular processes viz., RNA chaperone activity, translation, transcription, nucleoid condensation. During our studies on ribosomal protein S1 in moderate halophile Halomonas eurihalina, we observed the presence of two strong poly (U binding proteins in abundance in cell extracts from cells grown under normal growth conditions. The proteins can be isolated in a single step using Poly (U cellulose chromatography. The proteins were identified as major cold shock proteins belonging to Csp A family by MALDI-TOF and bioinformatic analysis. Csp 12 kDa was found in both exponential and stationary phases whereas Csp 8 kDa is found only in exponential phase.

  1. Bioinformatic prediction of the exportome of Babesia bovis and identification of novel proteins in parasite-infected red blood cells.

    Science.gov (United States)

    Gohil, Sejal; Kats, Lev M; Seemann, Torsten; Fernandez, Kate M; Siddiqui, Ghizal; Cooke, Brian M

    2013-04-01

    Babesia bovis is a pathogen of considerable economic significance to the livestock industry worldwide but the precise mechanisms by which this parasite causes disease in susceptible cattle remain poorly understood. It is clear, however, that alterations to the structure and function of red blood cells in which the parasites reside and replicate play an important role in pathogenesis and that these are secondary to the export of numerous, currently unknown and uncharacterised parasite-encoded proteins. Using a rational bioinformatic approach, we have identified a set of 362 proteins (117 of which are hypothetical) that we predict encompasses the B. bovis exportome. These exported proteins are likely to be trafficked to various cellular locations, with a subset destined for the red blood cell cytosol or the red blood cell cytoskeleton. These proteins are likely to play important roles in mediating the pathogenesis of babesiosis. We have selected three novel proteins and confirmed their predicted export and localisation within the host red blood cell by immunofluorescence using specific antibodies raised against these proteins. Complete characterisation of these novel exported parasite proteins will help elucidate their function within the host red blood cell and assist in identification of new therapeutic targets for babesiosis.

  2. Purification and identification of an antibacterial protein from the symbiotic bacteria associated with novel entomopathogenic nematode, Rhabditis (Oscheius) sp.

    Science.gov (United States)

    Anju, K M; Archana, M M; Mohandas, C; Nambisan, Bala

    2015-04-01

    Entomopathogenic nematodes (EPN) belonging to the families steinernematidae and heterorhabditidae and their symbiotic bacteria Xenorhabdus and Photorhabdus are well-known as biological control agents and are found to produce a wide range of bioactive secondary metabolites. Studies carried out at the Central Tuber Crops Research Institute (CTCRI) on entomopathogenic nematodes resulted in the identification of novel EPN belonging to the family Rhabditidae. This study reports the purification of a high molecular weight antibacterial protein from culture filtrates of a bacterium (Bacillus cereus) symbiotically associated with a novel entomopathogenic nematode Rhabditis (Oscheius) species, maintained at CTCRI laboratory. Fermentation conditions were standardized and optimum antibacterial activity was observed in tryptic soy broth after 48 h incubation at 30 °C. The aqueous extracts yielded antibacterial proteins which were purified by ammonium sulfate precipitation followed by ion exchange chromatography and size exclusion chromatography. Native gel electrophoresis indicated an active protein of molecular mass 220KDa which resolved into a major band of 90 kDa and a minor band of about 40 kDa on SDS-PAGE. The 90 kDa protein showed antibacterial activity and was further analysed by MALDI TOF-MS/MS. The protein was identified as a TQXA (Threonine-glutamine dipeptide) domain containing protein from Bacillus cereus. The protein was found to be active against Bacillus subtilis MTCC2756, Staphylococus aureus MTCC902 and Escherichia coli MTCC 2622 and was thermally stable.

  3. Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

    Institute of Scientific and Technical Information of China (English)

    Chao FAN; Lan-lan ZHANG; Cheng-feng LEI; Qin FANG

    2009-01-01

    Grass carp reovirus (GCRV), a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstructural protein NS80, encoded by GCRV segment 4, has a high similarity with uNS in MRV(Mammalian orthoreoviruses), which may be associated with viral factory formation. To understand the function of the uNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region (335.742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80(335.742) protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody (mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

  4. Proteome-scale identification of outer membrane proteins in Mycobacterium avium subspecies paratuberculosis using a structure based combined hierarchical approach.

    Science.gov (United States)

    Rana, Aarti; Rub, Abdur; Akhter, Yusuf

    2014-07-29

    Outer membrane proteins (OMPs) in eubacteria have several important roles, which range from membrane transport to the host-pathogen interactions. These are directly involved in pathogen attachment, entry and activation of several pathogen-induced signaling cascades in the cell. The cardinal structural features of OMPs include the presence of a β-barrel, a signal peptide and the absence of the transmembrane helix. This is the first report on proteome-wide identification of OMPs of ruminant pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). The complete proteome of MAP was analyzed using a pipeline of algorithms, which screens the amino acid sequences and structural features shared by OMPs in other bacteria. Secondary structure of these proteins is also analyzed and scores are calculated for amphiphilic β-strands. From the set of 588 exported proteins, 264 proteins are predicted to be inner membrane proteins while 83 proteins are identified as potential OMPs in MAP. Finally, this study identified 57 proteins as top candidates, on the basis of computed isoelectric points, as the core set of OMPs. Significantly, the resulting data for OMPs are not only useful in designing novel vaccines but may also open avenues for the development of early serodiagnostic tools for MAP.

  5. Buffalo cheese whey proteins, identification of a 24 kda protein and characterization of their hydrolysates: in vitro gastrointestinal digestion

    OpenAIRE

    Juliana C Bassan; Goulart,Antonio J.; Nasser, Ana L. M.; Bezerra, Thaís M. S. [UNESP; Garrido, Saulo S.; Rustiguel, Cynthia B.; Guimarães, Luis H. S.; Rubens Monti

    2015-01-01

    Milk whey proteins are well known for their high biological value and versatile functional properties, characteristics that allow its wide use in the food and pharmaceutical industries. In this work, a 24 kDa protein from buffalo cheese whey was analyzed by mass spectrometry and presented homology with Bos taurus beta-lactoglobulin. In addition, the proteins present in buffalo cheese whey were hydrolyzed with pepsin and with different combinations of trypsin, chymotrypsin and carboxypeptidase...

  6. Continuous nanoflow-scanning electrochemical microscopy: voltammetric characterization and application for accurate and reproducible imaging of enzyme-labeled protein microarrays.

    Science.gov (United States)

    Kai, Tianhan; Chen, Shu; Monterroso, Estuardo; Zhou, Feimeng

    2015-04-21

    The coupling of scanning electrochemical microscopy (SECM) to a continuous nanoflow (CNF) system is accomplished with the use of a microconcentric ring electrode/injector probe. The gold microring electrode encapsulated by a glass sheath is robust and can be beveled and polished. The CNF system, comprising a precision gas displacement pump and a rotary valve, is capable of delivering solution to the center of the SECM probe in the range of 1-150 nL/min. Major advantages of the CNF-SECM imaging mode over the conventional SECM generation/collection (G/C) mode include higher imaging resolution, immunity from interferences by species in the bulk solution or at other sites of the substrate, elimination of the feedback current that could interfere with the G/C data interpretation, and versatility of initiating surface reactions/processes via introducing different reactants into the flowing stream. Parameters such as flow rates, probe/substrate separations, and collection efficiencies are examined and optimized. Higher resolution, reproducibility, and accuracy are demonstrated through the application of CNF-SECM to horseradish peroxidase (HRP)-amplified imaging of protein microarrays. By flowing H2O2 and ferrocenemethanol through the injector and detecting the surface-generated ferriceniummethanol, human IgG spots covered with HPR-labeled antihuman IgG can be detected in the range of 13 nM-1.333 μM with a detection limit of 3.0 nM. In addition, consistent images of microarray spots for selective and high-density detection of analytes can be attained.

  7. Metallomics approach for the identification of the iron transport protein transferrin in the blood of harbour seals (Phoca vitulina).

    Science.gov (United States)

    Grebe, Mechthild; Pröfrock, Daniel; Kakuschke, Antje; Broekaert, Jose A C; Prange, Andreas

    2010-10-01

    The health status of marine mammals such as harbour seals (Phoca vitulina) represents an indirect but powerful way for the assessment of environmental changes. The present work illustrates the first investigation and characterisation of Tf isolated from blood samples of North Sea harbour seals with a view to using changes in Tf isoform patterns as an additional parameter in extended studies of their health status. Therefore, an HPLC-ICP-MS approach has been developed which allows the highly resolved separation and fractionation of up to eight different Tf isoforms, as well as their sensitive and specific detection on the basis of their characteristic iron content. Molecule-specific detection techniques such as nanoLC-ESI-QTRAP-MS or MALDI-TOF-MS were used as complementary techniques to unambiguously identify the isolated proteins as Tf via cross species protein identification and to further characterise the molecular weight as well as the sialic acid content, which is responsible for the elution behaviour of the different isoforms during their ion exchange separation. A molecular mass above 80 kDa has been measured for the different seal Tf isoforms, which is in good agreement with the known molecular mass in other mammalian species, while the estimated pI of the different isoforms indicates some differences in comparison to other species. A number of homologies to known Tf sequences have been observed, which finally allows the cross species protein identification. The combined metallomics orientated analytical approach, which includes the complementary application of element and molecule-specific detection techniques, opens up interesting possibilities for the fast and targeted isolation and identification of a diagnostically relevant metal containing protein from an un-sequenced mammalian species prior to its utilisation in extended studies.

  8. Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export.

    Science.gov (United States)

    Heiber, Arlett; Kruse, Florian; Pick, Christian; Grüring, Christof; Flemming, Sven; Oberli, Alexander; Schoeler, Hanno; Retzlaff, Silke; Mesén-Ramírez, Paolo; Hiss, Jan A; Kadekoppala, Madhusudan; Hecht, Leonie; Holder, Anthony A; Gilberger, Tim-Wolf; Spielmann, Tobias

    2013-01-01

    Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.

  9. Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens

    DEFF Research Database (Denmark)

    Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria;

    2012-01-01

    deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins...

  10. Identification of Putative ORF5 Protein of Porcine Circovirus Type 2 and Functional Analysis of GFP-Fused ORF5 Protein.

    Directory of Open Access Journals (Sweden)

    Qizhuang Lv

    Full Text Available Porcine circovirus type 2 (PCV2 is the essential infectious agent responsible for causing porcine circovirus-associated diseases in pigs. To date, eleven RNAs and five viral proteins of PCV2 have been detected. Here, we identified a novel viral gene within the PCV2 genome, termed ORF5, that exists at both the transcriptional and translational level during productive infection of PCV2 in porcine alveolar macrophages 3D4/2 (PAMs. Northern blot analysis was used to demonstrate that the ORF5 gene measures 180 bp in length and overlaps completely with ORF1 when read in the same direction. Site-directed mutagenesis was used to show that the ORF5 protein is not essential for PCV2 replication. To investigate the biological functions of the novel protein, we constructed a recombinant eukaryotic expression plasmid capable of expressing PCV2 ORF5. The results show that the GFP-tagged PCV2 ORF5 protein localizes to the endoplasmic reticulum (ER, is degraded via the proteasome, inhibits PAM growth and prolongs the S-phase of the cell cycle. Further studies show that the GFP-tagged PCV2 ORF5 protein induces ER stress and activates NF-κB, which was further confirmed by a significant upregulation in IL-6, IL-8 and COX-2 expression. In addition, five cellular proteins (GPNMB, CYP1A1, YWHAB, ZNF511 and SRSF3 were found to interact with ORF5 via yeast two-hybrid assay. These findings provide novel information on the identification and functional analysis of the PCV2 ORF5 protein and are likely to be of benefit in elucidating the molecular mechanisms of PCV2 pathogenicity. However, additional experiments are needed to validate the expression and function of the ORF5 protein during PCV2 infection in vitro before any definitive conclusion can be drawn.

  11. Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer

    Institute of Scientific and Technical Information of China (English)

    Ferdous Rastgar Jazii; Zahra Najafi; Reza Malekzadeh; Thomas P Conrads; Abed Ali Ziaee; Christian Abnet; Mansour Yazdznbod; Ali Asghar Karkhane; Ghasem H Salekdeh

    2006-01-01

    AIM: To assess the proteome of normal versus tumor tissue in squamous cell carcinoma of the esophagus(SCCE) in Iranian patients and compare our results with former reports by using proteomics.METHODS: Protein was extracted from normal and tumor tissues. Two dimensional electrophoresis was carried out and spots with differential expression were identified with mass spectrometry. RNA extraction and RT-PCR along with immunodetection were performed.RESULTS: Fourteen proteins were found whose expression levels differed in tumor compared to normal tissues. Mass spectrometric analysis resulted in the identification of β-tropomyosin (TMβ), myosin light chain 2 (and its isoform), myosin regulatory light chain 2,peroxyredoxin 2, annexin I and an unknown polypeptide as the down regulated polypeptides in tumor tissue. Heat shock protein 70 (HSP70), TPM4-ALK fusion oncoprotein 2, myosin light polypeptide 6, keratin I, GH16431p and calreticulin were the up-regulated polypeptides found in tumor tissue. Several of these proteins, such as TMβ,HSP70, annexin I, calreticulin, TPM4-ALK and isoforms of myosins, have been well recognized in tumorigenesis of esophageal or other types of cancers.CONCLUSION: Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCF, including TMβ.

  12. QUICK identification and SPR validation of signal transducers and activators of transcription 3 (Stat3) interacting proteins.

    Science.gov (United States)

    Zheng, Peng; Zhong, Qiu; Xiong, Qian; Yang, Mingkun; Zhang, Jia; Li, Chongyang; Bi, Li-Jun; Ge, Feng

    2012-01-04

    Signal transducers and activators of transcription 3 (Stat3) has been reported to be involved in the pathogenesis of various human diseases and is constitutively active in human multiple myeloma (MM) U266 cells. The Stat3-regulated mechanisms involved in these processes, however, are not fully defined. To further understand the regulation of Stat3 activity, we performed a systematic proteomic analysis of Stat3 interacting proteins in U266 cells. This analysis, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture (SILAC), immunoprecipitation, and quantitative MS. As a result, quantitative mass spectrometry analysis allowed us to distinguish specific Stat3 interacting proteins from background proteins and led to the identification of a total of 38 proteins. Three Stat3 interacting proteins - 14-3-3ζ, PRKCB and Hsp90 - were further confirmed by reciprocal co-immunoprecipitations and surface plasmon resonance (SPR) analysis. Our results therefore not only uncover a number of Stat3 interacting proteins that possess a variety of cellular functions, but also provide new insight into the mechanisms that regulate Stat3 activity and function in MM cells.

  13. Identification of protein secondary structures by laser induced autofluorescence: A study of urea and GnHCl induced protein denaturation

    Science.gov (United States)

    Siddaramaiah, Manjunath; Satyamoorthy, Kapaettu; Rao, Bola Sadashiva Satish; Roy, Suparna; Chandra, Subhash; Mahato, Krishna Kishore

    2017-03-01

    In the present study an attempt has been made to interrogate the bulk secondary structures of some selected proteins (BSA, HSA, lysozyme, trypsin and ribonuclease A) under urea and GnHCl denaturation using laser induced autofluorescence. The proteins were treated with different concentrations of urea (3 M, 6 M, 9 M) and GnHCl (2 M, 4 M, 6 M) and the corresponding steady state autofluorescence spectra were recorded at 281 nm pulsed laser excitations. The recorded fluorescence spectra of proteins were then interpreted based on the existing PDB structures of the proteins and the Trp solvent accessibility (calculated using "Scratch protein predictor" at 30% threshold). Further, the influence of rigidity and conformation of the indole ring (caused by protein secondary structures) on the intrinsic fluorescence properties of proteins were also evaluated using fluorescence of ANS-HSA complexes, CD spectroscopy as well as with trypsin digestion experiments. The outcomes obtained clearly demonstrated GnHCl preferably disrupt helix as compared to the beta β-sheets whereas, urea found was more effective in disrupting β-sheets as compared to the helices. The other way round the proteins which have shown detectable change in the intrinsic fluorescence at lower concentrations of GnHCl were rich in helices whereas, the proteins which showed detectable change in the intrinsic fluorescence at lower concentrations of urea were rich in β-sheets. Since high salt concentrations like GnHCl and urea interfere in the secondary structure analysis by circular dichroism Spectrometry, the present method of analyzing secondary structures using laser induced autofluorescence will be highly advantageous over existing tools for the same.

  14. Identification of an abundant 56 kDa protein implicated in food allergy as granule-bound starch synthase.

    Science.gov (United States)

    Krishnan, Hari B; Chen, Ming-Hsuan

    2013-06-01

    Rice, the staple food of south and east Asian counties, is considered to be hypoallergenic. However, several clinical studies have documented rice-induced allergy in sensitive patients. Rice proteins with molecular weights of 14-16, 26, 33, and 56 kDa have been identified as allergens. Recently, it was documented that the 56 kDa rice allergen was responsible for rice-induced anaphylaxis. The 14-16 kDa allergens have been identified as α-amylase inhibitors; the 26 kDa protein has been identified as α-globulin; and the 33 kDa protein has been identified as glyoxalase I. However, the identity of the 56 kDa rice allergen has not yet been determined. In this study, we demonstrate that serum from patients allergic to maize shows IgE binding to a 56 kDa protein that was present in both maize and rice but not in the oil seeds soybean and peanut. The 56 kDa IgE-binding protein was abundant in the rice endosperm. We have purified this protein from rice endosperm and demonstrated its reactivity to IgE antibodies from the serum of maize-allergic patients. The purified protein was subjected to matrix-assisted laser desorption ionization-time of flight-tandem mass spectrometry analysis, resulting in identification of this rice allergen as granule-bound starch synthase, a product of the Waxy gene. Immunoblot analysis using protein extracts from a waxy mutant of rice revealed the absence of the 56 kDa IgE-binding protein. Our results demonstrate that the 56 kDa rice allergen is granule-bound starch synthase and raise the possibility of using waxy mutants of rice as a potential source of the hypoallergenic diet for patients sensitized to the 56 kDa rice allergen.

  15. Identification of a mammalian mitochondrial homolog of ribosomal protein S7.

    Science.gov (United States)

    Cavdar Koc, E; Blackburn, K; Burkhart, W; Spremulli, L L

    1999-12-01

    Bovine mitochondrial small subunit ribosomal proteins were separated by two-dimensional electrophoresis. The region containing the most basic protein(s) was excised and the protein(s) present subjected to in-gel digestion with trypsin. Electrospray tandem mass spectrometry was used to provide sequence information on some of the peptide products. Searches of the human EST database using the sequence of the longest peptide analyzed indicated that this peptide was from the mammalian mitochondrial homolog of prokaryotic ribosomal protein S7 (MRP S7(human)). MRP S7(human) is a 28-kDa protein with a pI of 10. Significant homology to bacterial S7 is observed especially in the C-terminal half of the protein. Surprisingly, MRP S7(human) shows less homology to the corresponding mitochondrial proteins from plants and fungi than to bacterial S7.

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

    Science.gov (United States)

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

    2016-09-01

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

  17. 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 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...... and substantiate these networks, we performed expressional profiling in human pancreatic islets exposed to proinflammatory cytokines. Three networks were significantly enriched for cytokine-regulated genes and, thus, likely to play an important role for type 1 diabetes in pancreatic islets. Eight of the regulated...

  18. 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.

  19. Identification and in silico analysis of helical lipid binding regions in proteins belonging to the amphitropic protein family

    Indian Academy of Sciences (India)

    Rob C A Keller

    2014-12-01

    The role of protein–lipid interactions is increasingly recognized to be of importance in numerous biological processes. Bioinformatics is being increasingly used as a helpful tool in studying protein–lipid interactions. Especially recently developed approaches recognizing lipid binding regions in proteins can be implemented. In this study one of those bioinformatics approaches specialized in identifying lipid binding helical regions in proteins is expanded. The approach is explored further by features which can be easily obtained manually. Some interesting examples of members of the amphitropic protein family have been investigated in order to demonstrate the additional features of this bioinformatics approach. The results in this study seem to indicate interesting characteristics of amphitropic proteins and provide insight into the mechanistic functioning and overall understanding of this intriguing class of proteins. Additionally, the results demonstrate that the presented bioinformatics approach might be either an interesting starting point in protein–lipid interactions studies or a good tool for selecting new focus points for more detailed experimental research of proteins with known overall protein–lipid binding abilities.

  20. Identification of C5+ extraband of butyrylcholinesterase and two protein bands cathodic to it

    Directory of Open Access Journals (Sweden)

    F. D. Suyatna

    2001-09-01

    Full Text Available Electrophoresis of human plasma yields 4 butyrylcholinesterase (BChE protein bands, i.e. C1, C2, C3, C4 and in some individuals also an extraband C5+. In addition to that other protein bands called "S" bands are also invariably detected. In order to know whether the C5+ and the "S" bands are related to the BChE protein, we have carried out immunological and peptide mapping studies on these proteins. The immunology approach was done by raising polyclonal antibodies against each protein bands (S1, S2, C4 and C5+ and reacted to the plasma protein bands transferred on nitrocellulose papers. Individual raised antibodies recognized all protein bands studied including the C4, an isozyme of BChE, indicating that the protein bands contain similar epitopes. Several protein bands cathodic to S1 also reacted with the antibodies, suggesting that they are probably fractions of the BChE protein, as well. When individual protein bands were digested with S. aureus V8 toxin and α-chymotrypsin, they revealed a striking similarity in peptide pattern among each other. These studies indicate that the S1, S2 and C5+ protein bands belong to the BChE protein. (Med J Indones 2001; 10: 144-9Keywords: Butyrylcholinesterase, C5+ variant, immunodetection, peptide mapping

  1. 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...

  2. Identification and evaluation of metastasis-related proteins, oxysterol binding protein-like 5 and calumenin, in lung tumors.

    Science.gov (United States)

    Nagano, Kazuya; Imai, Sunao; Zhao, Xiluli; Yamashita, Takuya; Yoshioka, Yasuo; Abe, Yasuhiro; Mukai, Yohei; Kamada, Haruhiko; Nakagawa, Shinsaku; Tsutsumi, Yasuo; Tsunoda, Shin-Ichi

    2015-07-01

    Metastasis is an important prognosis factor in lung cancer, therefore, it is imperative to identify target molecules and elucidate molecular mechanism of metastasis for developing new therapeutics and diagnosis methods. We searched for metastasis-related proteins by utilizing a novel antibody proteome technology developed in our laboratory that facilitated efficient screening of useful target proteins. Two-dimensional differential in-gel electrophoresis (2D-DIGE) analysis identified sixteen proteins, which were highly expressed in metastatic lung cancer cells, as protein candidates. Monoclonal single-chain variable fragments (scFvs) binding to candidates were isolated from a scFv-displaying phage library by affinity selection. Tissue microarray analysis of scFvs binding to candidates revealed that oxysterol binding protein-like 5 (OSBPL5) and calumenin (CALU) were expressed at a significantly higher levels in the lung tissues of metastasis-positive cases than that in the metastasis-negative cases (OSBPL5; p=0.0156, CALU; p=0.0055). Furthermore, 80% of OSBPL5 and CALU double-positive cases were positive for lymph node metastasis. Consistent with these observations, overexpression of OSBPL5 and CALU promoted invasiveness of lung cancer cells. Conversely, knockdown of these proteins using respective siRNAs reversed the invasiveness of the lung cancer cells. Moreover, these proteins were expressed in lung tumor tissues, but not in normal lung tissues. In conclusion, OSBPL5 and CALU are related to metastatic potential of lung cancer cells, and they could be useful targets for cancer diagnosis and also for development of drugs against metastasis.

  3. Identification of Novel Targets of the Human Cell Cycle Regulatory Protein Cdc34

    Science.gov (United States)

    1997-07-01

    human Cdc34 and its interacting proteins using Southern, Northern and Western blot analysis. 11 PROPRIETARY Conclusion Knowledge gained about...expression in yeast. Task 2: Month 2-3: Excision of the library (the prey) encoding candidate interacting proteins fused to the activation domain from...Cdc34 and its interacting proteins in carcinogenesis. Task 7: Month 18-28: Study of the structure of human CDC34 and its novel partner proteins in

  4. 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.

  5. 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 b...

  6. Proteomics Identification of Differentially Expressed Leaf Proteins in Response to Setosphaeria turcica Infection in Resistant Maize

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-li; SI Bing-wen; FAN Cheng-ming; LI Hong-jie; WANG Xiao-ming

    2014-01-01

    Northern corn leaf blight (NCLB), caused by the heterothallic ascomycete fungus Setosphaeria turcica, is a destructive foliar disease of maize and represents a serious threat to maize production worldwide. A comparative proteomic study was conducted to explore the molecular mechanisms underlying the defense responses of the maize resistant line A619 Ht2 to S. turcica race 13. Leaf proteins were extracted from mock and S. turcica-infected leaves after inoculated for 72 h and analyzed for differentially expressed proteins using two-dimensional electrophoresis and mass spectrometry identiifcation. 137 proteins showed reproducible differences in abundance by more than 2-fold at least, including 50 up-regulated proteins and 87 down-regulated proteins. 48 protein spots were successfully identiifed by MS analysis, which included 10 unique, 6 up-regulated, 20 down-regulated and 12 disappeared protein spots. These identiifed proteins were classiifed into 9 functional groups and involved in multiple functions, particularly in energy metabolism (46%), protein destination and storage (12%), and disease defense (18%). Some defense-related proteins were upregulated such asβ-glucosidase, SOD, polyamines oxidase, HSC 70 and PPIases; while the expressions of photosynthesis- and metabolism-related proteins were down-regulated, by inoculation with S. turcica. The results indicated that a complex regulatory network was functioned in interaction between the resistant line A619 Ht2 and S. turcica. The resistance processes of A619 Ht2 mainly resided on directly releasing defense proteins, modulation of primary metabolism, affecting photosyntesis and carbohydrate metabolism.

  7. Identification of the FTBL protein of Sumner and Dounce as a leucine aminopeptidase.

    Science.gov (United States)

    Dounce, A L; Allen, P Z

    1987-08-15

    The crystalline beef liver protein of Sumner and Dounce (A. L. Dounce, P. Z. Allen, and G. A. Mourtzikos (1978) Arch. Biochem. Biophys. 188, 251-265) termed FTBL (football) protein because of the shape of its crystals, has been identified as a crystalline leucine aminopeptidase (LAP), on the basis of its high specific LAP activity and coincidence of its N terminal amino acid sequence (30 amino acids) with that of beef eye lens LAP. Amino acid analyses of the two proteins are also in reasonable agreement when based on the exact monomer molecular weight of beef eye lens protein obtained by the van Loon group ((1982) J. Biol. Chem. 257, 7077-7081). Our previously published monomer molecular weight of the FTBL protein was 25% too high, leading to the erroneous conclusion that the beef liver FTBL-LAP protein was a tetramer rather than a hexamer, as found by the van Loon group for beef lens LAP. The present report, taken together with our first paper on the FTBL protein establishes that the FTBL-LAP protein has been isolated from beef kidney and beef spleen as well as from beef liver. We now find that the properties of FTBL-LAP protein indicate that it is the same protein as beef eye lens LAP. The cellular and intracellular distributions of the FTBL-LAP protein have been considered in our first publication on the FTBL protein.

  8. Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination

    Energy Technology Data Exchange (ETDEWEB)

    Witt, P.L.; Bownds, M.D.

    1987-03-24

    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.

  9. Enhancements to the Rosetta Energy Function Enable Improved Identification of Small Molecules that Inhibit Protein-Protein Interactions.

    Directory of Open Access Journals (Sweden)

    Andrea Bazzoli

    Full Text Available Protein-protein interactions are among today's most exciting and promising targets for therapeutic intervention. To date, identifying small-molecules that selectively disrupt these interactions has proven particularly challenging for virtual screening tools, since these have typically been optimized to perform well on more "traditional" drug discovery targets. Here, we test the performance of the Rosetta energy function for identifying compounds that inhibit protein interactions, when these active compounds have been hidden amongst pools of "decoys." Through this virtual screening benchmark, we gauge the effect of two recent enhancements to the functional form of the Rosetta energy function: the new "Talaris" update and the "pwSHO" solvation model. Finally, we conclude by developing and validating a new weight set that maximizes Rosetta's ability to pick out the active compounds in this test set. Looking collectively over the course of these enhancements, we find a marked improvement in Rosetta's ability to identify small-molecule inhibitors of protein-protein interactions.

  10. Identification of Cry1Ac and Cry2Ab proteins in transgenic cotton seeds available in Gujarat (India by ELISA method

    Directory of Open Access Journals (Sweden)

    Alka Dohare

    2014-02-01

    Full Text Available Along with the increase market of the transgenic crops, the demand for testing GMOs and for certifying non-GMO foodstuffs has increased dramatically. Within the arena of expanding techniques for identification and quantification of transgenic crops, two major approaches for detecting GMOs are still applicable on large scale, namely ELISA based protein detection and PCR based gene identification. In present study, ELISA techniques was adopted to identify the specific Cry1Ac and Cry2Ab proteins in some transgenic cotton plants seed samples viz., Gujarat cotton hybrid – 6 (BG II, Gujarat cotton hybrid – 8 (BG II and Gujarat cotton hybrid – 10 (BG II from the Gujarat state of India. The study reveals the presence of both Cry1Ac and Cry2Ab proteins in the transgenic seed samples and also demonstrated that the technique of ELISA for identification of Cry1Ac and Cry2Ab proteins is quite handy and easily adoptable.

  11. Simplified sample preparation method for protein identification by matrix-assisted laser desorption/ionization mass spectrometry: in-gel digestion on the probe surface

    DEFF Research Database (Denmark)

    Stensballe, A; Jensen, Ole Nørregaard

    2001-01-01

    Identification and detailed characterization of complex mixtures of proteins separated by polyacrylamide gel electrophoresis (PAGE) require optimized and robust methods for interfacing electrophoretic techniques to mass spectrometry. Peptide mapping by matrix-assisted laser desorption/ionization-......Identification and detailed characterization of complex mixtures of proteins separated by polyacrylamide gel electrophoresis (PAGE) require optimized and robust methods for interfacing electrophoretic techniques to mass spectrometry. Peptide mapping by matrix-assisted laser desorption...... for protein identification similar to that obtained by the traditional protocols for in-gel digestion and MALDI peptide mass mapping of human proteins, i.e. approximately 60%. The overall performance of the novel on-probe digestion method is comparable with that of the standard in-gel sample preparation...

  12. In silico identification of specialized secretory-organelle proteins in apicomplexan parasites and in vivo validation in Toxoplasma gondii.

    Directory of Open Access Journals (Sweden)

    Zhongqiang Chen

    Full Text Available Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions.

  13. Fatty Acid-binding Proteins Interact with Comparative Gene Identification-58 Linking Lipolysis with Lipid Ligand Shuttling.

    Science.gov (United States)

    Hofer, Peter; Boeszoermenyi, Andras; Jaeger, Doris; Feiler, Ursula; Arthanari, Haribabu; Mayer, Nicole; Zehender, Fabian; Rechberger, Gerald; Oberer, Monika; Zimmermann, Robert; Lass, Achim; Haemmerle, Guenter; Breinbauer, Rolf; Zechner, Rudolf; Preiss-Landl, Karina

    2015-07-24

    The coordinated breakdown of intracellular triglyceride (TG) stores requires the exquisitely regulated interaction of lipolytic enzymes with regulatory, accessory, and scaffolding proteins. Together they form a dynamic multiprotein network designated as the "lipolysome." Adipose triglyceride lipase (Atgl) catalyzes the initiating step of TG hydrolysis and requires comparative gene identification-58 (Cgi-58) as a potent activator of enzyme activity. Here, we identify adipocyte-type fatty acid-binding protein (A-Fabp) and other members of the fatty acid-binding protein (Fabp) family as interaction partners of Cgi-58. Co-immunoprecipitation, microscale thermophoresis, and solid phase assays proved direct protein/protein interaction between A-Fabp and Cgi-58. Using nuclear magnetic resonance titration experiments and site-directed mutagenesis, we located a potential contact region on A-Fabp. In functional terms, A-Fabp stimulates Atgl-catalyzed TG hydrolysis in a Cgi-58-dependent manner. Additionally, transcriptional transactivation assays with a luciferase reporter system revealed that Fabps enhance the ability of Atgl/Cgi-58-mediated lipolysis to induce the activity of peroxisome proliferator-activated receptors. Our studies identify Fabps as crucial structural and functional components of the lipolysome.

  14. 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.

  15. Identification of a novel conserved HLA-A*0201-restricted epitope from the spike protein of SARS-CoV

    Directory of Open Access Journals (Sweden)

    Ni Bing

    2009-12-01

    Full Text Available Abstract Background The spike (S protein is a major structural glycoprotein of coronavirus (CoV, the causal agent of severe acute respiratory syndrome (SARS. The S protein is a potent target for SARS-specific cell-mediated immune responses. However, the mechanism CoV pathogenesis in SARS and the role of special CTLs in virus clearance are still largely uncharacterized. Here, we describe a study that leads to the identification of a novel HLA-A*0201-restricted epitope from conserved regions of S protein. Results First, different SARS-CoV sequences were analyzed to predict eight candidate peptides from conserved regions of the S protein based upon HLA-A*0201 binding and proteosomal cleavage. Four of eight candidate peptides were tested by HLA-A*0201 binding assays. Among the four candidate peptides, Sp8 (S958-966, VLNDILSRL induced specific CTLs both ex vivo in PBLs of healthy HLA-A2+ donors and in HLA-A2.1/Kb transgenic mice immunized with a plasmid encoding full-length S protein. The immunized mice released IFN-γ and lysed target cells upon stimulation with Sp8 p