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Sample records for improves protein identification

  1. Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor.

    PubMed

    Zhou, Hu; Wang, Fangjun; Wang, Yuwei; Ning, Zhibin; Hou, Weimin; Wright, Theodore G; Sundaram, Meenakshi; Zhong, Shumei; Yao, Zemin; Figeys, Daniel

    2011-10-01

    Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

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

    PubMed

    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.

  3. Separating the wheat from the chaff: unbiased filtering of background tandem mass spectra improves protein identification.

    PubMed

    Junqueira, Magno; Spirin, Victor; Santana Balbuena, Tiago; Waridel, Patrice; Surendranath, Vineeth; Kryukov, Grigoriy; Adzhubei, Ivan; Thomas, Henrik; Sunyaev, Shamil; Shevchenko, Andrej

    2008-08-01

    Only a small fraction of spectra acquired in LC-MS/MS runs matches peptides from target proteins upon database searches. The remaining, operationally termed background, spectra originate from a variety of poorly controlled sources and affect the throughput and confidence of database searches. Here, we report an algorithm and its software implementation that rapidly removes background spectra, regardless of their precise origin. The method estimates the dissimilarity distance between screened MS/MS spectra and unannotated spectra from a partially redundant background library compiled from several control and blank runs. Filtering MS/MS queries enhanced the protein identification capacity when searches lacked spectrum to sequence matching specificity. In sequence-similarity searches it reduced by, on average, 30-fold the number of orphan hits, which were not explicitly related to background protein contaminants and required manual validation. Removing high quality background MS/MS spectra, while preserving in the data set the genuine spectra from target proteins, decreased the false positive rate of stringent database searches and improved the identification of low-abundance proteins. PMID:18558732

  4. Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

    NASA Astrophysics Data System (ADS)

    Li, Xiaoyan; Li, Zixuan; Xie, Boer; Sharp, Joshua S.

    2013-11-01

    Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein-ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric "oxidized" peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

  5. Protein-energy malnutrition in the rehabilitation setting: Evidence to improve identification.

    PubMed

    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.

  6. Protein-energy malnutrition in the rehabilitation setting: Evidence to improve identification.

    PubMed

    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. PMID:26921933

  7. Improving protein identification using complementary fragmentation techniques in fourier transform mass spectrometry.

    PubMed

    Nielsen, Michael L; Savitski, Mikhail M; Zubarev, Roman A

    2005-06-01

    Identification of proteins by MS/MS is performed by matching experimental mass spectra against calculated spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the experimental data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-positive identifications, which arise from incomplete data as well as from the presence of misleading ions in experimental mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. We employed a novel technique of reduction of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the analysis specificity, which was further strengthened by the high mass accuracy (approximately 1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Analysis was made using the data-dependent acquisition mode. Extraction of complementary sequence information was performed prior to data base search using in-house written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on average a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on average 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amount of false-positive identifications was dramatically reduced by the combined use of CAD and ECD.

  8. Method optimization for proteomic analysis of soybean leaf: Improvements in identification of new and low-abundance proteins

    PubMed Central

    Mesquita, Rosilene Oliveira; de Almeida Soares, Eduardo; de Barros, Everaldo Gonçalves; Loureiro, Marcelo Ehlers

    2012-01-01

    The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins. PMID:22802721

  9. Improved enrichment and proteomic identification of outer membrane proteins from a Gram-negative bacterium: focus on Caulobacter crescentus.

    PubMed

    Cao, Yuan; Johnson, Helen M; Bazemore-Walker, Carthene R

    2012-01-01

    Efforts to characterize proteins found in the outer membrane (OM) of Gram-negative bacteria have been steadily increasing due to the promise of expanding our understanding of fundamental bacterial processes such as cell adhesion or cell wall biogenesis as well as the promise of finding potential vaccine- or drug-targets for virulent bacteria. We have developed a mass spectrometry-compatible experimental strategy that resulted in increased coverage of the OM proteome of a model organism, Caulobacter crescentus. The specificity of the OM enrichment step was improved by using detergent solubilization of the protein pellet, low-density cell culture conditions, and a surface-layer deficient cell line. Additionally, efficient gel-assisted digestion, high-resolution RP/RP-MS/MS, and rigorous bioinformatic analysis led to the identification of 234 proteins using strict identification criteria (≥ two unique peptides per protein; peptide false discovery rate <2%). Eighty-four of the detected proteins were predicted to localize to the OM or extracellular space. These results represent ~70% coverage of the predicted OM/extracellular proteome of C. crescentus. This analytical approach, which considers important experimental variables not previously explored in published OM protein studies, can be applied to other OM proteomic endeavors "as is" or with slight modification and should improve the large-scale study of this especially challenging subproteome.

  10. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry

    PubMed Central

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan A.; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization - tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT. PMID:23880742

  11. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry.

    PubMed

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan A; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-containing peptides in enzymic prolamin digests by electrospray ionization--tandem mass spectrometry. Both chymotrypsin and thermolysin yielded cysteine-containing peptides from different gluten proteins, but more proteins could be identified after chymotryptic digestion. In addition, to the best of our knowledge, we were the first to label prolamins with isotope coded affinity tags (ICAT), which are commonly used for quantitative proteomics. However, more peptides were detected after labeling gluten proteins with 4-VP and IDAM than with ICAT.

  12. Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

    PubMed

    Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

    2010-01-01

    Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions.

  13. Preprocessing significantly improves the peptide/protein identification sensitivity of high-resolution isobarically labeled tandem mass spectrometry data.

    PubMed

    Sheng, Quanhu; Li, Rongxia; Dai, Jie; Li, Qingrun; Su, Zhiduan; Guo, Yan; Li, Chen; Shyr, Yu; Zeng, Rong

    2015-02-01

    Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994. PMID:25435543

  14. Preprocessing significantly improves the peptide/protein identification sensitivity of high-resolution isobarically labeled tandem mass spectrometry data.

    PubMed

    Sheng, Quanhu; Li, Rongxia; Dai, Jie; Li, Qingrun; Su, Zhiduan; Guo, Yan; Li, Chen; Shyr, Yu; Zeng, Rong

    2015-02-01

    Isobaric labeling techniques coupled with high-resolution mass spectrometry have been widely employed in proteomic workflows requiring relative quantification. For each high-resolution tandem mass spectrum (MS/MS), isobaric labeling techniques can be used not only to quantify the peptide from different samples by reporter ions, but also to identify the peptide it is derived from. Because the ions related to isobaric labeling may act as noise in database searching, the MS/MS spectrum should be preprocessed before peptide or protein identification. In this article, we demonstrate that there are a lot of high-frequency, high-abundance isobaric related ions in the MS/MS spectrum, and removing isobaric related ions combined with deisotoping and deconvolution in MS/MS preprocessing procedures significantly improves the peptide/protein identification sensitivity. The user-friendly software package TurboRaw2MGF (v2.0) has been implemented for converting raw TIC data files to mascot generic format files and can be downloaded for free from https://github.com/shengqh/RCPA.Tools/releases as part of the software suite ProteomicsTools. The data have been deposited to the ProteomeXchange with identifier PXD000994.

  15. An improved in-gel digestion method for efficient identification of protein and glycosylation analysis of glycoproteins using guanidine hydrochloride.

    PubMed

    Takakura, Daisuke; Hashii, Noritaka; Kawasaki, Nana

    2014-02-01

    In-gel digestion followed by LC/MS/MS is widely used for the identification of trace amounts of proteins and for the site-specific glycosylation analysis of glycoproteins in cells and tissues. A major limitation of this technique is the difficulty in acquiring reliable mass spectra for peptides present in minute quantities and glycopeptides with high heterogeneity and poor hydrophobicity. It is considered that the SDS used in electrophoresis can interact with proteins noncovalently and impede the ionization of peptides/glycopeptides. In this study, we report an improved in-gel digestion method to acquire reliable mass spectra of a trace amount of peptides/glycopeptides. A key innovation of our improved method is the use of guanidine hydrochloride, which forms complexes with the residual SDS molecules in the sample. The precipitation and removal of SDS by addition of the guanidine hydrochloride was successful in improving the S/N of peptides/glycopeptides in mass spectra and acquiring a more comprehensive MS/MS data set for the various glycoforms of each glycopeptide. PMID:24272977

  16. 3PFDB+: improved search protocol and update for the identification of representatives of protein sequence domain families.

    PubMed

    Joseph, Agnel P; Shingate, Prashant; Upadhyay, Atul K; Sowdhamini, R

    2014-01-01

    Protein domain families are usually classified on the basis of similarity of amino acid sequences. Selection of a single representative sequence for each family provides targets for structure determination or modeling and also enables fast sequence searches to associate new members to a family. Such a selection could be challenging since some of these domain families exhibit huge variation depending on the number of members in the family, the average family sequence length or the extent of sequence divergence within a family. We had earlier created 3PFDB database as a repository of best representative sequences, selected from each PFAM domain family on the basis of high coverage. In this study, we have improved the database using more efficient strategies for the initial generation of sequence profiles and implement two independent methods, FASSM and HMMER, for identifying family members. HMMER employs a global sequence similarity search, while FASSM relies on motif identification and matching. This improved and updated database, 3PFDB+ generated in this study, provides representative sequences and profiles for PFAM families, with 13 519 family representatives having more than 90% family coverage. The representative sequence is also highlighted in a two-dimensional plot, which reflects the relative divergence between family members. Representatives belonging to small families with short sequences are mainly associated with low coverage. The set of sequences not recognized by the family representative profiles, highlight several potential false or weak family associations in PFAM. Partial domains and fragments dominate such cases, along with sequences that are highly diverged or different from other family members. Some of these outliers were also predicted to have different secondary structure contents, which reflect different putative structure or functional roles for these domain sequences. Database URL: http://caps.ncbs.res.in/3pfdbplus/.

  17. Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence.

    PubMed

    Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

    2016-07-01

    Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain

  18. Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence

    PubMed Central

    Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

    2016-01-01

    Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain

  19. Improvement in Protein Domain Identification Is Reached by Breaking Consensus, with the Agreement of Many Profiles and Domain Co-occurrence.

    PubMed

    Bernardes, Juliana; Zaverucha, Gerson; Vaquero, Catherine; Carbone, Alessandra

    2016-07-01

    Traditional protein annotation methods describe known domains with probabilistic models representing consensus among homologous domain sequences. However, when relevant signals become too weak to be identified by a global consensus, attempts for annotation fail. Here we address the fundamental question of domain identification for highly divergent proteins. By using high performance computing, we demonstrate that the limits of state-of-the-art annotation methods can be bypassed. We design a new strategy based on the observation that many structural and functional protein constraints are not globally conserved through all species but might be locally conserved in separate clades. We propose a novel exploitation of the large amount of data available: 1. for each known protein domain, several probabilistic clade-centered models are constructed from a large and differentiated panel of homologous sequences, 2. a decision-making protocol combines outcomes obtained from multiple models, 3. a multi-criteria optimization algorithm finds the most likely protein architecture. The method is evaluated for domain and architecture prediction over several datasets and statistical testing hypotheses. Its performance is compared against HMMScan and HHblits, two widely used search methods based on sequence-profile and profile-profile comparison. Due to their closeness to actual protein sequences, clade-centered models are shown to be more specific and functionally predictive than the broadly used consensus models. Based on them, we improved annotation of Plasmodium falciparum protein sequences on a scale not previously possible. We successfully predict at least one domain for 72% of P. falciparum proteins against 63% achieved previously, corresponding to 30% of improvement over the total number of Pfam domain predictions on the whole genome. The method is applicable to any genome and opens new avenues to tackle evolutionary questions such as the reconstruction of ancient domain

  20. Improved autonomous star identification algorithm

    NASA Astrophysics Data System (ADS)

    Luo, Li-Yan; Xu, Lu-Ping; Zhang, Hua; Sun, Jing-Rong

    2015-06-01

    The log-polar transform (LPT) is introduced into the star identification because of its rotation invariance. An improved autonomous star identification algorithm is proposed in this paper to avoid the circular shift of the feature vector and to reduce the time consumed in the star identification algorithm using LPT. In the proposed algorithm, the star pattern of the same navigation star remains unchanged when the stellar image is rotated, which makes it able to reduce the star identification time. The logarithmic values of the plane distances between the navigation and its neighbor stars are adopted to structure the feature vector of the navigation star, which enhances the robustness of star identification. In addition, some efforts are made to make it able to find the identification result with fewer comparisons, instead of searching the whole feature database. The simulation results demonstrate that the proposed algorithm can effectively accelerate the star identification. Moreover, the recognition rate and robustness by the proposed algorithm are better than those by the LPT algorithm and the modified grid algorithm. Project supported by the National Natural Science Foundation of China (Grant Nos. 61172138 and 61401340), the Open Research Fund of the Academy of Satellite Application, China (Grant No. 2014_CXJJ-DH_12), the Fundamental Research Funds for the Central Universities, China (Grant Nos. JB141303 and 201413B), the Natural Science Basic Research Plan in Shaanxi Province, China (Grant No. 2013JQ8040), the Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20130203120004), and the Xi’an Science and Technology Plan, China (Grant. No CXY1350(4)).

  1. Protein-protein binding site identification by enumerating the configurations

    PubMed Central

    2012-01-01

    Background The ability to predict protein-protein binding sites has a wide range of applications, including signal transduction studies, de novo drug design, structure identification and comparison of functional sites. The interface in a complex involves two structurally matched protein subunits, and the binding sites can be predicted by identifying structural matches at protein surfaces. Results We propose a method which enumerates “all” the configurations (or poses) between two proteins (3D coordinates of the two subunits in a complex) and evaluates each configuration by the interaction between its components using the Atomic Contact Energy function. The enumeration is achieved efficiently by exploring a set of rigid transformations. Our approach incorporates a surface identification technique and a method for avoiding clashes of two subunits when computing rigid transformations. When the optimal transformations according to the Atomic Contact Energy function are identified, the corresponding binding sites are given as predictions. Our results show that this approach consistently performs better than other methods in binding site identification. Conclusions Our method achieved a success rate higher than other methods, with the prediction quality improved in terms of both accuracy and coverage. Moreover, our method is being able to predict the configurations of two binding proteins, where most of other methods predict only the binding sites. The software package is available at http://sites.google.com/site/guofeics/dobi for non-commercial use. PMID:22768846

  2. Proteome-wide identification of proteins and their modifications with decreased ambiguities and improved false discovery rates using unique sequence tags

    SciTech Connect

    Shen, Yufeng; Tolic, Nikola; Hixson, Kim K.; Purvine, Samuel O.; Pasa-Tolic, Liljiana; Qian, Weijun; Adkins, Joshua N.; Moore, Ronald J.; Smith, Richard D.

    2008-03-15

    Identifying proteins correctly and with known levels of confidence remain as significant challenges for proteomics. Random or decoy peptide databases are increasingly being used to estimate the false discovery rate (FDR), e.g., from liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of tryptic digests. We show that this approach can significantly underestimate the FDR, and describe an approach for more confident protein identifications that uses unique partial sequences derived from a combination of database searching and de novo-style data analyses of high precision MS/MS data. Applied to a Saccharomyces cerevisiae tryptic digest, the approach provided 3,132 confident peptide identifications (~5% modified in some fashion), covering 575 proteins with an estimated zero FDR. The conventional approach provided 3,359 peptide identifications and 656 proteins with 0.3% FDR based upon a decoy database analysis. However, the present approach revealed ~5% of the 3,359 identifications to be incorrect, and many more as potentially ambiguous, (e.g., due to not considering certain amino acid substitutions and modifications). In addition, 677 peptides and 39 proteins were identified that had been missed by conventional analysis, including non-tryptic peptides, peptides with various expected/unexpected chemical modifications, known/unknown posttranslational modifications, single nucleotide polymorphisms or gene encoding errors, and multiple modifications of individual peptides.

  3. Improved method for identification of low abundance proteins using 2D-gel electrophoresis, MALDI-TOF and TOF/TOF

    EPA Science Inventory

    Introduction: Differential protein expression studies have been routinely performed in our laboratory to determine the health effects of environmentally-important chemicals. In this abstract, improvements in the in-gel protein digestion, MALDI plate spotting and data acquisition...

  4. Protein Identification Using Top-Down

    SciTech Connect

    Liu, Xiaowen; Sirotkin, Yakov; Shen, Yufeng; Anderson, Gordon A.; Tsai, Yi-Hsuan S.; Ting, Ying S.; Goodlett, David R.; Smith, Richard D.; Bafna, Vineet; Pevzner, Pavel A.

    2012-06-01

    In the last two years, due to advances in protein separation and mass spectrometry, top-down mass spectrometry moved from analyzing single proteins to analyzing complex samples and identifying hundreds and even thousands of proteins. However, computational tools for database search of top-down spectra against protein databases are still in infancy. We describe MS-Align+, a fast algorithm for top-down protein identification based on spectral alignment that enables searches for unexpected post-translational modifications (PTMs). We also propose a method for evaluating statistical significance of top-down protein identifications and further benchmark MS-Align+ along with PIITA, ProSightPTM and SEQUEST, which were previously used for top-down MS/MS database searches. We demonstrate that MS-Align+ and PIITA significantly increase the number of identified proteins as compared to ProSightPTM and SEQUEST.

  5. Identification of HUGT1 as a potential BiP activator and a cellular target for improvement of recombinant protein production using a cDNA screening system.

    PubMed

    Ku, Sebastian Chih Yuan; Lwa, Teng Rhui; Giam, Maybelline; Yap, Miranda Gek Sim; Chao, Sheng-Hao

    2009-05-31

    The development of a high-throughput functional genomic screening provides a novel and expeditious approach in identifying critical genes involved in specific biological processes. Here we describe a cell-based cDNA screening system to identify the transcription activators of BiP, an endoplasmic reticulum (ER) chaperone protein. BiP promoter contains the ER stress element which is commonly present in the genes involved in unfolded protein response (UPR) that regulates protein secretion in cells. Therefore, the positive regulators of BiP may also be utilized to improve the recombinant protein production through modulation of UPR. Four BiP activators, including human UDP-glucose:glycoprotein glucosyltransferase 1 (HUGT1), are identified by the cDNA screening. Overexpression of HUGT1 leads to a significant increase in the production of recombinant erythropoietin, interferon gamma, and monoclonal antibody in HEK293 cells. Our results demonstrate that the cDNA screening for BiP activators may be effective to identify the novel BiP regulators and HUGT1 may serve as an ideal target gene for improving the recombinant protein production in mammalian cells. PMID:19466607

  6. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling.

  7. Improvement of genome assembly completeness and identification of novel full-length protein-coding genes by RNA-seq in the giant panda genome.

    PubMed

    Chen, Meili; Hu, Yibo; Liu, Jingxing; Wu, Qi; Zhang, Chenglin; Yu, Jun; Xiao, Jingfa; Wei, Fuwen; Wu, Jiayan

    2015-12-11

    High-quality and complete gene models are the basis of whole genome analyses. The giant panda (Ailuropoda melanoleuca) genome was the first genome sequenced on the basis of solely short reads, but the genome annotation had lacked the support of transcriptomic evidence. In this study, we applied RNA-seq to globally improve the genome assembly completeness and to detect novel expressed transcripts in 12 tissues from giant pandas, by using a transcriptome reconstruction strategy that combined reference-based and de novo methods. Several aspects of genome assembly completeness in the transcribed regions were effectively improved by the de novo assembled transcripts, including genome scaffolding, the detection of small-size assembly errors, the extension of scaffold/contig boundaries, and gap closure. Through expression and homology validation, we detected three groups of novel full-length protein-coding genes. A total of 12.62% of the novel protein-coding genes were validated by proteomic data. GO annotation analysis showed that some of the novel protein-coding genes were involved in pigmentation, anatomical structure formation and reproduction, which might be related to the development and evolution of the black-white pelage, pseudo-thumb and delayed embryonic implantation of giant pandas. The updated genome annotation will help further giant panda studies from both structural and functional perspectives.

  8. Improvement of genome assembly completeness and identification of novel full-length protein-coding genes by RNA-seq in the giant panda genome.

    PubMed

    Chen, Meili; Hu, Yibo; Liu, Jingxing; Wu, Qi; Zhang, Chenglin; Yu, Jun; Xiao, Jingfa; Wei, Fuwen; Wu, Jiayan

    2015-01-01

    High-quality and complete gene models are the basis of whole genome analyses. The giant panda (Ailuropoda melanoleuca) genome was the first genome sequenced on the basis of solely short reads, but the genome annotation had lacked the support of transcriptomic evidence. In this study, we applied RNA-seq to globally improve the genome assembly completeness and to detect novel expressed transcripts in 12 tissues from giant pandas, by using a transcriptome reconstruction strategy that combined reference-based and de novo methods. Several aspects of genome assembly completeness in the transcribed regions were effectively improved by the de novo assembled transcripts, including genome scaffolding, the detection of small-size assembly errors, the extension of scaffold/contig boundaries, and gap closure. Through expression and homology validation, we detected three groups of novel full-length protein-coding genes. A total of 12.62% of the novel protein-coding genes were validated by proteomic data. GO annotation analysis showed that some of the novel protein-coding genes were involved in pigmentation, anatomical structure formation and reproduction, which might be related to the development and evolution of the black-white pelage, pseudo-thumb and delayed embryonic implantation of giant pandas. The updated genome annotation will help further giant panda studies from both structural and functional perspectives. PMID:26658305

  9. Improvement of genome assembly completeness and identification of novel full-length protein-coding genes by RNA-seq in the giant panda genome

    PubMed Central

    Chen, Meili; Hu, Yibo; Liu, Jingxing; Wu, Qi; Zhang, Chenglin; Yu, Jun; Xiao, Jingfa; Wei, Fuwen; Wu, Jiayan

    2015-01-01

    High-quality and complete gene models are the basis of whole genome analyses. The giant panda (Ailuropoda melanoleuca) genome was the first genome sequenced on the basis of solely short reads, but the genome annotation had lacked the support of transcriptomic evidence. In this study, we applied RNA-seq to globally improve the genome assembly completeness and to detect novel expressed transcripts in 12 tissues from giant pandas, by using a transcriptome reconstruction strategy that combined reference-based and de novo methods. Several aspects of genome assembly completeness in the transcribed regions were effectively improved by the de novo assembled transcripts, including genome scaffolding, the detection of small-size assembly errors, the extension of scaffold/contig boundaries, and gap closure. Through expression and homology validation, we detected three groups of novel full-length protein-coding genes. A total of 12.62% of the novel protein-coding genes were validated by proteomic data. GO annotation analysis showed that some of the novel protein-coding genes were involved in pigmentation, anatomical structure formation and reproduction, which might be related to the development and evolution of the black-white pelage, pseudo-thumb and delayed embryonic implantation of giant pandas. The updated genome annotation will help further giant panda studies from both structural and functional perspectives. PMID:26658305

  10. An improved PCR method for gender identification of eagles.

    PubMed

    Chang, Hsueh-Wei; Chou, Ta-Ching; Gu, De-Leung; Cheng, Chun-An; Chang, Chia-Che; Yao, Cheng-Te; Chuang, Li-Yeh; Wen, Cheng-Hao; Chou, Yii-Cheng; Tan, Kock-Yee; Cheng, Chien-Chung

    2008-06-01

    Eagles are sexually monomorphic and therefore it is difficult to determine their gender, which is a crucial need for management purposes. In this study, we have developed an improved gender identification method by exploiting length differences between the Chromo-Helicase-DNA binding protein (CHD)-Z and CHD-W genes of Spilornis cheela hoya. By comparing DNA sequences for CHD-W and CHD-Z from 10 species of Falconiformes eagles we designed universal gender identification PCR primers that exploit differences in product size. Standard agarose gels were shown to easily distinguish between the 148-bp CHD-ZW and the 258-bp CHD-W PCR products. When used with 28 samples of S. cheela hoya, our improved universal primers provided a fast and precise gender identification assay. PMID:18385011

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

    PubMed Central

    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

  12. A Perl procedure for protein identification by Peptide Mass Fingerprinting

    PubMed Central

    Tiengo, Alessandra; Barbarini, Nicola; Troiani, Sonia; Rusconi, Luisa; Magni, Paolo

    2009-01-01

    Background One of the topics of major interest in proteomics is protein identification. Protein identification can be achieved by analyzing the mass spectrum of a protein sample through different approaches. One of them, called Peptide Mass Fingerprinting (PMF), combines mass spectrometry (MS) data with searching strategies in a suitable database of known protein to provide a list of candidate proteins ranked by a score. To this aim, several algorithms and software tools have been proposed. However, the scoring methods and mainly the statistical evaluation of the results can be significantly improved. Results In this work, a Perl procedure for protein identification by PMF, called MsPI (Mass spectrometry Protein Identification), is presented. The implemented scoring methods were derived from the literature. MsPI implements a strategy to remove the contaminant masses present in the acquired spectra. Moreover, MsPI includes a statistical method to assign to each candidate protein, in addition to the scoring value, a p-value. Results obtained by MsPI on a dataset of 10 protein samples were compared with those achieved using two other software tools, i.e. Piums and Mascot. Piums implements one of the scoring methods available in MsPI, while Mascot is one of the most frequently used software tools in the protein identification field. MsPI scripts are available for downloading on the web site . Conclusion The performances of MsPI seem to be better than those of Piums and Mascot. In fact, on the considered dataset, MsPI includes in its candidate proteins list, the "true" proteins nine times over ten, whereas Piums includes in its list the "true" proteins only four time over ten. Even if Mascot also correctly includes in the candidates list the "true" proteins nine times over ten, it provides longer candidate lists, therefore increasing the number of false positives when the molecular weight of the proteins in the sample is approximatively known (e.g. by the 1-D/2-D

  13. Improved corn protein based articles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Developing higher value uses for zein (corn protein), a potential major co-product of the bio-ethanol industry, will improve the economics of this business. Historically, zein was predominantly used in the textile fiber industry. Unfortunately the techniques used at that time to modify the zein cann...

  14. Insect Seminal Fluid Proteins: Identification and Function

    PubMed Central

    Avila, Frank W.; Sirot, Laura K.; LaFlamme, Brooke A.; Rubinstein, C. Dustin; Wolfner, Mariana F.

    2014-01-01

    Seminal fluid proteins (SFPs) produced in reproductive tract tissues of male insects and transferred to females during mating induce numerous physiological and behavioral post-mating changes in females. These changes include decreasing receptivity to re-mating, affecting sperm storage parameters, increasing egg production, modulating sperm competition, feeding behaviors, and mating plug formation. In addition, SFPs also have anti-microbial functions and induce expression of anti-microbial peptides in at least some insects. Here, we review recent identification of insect SFPs and discuss the multiple roles these proteins play in the post-mating processes of female insects. PMID:20868282

  15. Protein expression strategies for identification of novel target proteins.

    PubMed

    Schuster, M; Wasserbauer, E; Einhauer, A; Ortner, C; Jungbauer, A; Hammerschmid, F; Werner, G

    2000-04-01

    Identification of new target proteins is a novel paradigm in drug discovery. A major bottleneck of this strategy is the rapid and simultaneous expression of proteins from differential gene expression to identify eligible candidates. By searching for a generic system enabling high throughput expression analysis and purification of unknown cDNAs, we evaluated the YEpFLAG-1 yeast expression system. We have selected cDNAs encoding model proteins (eukaryotic initiation factor-5A [eIF-5A] and Homo sapiens differentiation-dependent protein-A4) and cDNA encoding an unknown protein (UP-1) for overexpression in Saccharomyces cerevisiae using fusions with a peptide that changes its conformation in the presence of Ca2+ ions, the FLAG tag (Eastman Kodak, Rochester, NY). The cDNAs encoding unknown proteins originating from a directionally cloned cDNA library were expressed in all three possible reading frames. The expressed proteins were detected by an antibody directed against the FLAG tag and/or by antibodies against the model proteins. The alpha-leader sequence, encoding a yeast mating pheromone, upstream of the gene fusion site facilitates secretion into the culture supernatant. EIF-5A could be highly overexpressed and was secreted into the culture supernatant. In contrast, the Homo sapiens differentiation-dependent protein-A4 as well as the protein UP-1, whose cDNA did not match to any known gene, could not be detected in the culture supernatant. The expression product of the correct frame remained in the cells, whereas the FLAG-tagged proteins secreted into the supernatant were short, out-of-frame products. The presence of transmembrane domains or patches of hydrophobic amino acids may preclude secretion of these proteins into the culture supernatant. Subsequently, isolation and purification of the various proteins was accomplished by affinity chromatography or affinity extraction using magnetizable beads coated with the anti-FLAG monoclonal antibody. The purity of

  16. Proteomic identification of rainbow trout sperm proteins.

    PubMed

    Nynca, Joanna; Arnold, Georg J; Fröhlich, Thomas; Otte, Kathrin; Ciereszko, Andrzej

    2014-06-01

    Proteomics represents a powerful tool for the analysis of fish spermatozoa, since these cells are transcriptionally inactive. The aim of the present study was to generate an inventory of the most prominent rainbow trout sperm proteins by SDS-PAGE prefractionation combined with nano-LC-MS/MS based identification. This study provides the first in-depth analysis of the rainbow trout sperm proteome, with a total of 206 identified proteins. We found that rainbow trout spermatozoa are equipped with functionally diverse proteins related to energetic metabolism, signal transduction, protein turnover, transport, cytoskeleton, oxidative injuries, and stress and reproduction. The availability of a catalog of rainbow trout sperm proteins provides a crucial tool for the understanding of fundamental molecular processes in fish spermatozoa, for the ongoing development of novel markers of sperm quality and for the optimization of short- and long-term sperm preservation procedures. The MS data are available at ProteomeXchange with the dataset identifier PXD000355 and DOI 10.6019/PXD000355.

  17. 34 CFR 200.32 - Identification for school improvement.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Identification for school improvement. 200.32 Section... Improving Basic Programs Operated by Local Educational Agencies Lea and School Improvement § 200.32 Identification for school improvement. (a)(1)(i) An LEA must identify for school improvement any elementary...

  18. Identification of Topping Responsive Proteins in Tobacco Roots

    PubMed Central

    Li, Fei; Zhang, Huizhen; Wang, Shaoxin; Xiao, Wanfu; Ding, Chao; Liu, Weiqun; Guo, Hongxiang

    2016-01-01

    The process of topping elicits many responses in the tobacco plant, including an increase in nicotine biosynthesis, and the secondary growth of roots. Some topping responsive miRNAs and genes have been identified in our previous study, but the mechanism of the tobacco response to topping has not yet been fully elucidated. In this study, topping responsive proteins isolated from tobacco roots were screened using two-dimensional electrophoresis. Of the proteins identified, calreticulin and auxin-responsive protein indole acetic acid (IAA9) were involved in the secondary growth of roots; leucine-rich repeat disease resistance, heat shock protein 70, and farnesyl pyrophosphate synthase 1 were involved in the wounding stress response; and F-box protein played an important role in promoting the ability of nicotine synthesis after topping. In addition, we identified five tobacco bHLH proteins (NtbHLH, NtMYC1a, NtMYC1b, NtMYC2a, and NtMYC2b) related to nicotine biosynthesis. NtMYC2 was suggested to be the main positive transcription factor, with NtbHLH protein being a negative regulator in the jasmonic acid (JA)-mediated activation of nicotine biosynthesis after topping. Tobacco topping activates a comprehensive range of biological processes involving the IAA and JA signaling pathways, and the identification of proteins involved in these processes will improve our understanding of the topping response. PMID:27200055

  19. Improved target identification using synthetic infrared images

    NASA Astrophysics Data System (ADS)

    Weber, Bruce A.; Penn, Joseph A.

    2002-07-01

    The performance of infrared (IR) target identification classifiers, trained on randomly selected subsets of target chips taken from larger databases of either synthetic or measured data, is shown to improve rapidly with increasing subset size. This increase continues until the new data no longer provides additional information, or the classifier can not handle the information, at which point classifier performance levels off. It will also be shown that subsets of data selected with advanced knowledge can significantly outperform randomly selected sets, suggesting that classifier training-sets must be carefully selected if optimal performance is desired. Performance will also be shown to be subject to the quality of data used to train the classifier. Thus while increasing training set size generally improves classifier performance, the level to which the classifier performance can be raised will be shown to depend on the similarity between the training data and testing data. In fact, if the training data to be added to a given set of training data is unlike the testing data, performance will often not improve and may possibly diminish. Having too much data can reduce performance as much as having too little. Our results again demonstrate that an infrared (IR) target-identification classifier, trained on synthetic images of targets and tested on measured images, can perform as well as a classifier trained on measured images alone. We also demonstrate that the combination of the measured and the synthetic image databases can be used to train a classifier whose performance exceeds that of classifiers trained on either database alone. Results suggest that it may be possible to select data subsets from image databases that can optimize target classifiers performance for specific locations and operational scenarios.

  20. Quantitative identification of protein nitration sites.

    PubMed

    Chiappetta, Giovanni; Corbo, Claudia; Palmese, Angelo; Galli, Francesco; Piroddi, Marta; Marino, Gennaro; Amoresano, Angela

    2009-03-01

    Several labelling strategies have been developed targeting specific amino acid residues and/or PTMs. Methods specifically tailored for the qualitative and sometimes quantitative determination of PTMs have emerged. Many research groups have focused their attention towards o-nitrotyrosine residues, developing various methodologies for their identification, while direct quantification has remained elusive. So far the iTRAQ chemistry has been limited to primary amines. Here, we report a new strategy based on the use of iTRAQ reagents coupled to MS analysis for the selective labelling of o-nitrotyrosine residues. This method was proved to lead to the simultaneous localisation and quantification of nitration sites both in model proteins and in biological systems. PMID:19242934

  1. Identification of secreted bacterial proteins by noncanonical amino acid tagging

    PubMed Central

    Mahdavi, Alborz; Szychowski, Janek; Ngo, John T.; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Schneewind, Olaf; Mazmanian, Sarkis K.; Tirrell, David A.

    2014-01-01

    Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy. PMID:24347637

  2. Byonic: Advanced Peptide and Protein Identification Software

    PubMed Central

    Bern, Marshall; Kil, Yong J.; Becker, Christopher

    2013-01-01

    Byonic™ is the name of a software package for peptide and protein identification by tandem mass spectrometry. This software, which has only recently become commercially available, facilitates a much wider range of search possibilities than previous search software such as SEQUEST and Mascot. Byonic allows the user to define an essentially unlimited number of variable modification types. Byonic also allows the user to set a separate limit on the number of occurrences of each modification type, so that a search may consider only one or two chance modifications such as oxidations and deamidations per peptide, yet allow three or four biological modifications such as phosphorylations, which tend to cluster together. Hence Byonic can search for 10s or even 100s of modification types simultaneously without a prohibitively large combinatorial explosion. Byonic’s Wildcard Search™ allows the user to search for unanticipated or even unknown modifications alongside known modifications. Finally, Byonic’s Glycopeptide Search allows the user to identify glycopeptides without prior knowledge of glycan masses or glycosylation sites. PMID:23255153

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

    NASA Astrophysics Data System (ADS)

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-10-01

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

  4. Identification of Substrates of Protein-Group SUMOylation.

    PubMed

    Psakhye, Ivan; Jentsch, Stefan

    2016-01-01

    Protein modification by conjugation to the ubiquitin-related protein SUMO (SUMOylation) regulates numerous cellular functions and is reversible. However, unlike typical posttranslational modifications, SUMOylation often targets and regulates proteins of functionally and physically linked protein groups, rather than individual proteins. Functional studies of protein-group SUMOylation are thus particularly challenging, as they require the identification of ideally all members of a modified protein group. Here, we describe mass spectrometric approaches to detect SUMOylated protein groups in Saccharomyces cerevisiae, yet the protocols can be readily adapted for studies of SUMOylation in mammalian cells. PMID:27631809

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

    ERIC Educational Resources Information Center

    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,…

  6. Identification of differentially expressed serum proteins in gastric adenocarcinoma☆

    PubMed Central

    Subbannayya, Yashwanth; Mir, Sartaj Ahmad; Renuse, Santosh; Manda, Srikanth S.; Pinto, Sneha M.; Puttamallesh, Vinuth N.; Solanki, Hitendra Singh; Manju, H.C.; Syed, Nazia; Sharma, Rakesh; Christopher, Rita; Vijayakumar, M.; Kumar, K.V. Veerendra; Prasad, T.S. Keshava; Ramaswamy, Girija; Kumar, Rekha V.; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Gastric adenocarcinoma is an aggressive cancer with poor prognosis. Blood based biomarkers of gastric cancer have the potential to improve diagnosis and monitoring of these tumors. Proteins that show altered levels in the circulation of gastric cancer patients could prove useful as putative biomarkers. We used an iTRAQ-based quantitative proteomic approach to identify proteins that show altered levels in the sera of patients with gastric cancer. Our study resulted in identification of 643 proteins, of which 48 proteins showed increased levels and 11 proteins showed decreased levels in serum from gastric cancer patients compared to age and sex matched healthy controls. Proteins that showed increased expression in gastric cancer included inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Mannose-binding protein C (MBL2), sex hormone-binding globulin (SHBG), insulin-like growth factor-binding protein 2 (IGFBP2), serum amyloid A protein (SAA1), Orosomucoid 1 (ORM1) and extracellular superoxide dismutase [Cu–Zn] (SOD3). We used multiple reaction monitoring assays and validated elevated levels of ITIH4 and SAA1 proteins in serum from gastric cancer patients. Biological significance Gastric cancer is a highly aggressive cancer associated with high mortality. Serum-based biomarkers are of considerable interest in diagnosis and monitoring of various diseases including cancers. Gastric cancer is often diagnosed at advanced stages resulting in poor prognosis and high mortality. Pathological diagnosis using biopsy specimens remains the gold standard for diagnosis of gastric cancer. Serum-based biomarkers are of considerable importance as they are minimally invasive. In this study, we carried out quantitative proteomic profiling of serum from gastric cancer patients to identify proteins that show altered levels in gastric cancer patients. We identified more than 50 proteins that showed altered levels in gastric cancer patient sera. Validation in a large cohort of well

  7. Improved fingerprint identification with supervised filtering enhancement.

    PubMed

    Bal, Abdullah; El-Saba, Aed M; Alam, Mohammad S

    2005-02-10

    An important step in the fingerprint identification system is the reliable extraction of distinct features from fingerprint images. Identification performance is directly related to the enhancement of fingerprint images during or after the enrollment phase. Among the various enhancement algorithms, artificial-intelligence-based feature-extraction techniques are attractive owing to their adaptive learning properties. We present a new supervised filtering technique that is based on a dynamic neural-network approach to develop a robust fingerprint enhancement algorithm. For pattern matching, a joint transform correlation (JTC) algorithm has been incorporated that offers high processing speed for real-time applications. Because the fringe-adjusted JTC algorithm has been found to yield a significantly better correlation output compared with alternate JTCs, we used this algorithm for the identification process. Test results are presented to verify the effectiveness of the proposed algorithm.

  8. Compositions and methods for improved protein production

    DOEpatents

    Bodie, Elizabeth A.; Kim, Steve Sungjin

    2014-06-03

    The present invention relates to the identification of novel nucleic acid sequences, designated herein as 7p, 8k, 7E, 9G, 8Q and 203, in a host cell which effect protein production. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding 7p, 8k, 7E, 9G, 8Q and 203, which are presented in FIG. 1, and are SEQ ID NOS.: 1-6, respectively. The present invention also provides host cells further comprising a nucleic acid encoding a desired heterologous protein such as an enzyme.

  9. Compositions and methods for improved protein production

    DOEpatents

    Bodie, Elizabeth A.; Kim, Steve

    2012-07-10

    The present invention relates to the identification of novel nucleic acid sequences, designated herein as 7p, 8k, 7E, 9G, 8Q and 203, in a host cell which effect protein production. The present invention also provides host cells having a mutation or deletion of part or all of the gene encoding 7p, 8k, 7E, 9G, 8Q and 203, which are presented in FIG. 1, and are SEQ ID NOS.: 1-6, respectively. The present invention also provides host cells further comprising a nucleic acid encoding a desired heterologous protein such as an enzyme.

  10. Detection and identification of protein interactions of S100 proteins by ProteinChip technology.

    PubMed

    Lehmann, Roland; Melle, Christian; Escher, Niko; von Eggeling, Ferdinand

    2005-01-01

    The aim of this work was to establish an approach for identification of protein interactions. This assay used an anti-S100A8 antibody coupled on beads and incubated with cell extract. The bead eluates were analyzed using ProteinChip technology and subsequently subjected to an appropriate digestion. Molecular masses of digestion fragments were determined by SELDI-MS, and database analysis revealed S100A10 as interacting protein. This result was confirmed by co-immunoprecipitation and immunocapturing. Using S100A10 as new bait, a specific interaction with S100A7 was detectable. PMID:16212425

  11. Improving Pharmaceutical Protein Production in Oryza sativa

    PubMed Central

    Kuo, Yu-Chieh; Tan, Chia-Chun; Ku, Jung-Ting; Hsu, Wei-Cho; Su, Sung-Chieh; Lu, Chung-An; Huang, Li-Fen

    2013-01-01

    Application of plant expression systems in the production of recombinant proteins has several advantages, such as low maintenance cost, absence of human pathogens, and possession of complex post-translational glycosylation capabilities. Plants have been successfully used to produce recombinant cytokines, vaccines, antibodies, and other proteins, and rice (Oryza sativa) is a potential plant used as recombinant protein expression system. After successful transformation, transgenic rice cells can be either regenerated into whole plants or grown as cell cultures that can be upscaled into bioreactors. This review summarizes recent advances in the production of different recombinant protein produced in rice and describes their production methods as well as methods to improve protein yield and quality. Glycosylation and its impact in plant development and protein production are discussed, and several methods of improving yield and quality that have not been incorporated in rice expression systems are also proposed. Finally, different bioreactor options are explored and their advantages are analyzed. PMID:23615467

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

    PubMed

    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.

  13. Human tissue profiling with multidimensional protein identification technology.

    PubMed

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

    2005-01-01

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

  14. Detection and proteomic identification of S-nitrosated proteins in human hepatocytes.

    PubMed

    López-Sánchez, Laura M; Corrales, Fernando J; De La Mata, Manuel; Muntané, Jordi; Rodríguez-Ariza, Antonio

    2008-01-01

    The S-nitrosation of protein thiols is a redox-based posttranslational modification that modulates protein function and cell phenotype. Although the detection of S-nitrosated proteins is problematical because of the lability of S-nitrosothiols, an increasing range of proteins has been shown to undergo S-nitrosation with the improvement of molecular tools. This chapter describes the methodology used to identify potential targets of S-nitrosation in cultured primary human hepatocytes using proteomic approaches. This methodology is based on the biotin switch method, which labels S-nitrosated proteins with an affinity tag, allowing their selective detection and proteomic identification.

  15. Genetically modified proteins: functional improvement and chimeragenesis

    PubMed Central

    Balabanova, Larissa; Golotin, Vasily; Podvolotskaya, Anna; Rasskazov, Valery

    2015-01-01

    This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand nature's repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the reader's benefit. PMID:26211369

  16. Dealing with the identification of protein species in ancient amphorae.

    PubMed

    Dallongeville, Sophie; Garnier, Nicolas; Casasola, Dario Bernal; Bonifay, Michel; Rolando, Christian; Tokarski, Caroline

    2011-03-01

    This manuscript deals with the identification of protein residues in amphorae, including particularly identification of protein species. The work described was performed on fishes, the anchovy (Engraulis encrasicolus) and bonito (Sarda sarda) species frequently found in the Mediterranean area. Based on proteomic techniques, the analytical strategy was adapted to analysis of protein residues from tiny ceramic fragments. The major difficulty was to extract proteins and limit their hydrolysis during the sample preparation; consequently, multiple soft extraction techniques were evaluated. The most valuable results were obtained using a solution containing high amounts of denaturing agents, urea and thiourea, reducing agent, dithiothreitol, and detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The analysis using nano liquid chromatography-nano electrospray ionization double quadrupole time-of-flight mass spectrometry resulted in the identification of up to 200 proteins for the anchovy and bonito species, among which 73 peptides were found to be fish-specific. Because bonito and anchovy species are not documented and fully sequenced in genomic databases, the preliminary protein identification was realized via sequence homology to other fish sequenced species. Amino acid substitutions of peptides were assigned on the basis of the interpretation of tandem mass spectrometry spectra using de novo sequencing; these peptides, not reported up to now in databases, constitute species-specific markers. The method developed was finally applied to an archaeological sample replica impregnated with a mixture of fish tissue from both species; this experiment successfully led to the identification of 17 fish proteins, including 33 fish-specific peptides. This work shows that the analytical method developed has great potential for the identification of protein species in complex archaeological samples. PMID:20890751

  17. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

    Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. PMID:25930701

  18. Stable isotope, site-specific mass tagging for protein identification

    DOEpatents

    Chen, Xian

    2006-10-24

    Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.

  19. Identification of four plastid-localized protein kinases.

    PubMed

    Richter, Andreas S; Gartmann, Hans; Fechler, Mona; Rödiger, Anja; Baginsky, Sacha; Grimm, Bernhard

    2016-06-01

    In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. PMID:27214872

  20. CDvist: A webserver for identification and visualization of conserved domains in protein sequences

    DOE PAGES

    Adebali, Ogun; Ortega, Davi R.; Zhulin, Igor B.

    2014-12-18

    Identification of domains in protein sequences allows their assigning to biological functions. Several webservers exist for identification of protein domains using similarity searches against various databases of protein domain models. However, none of them provides comprehensive domain coverage while allowing bulk querying and their visualization schemes can be improved. To address these issues, we developed CDvist (a comprehensive domain visualization tool), which combines the best available search algorithms and databases into a user-friendly framework. First, a given protein sequence is matched to domain models using high-specificity tools and only then unmatched segments are subjected to more sensitive algorithms resulting inmore » a best possible comprehensive coverage. In conclusion, bulk querying and rich visualization and download options provide improved functionality to domain architecture analysis.« less

  1. CDvist: A webserver for identification and visualization of conserved domains in protein sequences

    SciTech Connect

    Adebali, Ogun; Ortega, Davi R.; Zhulin, Igor B.

    2014-12-18

    Identification of domains in protein sequences allows their assigning to biological functions. Several webservers exist for identification of protein domains using similarity searches against various databases of protein domain models. However, none of them provides comprehensive domain coverage while allowing bulk querying and their visualization schemes can be improved. To address these issues, we developed CDvist (a comprehensive domain visualization tool), which combines the best available search algorithms and databases into a user-friendly framework. First, a given protein sequence is matched to domain models using high-specificity tools and only then unmatched segments are subjected to more sensitive algorithms resulting in a best possible comprehensive coverage. In conclusion, bulk querying and rich visualization and download options provide improved functionality to domain architecture analysis.

  2. Proteomics: Protein Identification Using Online Databases

    ERIC Educational Resources Information Center

    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…

  3. Identification of protein stability determinants in chloroplasts

    PubMed Central

    Apel, Wiebke; Schulze, Waltraud X; Bock, Ralph

    2010-01-01

    Although chloroplast protein stability has long been recognised as a major level of post-translational regulation in photosynthesis and gene expression, the factors determining protein stability in plastids are largely unknown. Here, we have identified stability determinants in vivo by producing plants with transgenic chloroplasts that express a reporter protein whose N- and C-termini were systematically modified. We found that major stability determinants are located in the N-terminus. Moreover, testing of all 20 amino acids in the position after the initiator methionine revealed strong differences in protein stability and indicated an important role of the penultimate N-terminal amino acid residue in determining the protein half life. We propose that the stability of plastid proteins is largely determined by three factors: (i) the action of methionine aminopeptidase (the enzyme that removes the initiator methionine and exposes the penultimate N-terminal amino acid residue), (ii) an N-end rule-like protein degradation pathway, and (iii) additional sequence determinants in the N-terminal region. PMID:20545891

  4. Identification of Ina proteins from Fusarium acuminatum

    NASA Astrophysics Data System (ADS)

    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.

  5. Identification of ABCC2 as a binding protein of Cry1Ac on brush border membrane vesicles from Helicoverpa armigera by an improved pull-down assay.

    PubMed

    Zhou, Zishan; Wang, Zeyu; Liu, Yuxiao; Liang, Gemei; Shu, Changlong; Song, Fuping; Zhou, Xueping; Bravo, Alejandra; Soberón, Mario; Zhang, Jie

    2016-08-01

    Cry1Ac toxin-binding proteins from Helicoverpa armigera brush border membrane vesicles were identified by an improved pull-down method that involves coupling Cry1Ac to CNBr agarose combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). According to the LC-MS/MS results, Cry1Ac toxin could bind to six classes of aminopeptidase-N, alkaline phosphatase, cadherin-like protein, ATP-binding cassette transporter subfamily C protein (ABCC2), actin, ATPase, polycalin, and some other proteins not previously characterized as Cry toxin-binding molecules such as dipeptidyl peptidase or carboxyl/choline esterase and some serine proteases. This is the first report that suggests the direct binding of Cry1Ac toxin to ABCC2 in H. armigera. PMID:27037552

  6. Identification of Post-translational Modifications of Plant Protein Complexes

    PubMed Central

    Piquerez, Sophie J. M.; Balmuth, Alexi L.; Sklenář, Jan; Jones, Alexandra M.E.; Rathjen, John P.; Ntoukakis, Vardis

    2014-01-01

    Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved. Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs. This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein. PMID:24637539

  7. BioID Identification of Lamin-Associated Proteins.

    PubMed

    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.

  8. Detection and identification of protein citrullination in complex biological systems.

    PubMed

    Clancy, Kathleen W; Weerapana, Eranthie; Thompson, Paul R

    2016-02-01

    Protein citrullination is a post-translational modification of arginine that is catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Aberrantly increased citrullination is associated with a host of inflammatory diseases and cancer and PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis. In rheumatoid arthritis, citrullinated proteins serve as key antigens for rheumatoid arthritis-associated autoantibodies. These data suggest that citrullinated proteins may serve more generally as biomarkers of specific disease states, however, the identification of citrullinated residues remains challenging due to the small 1Da mass change that occurs upon citrullination. Herein, we highlight the available techniques to identify citrullinated proteins/residues focusing on advanced MS techniques as well as chemical derivatization strategies that are currently being employed to identify citrullinated proteins as well as the specific residues modified within those proteins.

  9. Protein nanopatterns for improved immunodetection sensitivity.

    PubMed

    Valsesia, A; Mannelli, I; Colpo, P; Bretagnol, F; Rossi, F

    2008-10-01

    In this work, we clearly demonstrate the capability of protein nanopatterns of improving the quality factors of immunosensing devices, such as lowering of the limit of detection and increase of sensitivity. This beneficial effect is obtained by the formation on the sensor's surface of bioadhesive domains of nanometric dimensions in a nonadhesive matrix by means of colloidal lithography.

  10. Recovery and identification of mature enamel proteins in ancient teeth.

    PubMed

    Porto, Isabel M; Laure, Helen J; Tykot, Robert H; de Sousa, Frederico B; Rosa, Jose C; Gerlach, Raquel F

    2011-12-01

    Proteins in mineralized tissues provide a window to the past, and dental enamel is peculiar in being highly resistant to diagenesis and providing information on a very narrow window of time, such as the developing period; however, to date, complete proteins have not been extracted successfully from ancient teeth. In this work we tested the ability of a whole-crown micro-etch technique to obtain enamel protein samples from mature enamel of recently extracted (n = 2) and ancient (n = 2; ad 800 to 1100) third molars. Samples were analyzed using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry, and the resulting spectra were searched against the Swiss-Prot protein database using the Mascot software for protein identification. In our protocol, the separation of proteins in gel is not necessary. Successful identification of specific enamel proteins was obtained after whole-crown superficial enamel etching with 10% HCl. Most protein fragments recovered from dry teeth and mummy teeth contained amino-terminal amelogenin peptides. Only one peptide specific for the amelogenin X-isoform was identified. In conclusion, the reported techniques allowed the successful recovery of proteins specific to dental enamel from samples obtained in a very conservative manner, which may also be important in forensic and/or archeological science. PMID:22243232

  11. Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

    PubMed Central

    Enyaru, John C.; Carr, Steven A.; Pearson, Terry W.

    2013-01-01

    Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification. PMID:23951171

  12. The tandem affinity purification method: an efficient system for protein complex purification and protein interaction identification.

    PubMed

    Xu, Xiaoli; Song, Yuan; Li, Yuhua; Chang, Jianfeng; Zhang, Hua; An, Lizhe

    2010-08-01

    Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein-protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method. PMID:20399864

  13. Support Vector Machine Classification of Probability Models and Peptide Features for Improved Peptide Identification from Shotgun Proteomics

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Oehmen, Chris S.; Cannon, William R.

    2007-12-01

    Proteomics is a rapidly advancing field offering a new perspective to biological systems. Mass spectrometry (MS) is a popular experimental approach because it allows global protein characterization of a sample in a high-throughput manner. The identification of a protein is based on the spectral signature of fragments of the constituent proteins, i.e., peptides. This peptide identification is typically performed with a computational database search algorithm; however, these database search algorithms return a large number of false positive identifications. We present a new scoring algorithm that uses a SVM to integrate database scoring metrics with peptide physiochemical properties, resulting in an improved ability to separate true from false peptide identification from MS. The Peptide Identification Classifier SVM (PICS) score using only five variables is significantly more accurate than the single best database metric, quantified as the area under a Receive Operating Characteristic curve of ~0.94 versus ~0.90.

  14. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

  15. Identification & Characterization of Fungal Ice Nucleation Proteins

    NASA Astrophysics Data System (ADS)

    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.

  16. Using protein binding site prediction to improve protein docking.

    PubMed

    Huang, Bingding; Schroeder, Michael

    2008-10-01

    Predicting protein interaction interfaces and protein complexes are two important related problems. For interface prediction, there are a number of tools, such as PPI-Pred, PPISP, PINUP, Promate, and SPPIDER, which predict enzyme-inhibitor interfaces with success rates of 23% to 55% and other interfaces with 10% to 28% on a benchmark dataset of 62 complexes. Here, we develop, metaPPI, a meta server for interface prediction. It significantly improves prediction success rates to 70% for enzyme-inhibitor and 44% for other interfaces. As shown with Promate, predicted interfaces can be used to improve protein docking. Here, we follow this idea using the meta server instead of individual predictions. We confirm that filtering with predicted interfaces significantly improves candidate generation in rigid-body docking based on shape complementarity. Finally, we show that the initial ranking of candidate solutions in rigid-body docking can be further improved for the class of enzyme-inhibitor complexes by a geometrical scoring which rewards deep pockets. A web server of metaPPI is available at scoppi.tu-dresden.de/metappi. The source code of our docking algorithm BDOCK is also available at www.biotec.tu-dresden.de /approximately bhuang/bdock.

  17. Bioinformatics pipeline for functional identification and characterization of proteins

    NASA Astrophysics Data System (ADS)

    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.

  18. Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita; Payne, Samuel H.; Kang, Jiyun; Bramer, Lisa M.; Nicora, Carrie D.; Shukla, Anil K.; Metz, Thomas O.; Rodland, Karin D.; Smith, Richard D.; Tardiff, Mark F.; McDermott, Jason E.; Pounds, Joel G.; Waters, Katrina M.

    2014-12-01

    As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.

  19. Seed storage proteins as a system for teaching protein identification by mass spectrometry in biochemistry laboratory.

    PubMed

    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, requiring more time and expertise than instructors of large laboratory classes can devote. We have developed an experimental module for our Biochemistry Laboratory course that engages students in MS-based protein identification following protein separation by one-dimensional SDS-PAGE, a technique that is usually taught in this type of course. The module is based on soybean seed storage proteins, a relatively simple mixture of proteins present in high levels in the seed, allowing the identification of the main protein bands by MS/MS and in some cases, even by peptide mass fingerprinting. Students can identify their protein bands using software available on the Internet, and are challenged to deduce post-translational modifications that have occurred upon germination. A collection of mass spectral data and tutorials that can be used as a stand-alone computer-based laboratory module were also assembled.

  20. Proteomic identification of erythrocyte membrane protein deficiency in hereditary spherocytosis.

    PubMed

    Peker, Selen; Akar, Nejat; Demiralp, Duygu Ozel

    2012-03-01

    Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia in Caucasians, with an estimated prevalence ranging from 1:2000 to 1:5000. The molecular defect in one of the erythrocytes (RBC) membrane proteins underlying HS like; spectrin-α, spectrin-β, ankyrin, band 3 and protein 4.2 that lead to membrane destabilization and vesiculation, may change the RBCs into denser and more rigid cells (spherocytes), which are removed by the spleen, leading to the development of hemolytic anemia. It is classified as mild, moderate and severe, according to the degree of the hemolytic anemia and the associated symptoms. Two-dimensional gel electrophoresis (2-DE) is potentially valuable method for studying heritable disorders as HS that involve membrane proteins. This separation technique of proteins based upon two biophysically unrelated parameters; molecular weight and charge, is a good option in clinical proteomics in terms of ability to separate complex mixtures, display post-translational modifications and changes after phosphorylation. In this study, we have used contemporary methods with some modifications for the solubilisation, separation and identification of erythrocyte membrane proteins in normal and in HS RBCs. Spectrin alpha and beta chain, ankyrin and band 3 proteins expression differences were found with PDQuest software 8.0.1. and peptide mass fingerprinting (PMF) analysis performed for identification of proteins in this study.

  1. Identification of shed proteins from Chinese hamster ovary cells: Application of statistical confidence using human and mouse protein databases

    SciTech Connect

    Ahram, Mamoun; Strittmatter, Eric F.; Monroe, Matthew E.; Adkins, Joshua N.; Hunter, Joel C.; Miller, John H.; Springer, David L.

    2005-05-01

    The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation in an effort to develop a fundamental understanding of the bystander response. CHO cells were chosen for this study because they have been widely used for radiation studies and since they have been reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and FTICR-mass spectrometry analysis. Since the hamster genome has not been sequenced, mass spectrometry data was searched against the mouse and human proteins databases. Nearly 150 proteins that were identified by tandem mass spectrometry were confirmed by FTICR. When both types of mass spectrometry data were evaluated with a new confidence scoring tool, which is based on discriminant analyses, about 500 protein were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface, hence were likely shed. However, estimates of quantitative changes, based on two independent mass spectrometry approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using mass spectrometry in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.

  2. Identification and verification of novel rodent postsynaptic density proteins.

    PubMed

    Jordan, Bryen A; Fernholz, Brian D; Boussac, Muriel; Xu, Chongfeng; Grigorean, Gabriela; Ziff, Edward B; Neubert, Thomas A

    2004-09-01

    The postsynaptic density (PSD) is a cellular structure specialized in receiving and transducing synaptic information. Here we describe the identification of 452 proteins isolated from biochemically purified PSD fractions of rat and mouse brains using nanoflow HPLC coupled to electrospray tandem mass spectrometry (LC-MS/MS). Fluorescence microscopy and Western blotting were used to verify that many of the novel proteins identified exhibit subcellular distributions consistent with those of PSD-localized proteins. In addition to identifying most previously described PSD components, we also detected proteins involved in signaling to the nucleus as well as regulators of ADP-ribosylation factor signaling, ubiquitination, RNA trafficking, and protein translation. These results suggest new mechanisms by which the PSD helps regulate synaptic strength and transmission.

  3. Identification and Validation of ISG15 Target Proteins.

    PubMed

    Durfee, Larissa A; Huibregtse, Jon M

    2010-01-01

    ISG15 is an interferon-induced ubiquitin-like protein (Ubl) that has antiviral properties. The core E1, E2 and E3 enzymes for conjugation of human ISG15 are Ube1L, UbcH8 and Herc5, all of which are induced at the transcriptional level by Type 1 interferon signaling. Several proteomics studies have, together, identified over 300 cellular proteins as ISG15 targets. These targets include a broad range of constitutively expressed proteins and approximately 15 interferon-induced proteins. This chapter provides an overview of the target identification process and the validation of these targets. We also discuss the limited number of examples where the biochemical effect of ISG15 conjugation on target proteins has been characterized. PMID:21222286

  4. Label-Free Identification and Quantification of SUMO Target Proteins.

    PubMed

    Hendriks, Ivo A; Vertegaal, Alfred C O

    2016-01-01

    Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism. The His10-tag allows for a single-step, high-yield, and high-purity enrichment of SUMOylated proteins, which are then digested and analyzed by high-resolution mass spectrometry. Matching between runs and label-free quantification integrated in the freely available MaxQuant software allow for a high rate and accuracy of quantification, providing a strong alternative to laborious sample or cell labeling techniques. The method described here allows for identification of >1000 SUMO target proteins, and characterization of their SUMOylation dynamics, without requiring sample fractionation. The purification procedure, starting from total lysate, can be performed in ~4 days. PMID:27631806

  5. Identification of an Oncogenic RAB Protein

    PubMed Central

    Wheeler, Douglas B.; Zoncu, Roberto; Root, David E.; Sabatini, David M.; Sawyers, Charles L.

    2015-01-01

    In an shRNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small GTPase—a protein previously implicated in endomembrane trafficking—as a new regulator of the PI3K pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and co-purifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive PDGFRα to LAMP2-positive endomembranes in the absence of ligand, suggesting there may be latent oncogenic potential in dysregulated endomembrane trafficking. PMID:26338797

  6. Identification of an oncogenic RAB protein.

    PubMed

    Wheeler, Douglas B; Zoncu, Roberto; Root, David E; Sabatini, David M; Sawyers, Charles L

    2015-10-01

    In a short hairpin RNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small guanosine triphosphatase (GTPase)-a protein previously implicated in endomembrane trafficking-as a regulator of the phosphatidylinositol 3'-OH kinase (PI3K) pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and copurifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive platelet-derived growth factor receptor α to LAMP2-positive endomembranes in the absence of ligand, suggesting that there may be latent oncogenic potential in dysregulated endomembrane trafficking.

  7. Identification of local variations within secondary structures of proteins.

    PubMed

    Kumar, Prasun; Bansal, Manju

    2015-05-01

    Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method, ASSP (Assignment of Secondary Structure in Proteins), using only the path traversed by the C(α) atoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely α-helices, 310-helices, π-helices, extended β-strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from α-helices and extended β-strands, 310-helices and π-helices were also found to occur in substantial numbers. ASSP was able to discriminate non-α-helical segments from flanking α-helices, which were often identified as part of α-helices by other algorithms. ASSP can also lead to the identification of novel SSEs. It is believed that ASSP could provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at http://nucleix.mbu.iisc.ernet.in/assp/index.php.

  8. Identification of local variations within secondary structures of proteins.

    PubMed

    Kumar, Prasun; Bansal, Manju

    2015-05-01

    Secondary-structure elements (SSEs) play an important role in the folding of proteins. Identification of SSEs in proteins is a common problem in structural biology. A new method, ASSP (Assignment of Secondary Structure in Proteins), using only the path traversed by the C(α) atoms has been developed. The algorithm is based on the premise that the protein structure can be divided into continuous or uniform stretches, which can be defined in terms of helical parameters, and depending on their values the stretches can be classified into different SSEs, namely α-helices, 310-helices, π-helices, extended β-strands and polyproline II (PPII) and other left-handed helices. The methodology was validated using an unbiased clustering of these parameters for a protein data set consisting of 1008 protein chains, which suggested that there are seven well defined clusters associated with different SSEs. Apart from α-helices and extended β-strands, 310-helices and π-helices were also found to occur in substantial numbers. ASSP was able to discriminate non-α-helical segments from flanking α-helices, which were often identified as part of α-helices by other algorithms. ASSP can also lead to the identification of novel SSEs. It is believed that ASSP could provide a better understanding of the finer nuances of protein secondary structure and could make an important contribution to the better understanding of comparatively less frequently occurring structural motifs. At the same time, it can contribute to the identification of novel SSEs. A standalone version of the program for the Linux as well as the Windows operating systems is freely downloadable and a web-server version is also available at http://nucleix.mbu.iisc.ernet.in/assp/index.php. PMID:25945573

  9. Identification of immunogenic maize proteins in a casein hydrolysate formula.

    PubMed

    Frisner, H; Rosendal, A; Barkholt, V

    2000-05-01

    Cow's milk-based formulas used for infants with cow's milk allergy are based on hydrolyzed proteins. The formulas that are successful in preventing allergic responses are extensively hydrolyzed. Nevertheless, reactions to such formulas are occasionally reported, and protein material of higher molecular weight than expected has been detected by binding immunoglobulin E (IgE) from patients' sera. This paper presents the identification of high-molecular-weight material in the extensively hydrolyzed casein formula, Nutramigen. The material was concentrated by simple centrifugation. The proteins in the pellet were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein-containing bands were analyzed by protein sequencing after electroblotting. The proteins were identified as maize zeins, which are water-insoluble proteins of apparent M(r) 20,000 and 23,000, presumably originating from the maize starch in Nutramigen. Rabbits immunized with this formula developed antibodies against zeins but not against milk proteins. The maize zeins are probably identical to the recently reported components in Nutramigen (1), detected by binding of IgE from milk allergic patients, but not correlated to clinical allergic reactivity. The clinical relevance of maize proteins in Nutramigen remains to be established. PMID:10893013

  10. Identification of differential protein interactors of lamin A and progerin

    PubMed Central

    Kubben, Nard; Voncken, Jan Willem; Demmers, Jeroen; Calis, Chantal; van Almen, Geert

    2010-01-01

    The nuclear lamina is an interconnected meshwork of intermediate filament proteins underlying the nuclear envelope. The lamina is an important regulator of nuclear structural integrity as well as nuclear processes, including transcription, DNA replication and chromatin remodeling. The major components of the lamina are A- and B-type lamins. Mutations in lamins impair lamina functions and cause a set of highly tissue-specific diseases collectively referred to as laminopathies. The phenotypic diversity amongst laminopathies is hypothesized to be caused by mutations affecting specific protein interactions, possibly in a tissue-specific manner. Current technologies to identify interaction partners of lamin A and its mutants are hampered by the insoluble nature of lamina components. To overcome the limitations of current technologies, we developed and applied a novel, unbiased approach to identify lamin A-interacting proteins. This approach involves expression of the high-affinity OneSTrEP-tag, precipitation of lamin-protein complexes after reversible protein cross-linking and subsequent protein identification by mass spectrometry. We used this approach to identify in mouse embryonic fibroblasts and cardiac myocyte NklTAg cell lines proteins that interact with lamin A and its mutant isoform progerin, which causes the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identified a total of 313 lamina-interacting proteins, including several novel lamin A interactors, and we characterize a set of 35 proteins which preferentially interact with lamin A or progerin. PMID:21327095

  11. Systematic identification of protein combinations mediating chromatin looping

    PubMed Central

    Zhang, Kai; Li, Nan; Ainsworth, Richard I.; Wang, Wei

    2016-01-01

    Chromatin looping plays a pivotal role in gene expression and other biological processes through bringing distal regulatory elements into spatial proximity. The formation of chromatin loops is mainly mediated by DNA-binding proteins (DBPs) that bind to the interacting sites and form complexes in three-dimensional (3D) space. Previously, identification of DBP cooperation has been limited to those binding to neighbouring regions in the proximal linear genome (1D cooperation). Here we present the first study that integrates protein ChIP-seq and Hi-C data to systematically identify both the 1D- and 3D-cooperation between DBPs. We develop a new network model that allows identification of cooperation between multiple DBPs and reveals cell-type-specific and -independent regulations. Using this framework, we retrieve many known and previously unknown 3D-cooperations between DBPs in chromosomal loops that may be a key factor in influencing the 3D organization of chromatin. PMID:27461729

  12. Establishment of a Protein Frequency Library and Its Application in the Reliable Identification of Specific Protein Interaction Partners*

    PubMed Central

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

    2010-01-01

    The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static “bead proteomes.” The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background. PMID:20023298

  13. Effective Leveraging of Targeted Search Spaces for Improving Peptide Identification in Tandem Mass Spectrometry Based Proteomics.

    PubMed

    Shanmugam, Avinash K; Nesvizhskii, Alexey I

    2015-12-01

    In shotgun proteomics, peptides are typically identified using database searching, which involves scoring acquired tandem mass spectra against peptides derived from standard protein sequence databases such as Uniprot, Refseq, or Ensembl. In this strategy, the sensitivity of peptide identification is known to be affected by the size of the search space. Therefore, creating a targeted sequence database containing only peptides likely to be present in the analyzed sample can be a useful technique for improving the sensitivity of peptide identification. In this study, we describe how targeted peptide databases can be created based on the frequency of identification in the global proteome machine database (GPMDB), the largest publicly available repository of peptide and protein identification data. We demonstrate that targeted peptide databases can be easily integrated into existing proteome analysis workflows and describe a computational strategy for minimizing any loss of peptide identifications arising from potential search space incompleteness in the targeted search spaces. We demonstrate the performance of our workflow using several data sets of varying size and sample complexity.

  14. Effective Leveraging of Targeted Search Spaces for Improving Peptide Identification in Tandem Mass Spectrometry Based Proteomics.

    PubMed

    Shanmugam, Avinash K; Nesvizhskii, Alexey I

    2015-12-01

    In shotgun proteomics, peptides are typically identified using database searching, which involves scoring acquired tandem mass spectra against peptides derived from standard protein sequence databases such as Uniprot, Refseq, or Ensembl. In this strategy, the sensitivity of peptide identification is known to be affected by the size of the search space. Therefore, creating a targeted sequence database containing only peptides likely to be present in the analyzed sample can be a useful technique for improving the sensitivity of peptide identification. In this study, we describe how targeted peptide databases can be created based on the frequency of identification in the global proteome machine database (GPMDB), the largest publicly available repository of peptide and protein identification data. We demonstrate that targeted peptide databases can be easily integrated into existing proteome analysis workflows and describe a computational strategy for minimizing any loss of peptide identifications arising from potential search space incompleteness in the targeted search spaces. We demonstrate the performance of our workflow using several data sets of varying size and sample complexity. PMID:26569054

  15. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  16. Identification of AOSC-binding proteins in neurons

    NASA Astrophysics Data System (ADS)

    Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

    2008-11-01

    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.

  17. Identification of novel CBP interacting proteins in embryonic orofacial tissue

    SciTech Connect

    Yin Xiaolong; Warner, Dennis R.; Roberts, Emily A.; Pisano, M. Michele; Greene, Robert M. . E-mail: greene@louisville.edu

    2005-04-15

    cAMP response element-binding protein (CREB)-binding protein (CBP) plays an important role as a general co-integrator of multiple signaling pathways and interacts with a large number of transcription factors and co-factors, through its numerous protein-binding domains. To identify nuclear factors associated with CBP in developing orofacial tissue, a yeast two-hybrid screen of a cDNA library derived from orofacial tissue from gestational day 11 to 13 mouse embryos was conducted. Using the carboxy terminus (amino acid residues 1676-2441) of CBP as bait, several novel proteins that bind CBP were identified, including an Msx-interacting-zinc finger protein, CDC42 interaction protein 4/thyroid hormone receptor interactor 10, SH3-domain GRB2-like 1, CCR4-NOT transcription complex subunit 3, adaptor protein complex AP-1 {beta}1 subunit, eukaryotic translation initiation factor 2B subunit 1 ({alpha}), and cyclin G-associated kinase. Results of the yeast two-hybrid screen were confirmed by glutathione S-transferase pull-down assays. The identification of these proteins as novel CBP-binding partners allows exploration of new mechanisms by which CBP regulates and integrates diverse cell signaling pathways.

  18. Identification of Immunogenic and Serum Binding Proteins of Staphylococcus epidermidis

    PubMed Central

    Sellman, Bret R.; Howell, Alan P.; Kelly-Boyd, Cari; Baker, Steve M.

    2005-01-01

    Staphylococcus epidermidis is a commensal of human skin and a leading cause of nosocomial bloodstream infections. Limited information is available about S. epidermidis proteins that are expressed upon transition to the bloodstream or those involved in host-pathogen interactions. A cell surface fraction from S. epidermidis 0-47 grown in rabbit serum to mimic environmental signals encountered during a bloodstream infection was separated by two-dimensional (2D) gel electrophoresis. Following 2D separation, the proteins were transferred to nitrocellulose and detected with either pooled sera generated in rabbits immunized with live S. epidermidis 0-47 or with biotin-labeled serum proteins eluted from the surface of bacteria grown in rabbit serum. Twenty-nine immunoreactive or serum binding proteins of S. epidermidis were identified by mass spectrometry. Twenty-seven of the corresponding genes were expressed in Escherichia coli, and the purified recombinant proteins were used to immunize mice. In a preliminary screen, 12 of the 27 recombinant proteins induced a response that reduced the number of bacteria recovered from the spleen or bloodstream of infected mice. In subsequent vaccination studies, 5 of the 12 proteins resulted in a statistically significant reduction in the number of bacteria. The identification of five candidate vaccine antigens from the initial screen of only 29 proteins demonstrates the utility of this approach. PMID:16177335

  19. Prediction of structural features and application to outer membrane protein identification

    NASA Astrophysics Data System (ADS)

    Yan, Renxiang; Wang, Xiaofeng; Huang, Lanqing; Yan, Feidi; Xue, Xiaoyu; Cai, Weiwen

    2015-06-01

    Protein three-dimensional (3D) structures provide insightful information in many fields of biology. One-dimensional properties derived from 3D structures such as secondary structure, residue solvent accessibility, residue depth and backbone torsion angles are helpful to protein function prediction, fold recognition and ab initio folding. Here, we predict various structural features with the assistance of neural network learning. Based on an independent test dataset, protein secondary structure prediction generates an overall Q3 accuracy of ~80%. Meanwhile, the prediction of relative solvent accessibility obtains the highest mean absolute error of 0.164, and prediction of residue depth achieves the lowest mean absolute error of 0.062. We further improve the outer membrane protein identification by including the predicted structural features in a scoring function using a simple profile-to-profile alignment. The results demonstrate that the accuracy of outer membrane protein identification can be improved by ~3% at a 1% false positive level when structural features are incorporated. Finally, our methods are available as two convenient and easy-to-use programs. One is PSSM-2-Features for predicting secondary structure, relative solvent accessibility, residue depth and backbone torsion angles, the other is PPA-OMP for identifying outer membrane proteins from proteomes.

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

    PubMed

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

    2012-01-01

    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

  1. An improved machine learning protocol for the identification of correct Sequest search results

    PubMed Central

    2010-01-01

    Background Mass spectrometry has become a standard method by which the proteomic profile of cell or tissue samples is characterized. To fully take advantage of tandem mass spectrometry (MS/MS) techniques in large scale protein characterization studies robust and consistent data analysis procedures are crucial. In this work we present a machine learning based protocol for the identification of correct peptide-spectrum matches from Sequest database search results, improving on previously published protocols. Results The developed model improves on published machine learning classification procedures by 6% as measured by the area under the ROC curve. Further, we show how the developed model can be presented as an interpretable tree of additive rules, thereby effectively removing the 'black-box' notion often associated with machine learning classifiers, allowing for comparison with expert rule-of-thumb. Finally, a method for extending the developed peptide identification protocol to give probabilistic estimates of the presence of a given protein is proposed and tested. Conclusions We demonstrate the construction of a high accuracy classification model for Sequest search results from MS/MS spectra obtained by using the MALDI ionization. The developed model performs well in identifying correct peptide-spectrum matches and is easily extendable to the protein identification problem. The relative ease with which additional experimental parameters can be incorporated into the classification framework, to give additional discriminatory power, allows for future tailoring of the model to take advantage of information from specific instrument set-ups. PMID:21138573

  2. A standardized framing for reporting protein identifications in mzIdentML 1.2.

    PubMed

    Seymour, Sean L; Farrah, Terry; Binz, Pierre-Alain; Chalkley, Robert J; Cottrell, John S; Searle, Brian C; Tabb, David L; Vizcaíno, Juan Antonio; Prieto, Gorka; Uszkoreit, Julian; Eisenacher, Martin; Martínez-Bartolomé, Salvador; Ghali, Fawaz; Jones, Andrew R

    2014-11-01

    Inferring which protein species have been detected in bottom-up proteomics experiments has been a challenging problem for which solutions have been maturing over the past decade. While many inference approaches now function well in isolation, comparing and reconciling the results generated across different tools remains difficult. It presently stands as one of the greatest barriers in collaborative efforts such as the Human Proteome Project and public repositories such as the PRoteomics IDEntifications (PRIDE) database. Here we present a framework for reporting protein identifications that seeks to improve capabilities for comparing results generated by different inference tools. This framework standardizes the terminology for describing protein identification results, associated with the HUPO-Proteomics Standards Initiative (PSI) mzIdentML standard, while still allowing for differing methodologies to reach that final state. It is proposed that developers of software for reporting identification results will adopt this terminology in their outputs. While the new terminology does not require any changes to the core mzIdentML model, it represents a significant change in practice, and, as such, the rules will be released via a new version of the mzIdentML specification (version 1.2) so that consumers of files are able to determine whether the new guidelines have been adopted by export software.

  3. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays

    PubMed Central

    Yu, Xiaobo; LaBaer, Joshua

    2015-01-01

    Summary AMPylation (adenylylation) has been recognized as an important post translational modification employed by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes and is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method to identify new substrates using protein microarrays, which can significantly expand the list of potential substrates. Here, we describe procedures to detect AMPylated and auto-AMPylated proteins in a sensitive, high throughput, and non-radioactive manner. The approach employs high-density protein microarrays fabricated using NAPPA (Nucleic Acid Programmable Protein Arrays) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide–alkyne cycloaddition. The assay can be accomplished within 11 hours. PMID:25881200

  4. Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

    PubMed

    Kapphahn, Rebecca J; Giwa, Babatomiwa M; Berg, Kristin M; Roehrich, Heidi; Feng, Xiao; Olsen, Timothy W; Ferrington, Deborah A

    2006-07-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification. PMID:16530755

  5. Identification of contractile vacuole proteins in Trypanosoma cruzi.

    PubMed

    Ulrich, Paul N; Jimenez, Veronica; Park, Miyoung; Martins, Vicente P; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N J; Orlando, Ron; Docampo, Roberto

    2011-03-18

    Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism

  6. Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins

    PubMed Central

    Butterfield, Erin R.; Howe, Christopher J.; Nisbet, R. Ellen R.

    2016-01-01

    The dinoflagellates are an extremely diverse group of algae closely related to the Apicomplexa and the ciliates. Much work has previously been undertaken to determine the presence of various biochemical pathways within dinoflagellate mitochondria. However, these studies were unable to identify several key transcripts including those encoding proteins involved in the pyruvate dehydrogenase complex, iron–sulfur cluster biosynthesis, and protein import. Here, we analyze the draft nuclear genome of the dinoflagellate Symbiodinium minutum, as well as RNAseq data to identify nuclear genes encoding mitochondrial proteins. The results confirm the presence of a complete tricarboxylic acid cycle in the dinoflagellates. Results also demonstrate the difficulties in using the genome sequence for the identification of genes due to the large number of introns, but show that it is highly useful for the determination of gene duplication events. PMID:26798115

  7. Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins.

    PubMed

    Butterfield, Erin R; Howe, Christopher J; Nisbet, R Ellen R

    2016-01-21

    The dinoflagellates are an extremely diverse group of algae closely related to the Apicomplexa and the ciliates. Much work has previously been undertaken to determine the presence of various biochemical pathways within dinoflagellate mitochondria. However, these studies were unable to identify several key transcripts including those encoding proteins involved in the pyruvate dehydrogenase complex, iron-sulfur cluster biosynthesis, and protein import. Here, we analyze the draft nuclear genome of the dinoflagellate Symbiodinium minutum, as well as RNAseq data to identify nuclear genes encoding mitochondrial proteins. The results confirm the presence of a complete tricarboxylic acid cycle in the dinoflagellates. Results also demonstrate the difficulties in using the genome sequence for the identification of genes due to the large number of introns, but show that it is highly useful for the determination of gene duplication events.

  8. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.

    PubMed

    Banks, Charles A S; Kong, Stephanie E; Washburn, Michael P

    2012-12-01

    Characterizing protein complexes and identifying their subunits promote our understanding of the machinery involved in many in vivo processes. Proteomic studies can identify a protein's binding partners, and this can provide insight into how protein complexes function and how they are regulated. In addition, the composition of a protein complex within an organism can be investigated as a function of time, as a function of location, or during the response of an organism to a change in environment. There are many ways to isolate a complex and identify its constituents. This review will focus on complex isolation using affinity purification and will address issues that biochemists should bear in mind as they isolate protein complexes for mass spectrometric analysis by multidimensional protein identification technology (MudPIT)(1). Protein complex analysis by mass spectrometry frequently involves the collaborative efforts of biochemists or biologists who purify protein complexes and proteomic specialists who analyze the samples - for fruitful collaborations it can be helpful for these specialized groups to be acquainted with basic principles of their collaborator's discipline. With this in mind, we first review the variety of affinity purification methods which might be considered for preparing complexes for analysis, and then provide brief primers on the principles of MudPIT mass spectrometry and data analysis. From this foundation, we then discuss how these techniques are integrated and optimized and suggest salient points to consider when preparing purified samples for protein identification, performing mass spectrometry runs, and analyzing the resulting data.

  9. Holistic processing improves change detection but impairs change identification.

    PubMed

    Mathis, Katherine M; Kahan, Todd A

    2014-10-01

    It has been just over a century since Gestalt psychologists described the factors that contribute to the holistic processing of visually presented stimuli. Recent research indicates that holistic processing may come at a cost; specifically, the perception of holistic forms may reduce the visibility of constituent parts. In the present experiment, we examined change detection and change identification accuracy with Kanizsa rectangle patterns that were arranged to either form a Gestalt whole or not. Results from an experiment with 62 participants support this trade-off in processing holistic forms. Holistic processing improved the detection of change but obstructed its identification. Results are discussed in terms of both their theoretical significance and their application in areas ranging from baggage screening and the detection of changes in radiological images to the systems that are used to generate composite images of perpetrators on the basis of eyewitness reports.

  10. Improved Identification and Analysis of Small Open Reading Frame Encoded Polypeptides.

    PubMed

    Ma, Jiao; Diedrich, Jolene K; Jungreis, Irwin; Donaldson, Cynthia; Vaughan, Joan; Kellis, Manolis; Yates, John R; Saghatelian, Alan

    2016-04-01

    Computational, genomic, and proteomic approaches have been used to discover nonannotated protein-coding small open reading frames (smORFs). Some novel smORFs have crucial biological roles in cells and organisms, which motivates the search for additional smORFs. Proteomic smORF discovery methods are advantageous because they detect smORF-encoded polypeptides (SEPs) to validate smORF translation and SEP stability. Because SEPs are shorter and less abundant than average proteins, SEP detection using proteomics faces unique challenges. Here, we optimize several steps in the SEP discovery workflow to improve SEP isolation and identification. These changes have led to the detection of several new human SEPs (novel human genes), improved confidence in the SEP assignments, and enabled quantification of SEPs under different cellular conditions. These improvements will allow faster detection and characterization of new SEPs and smORFs. PMID:27010111

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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 24 h post attachment to be transmitted. This study describes identification of 24 h 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 24 h fed female I. scapularis phage display cDNA expression library was biopanned using rabbit antibodies to 24 h 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 24 h, before the majority of TBD agents can be transmitted. PMID:25825233

  13. Effective identification of essential proteins based on priori knowledge, network topology and gene expressions.

    PubMed

    Li, Min; Zheng, Ruiqing; Zhang, Hanhui; Wang, Jianxin; Pan, Yi

    2014-06-01

    Identification of essential proteins is very important for understanding the minimal requirements for cellular life and also necessary for a series of practical applications, such as drug design. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which makes it possible to detect proteins' essentialities from the network level. Considering that most species already have a number of known essential proteins, we proposed a new priori knowledge-based scheme to discover new essential proteins from protein interaction networks. Based on the new scheme, two essential protein discovery algorithms, CPPK and CEPPK, were developed. CPPK predicts new essential proteins based on network topology and CEPPK detects new essential proteins by integrating network topology and gene expressions. The performances of CPPK and CEPPK were validated based on the protein interaction network of Saccharomyces cerevisiae. The experimental results showed that the priori knowledge of known essential proteins was effective for improving the predicted precision. The predicted precisions of CPPK and CEPPK clearly exceeded that of the other 10 previously proposed essential protein discovery methods: Degree Centrality (DC), Betweenness Centrality (BC), Closeness Centrality (CC), Subgraph Centrality (SC), Eigenvector Centrality (EC), Information Centrality (IC), Bottle Neck (BN), Density of Maximum Neighborhood Component (DMNC), Local Average Connectivity-based method (LAC), and Network Centrality (NC). Especially, CPPK achieved 40% improvement in precision over BC, CC, SC, EC, and BN, and CEPPK performed even better. CEPPK was also compared to four other methods (EPC, ORFL, PeC, and CoEWC) which were not node centralities and CEPPK was showed to achieve the best results. PMID:24565748

  14. Effective identification of essential proteins based on priori knowledge, network topology and gene expressions.

    PubMed

    Li, Min; Zheng, Ruiqing; Zhang, Hanhui; Wang, Jianxin; Pan, Yi

    2014-06-01

    Identification of essential proteins is very important for understanding the minimal requirements for cellular life and also necessary for a series of practical applications, such as drug design. With the advances in high throughput technologies, a large number of protein-protein interactions are available, which makes it possible to detect proteins' essentialities from the network level. Considering that most species already have a number of known essential proteins, we proposed a new priori knowledge-based scheme to discover new essential proteins from protein interaction networks. Based on the new scheme, two essential protein discovery algorithms, CPPK and CEPPK, were developed. CPPK predicts new essential proteins based on network topology and CEPPK detects new essential proteins by integrating network topology and gene expressions. The performances of CPPK and CEPPK were validated based on the protein interaction network of Saccharomyces cerevisiae. The experimental results showed that the priori knowledge of known essential proteins was effective for improving the predicted precision. The predicted precisions of CPPK and CEPPK clearly exceeded that of the other 10 previously proposed essential protein discovery methods: Degree Centrality (DC), Betweenness Centrality (BC), Closeness Centrality (CC), Subgraph Centrality (SC), Eigenvector Centrality (EC), Information Centrality (IC), Bottle Neck (BN), Density of Maximum Neighborhood Component (DMNC), Local Average Connectivity-based method (LAC), and Network Centrality (NC). Especially, CPPK achieved 40% improvement in precision over BC, CC, SC, EC, and BN, and CEPPK performed even better. CEPPK was also compared to four other methods (EPC, ORFL, PeC, and CoEWC) which were not node centralities and CEPPK was showed to achieve the best results.

  15. Protein complex prediction via improved verification methods using constrained domain-domain matching.

    PubMed

    Zhao, Yang; Hayashida, Morihiro; Nacher, Jose C; Nagamochi, Hiroshi; Akutsu, Tatsuya

    2012-01-01

    Identification of protein complexes within protein-protein interaction networks is one of the important objectives in functional genomics. Ozawa et al. proposed a verification method of protein complexes by introducing a structural constraint. In this paper, we propose an improved integer programming-based method based on the idea that a candidate complex should not be divided into many small complexes, and combination methods with maximal components and extreme sets. The results of computational experiments suggest that our methods outperform the method by Ozawa et al. We prove that the verification problems are NP-hard, which justifies the use of integer programming. PMID:22961452

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

    SciTech Connect

    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.

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

    SciTech Connect

    Agarwal, Pratul K.

    2013-04-09

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

  18. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases. PMID:26577786

  19. Identification of Proteins that Modify Cataract of the Eye Lens

    PubMed Central

    Hoehenwarter, Wolfgang; Tang, Yajun; Ackermann, Renate; Pleissner, Klaus-Peter; Schmid, Monika; Stein, Robert; Zimny-Arndt, Ursula; Kumar, Nalin M.; Jungblut, Peter R.

    2010-01-01

    The occurrence of a nuclear cataract in the eye lens due to disruption of theα3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and post-translational modifications occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29 and syntaxin binding protein 6 in the eye lens. DNA polymorphisms resulting in non-conservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1 and possibly gamma N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat shock proteins have a major role for influencing cataract formation in humans. PMID:19003866

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

    PubMed Central

    Marasco, Daniela; Scognamiglio, Pasqualina Liana

    2015-01-01

    Protein–protein interactions involving disordered partners have unique features and represent prominent targets in drug discovery processes. Intrinsically Disordered Proteins (IDPs) are involved in cellular regulation, signaling and control: they bind to multiple partners and these high-specificity/low-affinity interactions play crucial roles in many human diseases. Disordered regions, terminal tails and flexible linkers are particularly abundant in DNA-binding proteins and play crucial roles in the affinity and specificity of DNA recognizing processes. Protein complexes involving IDPs are short-lived and typically involve short amino acid stretches bearing few “hot spots”, thus the identification of molecules able to modulate them can produce important lead compounds: in this scenario peptides and/or peptidomimetics, deriving from structure-based, combinatorial or protein dissection approaches, can play a key role as hit compounds. Here, we propose a panoramic review of the structural features of IDPs and how they regulate molecular recognition mechanisms focusing attention on recently reported drug-design strategies in the field of IDPs. PMID:25849651

  1. Identification of specific protein markers in microdissected hepatocellular carcinoma.

    PubMed

    Melle, Christian; Ernst, Günther; Scheibner, Olaf; Kaufmann, Roland; Schimmel, Bettina; Bleul, Annett; Settmacher, Utz; Hommann, Merten; Claussen, Uwe; von Eggeling, Ferdinand

    2007-01-01

    At present, the molecular mechanisms of hepatocellular carcinogenesis are not well-understood, and hepatocellular carcinoma (HCC) stays one of the most frequent and high-risk metastatic visceral neoplasms worldwide. For the identification of tumor-relevant proteins, we analyzed microdissected cells from nontumorous liver tissue (n = 28) and tissue derived from hepatic tumor center (n = 25), as well as tumor margin (n = 23). We unequivocally identified 53 proteins from hepatic tumor tissues by peptide fingerprint mapping and SELDI mass spectrometry that were separated using two-dimensional gel electrophoresis. Among a number of signals that were detected as significantly different in the protein profiling analysis, we identified for the first time ferritin light subunit (FLS) and adenylate kinase 3 alpha-like 1 (AK3), showing decreased expressions in hepatic tumor, as well as biliverdin reductase B (BVRB) that was upregulated in HCC. The use of ProteinChip technology in combination with tissue microdissection gives insight of the complex changes occurring at the protein level in hepatocellular cancer associated with tumor development and progression and resulted in three new potential diagnostically useful markers. PMID:17203974

  2. Extraction and identification of electroimmunoprecipitated proteins from agarose gels.

    PubMed

    Beyer, Natascha Helena; Schou, Christian; Houen, Gunnar; Heegaard, Niels H H

    2008-01-31

    A method for the identification of protein antigens captured in electroimmunoprecipitates was developed. Different antigen-antibody precipitates were generated by agarose gel immunoelectrophoresis. The immunoprecipitates were excised and various methods for extracting and dissociating the precipitates were systematically studied by analyzing for protein components of the extracts using peptide mass fingerprinting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimal recovery of antigen was obtained by 24-h extraction at 37 degrees C using a minimal volume of 0.06 M Tris-HCl, 10% SDS (pH 7). This simple and robust method is useful for the characterization of antibody specificity. It can also be used to identify antigens generating unknown precipitates in crossed immunoelectrophoresis with polyspecific antisera, including human IgG-antigen complexes electroimmunoprecipitated by secondary antibodies. Thus, the method may prove useful as an additional technique in biomarker discovery.

  3. Identification of protein coding regions in RNA transcripts

    PubMed Central

    Tang, Shiyuyun; Lomsadze, Alexandre; Borodovsky, Mark

    2015-01-01

    Massive parallel sequencing of RNA transcripts by next-generation technology (RNA-Seq) generates critically important data for eukaryotic gene discovery. Gene finding in transcripts can be done by statistical (alignment-free) as well as by alignment-based methods. We describe a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. The algorithm parameters are estimated by unsupervised training which makes unnecessary manually curated preparation of training sets. We demonstrate that (i) the unsupervised training is robust with respect to the presence of transcripts assembly errors and (ii) the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting translation initiation sites in modelled as well as in assembled transcripts compares favourably to other existing methods. PMID:25870408

  4. Using the global proteome machine for protein identification.

    PubMed

    Beavis, Ronald C

    2006-01-01

    This chapter describes the use of an open-source, freely available informatics system for the identification of proteins using tandem mass spectra of peptides derived from an enzymatic digest of a mixture of mature proteins. The chapter describes the use of features of the Global Proteome Machine (GPM) interface that assist in making comprehensive assignments between spectra and sequences, including the detection of point mutations, posttranslational modifications, and experimental artifacts. The use of this interface to validate results using the GPM Database is also described. This data repository allows analysts to compare their own results to those obtained by other scientists to determine the degree to which their data are consistent with previous measurements.

  5. Shedding light on black boxes in protein identification.

    PubMed

    Vaudel, Marc; Venne, A Saskia; Berven, Frode S; Zahedi, René P; Martens, Lennart; Barsnes, Harald

    2014-05-01

    Performing a well thought-out proteomics data analysis can be a daunting task, especially for newcomers to the field. Even researchers experienced in the proteomics field can find it challenging to follow existing publication guidelines for MS-based protein identification and characterization in detail. One of the primary goals of bioinformatics is to enable any researcher to interpret the vast amounts of data generated in modern biology, by providing user-friendly and robust end-user applications, clear documentation, and corresponding teaching materials. In that spirit, we here present an extensive tutorial for peptide and protein identification, available at http://compomics.com/bioinformatics-for-proteomics. The material is completely based on freely available and open-source tools, and has already been used and refined at numerous international courses over the past 3 years. During this time, it has demonstrated its ability to allow even complete beginners to intuitively conduct advanced bioinformatics workflows, interpret the results, and understand their context. This tutorial is thus aimed at fully empowering users, by removing black boxes in the proteomics informatics pipeline. PMID:24678044

  6. Shedding light on black boxes in protein identification.

    PubMed

    Vaudel, Marc; Venne, A Saskia; Berven, Frode S; Zahedi, René P; Martens, Lennart; Barsnes, Harald

    2014-05-01

    Performing a well thought-out proteomics data analysis can be a daunting task, especially for newcomers to the field. Even researchers experienced in the proteomics field can find it challenging to follow existing publication guidelines for MS-based protein identification and characterization in detail. One of the primary goals of bioinformatics is to enable any researcher to interpret the vast amounts of data generated in modern biology, by providing user-friendly and robust end-user applications, clear documentation, and corresponding teaching materials. In that spirit, we here present an extensive tutorial for peptide and protein identification, available at http://compomics.com/bioinformatics-for-proteomics. The material is completely based on freely available and open-source tools, and has already been used and refined at numerous international courses over the past 3 years. During this time, it has demonstrated its ability to allow even complete beginners to intuitively conduct advanced bioinformatics workflows, interpret the results, and understand their context. This tutorial is thus aimed at fully empowering users, by removing black boxes in the proteomics informatics pipeline.

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

    PubMed Central

    2010-01-01

    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. PMID:20459704

  8. Improved solution for system identification equations by Epsilon-Decomposition

    NASA Technical Reports Server (NTRS)

    Ojalvo, Irving U.

    1990-01-01

    Matrix eigenvalue theory is used to examine the source of ill-conditioning in linear algebraic equations. This approach highlights the crucial role played by the zero and near-zero eigenvalues and corresponding eigenvectors of poorly conditioned systems. Insight gained from this approach is used to significantly improve a recently developed solution procedure called Epsilon-Decomposition (E-D). E-D is an efficient alternative to Singular Value Decomposition (SVD) for ill-conditioned systems arising in parameter estimation and system identification studies. The efficiency of the improved E-D over SVD resides in the need to only obtain the zero and near-zero eigenvalues of the coefficient matrix as opposed to all of its eigenvalues and vectors (as required by SVD). Thus, the efficiency of E-D is significant for large matrices with small rank deficiency.

  9. Improvements to the LC Muon tracking and identification software

    SciTech Connect

    Milstene, C.; Fisk, G.; Para, A.

    2005-03-01

    This note summarizes the evolution of the Muon-ID package originally written by R. Markeloff at NIU. The original method used a helical swimmer to extrapolate the tracks from the interaction point and to collect hits in all sub-detectors: the electromagnetic and hadronic calorimeters and muon detector. The package was modified to replace the swimmer by a stepper which does account for both the effects of the magnetic field and for the losses by ionization in the material encountered by the particle. The modified package shows a substantial improvement in the efficiency of muon identification. Further improvement should be reached by accounting for stochastic processes via the utilization of a Kalman filter.

  10. Identification of tuberculosis-associated proteins in whole blood supernatant

    PubMed Central

    2011-01-01

    Background Biological parameters are useful tools for understanding and monitoring complicated disease processes. In this study, we attempted to identify proteins associated with active pulmonary tuberculosis (TB) using a proteomic approach. Methods To assess TB-associated changes in the composition of human proteins, whole blood supernatants were collected from patients with active TB and healthy control subjects. Two-dimensional difference gel electrophoresis (2D-DIGE) was performed to analyze proteins with high molecular weights (approximately >20 kDa). Baseline protein levels were initially compared between patients with active TB and control subjects. Possible changes of protein patterns in active TB were also compared ex vivo between whole blood samples incubated with Mycobacterium tuberculosis (Mtb)-specific antigens (stimulated condition) and under unstimulated conditions. Immunoblot and enzyme-linked immunosorbent assays (ELISA) were performed to confirm differences in identified proteins. Results Under the baseline condition, we found that the levels of retinol-binding protein 4 (RBP4), fetuin-A (also called α-HS-glycoprotein), and vitamin D-binding protein differed between patients with active TB and control subjects on 2D gels. Immunoblotting results confirmed differential expression of RBP4 and fetuin-A. ELISA results further confirmed significantly lower levels of these two proteins in samples from patients with active TB than in control subjects (P < 0.0001). Mtb-specific antigen stimulation ex vivo altered clusterin expression in whole blood samples collected from patients with active TB. Conclusions We identified TB-associated proteins in whole blood supernatants. The dynamics of protein expression during disease progression may improve our understanding of the pathogenesis of TB. PMID:21418657

  11. Special Enrichment Strategies Greatly Increase the Efficiency of Missing Proteins Identification from Regular Proteome Samples.

    PubMed

    Su, Na; Zhang, Chengpu; Zhang, Yao; Wang, Zhiqiang; Fan, Fengxu; Zhao, Mingzhi; Wu, Feilin; Gao, Yuan; Li, Yanchang; Chen, Lingsheng; Tian, Miaomiao; Zhang, Tao; Wen, Bo; Sensang, Na; Xiong, Zhi; Wu, Songfeng; Liu, Siqi; Yang, Pengyuan; Zhen, Bei; Zhu, Yunping; He, Fuchu; Xu, Ping

    2015-09-01

    As part of the Chromosome-Centric Human Proteome Project (C-HPP) mission, laboratories all over the world have tried to map the entire missing proteins (MPs) since 2012. On the basis of the first and second Chinese Chromosome Proteome Database (CCPD 1.0 and 2.0) studies, we developed systematic enrichment strategies to identify MPs that fell into four classes: (1) low molecular weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), and (4) nucleic acid-associated proteins. Of 8845 proteins identified in 7 data sets, 79 proteins were classified as MPs. Among data sets derived from different enrichment strategies, data sets for LMW and PTM yielded the most novel MPs. In addition, we found that some MPs were identified in multiple-data sets, which implied that tandem enrichments methods might improve the ability to identify MPs. Moreover, low expression at the transcription level was the major cause of the "missing" of these MPs; however, MPs with higher expression level also evaded identification, most likely due to other characteristics such as LMW, high hydrophobicity and PTM. By combining a stringent manual check of the MS2 spectra with peptides synthesis verification, we confirmed 30 MPs (neXtProt PE2 ∼ PE4) and 6 potential MPs (neXtProt PE5) with authentic MS evidence. By integrating our large-scale data sets of CCPD 2.0, the number of identified proteins has increased considerably beyond simulation saturation. Here, we show that special enrichment strategies can break through the data saturation bottleneck, which could increase the efficiency of MP identification in future C-HPP studies. All 7 data sets have been uploaded to ProteomeXchange with the identifier PXD002255.

  12. Identification and localization of the FMR-1 protein product

    SciTech Connect

    Verheij, C.; Hoogeveen, A.T.; Verkerk, A.J.M.H.; DeGraaf, E.; Bakker, C.; Reuser, A.J.J.

    1994-07-15

    The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene. As a first step in the identification and localization of the FMR-1 gene product, antibodies were raised against different regions of the FMR-1 protein (FMRP). These antibodies were used to analyze FMRP in lymphoblastoid cell lines from patients (n=5) and controls (n=3). FMRP was immunoprecipated and subsequently analyzed by immunoblotting. Four molecular species (67-74 kDa) were found which were absent in 4 of the 5 patients. The lack is in agreement with the absence of FMR-1 mRNA. The patient expressing FMRP`s shows a mosaic DNA pattern with part of the cells carrying a premutation and others carrying a full mutation. The premutation allele is preceded by an unmethylated CpG island and is expressed into FMR-1 mRNA which is subsequently translated into protein. The four different FMRPs most likely result from alternative splicing of the FMR-1 mRNA. Two splice products were mimicked in cDNA constructs transiently expressed in COS-1 cells. Both splice products appeared to encode for stable protein products and were recognized by the antibodies. The molecular weight of the protein products was in agreement with two of the protein products found in the lymphoblastoid cell lines, indicating that the FMRPs detected in lymphoblasts are the result of alternative splicing. The intracellular localization of FMRP in COS-1 cells was cytoplasmatic. The finding of four FMRPs of the same molecular weight in controls and the mosaic patient indicate that the CGG repeat is not translated.

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

    PubMed Central

    Huang, Bill X.; Kim, Hee-Yong

    2013-01-01

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

  14. Identification of protein secretion systems in bacterial genomes

    PubMed Central

    Abby, Sophie S.; Cury, Jean; Guglielmini, Julien; Néron, Bertrand; Touchon, Marie; Rocha, Eduardo P. C.

    2016-01-01

    Bacteria with two cell membranes (diderms) have evolved complex systems for protein secretion. These systems were extensively studied in some model bacteria, but the characterisation of their diversity has lagged behind due to lack of standard annotation tools. We built online and standalone computational tools to accurately predict protein secretion systems and related appendages in bacteria with LPS-containing outer membranes. They consist of models describing the systems’ components and genetic organization to be used with MacSyFinder to search for T1SS-T6SS, T9SS, flagella, Type IV pili and Tad pili. We identified ~10,000 candidate systems in bacterial genomes, where T1SS and T5SS were by far the most abundant and widespread. All these data are made available in a public database. The recently described T6SSiii and T9SS were restricted to Bacteroidetes, and T6SSii to Francisella. The T2SS, T3SS, and T4SS were frequently encoded in single-copy in one locus, whereas most T1SS were encoded in two loci. The secretion systems of diderm Firmicutes were similar to those found in other diderms. Novel systems may remain to be discovered, since some clades of environmental bacteria lacked all known protein secretion systems. Our models can be fully customized, which should facilitate the identification of novel systems. PMID:26979785

  15. Retroactive Streaming Fails to Improve Concurrent Vowel Identification

    PubMed Central

    2015-01-01

    The sequential organization of sound over time can interact with the concurrent organization of sounds across frequency. Previous studies using simple acoustic stimuli have suggested that sequential streaming cues can retroactively affect the perceptual organization of sounds that have already occurred. It is unknown whether such effects generalize to the perception of speech sounds. Listeners’ ability to identify two simultaneously presented vowels was measured in the following conditions: no context, a preceding context stream (precursors), and a following context stream (postcursors). The context stream was comprised of brief repetitions of one of the two vowels, and the primary measure of performance was listeners’ ability to identify the other vowel. Results in the precursor condition showed a significant advantage for the identification of the second vowel compared to the no-context condition, suggesting that sequential grouping mechanisms aided the segregation of the concurrent vowels, in agreement with previous work. However, performance in the postcursor condition was significantly worse compared to the no-context condition, providing no evidence for an effect of stream segregation, and suggesting a possible interference effect. Two additional experiments involving inharmonic (jittered) vowels were performed to provide additional cues to aid retroactive stream segregation; however, neither manipulation enabled listeners to improve their identification of the target vowel. Taken together with earlier studies, the results suggest that retroactive streaming may require large spectral differences between concurrent sources and thus may not provide a robust segregation cue for natural broadband sounds such as speech. PMID:26451598

  16. Basophile: Accurate Fragment Charge State Prediction Improves Peptide Identification Rates

    DOE PAGES

    Wang, Dong; Dasari, Surendra; Chambers, Matthew C.; Holman, Jerry D.; Chen, Kan; Liebler, Daniel; Orton, Daniel J.; Purvine, Samuel O.; Monroe, Matthew E.; Chung, Chang Y.; et al

    2013-03-07

    In shotgun proteomics, database search algorithms rely on fragmentation models to predict fragment ions that should be observed for a given peptide sequence. The most widely used strategy (Naive model) is oversimplified, cleaving all peptide bonds with equal probability to produce fragments of all charges below that of the precursor ion. More accurate models, based on fragmentation simulation, are too computationally intensive for on-the-fly use in database search algorithms. We have created an ordinal-regression-based model called Basophile that takes fragment size and basic residue distribution into account when determining the charge retention during CID/higher-energy collision induced dissociation (HCD) of chargedmore » peptides. This model improves the accuracy of predictions by reducing the number of unnecessary fragments that are routinely predicted for highly-charged precursors. Basophile increased the identification rates by 26% (on average) over the Naive model, when analyzing triply-charged precursors from ion trap data. Basophile achieves simplicity and speed by solving the prediction problem with an ordinal regression equation, which can be incorporated into any database search software for shotgun proteomic identification.« less

  17. Basophile: Accurate Fragment Charge State Prediction Improves Peptide Identification Rates

    SciTech Connect

    Wang, Dong; Dasari, Surendra; Chambers, Matthew C.; Holman, Jerry D.; Chen, Kan; Liebler, Daniel; Orton, Daniel J.; Purvine, Samuel O.; Monroe, Matthew E.; Chung, Chang Y.; Rose, Kristie L.; Tabb, David L.

    2013-03-07

    In shotgun proteomics, database search algorithms rely on fragmentation models to predict fragment ions that should be observed for a given peptide sequence. The most widely used strategy (Naive model) is oversimplified, cleaving all peptide bonds with equal probability to produce fragments of all charges below that of the precursor ion. More accurate models, based on fragmentation simulation, are too computationally intensive for on-the-fly use in database search algorithms. We have created an ordinal-regression-based model called Basophile that takes fragment size and basic residue distribution into account when determining the charge retention during CID/higher-energy collision induced dissociation (HCD) of charged peptides. This model improves the accuracy of predictions by reducing the number of unnecessary fragments that are routinely predicted for highly-charged precursors. Basophile increased the identification rates by 26% (on average) over the Naive model, when analyzing triply-charged precursors from ion trap data. Basophile achieves simplicity and speed by solving the prediction problem with an ordinal regression equation, which can be incorporated into any database search software for shotgun proteomic identification.

  18. Retroactive Streaming Fails to Improve Concurrent Vowel Identification.

    PubMed

    Brandewie, Eugene J; Oxenham, Andrew J

    2015-01-01

    The sequential organization of sound over time can interact with the concurrent organization of sounds across frequency. Previous studies using simple acoustic stimuli have suggested that sequential streaming cues can retroactively affect the perceptual organization of sounds that have already occurred. It is unknown whether such effects generalize to the perception of speech sounds. Listeners' ability to identify two simultaneously presented vowels was measured in the following conditions: no context, a preceding context stream (precursors), and a following context stream (postcursors). The context stream was comprised of brief repetitions of one of the two vowels, and the primary measure of performance was listeners' ability to identify the other vowel. Results in the precursor condition showed a significant advantage for the identification of the second vowel compared to the no-context condition, suggesting that sequential grouping mechanisms aided the segregation of the concurrent vowels, in agreement with previous work. However, performance in the postcursor condition was significantly worse compared to the no-context condition, providing no evidence for an effect of stream segregation, and suggesting a possible interference effect. Two additional experiments involving inharmonic (jittered) vowels were performed to provide additional cues to aid retroactive stream segregation; however, neither manipulation enabled listeners to improve their identification of the target vowel. Taken together with earlier studies, the results suggest that retroactive streaming may require large spectral differences between concurrent sources and thus may not provide a robust segregation cue for natural broadband sounds such as speech. PMID:26451598

  19. Selecting fillers on emotional appearance improves lineup identification accuracy.

    PubMed

    Flowe, Heather D; Klatt, Thimna; Colloff, Melissa F

    2014-12-01

    Mock witnesses sometimes report using criminal stereotypes to identify a face from a lineup, a tendency known as criminal face bias. Faces are perceived as criminal-looking if they appear angry. We tested whether matching the emotional appearance of the fillers to an angry suspect can reduce criminal face bias. In Study 1, mock witnesses (n = 226) viewed lineups in which the suspect had an angry, happy, or neutral expression, and we varied whether the fillers matched the expression. An additional group of participants (n = 59) rated the faces on criminal and emotional appearance. As predicted, mock witnesses tended to identify suspects who appeared angrier and more criminal-looking than the fillers. This tendency was reduced when the lineup fillers matched the emotional appearance of the suspect. Study 2 extended the results, testing whether the emotional appearance of the suspect and fillers affects recognition memory. Participants (n = 1,983) studied faces and took a lineup test in which the emotional appearance of the target and fillers was varied between subjects. Discrimination accuracy was enhanced when the fillers matched an angry target's emotional appearance. We conclude that lineup member emotional appearance plays a critical role in the psychology of lineup identification. The fillers should match an angry suspect's emotional appearance to improve lineup identification accuracy.

  20. A complex standard for protein identification, designed by evolution.

    PubMed

    Vaudel, Marc; Burkhart, Julia M; Breiter, Daniela; Zahedi, René P; Sickmann, Albert; Martens, Lennart

    2012-10-01

    Shotgun proteomic investigations rely on the algorithmic assignment of mass spectra to peptides. The quality of these matches is therefore a cornerstone in the analysis and has been the subject of numerous recent developments. In order to establish the benefits of novel algorithms, they are applied to reference samples of known content. However, these were recently shown to be either too simple to resemble typical real-life samples or as leading to results of lower accuracy as the method itself. Here, we describe how to use the proteome of Pyrococcus furiosus , a hyperthermophile, as a standard to evaluate proteomics identification workflows. Indeed, we prove that the Pyrococcus furiosus proteome provides a valid method for detecting random hits, comparable to the decoy databases currently in popular use, but we also prove that the Pyrococcus furiosus proteome goes squarely beyond the decoy approach by also providing many hundreds of highly reliable true positive hits. Searching the Pyrococcus furiosus proteome can thus be used as a unique test that provides the ability to reliably detect both false positives as well as proteome-scale true positives, allowing the rigorous testing of identification algorithms at the peptide and protein level.

  1. Improving protein fold recognition by random forest

    PubMed Central

    2014-01-01

    Background Recognizing the correct structural fold among known template protein structures for a target protein (i.e. fold recognition) is essential for template-based protein structure modeling. Since the fold recognition problem can be defined as a binary classification problem of predicting whether or not the unknown fold of a target protein is similar to an already known template protein structure in a library, machine learning methods have been effectively applied to tackle this problem. In our work, we developed RF-Fold that uses random forest - one of the most powerful and scalable machine learning classification methods - to recognize protein folds. Results RF-Fold consists of hundreds of decision trees that can be trained efficiently on very large datasets to make accurate predictions on a highly imbalanced dataset. We evaluated RF-Fold on the standard Lindahl's benchmark dataset comprised of 976 × 975 target-template protein pairs through cross-validation. Compared with 17 different fold recognition methods, the performance of RF-Fold is generally comparable to the best performance in fold recognition of different difficulty ranging from the easiest family level, the medium-hard superfamily level, and to the hardest fold level. Based on the top-one template protein ranked by RF-Fold, the correct recognition rate is 84.5%, 63.4%, and 40.8% at family, superfamily, and fold levels, respectively. Based on the top-five template protein folds ranked by RF-Fold, the correct recognition rate increases to 91.5%, 79.3% and 58.3% at family, superfamily, and fold levels. Conclusions The good performance achieved by the RF-Fold demonstrates the random forest's effectiveness for protein fold recognition. PMID:25350499

  2. Improved Proteomic Analysis Following Trichloroacetic Acid Extraction of Bacillus anthracis Spore Proteins

    SciTech Connect

    Kaiser, Brooke LD; Wunschel, David S.; Sydor, Michael A.; Warner, Marvin G.; Wahl, Karen L.; Hutchison, Janine R.

    2015-08-07

    Proteomic analysis of bacterial samples provides valuable information about cellular responses and functions under different environmental pressures. Proteomic analysis is dependent upon efficient extraction of proteins from bacterial samples without introducing bias toward extraction of particular protein classes. While no single method can recover 100% of the bacterial proteins, selected protocols can improve overall protein isolation, peptide recovery, or enrich for certain classes of proteins. The method presented here is technically simple and does not require specialized equipment such as a mechanical disrupter. Our data reveal that for particularly challenging samples, such as B. anthracis Sterne spores, trichloroacetic acid extraction improved the number of proteins identified within a sample compared to bead beating (714 vs 660, respectively). Further, TCA extraction enriched for 103 known spore specific proteins whereas bead beating resulted in 49 unique proteins. Analysis of C. botulinum samples grown to 5 days, composed of vegetative biomass and spores, showed a similar trend with improved protein yields and identification using our method compared to bead beating. Interestingly, easily lysed samples, such as B. anthracis vegetative cells, were equally as effectively processed via TCA and bead beating, but TCA extraction remains the easiest and most cost effective option. As with all assays, supplemental methods such as implementation of an alternative preparation method may provide additional insight to the protein biology of the bacteria being studied.

  3. Template-based identification of protein-protein interfaces using eFindSitePPI.

    PubMed

    Maheshwari, Surabhi; Brylinski, Michal

    2016-01-15

    Protein-protein interactions orchestrate virtually all cellular processes, therefore, their exhaustive exploration is essential for the comprehensive understanding of cellular networks. A reliable identification of interfacial residues is vital not only to infer the function of individual proteins and their assembly into biological complexes, but also to elucidate the molecular and physicochemical basis of interactions between proteins. With the exponential growth of protein sequence data, computational approaches for detecting protein interface sites have drawn an increased interest. In this communication, we discuss the major features of eFindSite(PPI), a recently developed template-based method for interface residue prediction available at http://brylinski.cct.lsu.edu/efindsiteppi. We describe the requirements and installation procedures for the stand-alone version, and explain the content and format of output data. Furthermore, the functionality of the eFindSite(PPI) web application that is designed to provide a simple and convenient access for the scientific community is presented with illustrative examples. Finally, we discuss common problems encountered in predicting protein interfaces and set forth directions for the future development of eFindSite(PPI). PMID:26235816

  4. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h. PMID:25881200

  5. High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

    PubMed

    Yu, Xiaobo; LaBaer, Joshua

    2015-05-01

    AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

  6. MALDI-TOF mass spectrometry for rapid identification of clinical fungal isolates based on ribosomal protein biomarkers.

    PubMed

    Panda, Ashutosh; Ghosh, Anup K; Mirdha, Bijay R; Xess, Immaculata; Paul, Saikat; Samantaray, Jyotish C; Srinivasan, Alagiri; Khalil, Shehla; Rastogi, Neha; Dabas, Yubhisha

    2015-02-01

    This study aimed to evaluate the identification of clinical fungal isolates (yeast and molds) by protein profiling using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS). A total of 125 clinical fungal culture isolates (yeast and filamentous fungi) were collected. The test set included 88 yeast isolates (Candida albicans, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida parapsilosis, Candida rugosa, Candida tropicalis and Cryptococcus neoformans) and 37 isolates of molds (Alternaria spp., Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Cunninghamella spp., Histoplasma capsulatum, Microsporum gypseum, Microsporum nanum, Rhizomucor spp. and Trichophyton spp.). The correlation between MALDI TOF MS and conventional identification for all these 125 fungal isolates included in the study was 87.2% at the species level and 90.4% at the genus level. MALDI TOF MS results revealed that the correlation in yeast (n=88) identification was 100% both at the genus and species levels whereas, the correlation in mold (n=37) identification was more heterogeneous i.e. 10.81% isolates had correct identification up to the genus level, 56.7% isolates had correct identification both at the genus and species levels, whereas 32.42% isolates were deemed Not Reliable Identification (NRI). But, with the modification in sample preparation protocol for molds, there was a significant improvement in identification. 86.4% isolates had correct identification till the genus and species levels whereas, only 2.7% isolates had Not Reliable Identification. In conclusion, this study demonstrates that MALDI-TOF MS could be a possible alternative to conventional techniques both for the identification and differentiation of clinical fungal isolates. However, the main limitation of this technique is that MS identification could be more precise only if the reference spectrum of the fungal species is available in the

  7. Protein S-nitrosylation: specificity and identification strategies in plants

    NASA Astrophysics Data System (ADS)

    Lamotte, Olivier; Bertoldo, Jean; Besson-Bard, Angélique; Rosnoblet, Claire; Aimé, Sébastien; Hichami, Siham; Terenzi, Hernan; Wendehenne, David

    2014-12-01

    The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the Biotin Switch Technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed.

  8. Identification of Protein-Protein Interactions by Detecting Correlated Mutation at the Interface.

    PubMed

    Guo, Fei; Ding, Yijie; Li, Zhao; Tang, Jijun

    2015-09-28

    Protein-protein interactions play key roles in a multitude of biological processes, such as de novo drug design, immune response, and enzymatic activity. It is of great interest to understand how proteins in a complex interact with each other. Here, we present a novel method for identifying protein-protein interactions, based on typical co-evolutionary information. Correlated mutation analysis can be used to predict interface residues. In this paper, we propose a non-redundant database to detect correlated mutation at the interface. First, we construct structure alignments for one input protein, based on all aligned proteins in the database. Evolutionary distance matrices, one for each input protein, can be calculated through geometric similarity and evolutionary information. Then, we use evolutionary distance matrices to estimate correlation coefficient between each pair of fragments from two input proteins. Finally, we extract interacting residues with high values of correlation coefficient, which can be grouped as interacting patches. Experiments illustrate that our method achieves better results than some existing co-evolution-based methods. Applied to SK/RR interaction between sensor kinase and response regulator proteins, our method has accuracy and coverage values of 53% and 45%, which improves upon accuracy and coverage values of 50% and 30% for DCA method. We evaluate interface prediction on four protein families, and our method has overall accuracy and coverage values of 34% and 30%, which improves upon overall accuracy and coverage values of 27% and 21% for PIFPAM. Our method has overall accuracy and coverage values of 59% and 63% on Benchmark v4.0, and 50% and 49% on CAPRI targets. Comparing to existing methods, our method improves overall accuracy value by at least 2%. PMID:26284382

  9. Hydrolysis of soybean protein improves iron bioavailability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Iron is an important trace metal element in human body. Iron deficiency affects human health, especially pregnant women and children. Soybean protein is a popular food in Asia and can contain a high amount of iron (145.70±0.74 ug/g); however, it is usually reported as an inhibitor of iron absorption...

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

    SciTech Connect

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

    1987-02-10

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

  11. Improvement in Isolation and Identification of Mouse Oogonial Stem Cells

    PubMed Central

    Lu, Zhiyong; Wu, Meng; Zhang, Jinjin; Xiong, Jiaqiang; Cheng, Jing; Shen, Wei; Luo, Aiyue; Fang, Li; Wang, Shixuan

    2016-01-01

    Female germline stem cells (FGSCs) or oogonial stem cells (OSCs) have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary results in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days and then were purified by magnetic-activated cell sorting (MACS). The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were also used as feeder cells to support the proliferation of OSCs. The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feeder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse. PMID:26635882

  12. A shotgun approach for the identification of platinum-protein complexes.

    PubMed

    Moraleja, Irene; Moreno-Gordaliza, Estefanía; Esteban-Fernández, Diego; Mena, M Luz; Linscheid, Michael W; Gómez-Gómez, M Milagros

    2015-03-01

    A shotgun approach including peptide-based OFFGEL-isoelectric focusing (IEF) fractionation has been developed with the aim of improving the identification of platinum-binding proteins in biological samples. The method is based on a filter-aided sample preparation (FASP) tryptic digestion under denaturing and reducing conditions of cisplatin-, oxaliplatin-, and carboplatin-protein complexes, followed by OFFGEL-IEF separation of the peptides. Any risk of platinum loss is minimized throughout the procedure due to the removal of the reagents used after each stage of the FASP method and the absence of thiol-based reagents in the focusing buffer employed in the IEF separation. The platinum-peptide complexes stability after the FASP digestion and the IEF separation was confirmed by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The suitability of peptide-based OFFGEL-IEF fractionation for reducing the sample complexity for further nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) analysis has been demonstrated, allowing the detection of platinum-containing peptides, with significantly lower abundance and ionization efficiency than unmodified peptides. nLC-MS/MS analysis of selected OFFGEL-IEF fractions from tryptic digests with different complexity degrees: standard human serum albumin (HSA), a mixture of five proteins (albumin, transferrin, carbonic anhydrase, myoglobin, and cytochrome-c) and human blood serum allowed the identification of several platinum-peptides from cisplatin-HSA. Cisplatin-binding sites in HSA were elucidated from the MS/MS spectra and assessed considering the protein three-dimensional structure. Most of the potential superficial binding sites available on HSA were identified for all the samples, including a biologically relevant cisplatin-cross-link of two protein domains, demonstrating the capabilities of the methodology.

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

    PubMed Central

    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

  14. 34 CFR 200.39 - Responsibilities resulting from identification for school improvement.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 1 2014-07-01 2014-07-01 false Responsibilities resulting from identification for school improvement. 200.39 Section 200.39 Education Regulations of the Offices of the Department of... Lea and School Improvement § 200.39 Responsibilities resulting from identification for...

  15. 34 CFR 200.39 - Responsibilities resulting from identification for school improvement.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Responsibilities resulting from identification for school improvement. 200.39 Section 200.39 Education Regulations of the Offices of the Department of... Lea and School Improvement § 200.39 Responsibilities resulting from identification for...

  16. 34 CFR 200.39 - Responsibilities resulting from identification for school improvement.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Responsibilities resulting from identification for school improvement. 200.39 Section 200.39 Education Regulations of the Offices of the Department of... Lea and School Improvement § 200.39 Responsibilities resulting from identification for...

  17. Towards a Black-Box for Biological EXAFS Data Analysis - I. Identification of Zinc Finger Proteins

    SciTech Connect

    Wellenreuther, Gerd; Meyer-Klaucke, Wolfram

    2007-02-02

    EXAFS allows the determination of metal binding motifs in proteins. Here we present an algorithm for the identification of one subgroup, the Zinc finger proteins, by an automated refinement of biological EXAFS data. In combination with scoring criteria inspired by high resolution crystal structures these refinements led to the identification of these motifs, that playing an important role in protein-DNA and protein-RNA interactions. Robust criteria were identified investigating several EXAFS data sets from different zinc binding proteins. For all structural zinc motifs the automated EXAFS refinement led to results consistent with published ones.

  18. Data requirements for protein identification using chemically-assisted fragmentation and tandem mass spectrometry.

    PubMed

    Juhlin, Kenton D; Swift, Dionne D; Lacey, Martin P; Correa, Paul E; Keough, Thomas W

    2005-01-01

    Many laboratories identify proteins by searching tandem mass spectrometry data against genomic or protein sequence databases. These database searches typically use the measured peptide masses or the derived peptide sequence and, in this paper, we focus on the latter. We study the minimum peptide sequence data requirements for definitive protein identification from protein sequence databases. Accurate mass measurements are not needed for definitive protein identification, even when a limited amount of sequence data is available for searching. This information has implications for the mass spectrometry performance (and cost), data base search strategies and proteomics research.

  19. P2P proteomics -- data sharing for enhanced protein identification

    PubMed Central

    2012-01-01

    Background In order to tackle the important and challenging problem in proteomics of identifying known and new protein sequences using high-throughput methods, we propose a data-sharing platform that uses fully distributed P2P technologies to share specifications of peer-interaction protocols and service components. By using such a platform, information to be searched is no longer centralised in a few repositories but gathered from experiments in peer proteomics laboratories, which can subsequently be searched by fellow researchers. Methods The system distributively runs a data-sharing protocol specified in the Lightweight Communication Calculus underlying the system through which researchers interact via message passing. For this, researchers interact with the system through particular components that link to database querying systems based on BLAST and/or OMSSA and GUI-based visualisation environments. We have tested the proposed platform with data drawn from preexisting MS/MS data reservoirs from the 2006 ABRF (Association of Biomolecular Resource Facilities) test sample, which was extensively tested during the ABRF Proteomics Standards Research Group 2006 worldwide survey. In particular we have taken the data available from a subset of proteomics laboratories of Spain's National Institute for Proteomics, ProteoRed, a network for the coordination, integration and development of the Spanish proteomics facilities. Results and Discussion We performed queries against nine databases including seven ProteoRed proteomics laboratories, the NCBI Swiss-Prot database and the local database of the CSIC/UAB Proteomics Laboratory. A detailed analysis of the results indicated the presence of a protein that was supported by other NCBI matches and highly scored matches in several proteomics labs. The analysis clearly indicated that the protein was a relatively high concentrated contaminant that could be present in the ABRF sample. This fact is evident from the information that

  20. Preparation of recombinant protein spotted arrays for proteome-wide identification of kinase targets.

    PubMed

    Im, Hogune; Snyder, Michael

    2013-04-01

    Protein microarrays allow unique approaches for interrogating global protein interaction networks. Protein arrays can be divided into two categories: antibody arrays and functional protein arrays. Antibody arrays consist of various antibodies and are appropriate for profiling protein abundance and modifications. Functional full-length protein arrays employ full-length proteins with various post-translational modifications. A key advantage of the latter is rapid parallel processing of large number of proteins for studying highly controlled biochemical activities, protein-protein interactions, protein-nucleic acid interactions, and protein-small molecule interactions. This unit presents a protocol for constructing functional yeast protein microarrays for global kinase substrate identification. This approach enables the rapid determination of protein interaction networks in yeast on a proteome-wide level. The same methodology can be readily applied to higher eukaryotic systems with careful consideration of overexpression strategy.

  1. Improving peptide identification sensitivity in shotgun proteomics by stratification of search space.

    PubMed

    Alves, Gelio; Yu, Yi-Kuo

    2013-06-01

    Because of its high specificity, trypsin is the enzyme of choice in shotgun proteomics. Nonetheless, several publications do report the identification of semitryptic and nontryptic peptides. Many of these peptides are thought to be signaling peptides or to have formed during sample preparation. It is known that only a small fraction of tandem mass spectra from a trypsin-digested protein mixture can be confidently matched to tryptic peptides. If other possibilities such as post-translational modifications and single-amino acid polymorphisms are ignored, this suggests that many unidentified spectra originate from semitryptic and nontryptic peptides. To include them in database searches, however, may not improve overall peptide identification because of the possible sensitivity reduction from search space expansion. To circumvent this issue for E-value-based search methods, we have designed a scheme that categorizes qualified peptides (i.e., peptides whose differences in molecular weight from the parent ion are within a specified error tolerance) into three tiers: tryptic, semitryptic, and nontryptic. This classification allows peptides that belong to different tiers to have different Bonferroni correction factors. Our results show that this scheme can significantly improve retrieval performance compared to those of search strategies that assign equal Bonferroni correction factors to all qualified peptides. PMID:23668635

  2. Parameter identification methods for improving structural dynamic models. Ph.D. Thesis

    NASA Technical Reports Server (NTRS)

    Lawrence, Charles

    1988-01-01

    There is an increasing need to develop Parameter Identification methods for improving structural dynamic models, based on the inability of engineers to produce mathematical models which correlate with experimental data. This research explores the efficiency of combining Component Mode Synthesis (substructuring) methods with Parameter Identification procedures in order to improve analytical modeling of structural components and their connections. Improvements are computed in terms of physical stiffness and damping parameters in order that the physical characteristics of the model can be better understood. Connections involving both viscous and friction damping are investigated. Substructuring methods are utilized to reduce the complexity of the identification problem. Component and inter-component structural connection properties are evaluated and identified independently, thus simplifying the identification problem. It is shown that modal test data is effective for identifying modeling problems associated with structural components, and for determining the stiffness and damping properties of intercomponent connections. In general, Parameter Identification is improved when greater quantities of experimental data are available.

  3. Identification and evolution of structurally dominant nodes in protein-protein interaction networks.

    PubMed

    Wang, Pei; Yu, Xinghuo; Lü, Jinhu

    2014-02-01

    It is well known that protein-protein interaction (PPI) networks are typical evolving complex networks. Identification of important nodes has been an emerging popular topic in complex networks. Many indexes have been proposed to measure the importance of nodes in complex networks, such as degree, closeness, betweenness, k-shell, clustering coefficient, semi-local centrality, eigenvector centrality. Based on multivariate statistical analysis, through integrating the above indexes and further considering the appearances of nodes in network motifs, this paper aims at developing a new measure to characterize the structurally dominant proteins (SDP) in PPI networks. Moreover, we will further investigate the evolution of the defined dominant nodes in temporal evolving real-world and artificial PPI networks. Our results indicate that the constructed artificial networks have some similar statistical properties as those of the real-world evolving networks. In this case, the artificial PPI networks can be used to further investigate the above evolution characteristics of the real-world evolving networks. Simulation results reveal that SDP in the yeast PPI networks are evolutionary conserved, however, the undominant nodes evolve rapidly. Furthermore, PPI networks are very robust against random mutations, while fragile yet with certain robustness to targeted mutations on SDP. Our investigations shed some light on the future applications of the evolving characteristics of bio-molecular networks, such as reengineering of particular networks for technological, synthetic or pharmacological purposes. PMID:24681922

  4. Small acid soluble proteins for rapid spore identification.

    SciTech Connect

    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.

  5. Latest improvements in CIEF: from proteins to microorganisms.

    PubMed

    Šalplachta, Jiří; Kubesová, Anna; Horká, Marie

    2012-10-01

    In recent years, characterization and identification of microorganisms has become very important in different fields of human activity. Conventional laboratory methods are time consuming, laborious, and they may provide both false positive or negative results, especially for closely related microorganisms. On that account, new methods for fast and reliable microbial characterization are of great interest. In particular, capillary electrophoretic techniques have a great potential for characterization of microorganisms due to their unique surface properties. Cell surface proteins play a key role in this respect. Since CIEF represents one of the most efficient techniques for protein separation, it was consequently applied to the analysis of microbial cells. This review describes, after a brief introduction to CIEF of proteins, recent developments in CIEF of diverse microorganisms (viruses, bacteria, yeasts, and fungi). Possible application schemes in human and veterinary medicine as well as in plant protection and in biosecurity are outlined.

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

    PubMed Central

    Brown, JB; Akutsu, Tatsuya

    2009-01-01

    Background DNA repair is the general term for the collection of critical mechanisms which repair many forms of DNA damage such as methylation or ionizing radiation. DNA repair has mainly been studied in experimental and clinical situations, and relatively few information-based approaches to new extracting DNA repair knowledge exist. As a first step, automatic detection of DNA repair proteins in genomes via informatics techniques is desirable; however, there are many forms of DNA repair and it is not a straightforward process to identify and classify repair proteins with a single optimal method. We perform a study of the ability of homology and machine learning-based methods to identify and classify DNA repair proteins, as well as scan vertebrate genomes for the presence of novel repair proteins. Combinations of primary sequence polypeptide frequency, secondary structure, and homology information are used as feature information for input to a Support Vector Machine (SVM). Results We identify that SVM techniques are capable of identifying portions of DNA repair protein datasets without admitting false positives; at low levels of false positive tolerance, homology can also identify and classify proteins with good performance. Secondary structure information provides improved performance compared to using primary structure alone. Furthermore, we observe that machine learning methods incorporating homology information perform best when data is filtered by some clustering technique. Analysis by applying these methodologies to the scanning of multiple vertebrate genomes confirms a positive correlation between the size of a genome and the number of DNA repair protein transcripts it is likely to contain, and simultaneously suggests that all organisms have a non-zero minimum number of repair genes. In addition, the scan result clusters several organisms' repair abilities in an evolutionarily consistent fashion. Analysis also identifies several functionally unconfirmed

  7. 34 CFR 200.39 - Responsibilities resulting from identification for school improvement.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... school improvement. 200.39 Section 200.39 Education Regulations of the Offices of the Department of... Lea and School Improvement § 200.39 Responsibilities resulting from identification for school improvement. (a) If an LEA identifies a school for school improvement under § 200.32— (1) The LEA must—...

  8. Polyacrylamide gel miniaturization improves protein visualization and autoradiographic detection

    SciTech Connect

    Mohamed, M.A.; Lerro, K.A.; Prestwich, G.D.

    1989-03-01

    Polyacrylamide gels shrink to one-quarter of their original area when soaked in a 50% (w/v) solution of polyethylene glycol. Gel miniaturization improves the contrast of protein bands, with four valuable consequences. (i) A 5- to 10-fold increase in sensitivity for Coomassie blue is observed. (ii) Gels are more durable; i.e., they resist tearing when wet and they do not crack during drying under vacuum. (iii) Shrunken gels give sharper photographic images and provide better interlane protein band comparisons. (iv) Condensed protein bands lead to an increased sensitivity for detecting low-abundance, radioactively-labeled proteins by fluorography.

  9. Improving protein-protein interactions prediction accuracy using protein evolutionary information and relevance vector machine model.

    PubMed

    An, Ji-Yong; Meng, Fan-Rong; You, Zhu-Hong; Chen, Xing; Yan, Gui-Ying; Hu, Ji-Pu

    2016-10-01

    Predicting protein-protein interactions (PPIs) is a challenging task and essential to construct the protein interaction networks, which is important for facilitating our understanding of the mechanisms of biological systems. Although a number of high-throughput technologies have been proposed to predict PPIs, there are unavoidable shortcomings, including high cost, time intensity, and inherently high false positive rates. For these reasons, many computational methods have been proposed for predicting PPIs. However, the problem is still far from being solved. In this article, we propose a novel computational method called RVM-BiGP that combines the relevance vector machine (RVM) model and Bi-gram Probabilities (BiGP) for PPIs detection from protein sequences. The major improvement includes (1) Protein sequences are represented using the Bi-gram probabilities (BiGP) feature representation on a Position Specific Scoring Matrix (PSSM), in which the protein evolutionary information is contained; (2) For reducing the influence of noise, the Principal Component Analysis (PCA) method is used to reduce the dimension of BiGP vector; (3) The powerful and robust Relevance Vector Machine (RVM) algorithm is used for classification. Five-fold cross-validation experiments executed on yeast and Helicobacter pylori datasets, which achieved very high accuracies of 94.57 and 90.57%, respectively. Experimental results are significantly better than previous methods. To further evaluate the proposed method, we compare it with the state-of-the-art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM-BiGP method is significantly better than the SVM-based method. In addition, we achieved 97.15% accuracy on imbalance yeast dataset, which is higher than that of balance yeast dataset. The promising experimental results show the efficiency and robust of the proposed method, which can be an automatic decision support tool for future

  10. Improving protein-protein interactions prediction accuracy using protein evolutionary information and relevance vector machine model.

    PubMed

    An, Ji-Yong; Meng, Fan-Rong; You, Zhu-Hong; Chen, Xing; Yan, Gui-Ying; Hu, Ji-Pu

    2016-10-01

    Predicting protein-protein interactions (PPIs) is a challenging task and essential to construct the protein interaction networks, which is important for facilitating our understanding of the mechanisms of biological systems. Although a number of high-throughput technologies have been proposed to predict PPIs, there are unavoidable shortcomings, including high cost, time intensity, and inherently high false positive rates. For these reasons, many computational methods have been proposed for predicting PPIs. However, the problem is still far from being solved. In this article, we propose a novel computational method called RVM-BiGP that combines the relevance vector machine (RVM) model and Bi-gram Probabilities (BiGP) for PPIs detection from protein sequences. The major improvement includes (1) Protein sequences are represented using the Bi-gram probabilities (BiGP) feature representation on a Position Specific Scoring Matrix (PSSM), in which the protein evolutionary information is contained; (2) For reducing the influence of noise, the Principal Component Analysis (PCA) method is used to reduce the dimension of BiGP vector; (3) The powerful and robust Relevance Vector Machine (RVM) algorithm is used for classification. Five-fold cross-validation experiments executed on yeast and Helicobacter pylori datasets, which achieved very high accuracies of 94.57 and 90.57%, respectively. Experimental results are significantly better than previous methods. To further evaluate the proposed method, we compare it with the state-of-the-art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM-BiGP method is significantly better than the SVM-based method. In addition, we achieved 97.15% accuracy on imbalance yeast dataset, which is higher than that of balance yeast dataset. The promising experimental results show the efficiency and robust of the proposed method, which can be an automatic decision support tool for future

  11. Identification of maize embryo-preferred promoters suitable for high-level heterologous protein production.

    PubMed

    Streatfield, Stephen J; Bray, Jeffrey; Love, Robert T; Horn, Michael E; Lane, Jeffrey R; Drees, Carol F; Egelkrout, Erin M; Howard, John A

    2010-01-01

    The production of heterologous proteins in plants at levels consistent with commercialization of protein products requires molecular tools to ensure high-level transgene expression. The identification of strong promoters, preferably specific to the target expression tissue, is a focus for improving foreign protein yields using transgenic cereals as a production system. Thus, there is a requirement for strong embryo preferred monocot promoters. We obtained the sequences of 500 randomly selected maize cDNA clones to determine gene expression profiles in embryo tissues at multiple stages during development. Promoters corresponding to the most abundant clones were identified and isolated. These promoters were fused to the b-glucuronidase reporter and their tissue specificity and developmental expression characteristics assessed in transgenic maize. All of the isolated promoters tested drove transgene expression predominantly in the embryo and were most active late in embryogenesis during storage protein deposition. One of the most active promoters assessed by transgene expression was associated with the globulin-1 protein. Sequence identified here extended approximately 1.6 kb distal to the previously identified extent of the globulin-1 promoter, and this additional sequence boosted expression over two-fold. The extended globulin-1 promoter sequence isolated in this study has the potential for driving transgene expression at higher levels than those previously reported for cereals. Also, other highly active embryo promoters identified here offer opportunities to express multiple foreign proteins simultaneously at high levels in embryo tissues, while avoiding concerns over gene silencing due to the repeated use of a single promoter. PMID:21844671

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

    SciTech Connect

    Rodibaugh, R.

    1989-01-01

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

  13. USING A TAPED INTERVENTION TO IMPROVE KINDERGARTEN STUDENTS' NUMBER IDENTIFICATION

    PubMed Central

    Krohn, Katherine R; Skinner, Christopher H; Fuller, Emily J; Greear, Corrine

    2012-01-01

    A multiple baseline design across students was used to evaluate the effects of a taped numbers (TN) intervention on the number-identification accuracy of 4 kindergarten students. During TN, students attempted to name the numbers 0 through 9 on randomized lists before each number was provided via a tape player 2 s later. All 4 students showed immediate increases and reached 100% in number-identification accuracy. One student reached 100% accuracy after TN was supplemented with performance feedback, reinforcement, and overcorrection. PMID:22844152

  14. Hide and seek: Identification and confirmation of small molecule protein targets.

    PubMed

    Ursu, Andrei; Waldmann, Herbert

    2015-08-15

    Target identification and confirmation for small molecules is often the rate limiting step in drug discovery. A robust method to identify proteins addressed by small molecules is affinity chromatography using chemical probes. These usually consist of the compound of interest equipped with a linker molecule and a proper tag. Recently, methods emerged that allow the identification of protein targets without prior functionalization of the small molecule of interest. The digest offers an update on the newest developments in the area of target identification with special focus on confirmation techniques. PMID:26115575

  15. Identification of Associated Proteins by Immunoprecipitation and Mass Spectrometry Analysis.

    PubMed

    Cao, Xiumei; Yan, Jianshe

    2016-01-01

    Protein-protein interactions play central roles in intercellular and intracellular signal transduction. Impairment of protein-protein interactions causes many diseases such as cancer, cardiomyopathies, diabetes, microbial infections, and genetic and neurodegenerative disorders. Immunoprecipitation is a technique in which a target protein of interest bound by an antibody is used to pull down the protein complex out of cell lysates, which can be identified by mass spectrometry. Here, we describe the protocol to immunoprecipitate and identify the components of the protein complexes of ElmoE in Dictyostelium discoideum cells. PMID:27271899

  16. Identification and characterization of secreted proteins in Eimeria tenella

    NASA Astrophysics Data System (ADS)

    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.

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

    SciTech Connect

    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.

  18. Combination of MS protein identification and bioassay of chromatographic fractions to identify biologically active substances from complex protein sources.

    PubMed

    Kuromitsu, Sadao; Yokota, Hiroyuki; Hiramoto, Masashi; Yuri, Masatoshi; Naitou, Masanori; Nakamura, Naoto; Kawabata, Shigeki; Kobori, Masato; Katoh, Masao; Furuchi, Kiyoshi; Mita, Haruhisa; Yamada, Tetsuo

    2009-06-01

    Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

  19. Using a Taped Intervention to Improve Kindergarten Students' Number Identification

    ERIC Educational Resources Information Center

    Krohn, Katherine R.; Skinner, Christopher H.; Fuller, Emily J.; Greear, Corrine

    2012-01-01

    A multiple baseline design across students was used to evaluate the effects of a taped numbers (TN) intervention on the number-identification accuracy of 4 kindergarten students. During TN, students attempted to name the numbers 0 through 9 on randomized lists before each number was provided via a tape player 2 s later. All 4 students showed…

  20. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    PubMed

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital. PMID:25768007

  1. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    PubMed

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital.

  2. Polymorphism Identification and Improved Genome Annotation of Brassica rapa Through Deep RNA Sequencing

    PubMed Central

    Devisetty, Upendra Kumar; Covington, Michael F.; Tat, An V.; Lekkala, Saradadevi; Maloof, Julin N.

    2014-01-01

    The mapping and functional analysis of quantitative traits in Brassica rapa can be greatly improved with the availability of physically positioned, gene-based genetic markers and accurate genome annotation. In this study, deep transcriptome RNA sequencing (RNA-Seq) of Brassica rapa was undertaken with two objectives: SNP detection and improved transcriptome annotation. We performed SNP detection on two varieties that are parents of a mapping population to aid in development of a marker system for this population and subsequent development of high-resolution genetic map. An improved Brassica rapa transcriptome was constructed to detect novel transcripts and to improve the current genome annotation. This is useful for accurate mRNA abundance and detection of expression QTL (eQTLs) in mapping populations. Deep RNA-Seq of two Brassica rapa genotypes—R500 (var. trilocularis, Yellow Sarson) and IMB211 (a rapid cycling variety)—using eight different tissues (root, internode, leaf, petiole, apical meristem, floral meristem, silique, and seedling) grown across three different environments (growth chamber, greenhouse and field) and under two different treatments (simulated sun and simulated shade) generated 2.3 billion high-quality Illumina reads. A total of 330,995 SNPs were identified in transcribed regions between the two genotypes with an average frequency of one SNP in every 200 bases. The deep RNA-Seq reassembled Brassica rapa transcriptome identified 44,239 protein-coding genes. Compared with current gene models of B. rapa, we detected 3537 novel transcripts, 23,754 gene models had structural modifications, and 3655 annotated proteins changed. Gaps in the current genome assembly of B. rapa are highlighted by our identification of 780 unmapped transcripts. All the SNPs, annotations, and predicted transcripts can be viewed at http://phytonetworks.ucdavis.edu/. PMID:25122667

  3. Polymorphism identification and improved genome annotation of Brassica rapa through Deep RNA sequencing.

    PubMed

    Devisetty, Upendra Kumar; Covington, Michael F; Tat, An V; Lekkala, Saradadevi; Maloof, Julin N

    2014-08-12

    The mapping and functional analysis of quantitative traits in Brassica rapa can be greatly improved with the availability of physically positioned, gene-based genetic markers and accurate genome annotation. In this study, deep transcriptome RNA sequencing (RNA-Seq) of Brassica rapa was undertaken with two objectives: SNP detection and improved transcriptome annotation. We performed SNP detection on two varieties that are parents of a mapping population to aid in development of a marker system for this population and subsequent development of high-resolution genetic map. An improved Brassica rapa transcriptome was constructed to detect novel transcripts and to improve the current genome annotation. This is useful for accurate mRNA abundance and detection of expression QTL (eQTLs) in mapping populations. Deep RNA-Seq of two Brassica rapa genotypes-R500 (var. trilocularis, Yellow Sarson) and IMB211 (a rapid cycling variety)-using eight different tissues (root, internode, leaf, petiole, apical meristem, floral meristem, silique, and seedling) grown across three different environments (growth chamber, greenhouse and field) and under two different treatments (simulated sun and simulated shade) generated 2.3 billion high-quality Illumina reads. A total of 330,995 SNPs were identified in transcribed regions between the two genotypes with an average frequency of one SNP in every 200 bases. The deep RNA-Seq reassembled Brassica rapa transcriptome identified 44,239 protein-coding genes. Compared with current gene models of B. rapa, we detected 3537 novel transcripts, 23,754 gene models had structural modifications, and 3655 annotated proteins changed. Gaps in the current genome assembly of B. rapa are highlighted by our identification of 780 unmapped transcripts. All the SNPs, annotations, and predicted transcripts can be viewed at http://phytonetworks.ucdavis.edu/.

  4. Identification of potential genetic markers for improved growth rate in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Identification of genetic polymorphism associated with muscle growth would improve selection efficiency of channel catfish broodstock. Because faster growth is typically associated with increased food intake, factors involved in food intake regulation may serve as potential gene markers for selecti...

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Identification of binary interactions between human cytomegalovirus virion proteins.

    PubMed

    Phillips, Stacia L; Bresnahan, Wade A

    2011-01-01

    Human cytomegalovirus (HCMV) virions are composed of a DNA-containing nucleocapsid surrounded by a tegument layer and host-derived lipid envelope studded with virally encoded glycoproteins. These complex virions are estimated to be composed of more than 50 viral proteins. Assembly of HCMV virions is poorly understood, especially with respect to acquisition of the tegument; however, it is thought to involve the stepwise addition of virion components through protein-protein interactions. We sought to identify interactions among HCMV virion proteins using yeast two-hybrid analysis. Using 33 known capsid and tegument proteins, we tested 1,089 pairwise combinations for binary interaction in the two-hybrid assay. We identified 24 interactions among HCMV virion proteins, including 13 novel interactions among tegument proteins and one novel interaction between capsid proteins. Several of these novel interactions were confirmed by coimmunoprecipitation of protein complexes from transfected cells. In addition, we demonstrate three of these interactions in the context of HCMV infection. This study reveals several new protein-protein interactions among HCMV tegument proteins, some of which are likely important for HCMV replication and pathogenesis. PMID:20962080

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

    PubMed

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

    2013-04-01

    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. PMID:23391412

  8. Robust enzyme design: bioinformatic tools for improved protein stability.

    PubMed

    Suplatov, Dmitry; Voevodin, Vladimir; Švedas, Vytas

    2015-03-01

    The ability of proteins and enzymes to maintain a functionally active conformation under adverse environmental conditions is an important feature of biocatalysts, vaccines, and biopharmaceutical proteins. From an evolutionary perspective, robust stability of proteins improves their biological fitness and allows for further optimization. Viewed from an industrial perspective, enzyme stability is crucial for the practical application of enzymes under the required reaction conditions. In this review, we analyze bioinformatic-driven strategies that are used to predict structural changes that can be applied to wild type proteins in order to produce more stable variants. The most commonly employed techniques can be classified into stochastic approaches, empirical or systematic rational design strategies, and design of chimeric proteins. We conclude that bioinformatic analysis can be efficiently used to study large protein superfamilies systematically as well as to predict particular structural changes which increase enzyme stability. Evolution has created a diversity of protein properties that are encoded in genomic sequences and structural data. Bioinformatics has the power to uncover this evolutionary code and provide a reproducible selection of hotspots - key residues to be mutated in order to produce more stable and functionally diverse proteins and enzymes. Further development of systematic bioinformatic procedures is needed to organize and analyze sequences and structures of proteins within large superfamilies and to link them to function, as well as to provide knowledge-based predictions for experimental evaluation.

  9. Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach.

    PubMed

    Alvarez, Angel H; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

    2015-10-01

    Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.

  10. Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach

    PubMed Central

    Alvarez, Angel H.; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

    2015-01-01

    Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD. PMID:26424916

  11. Data identification for improving gene network inference using computational algebra.

    PubMed

    Dimitrova, Elena; Stigler, Brandilyn

    2014-11-01

    Identification of models of gene regulatory networks is sensitive to the amount of data used as input. Considering the substantial costs in conducting experiments, it is of value to have an estimate of the amount of data required to infer the network structure. To minimize wasted resources, it is also beneficial to know which data are necessary to identify the network. Knowledge of the data and knowledge of the terms in polynomial models are often required a priori in model identification. In applications, it is unlikely that the structure of a polynomial model will be known, which may force data sets to be unnecessarily large in order to identify a model. Furthermore, none of the known results provides any strategy for constructing data sets to uniquely identify a model. We provide a specialization of an existing criterion for deciding when a set of data points identifies a minimal polynomial model when its monomial terms have been specified. Then, we relax the requirement of the knowledge of the monomials and present results for model identification given only the data. Finally, we present a method for constructing data sets that identify minimal polynomial models.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  14. Improved hybrid optimization algorithm for 3D protein structure prediction.

    PubMed

    Zhou, Changjun; Hou, Caixia; Wei, Xiaopeng; Zhang, Qiang

    2014-07-01

    A new improved hybrid optimization algorithm - PGATS algorithm, which is based on toy off-lattice model, is presented for dealing with three-dimensional protein structure prediction problems. The algorithm combines the particle swarm optimization (PSO), genetic algorithm (GA), and tabu search (TS) algorithms. Otherwise, we also take some different improved strategies. The factor of stochastic disturbance is joined in the particle swarm optimization to improve the search ability; the operations of crossover and mutation that are in the genetic algorithm are changed to a kind of random liner method; at last tabu search algorithm is improved by appending a mutation operator. Through the combination of a variety of strategies and algorithms, the protein structure prediction (PSP) in a 3D off-lattice model is achieved. The PSP problem is an NP-hard problem, but the problem can be attributed to a global optimization problem of multi-extremum and multi-parameters. This is the theoretical principle of the hybrid optimization algorithm that is proposed in this paper. The algorithm combines local search and global search, which overcomes the shortcoming of a single algorithm, giving full play to the advantage of each algorithm. In the current universal standard sequences, Fibonacci sequences and real protein sequences are certified. Experiments show that the proposed new method outperforms single algorithms on the accuracy of calculating the protein sequence energy value, which is proved to be an effective way to predict the structure of proteins. PMID:25069136

  15. Brachypodium distachyon grain: identification and subcellular localization of storage proteins

    PubMed Central

    Larré, C.; Penninck, S.; Bouchet, B.; Lollier, V.; Tranquet, O.; Denery-Papini, S.; Guillon, F.; Rogniaux, H.

    2010-01-01

    Seed storage proteins are of great importance in nutrition and in industrial transformation because of their functional properties. Brachypodium distachyon has been proposed as a new model plant to study temperate cereals. The protein composition of Brachypodium grain was investigated by separating the proteins on the basis of their solubility combined with a proteomic approach. Salt-soluble proteins as well as salt-insoluble proteins separated by two-dimensional gel electrophoresis revealed 284 and 120 spots, respectively. Proteins from the major spots were sequenced by mass spectrometry and identified by searching against a Brachypodium putative protein database. Our analysis detected globulins and prolamins but no albumins. Globulins were represented mainly by the 11S type and their solubility properties corresponded to the glutelin found in rice. An in silico search for storage proteins returned more translated genes than expressed products identified by mass spectrometry, particularly in the case of prolamin type proteins, reflecting a strong expression of globulins at the expense of prolamins. Microscopic examination of endosperm cells revealed scarce small-size starch granules surrounded by protein bodies containing 11S globulins. The presence of protein bodies containing glutelins makes B. distachyon closer to rice or oat than to wheat endosperm. PMID:20385545

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

    SciTech Connect

    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.

  17. INCREASING PROTEIN STABILITY BY IMPROVING BETA-TURNS

    PubMed Central

    Fu, Hailong; Grimsley, Gerald R.; Razvi, Abbas; Scholtz, J. Martin; Pace, C. Nick

    2009-01-01

    Our goal was to gain a better understanding of how protein stability can be increased by improving β-turns. We studied 22 β-turns in nine proteins with 66 to 370 residues by replacing other residues with proline and glycine and measuring the stability. These two residues are statistically preferred in some β-turn positions. We studied: Cold shock protein B (CspB), Histidine-containing phosphocarrier protein (HPr), Ubiquitin, Ribonucleases Sa2, Sa3, T1, and HI, Tryptophan synthetase α-subunit (TSα), and Maltose binding protein (MBP). Of the fifteen single proline mutations, 11increased stability (Average = 0.8 ± 0.3; Range = 0.3 – 1.5 kcal/mol), and the stabilizing effect of double proline mutants was additive. Based on this and our previous work, we conclude that proteins can generally be stabilized by replacing non-proline residues with proline residues at the i + 1 position of Type I and II β-turns and at the i position in Type II β-turns. Other turn positions can sometimes be used if the φ angle is near −60° for the residue replaced. It is important that the side chain of the residue replaced is less than 50% buried. Identical substitutions in β-turns in related proteins give similar results. Proline substitutions increase stability mainly by decreasing the entropy of the denatured state. In contrast, the large, diverse group of proteins considered here had almost no residues in β-turns that could be replaced by Gly to increase protein stability. Improving β-turns by substituting Pro residues is a generally useful way of increasing protein stability. PMID:19626709

  18. Identification of blood-protein carriers of the CA 19-9 antigen and characterization of prevalence in pancreatic diseases.

    PubMed

    Yue, Tingting; Partyka, Katie; Maupin, Kevin A; Hurley, Mary; Andrews, Philip; Kaul, Karen; Moser, A James; Zeh, Herbert; Brand, Randall E; Haab, Brian B

    2011-09-01

    The current best serum marker for pancreatic cancer, CA 19-9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19-9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19-9 antigen, we immunoprecipitated the CA 19-9 antigen from pooled sera and identified the associated proteins using MS. Among the high-confidence identifications, we confirmed the presence of the CA 19-9 antigen on Apolipoprotein B-100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19-9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10-25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19-9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

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

    PubMed

    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. PMID:26060076

  20. Identification of urinary protein biomarkers for tobacco smoking.

    PubMed

    Haniff, Aj Nabill; Gam, Lay-Harn

    2016-01-01

    Smoking, passive smoking, and nonsmoking are conditions that give different degrees of stress to the body. In this study, a proteomic technique was used to analyze differentially urinary protein expression between these three groups of subjects. Urinary proteins were precipitated using ammonium sulfate followed by separation according to molecular weights using SDS-PAGE. The gel was stained by Coommassie blue, and the image of the gel was captured for the comparison study. The protein bands that were consistently detected but expressed at different intensity between the smokers and nonsmokers were targeted for further analysis. Three targeted protein bands were excised from the gel, consisting of a unique protein band of smokers and a pair of differentially expressed protein bands from smokers and nonsmokers. The proteins were digested in gel by trypsin. The tryptic peptides were analyzed with ultra performance liquid chromatography-tandem mass spectrometry. Protein identity was determined by the product ion spectrum in the MS/MS scan. Four unique proteins from the smokers, namely, pancreatic alpha amylase, proepidermal growth factor, protein 4.1, and prostatic acid phosphatase, were found to be potential urinary biomarkers to indicate smoking status of a person. PMID:25640279

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

    PubMed

    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.

  2. Identification of human liver microsomal proteins adducted by a reactive metabolite using shotgun proteomics.

    PubMed

    Yang, Yanou; Xiao, Qing; Humphreys, W Griffith; Dongre, Ashok; Shu, Yue-Zhong

    2014-09-15

    Covalent modification of cellular proteins by chemically reactive compounds/metabolites has the potential to disrupt biological function and elicit serious adverse drug reactions. Information on the nature and binding patterns of protein targets are critical toward understanding the mechanism of drug induced toxicity. Protein covalent binding studies established in liver microsomes can quantitively estimate the extent of protein modification, but they provide little information on the nature of the modified proteins. In this article, we describe a label-free shotgun proteomic workflow for the identification of target proteins modified in situ by reactive metabolites in human liver microsome incubations. First, we developed a shotgun proteomic workflow for the characterization of the human liver microsomal subproteome, which consists of predominately membrane-bound proteins. Human liver microsomes were solubilized with a combination of MS-compatible organic solvents followed by protein reduction, alkylation, and tryptic digestion. The unmodified samples were analyzed by UHPLC-MS/MS, and the proteins were identified by database searching. This workflow led to the successful identification of 329 human liver microsomal subproteome proteins with 1% FDR (false discovery rate). The same method was then applied to identify the modifications of human liver microsomal proteins by a known reactive metabolite 2-(methylsulfonyl)benzo[d]thiazole (2), either after incubation directly with 2 or with its parent compound 2-(methylthio)benzo[d]thiazole (1). A total of 19 modified constituent peptides which could be mapped to 18 proteins were identified in human liver microsomes incubated directly with 2. Among these, 5 modified constituent peptides which could be mapped to 4 proteins were identified in incubation with 1, which is known to generate 2 in human liver microsomal incubations. This label-free workflow is generally applicable to the identification and characterization of

  3. Large-scale identification of yeast integral membrane protein interactions

    PubMed Central

    Miller, John P.; Lo, Russell S.; Ben-Hur, Asa; Desmarais, Cynthia; Stagljar, Igor; Noble, William Stafford; Fields, Stanley

    2005-01-01

    We carried out a large-scale screen to identify interactions between integral membrane proteins of Saccharomyces cerevisiae by using a modified split-ubiquitin technique. Among 705 proteins annotated as integral membrane, we identified 1,985 putative interactions involving 536 proteins. To ascribe confidence levels to the interactions, we used a support vector machine algorithm to classify interactions based on the assay results and protein data derived from the literature. Previously identified and computationally supported interactions were used to train the support vector machine, which identified 131 interactions of highest confidence, 209 of the next highest confidence, 468 of the next highest, and the remaining 1,085 of low confidence. This study provides numerous putative interactions among a class of proteins that have been difficult to analyze on a high-throughput basis by other approaches. The results identify potential previously undescribed components of established biological processes and roles for integral membrane proteins of ascribed functions. PMID:16093310

  4. Improved Quick Disconnect (QD) Interface Through Fail Safe Parts Identification

    NASA Technical Reports Server (NTRS)

    Blanch-Payne, Evelyn

    2001-01-01

    An extensive review of existing Quick Disconnects (QDs) mating and demating operations was performed to determine which shuttle part interface identifications and procedures contribute to human factor errors. The research methods used consisted of interviews with engineers and technicians, examination of incident reports, critiques of video and audio tapes of QD operations, and attendance of a Hyper QD operational course. The data strongly suggests that there are inherit human factor errors involved in QD operations. To promote fail-safe operations, QD interface problem areas and recommendations were outlined and reviewed. It is suggested that dialogue, investigations and recommendations continue.

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

    SciTech Connect

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

    2013-11-05

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

  6. Identification of ultramodified proteins using top-down spectra

    SciTech Connect

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

    2013-04-10

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

  7. Proteomic identification of germline proteins in Caenorhabditis elegans

    PubMed Central

    Turner, B Elizabeth; Basecke, Sophia M; Bazan, Grace C; Dodge, Eric S; Haire, Cassy M; Heussman, Dylan J; Johnson, Chelsey L; Mukai, Chelsea K; Naccarati, Adrianna M; Norton, Sunny-June; Sato, Jennifer R; Talavera, Chihara O; Wade, Michael V; Hillers, Kenneth J

    2015-01-01

    Sexual reproduction involves fusion of 2 haploid gametes to form diploid offspring with genetic contributions from both parents. Gamete formation represents a unique developmental program involving the action of numerous germline-specific proteins. In an attempt to identify novel proteins involved in reproduction and embryonic development, we have carried out a proteomic characterization of the process in Caenorhabditis elegans. To identify candidate proteins, we used 2D gel electrophoresis (2DGE) to compare protein abundance in nucleus-enriched extracts from wild-type C. elegans, and in extracts from mutant worms with greatly reduced gonads (glp-4(bn2) worms reared at 25°C); 84 proteins whose abundance correlated with germline presence were identified. To validate candidates, we used feeding RNAi to deplete candidate proteins, and looked for reduction in fertility and/or germline cytological defects. Of 20 candidates so screened for involvement in fertility, depletion of 13 (65%) caused a significant reduction in fertility, and 6 (30%) resulted in sterility (<5 % of wild-type fertility). Five of the 13 proteins with demonstrated roles in fertility have not previously been implicated in germline function. The high frequency of defects observed after RNAi depletion of candidate proteins suggests that this approach is effective at identifying germline proteins, thus contributing to our understanding of this complex organ. PMID:26435885

  8. Large-Scale Identification of Putative Exported Proteins in Candida albicans by Genetic Selection

    PubMed Central

    Monteoliva, L.; López Matas, M.; Gil, C.; Nombela, C.; Pla, J.

    2002-01-01

    In all living organisms, secreted proteins play essential roles in different processes. Of special interest is the construction of the fungal cell wall, since this structure is absent from mammalian cells. The identification of the proteins involved in its biogenesis is therefore a primary goal in antifungal research. To perform a systematic identification of such proteins in Candida albicans, we carried out a genetic screening in which in-frame fusions with an intracellular allele of invertase gene SUC2 of Saccharomyces cerevisiae can be used to select and identify putatively exported proteins in the heterologous host S. cerevisiae. Eighty-three clones were selected, including 11 previously identified genes from C. albicans as well as 41 C. albicans genes that encode proteins homologous to already described proteins from related organisms. They include enzymes involved in cell wall synthesis and protein secretion. We also found membrane receptors and transporters presumably related to the interaction of C. albicans with the environment as well as extracellular enzymes and proteins involved in different morphological transitions. In addition, 11 C. albicans open reading frames (ORFs) identified in this screening encode proteins homologous to unknown or putative proteins, while 5 ORFs encode novel secreted proteins without known homologues in other organisms. This screening procedure therefore not only identifies a set of targets of interest in antifungal research but also provides new clues for understanding the topological locations of many proteins involved in processes relevant to the pathogenicity of this microorganism. PMID:12456000

  9. Production of rice protein by alkaline extraction improves its digestibility.

    PubMed

    Kumagai, Takehisa; Kawamura, Hiroyuki; Fuse, Takao; Watanabe, Toshiyuki; Saito, Yuhi; Masumura, Takehiro; Watanabe, Reiko; Kadowaki, Motoni

    2006-12-01

    Rice seed endosperm has two types of protein bodies (PB). Type I protein body (PB-I) accumulates prolamin and is hard to digest, while type II protein body (PB-II) mainly consists of glutelin, an easily digestible protein. A simple method to process rice protein and improve its digestibility was tested from the viewpoint of its application to food manufacturing. Rice protein prepared by alkaline extraction followed by neutralization sedimentation (AE-RP) was compared with that prepared by starch degradation by alpha-amylase (SD-RP). The crude protein content of AE-RP and SD-RP was 84.7% and 78.2%, respectively. There were no major differences in protein composition among AE-RP, SD-RP and rice flour by SDS-PAGE, except 16 kDa polypeptide. With respect to amino acids, all the groups showed quite similar compositions, although cysteine and methionine were lower in AE-RP. In an in vitro digestion study with pepsin and pancreatin, both the SDS-PAGE analysis of protein pattern and the crude protein content of undigested residue clearly demonstrated that AE-RP has a higher digestibility than SD-RP. To find the cause of the difference in digestibility, the structural property of protein bodies by two production methods was compared using electron microscopy. PB-II of AE-RP was transformed into small, amorphous granules, while that of SD-RP was still kept partial protein body structures. PB-I of AE-RP kept its protein body structure, but produced double layers. From the finding that glutelin-gold was detected by immunochemistry not only in small, amorphous granules but also in PB-I, mainly the cortex layer, in AE-RP, it became clear that PB-I was swollen and fragile as a result of alkali treatment. These results strongly indicate that the improvement in digestibility of AE-RP is a result of the structural change of PB-I and -II caused by alkaline extraction.

  10. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry.

    PubMed

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application. PMID:26761849

  11. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry.

    PubMed

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application.

  12. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry

    PubMed Central

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutritional benefits in contrast to heat denaturation or native applications. WPHs improve solubility over a wide range of pH, create viscosity through water binding, and promote cohesion, adhesion, and elasticity. WPHs form stronger but more flexible edible films than WPC or WPI. WPHs enhance emulsification, bind fat, and facilitate whipping, compared to intact WPs. Extensive hydrolyzed WPHs with proper heat applications are the best emulsifiers and addition of polysaccharides improves the emulsification ability of WPHs. Also, WPHs improve the sensorial properties like color, flavor, and texture but impart a bitter taste in case where extensive hydrolysis (degree of hydrolysis greater than 8%). It is important to consider the type of enzyme, hydrolysis conditions, and WPHs production method based on the nature of food application. PMID:26761849

  13. Identification of new Palmitoylated Proteins in Toxoplasma gondii

    PubMed Central

    Caballero, Marina C.; Alonso, Andrés M.; Deng, Bin; Attias, Marcia; de Souza, Wanderley; Corvi, María M.

    2016-01-01

    Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In the recent years protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes. PMID:26825284

  14. Identification of a Fluorescent Protein from Rhacostoma Atlantica.

    PubMed

    Tota, Michael R; Allen, Jeanna M; Karolin, Jan O; Geddes, Chris D; Ward, William W

    2016-09-01

    We have cloned a novel fluorescent protein from the jellyfish Rhacostoma atlantica. The closest known related fluorescent protein is the Phialidium yellow fluorescent protein, with only a 55% amino acid sequence identity. A somewhat unusual alanine-tyrosine-glycine amino acid sequence forms the presumed chromophore of the novel protein. The protein has an absorption peak at 466 nm and a fluorescence emission peak at 498 nm. The fluorescence quantum yield was measured to be 0.77 and the extinction coefficient is 58 200 M(-1) cm(-1) . Several mutations were identified that shift the absorption peak to about 494 nm and the emission peak to between 512 and 514 nm. PMID:27288884

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

    PubMed

    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.

  16. Method for identification of rigid domains and hinge residues in proteins based on exhaustive enumeration.

    PubMed

    Sim, Jaehyun; Sim, Jun; Park, Eunsung; Lee, Julian

    2015-06-01

    Many proteins undergo large-scale motions where relatively rigid domains move against each other. The identification of rigid domains, as well as the hinge residues important for their relative movements, is important for various applications including flexible docking simulations. In this work, we develop a method for protein rigid domain identification based on an exhaustive enumeration of maximal rigid domains, the rigid domains not fully contained within other domains. The computation is performed by mapping the problem to that of finding maximal cliques in a graph. A minimal set of rigid domains are then selected, which cover most of the protein with minimal overlap. In contrast to the results of existing methods that partition a protein into non-overlapping domains using approximate algorithms, the rigid domains obtained from exact enumeration naturally contain overlapping regions, which correspond to the hinges of the inter-domain bending motion. The performance of the algorithm is demonstrated on several proteins.

  17. Identification of recombinant baculoviruses using green fluorescent protein as a selectable marker.

    PubMed

    Wilson, L E; Wilkinson, N; Marlow, S A; Possee, R D; King, L A

    1997-04-01

    A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties of the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperda cells resulted in the synthesis of a 30-kDa protein, which was confirmed as GFP by Western blotting and by the emission of green fluorescence when illuminated with longwave UV light (495 or 365 nm). To use GFP as a marker for the selection of recombinant baculoviruses, we prepared a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of lacZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfection to prepare recombinant baculoviruses, the virus DNA was linearized with Bsu361 to improve the recovery of non-parental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with the non-parental phenotype. Plaques were rapidly identified by simply exposing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains. PMID:9105619

  18. Large-scale identification of encystment-related proteins and genes in Pseudourostyla cristata

    PubMed Central

    Gao, Xiuxia; Chen, Fenfen; Niu, Tao; Qu, Ruidan; Chen, Jiwu

    2015-01-01

    The transformation of a ciliate into cyst is an advance strategy against an adverse situation. However, the molecular mechanism for the encystation of free-living ciliates is poorly understood. A large-scale identification of the encystment-related proteins and genes in ciliate would provide us with deeper insights into the molecular mechanisms for the encystations of ciliate. We identified the encystment-related proteins and genes in Pseudourostyla cristata with shotgun LC-MS/MS and scale qRT-PCR, respectively, in this report. A total of 668 proteins were detected in the resting cysts, 102 of these proteins were high credible proteins, whereas 88 high credible proteins of the 724 total proteins were found in the vegetative cells. Compared with the vegetative cell, 6 specific proteins were found in the resting cyst. However, the majority of high credible proteins in the resting cyst and the vegetative cell were co-expressed. We compared 47 genes of the co-expressed proteins with known functions in both the cyst and the vegetative cell using scale qRT-PCR. Twenty-seven of 47 genes were differentially expressed in the cyst compared with the vegetative cell. In our identifications, many uncharacterized proteins were also found. These results will help reveal the molecular mechanism for the formation of cyst in ciliates. PMID:26079518

  19. Proteomic identification of putative plasmodesmatal proteins from Chara corallina.

    PubMed

    Faulkner, Christine R; Blackman, Leila M; Cordwell, Stuart J; Overall, Robyn L

    2005-07-01

    Plasmodesmata are channels that bridge the cell walls of plant cells, allowing regulated transport of molecules between neighbouring cells. We have used a proteomic strategy to identify putative plasmodesmata-associated proteins in the giant-celled green alga Chara corallina. Proteins were extracted from the plasmodesmata-rich nodal complexes and the middle of the long internodal cells, which do not contain plasmodesmata. Comparison of protein spot patterns generated by two-dimensional gel electrophoresis of both the soluble and cell wall fractions from the two cell types was done. Fifty-eight spots that were common to the nodal and internodal soluble fractions were analysed by matrix assisted laser desorption/ionisation-time of flight mass spectrometry, and peptide mass fingerprint data were used to search the database. Matches were made to four of these spots, in each case to housekeeping proteins. Further, a number of nodal specific spots were identified, 11 from the soluble fraction and nine from the wall fraction. These spots were excised from the gels and analysed by liquid chromatography tandem mass spectrometry to obtain peptide sequence. Database searches suggest that these spots include homologues to previously identified plasmodesmata-associated proteins cp-wap13 and heat shock cognate 70, as well as RNA-binding proteins, eukaryotic initiation factor 4A and a beta-1,3-glucanase. Several spots remained unidentified providing exciting new candidate plasmodesmata-associated proteins.

  20. Can bioinformatics help in the identification of moonlighting proteins?

    PubMed

    Hernández, Sergio; Calvo, Alejandra; Ferragut, Gabriela; Franco, Luís; Hermoso, Antoni; Amela, Isaac; Gómez, Antonio; Querol, Enrique; Cedano, Juan

    2014-12-01

    Protein multitasking or moonlighting is the capability of certain proteins to execute two or more unique biological functions. This ability to perform moonlighting functions helps us to understand one of the ways used by cells to perform many complex functions with a limited number of genes. Usually, moonlighting proteins are revealed experimentally by serendipity, and the proteins described probably represent just the tip of the iceberg. It would be helpful if bioinformatics could predict protein multifunctionality, especially because of the large amounts of sequences coming from genome projects. In the present article, we describe several approaches that use sequences, structures, interactomics and current bioinformatics algorithms and programs to try to overcome this problem. The sequence analysis has been performed: (i) by remote homology searches using PSI-BLAST, (ii) by the detection of functional motifs, and (iii) by the co-evolutionary relationship between amino acids. Programs designed to identify functional motifs/domains are basically oriented to detect the main function, but usually fail in the detection of secondary ones. Remote homology searches such as PSI-BLAST seem to be more versatile in this task, and it is a good complement for the information obtained from protein-protein interaction (PPI) databases. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can be used only in very restricted situations, but can suggest how the evolutionary process of the acquisition of the second function took place. PMID:25399591

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

    PubMed

    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.

  2. Simple method for identification of plasmid-coded proteins.

    PubMed

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

    1979-01-01

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

  3. 34 CFR 200.37 - Notice of identification for improvement, corrective action, or restructuring.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 34 Education 1 2014-07-01 2014-07-01 false Notice of identification for improvement, corrective action, or restructuring. 200.37 Section 200.37 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  4. 34 CFR 200.37 - Notice of identification for improvement, corrective action, or restructuring.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 34 Education 1 2013-07-01 2013-07-01 false Notice of identification for improvement, corrective action, or restructuring. 200.37 Section 200.37 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  5. Identification of novel sweet protein for nutritional applications

    PubMed Central

    Gnanavel, Mutharasu; Serva Peddha, Muthukumar

    2011-01-01

    The prevalence of obesity and diabetes has increased exponentially in recent years around the globe, especially in India. Sweet proteins have the potential to substitute the sugars, by acting as natural, good and low calorie sweeteners. They also do not trigger a demand for insulin in diabetic patients unlike sucrose. In humans, the sweet taste perception is mainly due to taste-specific G protein-coupled heterodimeric receptors T1R2-T1R3. These receptors recognize diverse natural and synthetic sweeteners such as monelin, brazzein, thaumatin, curculin, mabinlin, miraculin and pentadin. Structural modeling of new sweetener proteins will be a great leap in further advancement of knowledge and their utility as sweeteners. We have explored the fingerprints of sweetness by studying the aminoacid composition and structure properties of the above proteins. The structural analysis of monellin revealed that the individual A or B chains of monellin are not contributing to its sweetness. However, the native conformation and ionic interaction between AspB7 of monellin with active site of T1R2-T1R3 receptor, along with hydrogen bonding stability of IleB6 and IleB8 are responsible for the sweet taste. Based on structural similarity search, we found a new hypothetical protein from Shewanella loihica, which has the presence of Asp32 with adjacent isoleucine residues. Further, we examined the lead protein by two-step docking for the study of interaction of functionally conserved residues with receptors. The identified protein showed similar ionic and hydrophobic interactions with monelin. This gives a promising opportunity to explore this protein for potential health application in the low calorie sweetener industry viz., soft drinks, snacks, food, chocolate industries etc. PMID:22125379

  6. Identification of novel sweet protein for nutritional applications.

    PubMed

    Gnanavel, Mutharasu; Serva Peddha, Muthukumar

    2011-01-01

    The prevalence of obesity and diabetes has increased exponentially in recent years around the globe, especially in India. Sweet proteins have the potential to substitute the sugars, by acting as natural, good and low calorie sweeteners. They also do not trigger a demand for insulin in diabetic patients unlike sucrose. In humans, the sweet taste perception is mainly due to taste-specific G protein-coupled heterodimeric receptors T1R2-T1R3. These receptors recognize diverse natural and synthetic sweeteners such as monelin, brazzein, thaumatin, curculin, mabinlin, miraculin and pentadin. Structural modeling of new sweetener proteins will be a great leap in further advancement of knowledge and their utility as sweeteners. We have explored the fingerprints of sweetness by studying the aminoacid composition and structure properties of the above proteins. The structural analysis of monellin revealed that the individual A or B chains of monellin are not contributing to its sweetness. However, the native conformation and ionic interaction between AspB7 of monellin with active site of T1R2-T1R3 receptor, along with hydrogen bonding stability of IleB6 and IleB8 are responsible for the sweet taste. Based on structural similarity search, we found a new hypothetical protein from Shewanella loihica, which has the presence of Asp(32) with adjacent isoleucine residues. Further, we examined the lead protein by two-step docking for the study of interaction of functionally conserved residues with receptors. The identified protein showed similar ionic and hydrophobic interactions with monelin. This gives a promising opportunity to explore this protein for potential health application in the low calorie sweetener industry viz., soft drinks, snacks, food, chocolate industries etc. PMID:22125379

  7. Identification and comparative analysis of accessory gland proteins in Orthoptera.

    PubMed

    Braswell, W Evan; Andrés, José A; Maroja, Luana S; Harrison, Richard G; Howard, Daniel J; Swanson, Willie J

    2006-09-01

    Accessory reproductive gland proteins (Acps) in Drosophila evolve quickly and appear to play an important role in ensuring the fertilization success of males. Moreover, Acps are thought to be involved in establishing barriers to fertilization between closely related species. While accessory glands are known to occur in the males of many insect groups, the proteins that are passed on to females by males during mating have not been well characterized outside of Drosophila. To gain a better understanding of these proteins, we characterized ESTs from the accessory glands of two cricket species, Allonemobius fasciatus and Gryllus firmus. Using an expressed sequence tag (EST) approach, followed by bioinformatic and evolutionary analyses, we found that many proteins are secreted and, therefore, available for transfer to the female during mating. Further, we found that most ESTs are novel, showing little sequence similarity between taxa. Evolutionary analyses suggest that cricket proteins are subject to diversifying selection and indicate that Allonemobius is much less polymorphic than Gryllus. Despite rapid nucleotide sequence divergence, there appears to be functional conservation of protein classes among Drosophila and cricket taxa.

  8. Identification of a mastigoneme protein from Phytophthora nicotianae.

    PubMed

    Blackman, Leila M; Arikawa, Mikihiko; Yamada, Shuhei; Suzaki, Toshinobu; Hardham, Adrienne R

    2011-01-01

    Tripartite tubular hairs (mastigonemes) on the anterior flagellum of protists in the stramenopile taxon are responsible for reversing the thrust of flagellar beat and for cell motility. Immunoprecipitation experiments using antibodies directed towards mastigonemes on the flagella of zoospores ofPhytophthora nicotianaehave facilitated the cloning of a gene encoding a mastigoneme shaft protein in this Oomycete. Expression of the gene, designatedPnMas2, is up-regulated during asexual sporulation, a period during which many zoospore components are synthesized. Analysis of the sequence of the PnMas2 protein has revealed that, like other stramenopile mastigoneme proteins, PnMas2 has an N-terminal secretion signal and contains four cysteine-rich epidermal growth factor (EGF)-like domains. Evidence from non-denaturing gels indicates that PnMas2 forms large oligomeric complexes, most likely through disulphide bridging. Bioinformatic analysis has revealed thatPhytophthoraspecies typically contain three or four putative mastigoneme proteins containing the four EGF-like domains. These proteins are similar in sequence to mastigoneme proteins in other stramenopile protists including the algaeOchromonas danica,Aureococcus anophagefferensandScytosiphon lomentariaand the diatomsThalassiosira pseudonana and T. weissflogii.

  9. Process correlation analysis model for process improvement identification.

    PubMed

    Choi, Su-jin; Kim, Dae-Kyoo; Park, Sooyong

    2014-01-01

    Software process improvement aims at improving the development process of software systems. It is initiated by process assessment identifying strengths and weaknesses and based on the findings, improvement plans are developed. In general, a process reference model (e.g., CMMI) is used throughout the process of software process improvement as the base. CMMI defines a set of process areas involved in software development and what to be carried out in process areas in terms of goals and practices. Process areas and their elements (goals and practices) are often correlated due to the iterative nature of software development process. However, in the current practice, correlations of process elements are often overlooked in the development of an improvement plan, which diminishes the efficiency of the plan. This is mainly attributed to significant efforts and the lack of required expertise. In this paper, we present a process correlation analysis model that helps identify correlations of process elements from the results of process assessment. This model is defined based on CMMI and empirical data of improvement practices. We evaluate the model using industrial data.

  10. Process Correlation Analysis Model for Process Improvement Identification

    PubMed Central

    Park, Sooyong

    2014-01-01

    Software process improvement aims at improving the development process of software systems. It is initiated by process assessment identifying strengths and weaknesses and based on the findings, improvement plans are developed. In general, a process reference model (e.g., CMMI) is used throughout the process of software process improvement as the base. CMMI defines a set of process areas involved in software development and what to be carried out in process areas in terms of goals and practices. Process areas and their elements (goals and practices) are often correlated due to the iterative nature of software development process. However, in the current practice, correlations of process elements are often overlooked in the development of an improvement plan, which diminishes the efficiency of the plan. This is mainly attributed to significant efforts and the lack of required expertise. In this paper, we present a process correlation analysis model that helps identify correlations of process elements from the results of process assessment. This model is defined based on CMMI and empirical data of improvement practices. We evaluate the model using industrial data. PMID:24977170

  11. The Identification of Perillyl Alcohol Glycosides with Improved Antiproliferative Activity

    PubMed Central

    2015-01-01

    A facile route to perillyl alcohol (POH) differential glycosylation and the corresponding synthesis of a set of 34 POH glycosides is reported. Subsequent in vitro studies revealed a sugar dependent antiproliferative activity and the inhibition of S6 ribosomal protein phosphorylation as a putative mechanism of representative POH glycosides. The most active glycoside from this cumulative study (4′-azido-d-glucoside, PG9) represents one of the most cytotoxic POH analogues reported to date. PMID:25121720

  12. Advancement in stationary phase for peptide separation helps in protein identification: application to atheroma plaque proteomics using nano-chip liquid chromatography and mass spectrometry.

    PubMed

    Delporte, Cédric; Noyon, Caroline; Raynal, Pierre; Dufour, Damien; Nève, Jean; Abts, Frederic; Haex, Martin; Zouaoui Boudjeltia, Karim; Van Antwerpen, Pierre

    2015-03-13

    In the last decades, proteomics has largely progressed. Mass spectrometry and liquid chromatography (LC) are generally used in proteomics. These techniques enable proper separation of peptides and good identification and/or quantification of them. Later, nano-scaled liquid chromatography, improvements of mass spectrometry resolution and sensitivity brought huge advancements. Enhancements in chemistry of chromatographic columns also brought interesting results. In the present work, the potency of identification of proteins by different nano-chip columns was studied and compared with classical LC column. The present study was applied to cardiovascular field where proteomics has shown to be highly helpful in research of new biomarkers. Protein extracts from atheroma plaques were used and proteomics data were compared. Results show that fewer spectra were acquired by the mass spectrometer when nano-chip columns were used instead of the classical ones. However, approximately 40% more unique peptides were identified by the recently optimized chip named Polaris-HR-chip-3C18 column, and 20% more proteins were identified. This fact leads to the identification of more low-abundance proteins. Many of them are involved in atheroma plaque development such as apolipoproteins, ceruloplasmin, etc. In conclusion, present data shows that recent developments of nanoLC column chemistry and dimensions enabled the improved detection and identification of low-abundance proteins in atheroma plaques. Several of them are of major interest in the field of cardiovascular disease. PMID:25680550

  13. Automatic annotation of protein function based on family identification.

    PubMed

    Abascal, Federico; Valencia, Alfonso

    2003-11-15

    Although genomes are being sequenced at an impressive rate, the information generated tells us little about protein function, which is slow to characterize by traditional methods. Automatic protein function annotation based on computational methods has alleviated this imbalance. The most powerful current approach for inferring the function of new proteins is by studying the annotations of their homologues, since their common origin is assumed to be reflected in their structure and function. Unfortunately, as proteins evolve they acquire new functions, so annotation based on homology must be carried out in the context of orthologues or subfamilies. Evolution adds new complications through domain shuffling: homology (or orthology) frequently corresponds to domains rather than complete proteins. Moreover, the function of a protein may be seen as the result of combining the functions of its domains. Additionally, automatic annotation has to deal with problems related to the annotations in the databases: errors (which are likely to be propagated), inconsistencies, or different degrees of function specification. We describe a method that addresses these difficulties for the annotation of protein function. Sequence relationships are detected and measured to obtain a map of the sequence space, which is searched for differentiated groups of proteins (similar to islands on the map), which are expected to have a common function and correspond to groups of orthologues or subfamilies. This mapmaking is done by applying a clustering algorithm based on Normalized cuts in graphs. The domain problem is addressed in a simple way: pairwise local alignments are analyzed to determine the extent to which they cover the entire sequence lengths of the two proteins. This analysis determines both what homologues are preferred for functional inheritance and the level of confidence of the annotation. To alleviate the problems associated with database annotations, the information on all the

  14. WISCOD: a statistical web-enabled tool for the identification of significant protein coding regions.

    PubMed

    Vilardell, Mireia; Parra, Genis; Civit, Sergi

    2014-01-01

    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. PMID:25313355

  15. WISCOD: a statistical web-enabled tool for the identification of significant protein coding regions.

    PubMed

    Vilardell, Mireia; Parra, Genis; Civit, Sergi

    2014-01-01

    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.

  16. SoyProDB: A database for the identification of soybean seed proteins

    PubMed Central

    Tavakolan, Mona; Alkharouf, Nadim W; Khan, Farooq H; Natarajan, Savithiry

    2013-01-01

    Soybean continues to serve as a rich and inexpensive source of protein for humans and animals. A substantial amount of information has been reported on the genotypic variation and beneficial genetic manipulation of soybeans. For better understanding of the consequences of genetic manipulation, elucidation of soybean protein composition is necessary, because of its direct relationship to phenotype. We have conducted studies to determine the composition of storage, allergen and anti-nutritional proteins in cultivated soybean using a combined proteomics approach. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) was implemented for the separation of proteins along with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS) for the identification of proteins. Our analysis resulted in the identification of several proteins, and a web based database named soybean protein database (SoyProDB) was subsequently built to house and allow scientists to search the data. This database will be useful to scientists who wish to genetically alter soybean with higher quality storage proteins, and also helpful for consumers to get a greater understanding about proteins that compose soy products available in the market. The database is freely accessible. Availability http://bioinformatics.towson.edu/Soybean_Seed_Proteins_2D_Gel_DB/Home.aspx PMID:23423175

  17. Differential protein mapping of ovarian serous adenocarcinomas: identification of potential markers for distinct tumor stage.

    PubMed

    Wang, Yanfei; Wu, Rong; Cho, Kathleen R; Thomas, Dafydd G; Gossner, Gabrielle; Liu, J Rebecca; Giordano, Thomas J; Shedden, Kerby A; Misek, David E; Lubman, David M

    2009-03-01

    Ovarian serous carcinomas (OSCs) comprise over half of all ovarian carcinomas and account for the majority of ovarian cancer-related deaths. We used a 2-dimensional liquid-based protein mapping strategy to characterize global protein expression patterns in 19 OSC tumor samples from 15 different patients to facilitate molecular classification of tumor stage. Protein expression profiles were produced, using pI-based separation in the first dimension and hydrophobicity-based separation in the second dimension, over a pH range of 4.0-7.0. Hierarchical clustering was applied to protein maps to indicate the tumor interrelationships. The 19 tumor samples could be classified into two different groups, one group associated with low stage (Stage 1) tumors and the other group associated with high stage (Stages 3/4) tumors. Proteins that were differentially expressed in different groups were selected for identification by LTQ-ESI-MS/MS. Fourteen of the selected proteins were overexpressed in the low stage tumors; 46 of the proteins were overexpressed in the high stage tumors. These proteins are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement and may serve as markers associated with different stages of OSCs. To further confirm the stage-dependent protein identifications, Lamin A/C and Vimentin expression in ovarian serous carcinomas was assessed by immunohistochemistry using ovarian tumor tissue microarrays for 66 samples.

  18. Identification of cancer protein biomarkers using proteomic techniques

    DOEpatents

    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.

  19. Identification of cancer protein biomarkers using proteomic techniques

    DOEpatents

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

    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.

  20. Identification of cancer protein biomarkers using proteomic techniques

    DOEpatents

    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.

  1. Protein Identification False Discovery Rates for Very Large Proteomics Data Sets Generated by Tandem Mass Spectrometry*

    PubMed Central

    Reiter, Lukas; Claassen, Manfred; Schrimpf, Sabine P.; Jovanovic, Marko; Schmidt, Alexander; Buhmann, Joachim M.; Hengartner, Michael O.; Aebersold, Ruedi

    2009-01-01

    Comprehensive characterization of a proteome is a fundamental goal in proteomics. To achieve saturation coverage of a proteome or specific subproteome via tandem mass spectrometric identification of tryptic protein sample digests, proteomics data sets are growing dramatically in size and heterogeneity. The trend toward very large integrated data sets poses so far unsolved challenges to control the uncertainty of protein identifications going beyond well established confidence measures for peptide-spectrum matches. We present MAYU, a novel strategy that reliably estimates false discovery rates for protein identifications in large scale data sets. We validated and applied MAYU using various large proteomics data sets. The data show that the size of the data set has an important and previously underestimated impact on the reliability of protein identifications. We particularly found that protein false discovery rates are significantly elevated compared with those of peptide-spectrum matches. The function provided by MAYU is critical to control the quality of proteome data repositories and thereby to enhance any study relying on these data sources. The MAYU software is available as standalone software and also integrated into the Trans-Proteomic Pipeline. PMID:19608599

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

    SciTech Connect

    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.

  3. Identification of disulfide bonds in protein proteolytic degradation products using de novo-protein unique sequence tags approach.

    PubMed

    Shen, Yufeng; Tolić, Nikola; Purvine, Samuel O; Smith, Richard D

    2010-08-01

    Disulfide bonds are a form of post-translational 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, 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 approximately 10 kDa. PMID:20590115

  4. Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

    PubMed Central

    Brayton, Kelly A.; Beare, Paul A.; Brown, Wendy C.; Heinzen, Robert A.; Broschat, Shira L.

    2011-01-01

    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. PMID:22140462

  5. Proteomic identification of secreted proteins of Propionibacterium acnes

    PubMed Central

    2010-01-01

    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 properties of P. acnes isolates

  6. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    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.

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

    PubMed Central

    Neuharth, Skyla; Kim, Dae In; Motamedchaboki, Khatereh; Roux, Kyle J.

    2016-01-01

    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. PMID:27437069

  8. Identification of giant Mimivirus protein functions using RNA interference

    PubMed Central

    Sobhy, Haitham; Scola, Bernard La; Pagnier, Isabelle; Raoult, Didier; Colson, Philippe

    2015-01-01

    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 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 three 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 four 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. PMID:25972846

  9. SPIDER: software for protein identification from sequence tags with de novo sequencing error.

    PubMed

    Han, Yonghua; Ma, Bin; Zhang, Kaizhong

    2005-06-01

    For the identification of novel proteins using MS/MS, de novo sequencing software computes one or several possible amino acid sequences (called sequence tags) for each MS/MS spectrum. Those tags are then used to match, accounting amino acid mutations, the sequences in a protein database. If the de novo sequencing gives correct tags, the homologs of the proteins can be identified by this approach and software such as MS-BLAST is available for the matching. However, de novo sequencing very often gives only partially correct tags. The most common error is that a segment of amino acids is replaced by another segment with approximately the same masses. We developed a new efficient algorithm to match sequence tags with errors to database sequences for the purpose of protein and peptide identification. A software package, SPIDER, was developed and made available on Internet for free public use. This paper describes the algorithms and features of the SPIDER software. PMID:16108090

  10. SPIDER: software for protein identification from sequence tags with de novo sequencing error.

    PubMed

    Han, Yonghua; Ma, Bin; Zhang, Kaizhong

    2004-01-01

    For the identification of novel proteins using MS/MS, de novo sequencing software computes one or several possible amino acid sequences (called sequence tags) for each MS/MS spectrum. Those tags are then used to match, accounting amino acid mutations, the sequences in a protein database. If the de novo sequencing gives correct tags, the homologs of the proteins can be identified by this approach and software such as MS-BLAST is available for the matching. However, de novo sequencing very often gives only partially correct tags. The most common error is that a segment of amino acids is replaced by another segment with approximately the same masses. We developed a new efficient algorithm to match sequence tags with errors to database sequences for the purpose of protein and peptide identification. A software package, SPIDER, was developed and made available on Internet for free public use. This paper describes the algorithms and features of the SPIDER software. PMID:16448014

  11. Identification of immunodominant proteins of the microalgae Prototheca by proteomic analysis.

    PubMed

    Irrgang, A; Weise, C; Murugaiyan, J; Roesler, U

    2015-01-01

    Prototheca zopfii associated with bovine mastitis and human protothecosis exists as two genotypes, of which genotype 1 is considered as non-infectious and genotype 2 as infectious. The mechanism of infection has not yet been described. The present study was aimed to identify genotype 2-specific immunodominant proteins. Prototheca proteins were separated using two-dimensional gel electrophoresis. Subsequent western blotting with rabbit hyperimmune serum revealed 28 protein spots. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis resulted in the identification of 15 proteins including malate dehydrogenase, elongation factor 1-alpha, heat shock protein 70, and 14-3-3 protein, which were previously described as immunogenic proteins of other eukaryotic pathogens. PMID:25755891

  12. Identification of immunodominant proteins of the microalgae Prototheca by proteomic analysis

    PubMed Central

    Irrgang, A.; Weise, C.; Murugaiyan, J.; Roesler, U.

    2014-01-01

    Prototheca zopfii associated with bovine mastitis and human protothecosis exists as two genotypes, of which genotype 1 is considered as non-infectious and genotype 2 as infectious. The mechanism of infection has not yet been described. The present study was aimed to identify genotype 2-specific immunodominant proteins. Prototheca proteins were separated using two-dimensional gel electrophoresis. Subsequent western blotting with rabbit hyperimmune serum revealed 28 protein spots. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis resulted in the identification of 15 proteins including malate dehydrogenase, elongation factor 1-alpha, heat shock protein 70, and 14-3-3 protein, which were previously described as immunogenic proteins of other eukaryotic pathogens. PMID:25755891

  13. The Improvement of Automated Spectral Identification Tool ASERA

    NASA Astrophysics Data System (ADS)

    Yuan, Hailong; zhang, Yanxia

    2015-08-01

    The regular survey of Large Sky Area Multi-Object Fiber Spectroscopic Telescope (LAMOST) has acquired over four millions spectra of celestial objects by the summer of 2014, covering about a third of the whole sky area. More spectra will be obtained as the survey projects (eg. LAMOST, SDSS) keeps going on. To effectively make use of the massive spectral data, various advanced data analysis methods and technologies are in great requirement. ASERA, A Spectrum Eye Recognition Assistant, provides a simple convenient solution for the user to access spectra from LAMOST and SDSS, identify their types (QSO, galaxy, and various types of stars) and estimate their redshifts in an interactive graphic interface. The toolkit is at first especially designed for quasar identification. By shifting the quasar template overlaping the target spectrum interactively, one can easily find out the best broad emission line position and the redshift value. Now, besides the quasar template, various templates for different types of galaxies (early type, later type, starburst, bulge, elliptical and luminous red galaxies) and stars (O, B, A, F, G, K, M, WD, CV, Double Stars and Emission-Line-Objects) are added. We also have developed many new useful functionalities for inspecting and analyzing spectra, such as zooming, line fitting, smoothing and automatic result saving. The target information from input catalogues and data processing result from the pipeline as well as fitting parameters for various types of templates, can be presented at the same time. Several volume processing components are developed to support the cooperation with MySQL database, internet resources and SSAP services. ASERA will be a strong helper for astronomers to recognize spectra.

  14. Improved packing of protein side chains with parallel ant colonies

    PubMed Central

    2014-01-01

    Introduction The accurate packing of protein side chains is important for many computational biology problems, such as ab initio protein structure prediction, homology modelling, and protein design and ligand docking applications. Many of existing solutions are modelled as a computational optimisation problem. As well as the design of search algorithms, most solutions suffer from an inaccurate energy function for judging whether a prediction is good or bad. Even if the search has found the lowest energy, there is no certainty of obtaining the protein structures with correct side chains. Methods We present a side-chain modelling method, pacoPacker, which uses a parallel ant colony optimisation strategy based on sharing a single pheromone matrix. This parallel approach combines different sources of energy functions and generates protein side-chain conformations with the lowest energies jointly determined by the various energy functions. We further optimised the selected rotamers to construct subrotamer by rotamer minimisation, which reasonably improved the discreteness of the rotamer library. Results We focused on improving the accuracy of side-chain conformation prediction. For a testing set of 442 proteins, 87.19% of X1 and 77.11% of X12 angles were predicted correctly within 40° of the X-ray positions. We compared the accuracy of pacoPacker with state-of-the-art methods, such as CIS-RR and SCWRL4. We analysed the results from different perspectives, in terms of protein chain and individual residues. In this comprehensive benchmark testing, 51.5% of proteins within a length of 400 amino acids predicted by pacoPacker were superior to the results of CIS-RR and SCWRL4 simultaneously. Finally, we also showed the advantage of using the subrotamers strategy. All results confirmed that our parallel approach is competitive to state-of-the-art solutions for packing side chains. Conclusions This parallel approach combines various sources of searching intelligence and energy

  15. Identification of extensin protein associated with sugar beet pectin.

    PubMed

    Nuñez, Alberto; Fishman, Marshall L; Fortis, Laurie L; Cooke, Peter H; Hotchkiss, Arland T

    2009-11-25

    Several studies have suggested that the emulsification properties associated with pectin obtained from sugar beet (Beta vulgaris) are due to the presence of a protein-pectin complex. Nevertheless, the identity of the protein has remained elusive. Pectin, extracted from sugar beet pulp by microwave-assisted extraction, and a commercial sample were both subjected to protease digestion with trypsin. The resulting peptides were separated from the pectin solution by ultrafiltration using a 3 kDa molecular weight cutoff (MWCO) membrane and analyzed using matrix-assisted laser desorption ionization with tandem time-of-flight mass spectrometry. The partial sequences derived from the mass spectrometry analyses of the resulting tryptic peptides are found to be highly consistent with extensin protein matched from the B. vulgaris Genetic Index database and also correspond to previously reported extensin peptides found in sugar beet cell suspension cultures. Further attempts were made to disassociate the protein from pectin using 1 M NaCl and a 100 kDa MWCO membrane; however, no peptides were observed following trypsin digestion of the permeate solution. This evidence suggests the existence of a complex between the pectin and extensin that is not due to ionic interactions. Trypsin digestion of commercial sugar beet pectin also produced the peptide profile observed with the microwave-assisted extracted pectin sample. Atomic force microscopy established that the number of rod-like elements decreased following protease treatment compared to the untreated sample.

  16. Identification, classification, and analysis of beta-bulges in proteins.

    PubMed Central

    Chan, A. W.; Hutchinson, E. G.; Harris, D.; Thornton, J. M.

    1993-01-01

    A beta-bulge is a region of irregularity in a beta-sheet involving two beta-strands. It usually involves two or more residues in the bulged strand opposite to a single residue on the adjacent strand. These irregularities in beta-sheets were identified and classified automatically, extending the definition of beta-bulges given by Richardson et al. (Richardson, J.S., Getzoff, E.D., & Richardson, D.C., 1978, Proc. Natl. Acad. Sci. USA 75, 2574-2578). A set of 182 protein chains (170 proteins) was used, and a total of 362 bulges were extracted. Five types of beta-bulges were found: classic, G1, wide, bent, and special. Their characteristic amino acid preferences were found for most classes of bulges. Basically, bulges occur frequently in proteins; on average there are more than two bulges per protein. In general, beta-bulges produce two main changes in the structure of a beta-sheet: (1) disrupt the normal alternation of side-chain direction; (2) accentuate the twist of the sheet, altering the direction of the surrounding strands. PMID:8251933

  17. Identification of a Plasmodium falciparum Phospholipid Transfer Protein*

    PubMed Central

    van Ooij, Christiaan; Withers-Martinez, Chrislaine; Ringel, Alessa; Cockcroft, Shamshad; Haldar, Kasturi; Blackman, Michael J.

    2013-01-01

    Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte. PMID:24043620

  18. Identification of lectin binding proteins in human tears.

    PubMed

    Kuizenga, A; van Haeringen, N J; Kijlstra, A

    1991-12-01

    The identity of glycoproteins in stimulated normal human tears was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of tears onto minigels, blotting, and subsequent incubation with different biotinylated lectins (concanavalin A [Con A], peanut agglutinin [PNA], glycine max agglutinin [SBA], Phaseolus vulgaris agglutinin, wheat germ agglutinin [WGA, native form], Artocarpus integrifolia agglutinin [Jacalin], and Pisum sativum agglutinin). Control proteins included purified secretory immunoglobulin A (sIgA) from human colostrum, human milk lactoferrin, and chicken-egg lysozyme. All samples were prepared in a denaturing (SDS) buffer under nonreducing and reducing conditions. The sIgA in tears and IgA (alpha) heavy chain fragments (reduced sample) were identified with most of the lectins tested. A particular high molecular weight (greater than 200 kD) protein fraction in tears that just entered the separation gel on SDS-PAGE was detected with WGA and Jacalin. This fraction stain poorly with silver. Tear lactoferrin was identified with all lectins used, although binding was low with SBA. Purified milk lactoferrin showed a poor reaction with Jacalin, but a protein in tears of similar mobility bound this lectin (nonreduced samples). Under both nonreducing and reducing conditions, tear-specific prealbumin in tears did not bind any of the lectins tested. Tear lysozyme only reacted with lectin after reduction. The techniques described may provide additional valuable information in addition to commonly used methods for tear protein analysis and further knowledge concerning the role of glycoproteins on the ocular surface.

  19. Identification of the major capsid protein gene of human cytomegalovirus.

    PubMed Central

    Chee, M; Rudolph, S A; Plachter, B; Barrell, B; Jahn, G

    1989-01-01

    The coding region for the major capsid protein (MCP) of human cytomegalovirus (HCMV) was identified by comparing the protein sequence with the respective sequences of herpes simplex virus (HSV), Epstein-Barr virus, and varicella-zoster virus. The predicted length of the HCMV MCP was 1,370 amino acids. Comparison of the MCP sequences of the different human herpesviruses showed a homology of 25% to the MCP of HSV type 1, a homology of 29% to the MCP of Epstein-Barr virus, and a homology of 23% to the MCP of varicella-zoster virus. A subfragment of the HSV type 1 KpnI i fragment encoding the MCP VP5 cross-hybridized with the HCMV HindIII U fragment containing part of the MCP gene. Northern (RNA) blot analyses with subclones out of the coding region for the HCMV MCP detected one large transcript of about 8 kilobases. A portion of the open reading frame was expressed in Escherichia coli plasmid pBD2 IC2OH as a beta-galactosidase fusion protein and was used to generate polyclonal antibodies in New Zealand White rabbits. The obtained antisera reacted in Western immunoblots with the MCP of purified HCMV virions. A monoclonal antibody against the human MCP and a monospecific rabbit antiserum against strain Colburn of simian cytomegalovirus detected the fusion protein as well as the MCP of purified virions in immunoblots. Images PMID:2536837

  20. Recombinant proteins in newly developed foods: identification of allergenic activity.

    PubMed

    Lehrer, S B; Reese, G

    1997-01-01

    A number of agricultural crops are being modified for various purposes using recombinant DNA technology. Since transferred genes may code for proteins that are ordinarily not present, there is concern about the potential allergenicity of these new varieties. The safety evaluation of transgenic foods is relatively easy when the allergenicity of the gene source is known. Recombinant allergens in genetically engineered or altered foods can be identified using traditional immunological assays such as RAST or ELISA inhibition or immunoblotting procedures. Our recent studies of two corn proteins (10 kD and HSZ) used to alter grain amino acid composition and of transgenic soybeans with an altered fatty acid profile are examples of this approach. Both 10 kD and HSZ did not bind IgE antibodies from sera of corn-reactive subjects by immunoblotting. Studies of wild-type and transgenic soybeans with high oleic acidic content by RAST inhibition and immunoblotting with pooled sera of soy-allergic individuals demonstrated no difference in the allergen content of both extracts. In contrast to these studies, a recent investigation by Nordlee et al. (1996) of transgenic soybeans which expressed a methionine/cysteine-rich protein from Brazil nuts identified this protein as a major Brazil nut allergen. These studies indicate that, when the gene source is from a known allergen or if the recipient contains allergens, it is possible to determine whether the allergen content of the transgenic line is altered relative to the nontransgenic varieties.

  1. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening

    PubMed Central

    Guazzaroni, María-Eugenia; Silva-Rocha, Rafael; Ward, Richard John

    2015-01-01

    There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods. PMID:25123225

  2. Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening.

    PubMed

    Guazzaroni, María-Eugenia; Silva-Rocha, Rafael; Ward, Richard John

    2015-01-01

    There is a growing demand for enzymes with improved catalytic performance or tolerance to process-specific parameters, and biotechnology plays a crucial role in the development of biocatalysts for use in industry, agriculture, medicine and energy generation. Metagenomics takes advantage of the wealth of genetic and biochemical diversity present in the genomes of microorganisms found in environmental samples, and provides a set of new technologies directed towards screening for new catalytic activities from environmental samples with potential biotechnology applications. However, biased and low level of expression of heterologous proteins in Escherichia coli together with the use of non-optimal cloning vectors for the construction of metagenomic libraries generally results in an extremely low success rate for enzyme identification. The bottleneck arising from inefficient screening of enzymatic activities has been addressed from several perspectives; however, the limitations related to biased expression in heterologous hosts cannot be overcome by using a single approach, but rather requires the synergetic implementation of multiple methodologies. Here, we review some of the principal constraints regarding the discovery of new enzymes in metagenomic libraries and discuss how these might be resolved by using synthetic biology methods.

  3. Identification of a novel nurr1-interacting protein.

    PubMed

    Luo, Yu; Xing, Feng; Guiliano, Rita; Federoff, Howard J

    2008-09-10

    The orphan nuclear receptor Nurr1 is required for the development of ventral mesencephalic dopaminergic neurons in mice. One of the possible mechanisms that might contribute to the regulation activity of Nurr1 is through interaction with other proteins. To identify potential partners of Nurr1, we screened a yeast two-hybrid library from developing mouse embryonic mesencephalon with the Nurr1 ligand-binding domain (NLBD). We identified a novel interacting protein, termed the Nurr1-interacting protein (NuIP). We demonstrate that it specifically interacts with NLBD using the mammalian two-hybrid assay and coimmunoprecipitation studies in MN9D cells. In addition, we show that NuIP interacts with Nurr1 in lysates from substantia nigra. Coexpression of NuIP with Nurr1 results in potentiation of the transcriptional activity of Nurr1 on an nerve growth factor inducible-B response element reporter, as well as reporters driven by the endogenous tyrosine hydroxylase promoter. The mechanism underlying the regulatory action of NuIP on Nurr1 is demonstrated to be through assembly of distinct helical domains of the NLBD. Using a NuIP specific antibody, we show that expression of NuIP protein is mainly colocalized with Nurr1 in adult midbrain dopaminergic neurons. Finally, we demonstrate that suppression of NuIP expression in MN9D cells by NuIP-specific small interfering RNA leads to decreased cell division and decreased expression of a Nurr1 target gene, the dopamine transporter. These results suggest NuIP interacts with and positively regulates the activity of Nurr1 protein and modulates the phenotype of dopaminergic cells. PMID:18784308

  4. An improved cyan fluorescent protein variant useful for FRET.

    PubMed

    Rizzo, Mark A; Springer, Gerald H; Granada, Butch; Piston, David W

    2004-04-01

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation. PMID:14990965

  5. An experimental strategy for the identification of AMPylation targets from complex protein samples.

    PubMed

    Pieles, Kathrin; Glatter, Timo; Harms, Alexander; Schmidt, Alexander; Dehio, Christoph

    2014-05-01

    AMPylation is a posttranslational modification (PTM) that has recently caught much attention in the context of bacterial infections as pathogens were shown to secrete Fic proteins that AMPylate Rho GTPases and thus interfere with host cell signaling processes. Although Fic proteins are widespread and found in all kingdoms of life, only a small number of AMPylation targets are known to date. A major obstacle to target identification is the limited availability of generic strategies allowing sensitive and robust identification of AMPylation events. Here, we present an unbiased MS-based approach utilizing stable isotope-labeled ATP. The ATP isotopes are transferred onto target proteins in crude cell lysates by in vitro AMPylation introducing specific reporter ion clusters that allow detection of AMPylated peptides in complex biological samples by MS analysis. Applying this strategy on the secreted Fic protein Bep2 of Bartonella rochalimae, we identified the filamenting protein vimentin as an AMPylation target that was confirmed by independent assays. Vimentin represents a new class of target proteins and its identification emphasizes our method as a valuable tool to systematically uncover AMPylation targets. Furthermore, the approach can be generically adapted to study targets of other PTMs that allow incorporation of isotopically labeled substrates.

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

    PubMed

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

    2003-08-15

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

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

    PubMed

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

    2003-08-15

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

  8. Health information exchange improves identification of frequent emergency department users.

    PubMed

    Shapiro, Jason S; Johnson, Sarah A; Angiollilo, John; Fleischman, William; Onyile, Arit; Kuperman, Gilad

    2013-12-01

    We hypothesized that using communitywide data from a health information exchange (HIE) could improve the ability to identify frequent emergency department (ED) users-those with four or more ED visits in thirty days-by allowing ED use to be measured across unaffiliated hospitals. When we analyzed HIE-wide data instead of site-specific data, we identified 20.3 percent more frequent ED users (5,756 versus 4,785) and 16.0 percent more visits by them to the ED (53,031 versus 45,771). Additionally, we found that 28.8 percent of frequent ED users visited multiple EDs during the twelve-month study period, versus 3.0 percent of all ED users. All three differences were significant ($$p ). An improved ability to identify frequent ED users allows better targeting of case management and other services that can improve frequent ED users' health and reduce their use of costly emergency medical services.

  9. Glycosylation: impact, control and improvement during therapeutic protein production.

    PubMed

    Costa, Ana Rita; Rodrigues, Maria Elisa; Henriques, Mariana; Oliveira, Rosário; Azeredo, Joana

    2014-12-01

    The emergence of the biopharmaceutical industry represented a major revolution for modern medicine, through the development of recombinant therapeutic proteins that brought new hope for many patients with previously untreatable diseases. There is a ever-growing demand for these therapeutics that forces a constant technological evolution to increase product yields while simultaneously reducing costs. However, the process changes made for this purpose may also affect the quality of the product, a factor that was initially overlooked but which is now a major focus of concern. Of the many properties determining product quality, glycosylation is regarded as one of the most important, influencing, for example, the biological activity, serum half-life and immunogenicity of the protein. Consequently, monitoring and control of glycosylation is now critical in biopharmaceutical manufacturing and a requirement of regulatory agencies. A rapid evolution is being observed in this context, concerning the influence of glycosylation in the efficacy of different therapeutic proteins, the impact on glycosylation of a diversity of parameters/processes involved in therapeutic protein production, the analytical methodologies employed for glycosylation monitoring and control, as well as strategies that are being explored to use this property to improve therapeutic protein efficacy (glycoengineering). This work reviews the main findings on these subjects, providing an up-to-date source of information to support further studies.

  10. Metabolic engineering of Escherichia coli to improve recombinant protein production.

    PubMed

    Liu, Min; Feng, Xinjun; Ding, Yamei; Zhao, Guang; Liu, Huizhou; Xian, Mo

    2015-12-01

    Escherichia coli is one of the most widely used strains for recombinant protein production. However, obstacles also exist in both academic researches and industrial applications, such as the metabolic burden, the carbon source waste, and the cells' physiological deterioration. This article reviews recent approaches for improving recombinant protein production in metabolic engineering, including workhorse selection, stress factor application, and carbon flux regulation. Selecting a suitable host is the first key point for recombinant protein production. In general, it all depends on characteristics of the strains and the target proteins. It will be triggered cells physiological deterioration when the medium is significantly different from the cell's natural environment. Coexpression of stress factors can help proteins to fold into their native conformation. Carbon flux regulation is a direct approach for redirecting more carbon flux toward the desirable pathways and products. However, some undesirable consequences are usually found in metabolic engineering, such as glucose transport inhibition, cell growth retardation, and useless metabolite accumulation. More efficient regulators and platform cell factories should be explored to meet a variety of production demands.

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

    EPA Science Inventory

    The Identification and Characterization of an IgE-Inducing Protein in Metarhizium anisopliae Extract

    Marsha D.W. Ward1, Lisa B. Copeland1, Maura J. Donahue2, and Jody A. Shoemaker3
    1ORD, NHEERL, US EPA, RTP, NC; 2Oak Ridge Institute for Science and Education, Cincinnati...

  12. Retinal identification based on an Improved Circular Gabor Filter and Scale Invariant Feature Transform.

    PubMed

    Meng, Xianjing; Yin, Yilong; Yang, Gongping; Xi, Xiaoming

    2013-01-01

    Retinal identification based on retinal vasculatures in the retina provides the most secure and accurate means of authentication among biometrics and has primarily been used in combination with access control systems at high security facilities. Recently, there has been much interest in retina identification. As digital retina images always suffer from deformations, the Scale Invariant Feature Transform (SIFT), which is known for its distinctiveness and invariance for scale and rotation, has been introduced to retinal based identification. However, some shortcomings like the difficulty of feature extraction and mismatching exist in SIFT-based identification. To solve these problems, a novel preprocessing method based on the Improved Circular Gabor Transform (ICGF) is proposed. After further processing by the iterated spatial anisotropic smooth method, the number of uninformative SIFT keypoints is decreased dramatically. Tested on the VARIA and eight simulated retina databases combining rotation and scaling, the developed method presents promising results and shows robustness to rotations and scale changes. PMID:23873409

  13. Retinal identification based on an Improved Circular Gabor Filter and Scale Invariant Feature Transform.

    PubMed

    Meng, Xianjing; Yin, Yilong; Yang, Gongping; Xi, Xiaoming

    2013-07-18

    Retinal identification based on retinal vasculatures in the retina provides the most secure and accurate means of authentication among biometrics and has primarily been used in combination with access control systems at high security facilities. Recently, there has been much interest in retina identification. As digital retina images always suffer from deformations, the Scale Invariant Feature Transform (SIFT), which is known for its distinctiveness and invariance for scale and rotation, has been introduced to retinal based identification. However, some shortcomings like the difficulty of feature extraction and mismatching exist in SIFT-based identification. To solve these problems, a novel preprocessing method based on the Improved Circular Gabor Transform (ICGF) is proposed. After further processing by the iterated spatial anisotropic smooth method, the number of uninformative SIFT keypoints is decreased dramatically. Tested on the VARIA and eight simulated retina databases combining rotation and scaling, the developed method presents promising results and shows robustness to rotations and scale changes.

  14. Identification and characterization of nonsedimentable lipid-protein microvesicles.

    PubMed Central

    Yao, K; Paliyath, G; Humphrey, R W; Hallett, F R; Thompson, J E

    1991-01-01

    Previously uncharacterized lipid-protein microvesicles have been isolated from young and senescing bean cotyledon tissue. The microvesicles are nonsedimentable and enriched in phospholipid degradation products (free fatty acids, long-chain aldehydes, and long-chain hydrocarbons). They range from 70 to 170 nm (radius) with a mean radius of 132 nm, and it is clear from freeze-fracture electron micrographs that they are bilayered in nature. Nonsedimentable lipid-protein microvesicles containing the same products of phospholipid degradation but smaller were also formed in vitro when smooth microsomal membranes from young cotyledon tissue were treated with Ca2+ to stimulate enzymatic degradation of phospholipids. The data suggest that these microvesicles comprise an intermediate stage of membrane lipid deterioration. They appear to serve as a vehicle for moving phospholipid degradation products out of membranes into the cytosol during senescence and perhaps also during normal membrane lipid turnover. Images PMID:11607164

  15. Computational identification of MoRFs in protein sequences

    PubMed Central

    Malhis, Nawar; Gsponer, Jörg

    2015-01-01

    Motivation: Intrinsically disordered regions of proteins play an essential role in the regulation of various biological processes. Key to their regulatory function is the binding of molecular recognition features (MoRFs) to globular protein domains in a process known as a disorder-to-order transition. Predicting the location of MoRFs in protein sequences with high accuracy remains an important computational challenge. Method: In this study, we introduce MoRFCHiBi, a new computational approach for fast and accurate prediction of MoRFs in protein sequences. MoRFCHiBi combines the outcomes of two support vector machine (SVM) models that take advantage of two different kernels with high noise tolerance. The first, SVMS, is designed to extract maximal information from the general contrast in amino acid compositions between MoRFs, their surrounding regions (Flanks), and the remainders of the sequences. The second, SVMT, is used to identify similarities between regions in a query sequence and MoRFs of the training set. Results: We evaluated the performance of our predictor by comparing its results with those of two currently available MoRF predictors, MoRFpred and ANCHOR. Using three test sets that have previously been collected and used to evaluate MoRFpred and ANCHOR, we demonstrate that MoRFCHiBi outperforms the other predictors with respect to different evaluation metrics. In addition, MoRFCHiBi is downloadable and fast, which makes it useful as a component in other computational prediction tools. Availability and implementation: http://www.chibi.ubc.ca/morf/. Contact: gsponer@chibi.ubc.ca. Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25637562

  16. Latest methods of fluorescence-based protein crystal identification

    SciTech Connect

    Meyer, Arne; Betzel, Christian

    2015-01-28

    Fluorescence, whether intrinsic or by using trace fluorescent labeling, can be a powerful aid in macromolecule crystallization. Its use in screening for crystals is discussed here. Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes. The introduction of fluorescence techniques has brought a powerful and versatile tool to the aid of the crystal grower. Trace fluorescent labeling, in which a fluorescent probe is covalently bound to a subpopulation (<0.5%) of the protein, enables the use of visible fluorescence. Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins. By the use of these techniques, crystals that had previously been obscured in the crystallization drop can readily be identified and distinguished from amorphous precipitate or salt crystals. Additionally, lead conditions that may not have been obvious as such under white-light illumination can be identified. In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity.

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

    SciTech Connect

    Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki . E-mail: yigarash@pharm.hokudai.ac.jp

    2006-05-12

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

  18. Characterization of quinone derived protein adducts and their selective identification using redox cycling based chemiluminescence assay.

    PubMed

    Elgawish, Mohamed Saleh; Kishikawa, Naoya; Ohyama, Kaname; Kuroda, Naotaka

    2015-07-17

    The cytotoxic mechanism of many quinones has been correlated to covalent modification of cellular proteins. However, the identification of relevant proteins targets is essential but challenging goals. To better understand the quinones cytotoxic mechanism, human serum albumin (HSA) was incubated in vitro with different concentration of menadione (MQ). In this respect, the initial nucleophilic addition of proteins to quinone converts the conjugates to redox-cycling quinoproteins with altered conformation and secondary structure and extended life span than the short lived, free quinones. The conjugation of MQ with nucleophilic sites likewise, free cysteine as well as ɛ-amino group of lysine residue of HSA has been found to be in concentration dependent manner. The conventional methods for modified proteins identification in complex mixtures are complicated and time consuming. Herein, we describe a highly selective, sensitive, simple, and fast strategy for quinoproteins identification. The suggested strategy exploited the unique redox-cycling capability of quinoproteins in presence of a reductant, dithiothreitol (DTT), to generate reactive oxygen species (ROS) that gave sufficient chemiluminescence (CL) when mixed with luminol. The CL approach is highly selective and sensitive to detect the quinoproteins in ten-fold molar excess of native proteins without adduct enrichment. The approach was also coupled with gel filtration chromatography (GFC) and used to identify adducts in complex mixture of proteins in vitro as well as in rat plasma after MQ administration. Albumin was identified as the main protein in human and rat plasma forming adduct with MQ. Overall, the identification of quinoproteins will encourage further studies of toxicological impact of quinones on human health. PMID:26044383

  19. Learning representations for improved target identification, scene classification, and information fusion

    NASA Astrophysics Data System (ADS)

    Flenner, Arjuna; Culp, Michael; McGee, Ryan; Flenner, Jennifer; Garcia-Cardona, Cristina

    2015-05-01

    Object representation is fundamental to Automated Target Recognition (ATR). Many ATR approaches choose a basis, such as a wavelet or Fourier basis, to represent the target. Recently, advancements in Image and Signal processing have shown that object recognition can be improved if, rather than a assuming a basis, a database of training examples is used to learn a representation. We discuss learning representations using Non-parametric Bayesian topic models, and demonstrate how to integrate information from other sources to improve ATR. We apply the method to EO and IR information integration for vehicle target identification and show that the learned representation of the joint EO and IR information improves target identification by 4%. Furthermore, we demonstrate that we can integrate text and imagery data to direct the representation for mission specific tasks and improve performance by 8%. Finally, we illustrate integrating graphical models into representation learning to improve performance by 2%.

  20. Identification of Epstein-Barr Virus Replication Proteins in Burkitt's Lymphoma Cells.

    PubMed

    Traylen, Chris; Ramasubramanyan, Sharada; Zuo, Jianmin; Rowe, Martin; Almohammad, Rajaei; Heesom, Kate; Sweet, Steve M M; Matthews, David A; Sinclair, Alison J

    2015-01-01

    The working model to describe the mechanisms used to replicate the cancer-associated virus Epstein-Barr virus (EBV) is partly derived from comparisons with other members of the Herpes virus family. Many genes within the EBV genome are homologous across the herpes virus family. Published transcriptome data for the EBV genome during its lytic replication cycle show extensive transcription, but the identification of the proteins is limited. We have taken a global proteomics approach to identify viral proteins that are expressed during the EBV lytic replication cycle. We combined an enrichment method to isolate cells undergoing EBV lytic replication with SILAC-labeling coupled to mass-spectrometry and identified viral and host proteins expressed during the OPEN ACCESS Pathogens 2015, 4 740 EBV lytic replication cycle. Amongst the most frequently identified viral proteins are two components of the DNA replication machinery, the single strand DNA binding protein BALF2, DNA polymerase accessory protein BMRF1 and both subunits of the viral ribonucleoside-diphosphate reductase enzyme (BORF2 and BaRF1). An additional 42 EBV lytic cycle proteins were also detected. This provides proteomic identification for many EBV lytic replication cycle proteins and also identifies post-translational modifications. PMID:26529022

  1. Protein markers for identification of Yersinia pestis and their variation related to culture

    SciTech Connect

    Wunschel, David S.; Engelmann, Heather E.; Victry, Kristin D.; Clowers, Brian H.; Sorensen, Christina M.; Valentine, Nancy B.; Mahoney Fahey, Christine M.; Wietsma, Thomas W.; Wahl, Karen L.

    2013-12-11

    The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y. pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.

  2. Identification of proteins bound to a thioaptamer probe on a proteomics array

    SciTech Connect

    Wang He; Yang, Xianbin; Bowick, Gavin C.; Herzog, Norbert K.; Luxon, Bruce A.; Lomas, Lee O. . E-mail: lomas@ciphergen.com; Gorenstein, David G. . E-mail: dggorens@utmb.edu

    2006-09-01

    A rapid method to screen and identify unknown bound proteins to specific nucleic acid probes anchored on ProteinChip array surfaces from crude biological samples has been developed in this paper. It was demonstrated with screening specific binding proteins from LPS-stimulated mouse 70Z/3 pre-B cell nuclear extracts by direct coupling of thioaptamer XBY-S2 to the pre-activated ProteinChip array surfaces. With pre-fractionation of crude nuclear extracts by ion exchange method, specific 'on-chip' captured proteins have been obtained that were pure enough to do 'on-chip' digestion and the subsequent identification of the 'on-chip' bound proteins by microsequencing of the trypsin digested peptide fragments through tandem MS. Five mouse heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2/B1, A3, A/B, and D0 were identified. To verify those bound hnRNPs, a novel thioaptamer/antibody sandwich assay provides highly sensitive and selective identification of proteins on ProteinChip arrays.

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

    PubMed Central

    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. PMID:26859666

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

    PubMed

    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 Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

    PubMed

    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. PMID:26859666

  6. Recent advances in protein prenyltransferases: substrate identification, regulation, and disease interventions.

    PubMed

    Zverina, Elaina A; Lamphear, Corissa L; Wright, Elia N; Fierke, Carol A

    2012-12-01

    Protein post-translational modifications increase the functional diversity of the proteome by covalently adding chemical moieties onto proteins thereby changing their activation state, cellular localization, interacting partners, and life cycle. Lipidation is one such modification that enables membrane association of naturally cytosolic proteins. Protein prenyltransferases irreversibly install isoprenoid units of varying length via a thioether linkage onto proteins that exert their cellular activity at membranes. Substrates of prenyltransferases are involved in countless signaling pathways and processes within the cell. Identification of new prenylation substrates, prenylation pathway regulators, and dynamic trafficking of prenylated proteins are all avenues of intense, ongoing research that are challenging, exciting, and have the potential to significantly advance the field in the near future.

  7. Identification of RNA-Protein Interactions Through In Vitro RNA Pull-Down Assays.

    PubMed

    Barnes, Claire; Kanhere, Aditi

    2016-01-01

    Recent advances in next-generation sequencing have revealed that majority of the human genome is transcribed into long and short RNA (ncRNA) transcripts. Many ncRNAs function by interacting with proteins and forming regulatory complexes. RNA-protein interactions are vital in controlling core cellular processes like transcription and translation. Therefore identifying proteins that interact with ncRNAs is central to deciphering ncRNA functions. Here we describe an RNA-protein pull-down assay, which enables the identification of proteins that interact with an RNA under study. As an example we describe pull-down of proteins interacting with ncRNA XIST, which assists in the recruitment of the polycomb-repressive complex-2 (PRC2) and drives X-chromosomal inactivation. PMID:27659978

  8. Identification of an ideal-like fingerprint for a protein fold using overlapped conserved residues based approach.

    PubMed

    Goyal, Amit; Sokalingam, Sriram; Hwang, Kyu-Suk; Lee, Sun-Gu

    2014-07-10

    Design of an efficient fingerprint that detects homologous proteins at distant sequence identity has been a great challenge. This paper proposes a strategy to extract an ideal-like fingerprint with high specificity and sensitivity from a group of sequences related to a fold. The approach is devised based on the assumptions that the critical residues for a protein fold may be conserved in three aspects, i.e. sequence, structure, and intramolecular interaction, and embedded in secondary structures. We hypothesized that the residues satisfying such conditions simultaneously may work as an efficient fingerprint. This idea was tested on protein folds of various classes, such as beta-strand rich, alpha + beta proteins and alpha/beta proteins with discrete sequence similarities. The fingerprint for each fold was generated by selecting the overlapped conserved residues (OCR) from the conserved residues obtained using independent three alignment methods, i.e. multiple sequence alignment, structure-based alignment, and alignment based on the interstrand hydrogen-bonds. The OCR fingerprints showed more than 90% detection efficiency for all the folds tested and were identified to be almost the minimal fingerprints composed of only critical residues. This study is expected to provide an important conceptual improvement in the identification or design of ideal fingerprints for a protein fold.

  9. Identification of cellular genes critical to recombinant protein production using a Gaussia luciferase-based siRNA screening system.

    PubMed

    Lwa, Teng Rhui; Tan, Chuan Hao; Lew, Qiao Jing; Chu, Kai Ling; Tan, Janice; Lee, Yih Yean; Chao, Sheng-Hao

    2010-04-15

    Development of high-throughput functional genomic screening, including siRNA screening, provides a novel approach for quick identification of critical factors involved in biological processes. Here, we apply this strategy to search for cellular genes involved in recombinant protein production. Since most of biopharmaceutical proteins are secreted proteins, we develop a cell-based reporter assay using a secreted luciferase, Gaussia luciferase (Gluc), as the reporter. Human embryonic kidney 293 (HEK293) cells transiently transfected with the Gluc reporter plasmid are used to screen our siRNA panel. Three cellular genes, CCAAT/enhancer binding protein gamma (CEBPG), potassium channel tetramerisation domain containing 2 (KCTD2), transmembrane protein 183A (TMEM183A), were isolated from the screening. Production of erythropoietin (EPO) was significantly inhibited when CEBPG, KCTD2, and TMEM183A were knocked down. Furthermore, overexpression of CEBPG is shown to significantly improve production of recombinant EPO, interferon gamma, and monoclonal antibody in HEK293 and Chinese hamster ovary cells. Collectively, this novel Gluc-based siRNA screening system is proven to be a useful tool for investigation of secreted protein production in mammalian cells. PMID:20188772

  10. Proteomic analysis of human aqueous humor using multidimensional protein identification technology

    PubMed Central

    Richardson, Matthew R.; Price, Marianne O.; Price, Francis W.; Pardo, Jennifer C.; Grandin, Juan C.; You, Jinsam; Wang, Mu

    2009-01-01

    Aqueous humor (AH) supports avascular tissues in the anterior segment of the eye, maintains intraocular pressure, and potentially influences the pathogenesis of ocular diseases. Nevertheless, the AH proteome is still poorly defined despite several previous efforts, which were hindered by interfering high abundance proteins, inadequate animal models, and limited proteomic technologies. To facilitate future investigations into AH function, the AH proteome was extensively characterized using an advanced proteomic approach. Samples from patients undergoing cataract surgery were pooled and depleted of interfering abundant proteins and thereby divided into two fractions: albumin-bound and albumin-depleted. Multidimensional Protein Identification Technology (MudPIT) was utilized for each fraction; this incorporates strong cation exchange chromatography to reduce sample complexity before reversed-phase liquid chromatography and tandem mass spectrometric analysis. Twelve proteins had multi-peptide, high confidence identifications in the albumin-bound fraction and 50 proteins had multi-peptide, high confidence identifications in the albumin-depleted fraction. Gene ontological analyses were performed to determine which cellular components and functions were enriched. Many proteins were previously identified in the AH and for several their potential role in the AH has been investigated; however, the majority of identified proteins were novel and only speculative roles can be suggested. The AH was abundant in anti-oxidant and immunoregulatory proteins as well as anti-angiogenic proteins, which may be involved in maintaining the avascular tissues. This is the first known report to extensively characterize and describe the human AH proteome and lays the foundation for future work regarding its function in homeostatic and pathologic states. PMID:20019884

  11. Identification of Protein-Protein Interactions and Topologies in Living Cells with Chemical Cross-linking and Mass Spectrometry*S⃞

    PubMed Central

    Zhang, Haizhen; Tang, Xiaoting; Munske, Gerhard R.; Tolic, Nikola; Anderson, Gordon A.; Bruce, James E.

    2009-01-01

    We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno-/affinity purifications. The strategy consists of (i) a chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by two-dimensional LC/MSMS and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; and (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to Shewanella oneidensis bacterial cells, and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore most identified interactions involved membrane proteins, suggesting that the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely used approaches. PMID:18936057

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

    PubMed

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

    2016-05-01

    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.

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

    PubMed

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

    2016-05-01

    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. PMID:26964663

  14. Secondary Reactions and Strategies to Improve Quantitative Protein Footprinting

    SciTech Connect

    Xu,G.; Kiselar, J.; He, Q.; Chance, M.

    2005-01-01

    Hydroxyl radical-mediated footprinting permits detailed examination of structure and dynamic processes of proteins and large biological assemblies, as changes in the rate of reaction of radicals with target peptides are governed by changes in the solvent accessibility of the side-chain probe residues. The precise and accurate determination of peptide reaction rates is essential to successfully probing protein structure using footprinting. In this study, we specifically examine the magnitude and mechanisms of secondary oxidation occurring after radiolytic exposure and prior to mass spectrometric analysis. Secondary oxidation results from hydrogen peroxide and other oxidative species generated during radiolysis, significantly impacting the oxidation of Met and Cys but not aromatic or other reactive residues. Secondary oxidation of Met with formation of sulfoxide degrades data reproducibility and inflates the perceived solvent accessibility of Met-containing peptides. It can be suppressed by adding trace amounts of catalase or millimolar Met-NH{sub 2} (or Met-OH) buffer immediately after irradiation; this leads to greatly improved adherence to first-order kinetics and more precise observed oxidation rates. The strategy is shown to suppress secondary oxidation in model peptides and improve data quality in examining the reactivity of peptides within the Arp2/3 protein complex. Cysteine is also subject to secondary oxidation generating disulfide as the principal product. The disulfides can be reduced before mass spectrometric analysis by reducing agents such as TCEP, while methionine sulfoxide is refractory to reduction by this reagent under typical reducing conditions.

  15. Metals and (metallo)proteins identification in vitreous humor focusing on post-mortem biochemistry.

    PubMed

    Santos Júnior, Júlio César; Mollo Filho, Pedro Carlos; Felice Guidugli, Ruggero Bernardo; Eberlin, Marcos Nogueira; Pessôa, Gustavo de Souza; da Silva, Elidiane Gomes; Arruda, Marco Aurélio Zezzi; Höehr, Nelci Fenalti

    2014-10-01

    This work describes the evaluation of metals and (metallo)proteins in vitreous humor samples and their correlations with some biological aspects in different post-mortem intervals (1-7 days), taking into account both decomposing and non-decomposing bodies. After qualitative evaluation of the samples involving 26 elements, representative metal ions (Fe, Mg and Mo) are determined by inductively coupled plasma mass spectrometry after using mini-vial decomposition system for sample preparation. A significant trend for Fe is found with post-mortem time for decomposing bodies because of a significant increase of iron concentration when comparing samples from bodies presenting 3 and 7 days post-mortem interval. An important clue to elucidate the role of metals is the coupling of liquid chromatography with inductively coupled plasma mass spectrometry for identification of metals linked to proteins, as well as mass spectrometry for the identification of those proteins involved in the post-mortem interval.

  16. Identification, Nomenclature, and Evolutionary Relationships of Mitogen-Activated Protein Kinase (MAPK) Genes in Soybean

    PubMed Central

    Neupane, Achal; Nepal, Madhav P.; Piya, Sarbottam; Subramanian, Senthil; Rohila, Jai S.; Reese, R. Neil; Benson, Benjamin V.

    2013-01-01

    Mitogen-activated protein kinase (MAPK) genes in eukaryotes regulate various developmental and physiological processes including those associated with biotic and abiotic stresses. Although MAPKs in some plant species including Arabidopsis have been identified, they are yet to be identified in soybean. Major objectives of this study were to identify GmMAPKs, assess their evolutionary relationships, and analyze their functional divergence. We identified a total of 38 MAPKs, eleven MAPKKs, and 150 MAPKKKs in soybean. Within the GmMAPK family, we also identified a new clade of six genes: four genes with TEY and two genes with TQY motifs requiring further investigation into possible legume-specific functions. The results indicated the expansion of the GmMAPK families attributable to the ancestral polyploidy events followed by chromosomal rearrangements. The GmMAPK and GmMAPKKK families were substantially larger than those in other plant species. The duplicated GmMAPK members presented complex evolutionary relationships and functional divergence when compared to their counterparts in Arabidopsis. We also highlighted existing nomenclatural issues, stressing the need for nomenclatural consistency. GmMAPK identification is vital to soybean crop improvement, and novel insights into the evolutionary relationships will enhance our understanding about plant genome evolution. PMID:24137047

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

    SciTech Connect

    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.

  18. Probabilistic consensus scoring improves tandem mass spectrometry peptide identification.

    PubMed

    Nahnsen, Sven; Bertsch, Andreas; Rahnenführer, Jörg; Nordheim, Alfred; Kohlbacher, Oliver

    2011-08-01

    Database search is a standard technique for identifying peptides from their tandem mass spectra. To increase the number of correctly identified peptides, we suggest a probabilistic framework that allows the combination of scores from different search engines into a joint consensus score. Central to the approach is a novel method to estimate scores for peptides not found by an individual search engine. This approach allows the estimation of p-values for each candidate peptide and their combination across all search engines. The consensus approach works better than any single search engine across all different instrument types considered in this study. Improvements vary strongly from platform to platform and from search engine to search engine. Compared to the industry standard MASCOT, our approach can identify up to 60% more peptides. The software for consensus predictions is implemented in C++ as part of OpenMS, a software framework for mass spectrometry. The source code is available in the current development version of OpenMS and can easily be used as a command line application or via a graphical pipeline designer TOPPAS.

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

    PubMed

    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.

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

    PubMed

    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. PMID:26950937

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

    PubMed Central

    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. PMID:26950937

  2. Nonculture-based identification of bacteria in milk by protein fingerprinting.

    PubMed

    Barreiro, Juliana Regina; Braga, Patricia Aparecida Campos; Ferreira, Christina Ramires; Kostrzewa, Markus; Maier, Thomas; Wegemann, Beatrix; Böettcher, Viktoria; Eberlin, Marcos N; dos Santos, Marcos Veiga

    2012-08-01

    Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI Sepsityper(TM) Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3) -10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (10(4) cfu mL(-1) ), bacterial identification could be performed after initial incubation at 37°C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the

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

    PubMed Central

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

    2011-01-01

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

  4. Protein interaction network analysis--approach for potential drug target identification in Mycobacterium tuberculosis.

    PubMed

    Kushwaha, Sandeep K; Shakya, Madhvi

    2010-01-21

    In host-parasite diseases like tuberculosis, non-homologous proteins (enzymes) as drug target are first preference. Most potent drug target can be identified among large number of non-homologous protein through protein interaction network analysis. In this study, the entire promising dimension has been explored for identification of potential drug target. A comparative metabolic pathway analysis of the host Homo sapiens and the pathogen M. tuberculosis H37Rv has been performed with three level of analysis. In first level, the unique metabolic pathways of M. tuberculosis have been identified through its comparative study with H. sapiens and identification of non-homologous proteins has been done through BLAST similarity search. In second level, choke-point analysis has been performed with identified non-homologous proteins of metabolic pathways. In third level, two type of analysis have been performed through protein interaction network. First analysis has been done to find out the most potential metabolic functional associations among all identified choke point proteins whereas second analysis has been performed to find out the functional association of high metabolic interacting proteins to pathogenesis causing proteins. Most interactive metabolic proteins which have highest number of functional association with pathogenesis causing proteins have been considered as potential drug target. A list of 18 potential drug targets has been proposed which are various stages of progress at the TBSGC and proposed drug targets are also studied for other pathogenic strains. As a case study, we have built a homology model of identified drug targets histidinol-phosphate aminotransferase (HisC1) using MODELLER software and various information have been generated through molecular dynamics which will be useful in wetlab structure determination. The generated model could be further explored for insilico docking studies with suitable inhibitors.

  5. Identification and characterisation of a novel heat shock protein 90 inhibitor ONO4140.

    PubMed

    Eachkoti, Rafiqa; Reddy, M V Ramana; Lieu, Yen K; Cosenza, Stephen C; Reddy, E PremKumar

    2014-07-01

    Heat shock protein (Hsp) 90 is a key component of the super-chaperone complex that maintains functionally active conformation of various client proteins. Many of these client proteins regulate important nodal points in multiple signalling pathways that promote cancer cell growth and survival. Inhibitors of Hsp90, therefore, have the potential of functioning as anti-cancer agents with pleiotropic effects. Identification of novel Hsp90 inhibitors with more favourable pharmacological properties is a priority in cancer therapy. To achieve this goal, we screened a compound library using a biochemical assay based on refolding of denatured firefly luciferase. The assay revealed high sensitivity, reliability and reproducibility with a Z-factor of 0.81 ± 0.17. Six Hsp90 inhibitory compounds identified by this screening with IC50 values between 1.0 and 6 μM were further characterised for anti-proliferative activity by Cell Titer-Blue Cell Viability Assay using multiple tumour cell lines. Of particular interest was ONO4140 with lowest GI50 values in three different cancer cell lines viz; DU-145, BT-474 and K562 cell lines. This study also revealed that short-term exposure of tumour cells with ONO4140 is sufficient to inhibit the catalytic activity of Hsp90, evaluated through disruption of Hsp90-p23 association by immunoprecipitation. This short term exposure appears to initiate events like degradation of Hsp90 client proteins such as ErbB2/Her-2 and Akt with concomitant inhibition of survival signalling leading to the apoptotic death of tumour cells as seen by western blotting and Caspase Glow-3,7 assay. The study also reveals that apoptosis following Hsp90 inhibition with ONO4140 occurs via Caspase9-Caspase3 intrinsic apoptotic pathway, a process that is likely triggered by inactivation of Akt. In conclusion, we have identified a novel class of synthetic compounds which show potent Hsp90 inhibitory action in preclinical studies. The discovery of this novel class of

  6. Identification of Protein Succination as a Novel Modification of Tubulin

    PubMed Central

    Piroli, Gerardo G.; Manuel, Allison M.; Walla, Michael D.; Jepson, Matthew J.; Brock, Jonathan W.C.; Rajesh, Mathur P.; Tanis, Ross M.; Cotham, William E.; Frizzell, Norma

    2015-01-01

    Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). We demonstrate that both alpha (α) and beta (β) tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound dimethylfumarate (DMF, 500 μM) inhibited polymerization up to 35% and 59%, respectively. Using mass spectrometry we identified Cys347α, Cys376α, Cys12β and Cys303β as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteines increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose vs. normal glucose also had reduced reactivity with the anti-αtubulin antibody; suggesting that succination may interfere with tubulin:protein interactions. DMF reacted rapidly with 11 of the 20 cysteines in the αβ tubulin dimer, decreased the number of free sulfhydryls and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggests that inhibition of tubulin polymerization is an important, undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics. PMID:24909641

  7. Identification and characterization of merozoite surface protein 1 epitope

    PubMed Central

    Singh, Satarudra Prakash; Mishra, Bhartendu Nath

    2009-01-01

    Malaria is an important tropical infection which urgently requires intervention of an effective vaccine. Antigenic variations of the parasite and allelic diversity of the host are main problems in the development of an effective malaria vaccine. Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum­derived antigens are shown to play an important role for the protection against malaria. The merozoite surface protein 1 (MSP1) is expressed in all the four life-cycle stages of Plasmodium falciparum and did not find any sequence similarity to human and mouse reference proteins. MSP1 is a known target of the immune response and a single CTL epitope binding to the HLA­A*0201 is available for merozoite form. Here, we report the results from the computational characterization of MSP1, precursor (1720 residue) and screening of highest scoring potential CTL epitopes for 1712 overlapping peptides binding to thirty four HLA class­I alleles and twelve HLA class­I supertypes (5 HLA­A and 7 HLA­B) using bioinformatics tools. Supertypes are the clustered groups of HLA class­I molecules, representing a sets of molecules that share largely overlapping peptide binding specificity. The prediction results for MSP1 as adhesin and adhesin-like in terms of probability is 1.0. Results also show that MSP1 has orthologs to other related species as well as having non allergenicity and single transmembrane properties demonstrating its suitability as a vaccine candidate. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage. PMID:20011145

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

    PubMed Central

    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

  9. Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

  10. PPCM: Combing Multiple Classifiers to Improve Protein-Protein Interaction Prediction

    DOE PAGES

    Yao, Jianzhuang; Guo, Hong; Yang, Xiaohan

    2015-01-01

    Determining protein-protein interaction (PPI) in biological systems is of considerable importance, and prediction of PPI has become a popular research area. Although different classifiers have been developed for PPI prediction, no single classifier seems to be able to predict PPI with high confidence. We postulated that by combining individual classifiers the accuracy of PPI prediction could be improved. We developed a method called protein-protein interaction prediction classifiers merger (PPCM), and this method combines output from two PPI prediction tools, GO2PPI and Phyloprof, using Random Forests algorithm. The performance of PPCM was tested by area under the curve (AUC) using anmore » assembled Gold Standard database that contains both positive and negative PPI pairs. Our AUC test showed that PPCM significantly improved the PPI prediction accuracy over the corresponding individual classifiers. We found that additional classifiers incorporated into PPCM could lead to further improvement in the PPI prediction accuracy. Furthermore, cross species PPCM could achieve competitive and even better prediction accuracy compared to the single species PPCM. This study established a robust pipeline for PPI prediction by integrating multiple classifiers using Random Forests algorithm. This pipeline will be useful for predicting PPI in nonmodel species.« less

  11. Metal affinity enrichment increases the range and depth of proteome identification for extracellular microbial proteins

    SciTech Connect

    Wheeler, Korin; Erickson, Brian K; Mueller, Ryan; Singer, Steven; Verberkmoes, Nathan C; Hwang, Mona; Thelen, Michael P.; Hettich, Robert {Bob} L

    2012-01-01

    Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added 20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as hypothetical were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.

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

    PubMed

    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.

  13. Tenebrio molitor antifreeze protein gene identification and regulation.

    PubMed

    Qin, Wensheng; Walker, Virginia K

    2006-02-15

    The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

  14. Cholesteryl ester transfer protein deficiency: Identification in the chinese

    SciTech Connect

    Akita, Harukuni; Chiba, Hitoshi; Hui, Shu-Ping; Takahashi, Yukihiro; Matsuno, Kazuhiko; Kobayashi, Kunihiko

    1995-11-20

    Cholesteryl ester transfer protein (CETP) regulates cholesterol content in high-density lipoproteins (HDL) through the exchange of cholesteryl ester in HDL and triglyceride in triglyceride-rich lipoproteins. Three types of CETP deficiency have so far been reported, all in Japanese individuals. Among them, two types of mutation in the CETP gene are relatively common: (1) a guanine to adenine mutation in the intron 14 splicing donor site (114A), and (2) an Asp 442 to Gly (D442G) mutation. The 114A mutation has an estimated allele frequency of 0.81% in the Japanese. It elevates the plasma HDL-cholesterol level strikingly: typically, >120 mg/dl for homozygotes and 80-110 mg/dl for heterozygotes. In contrast, the D442G mutation has a higher estimated allele frequency (4.62%), but has a mild effect on the HDL-cholesterol level: typically, >80 mg/dl for homozygotes and 50-100 mg/dl for heterozygotes. We screened Chinese individuals for the two CETP gene mutations. 7 refs., 1 tab.

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

  18. A rapid and selective mass spectrometric method for the identification of nitrated proteins.

    PubMed

    Amoresano, Angela; Chiappetta, Giovanni; Pucci, Piero; Marino, Gennaro

    2008-01-01

    The nitration of protein tyrosine residues represents an important posttranslational modification during development, oxidative stress, and biological aging. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels, from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Identification of the specific targets of protein oxidation was previously accomplished using immunoprecipitation techniques followed by immunochemical detection. Here, we report a totally new approach involving dansyl chloride labeling of the nitration sites which relies on the enormous potential of MS(n) analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and a MS3 analysis. The novel labeling procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures. PMID:19082935

  19. Identification of human protein complexes from local sub-graphs of protein-protein interaction network based on random forest with topological structure features.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Zhou, Xuan; Dai, Zong; Zou, Xiao-Yong

    2012-03-01

    In the post-genomic era, one of the most important and challenging tasks is to identify protein complexes and further elucidate its molecular mechanisms in specific biological processes. Previous computational approaches usually identify protein complexes from protein interaction network based on dense sub-graphs and incomplete priori information. Additionally, the computational approaches have little concern about the biological properties of proteins and there is no a common evaluation metric to evaluate the performance. So, it is necessary to construct novel method for identifying protein complexes and elucidating the function of protein complexes. In this study, a novel approach is proposed to identify protein complexes using random forest and topological structure. Each protein complex is represented by a graph of interactions, where descriptor of the protein primary structure is used to characterize biological properties of protein and vertex is weighted by the descriptor. The topological structure features are developed and used to characterize protein complexes. Random forest algorithm is utilized to build prediction model and identify protein complexes from local sub-graphs instead of dense sub-graphs. As a demonstration, the proposed approach is applied to protein interaction data in human, and the satisfied results are obtained with accuracy of 80.24%, sensitivity of 81.94%, specificity of 80.07%, and Matthew's correlation coefficient of 0.4087 in 10-fold cross-validation test. Some new protein complexes are identified, and analysis based on Gene Ontology shows that the complexes are likely to be true complexes and play important roles in the pathogenesis of some diseases. PCI-RFTS, a corresponding executable program for protein complexes identification, can be acquired freely on request from the authors.

  20. Using support vector machine for improving protein-protein interaction prediction utilizing domain interactions

    SciTech Connect

    Singhal, Mudita; Shah, Anuj R.; Brown, Roslyn N.; Adkins, Joshua N.

    2010-10-02

    Understanding protein interactions is essential to gain insights into the biological processes at the whole cell level. The high-throughput experimental techniques for determining protein-protein interactions (PPI) are error prone and expensive with low overlap amongst them. Although several computational methods have been proposed for predicting protein interactions there is definite room for improvement. Here we present DomainSVM, a predictive method for PPI that uses computationally inferred domain-domain interaction values in a Support Vector Machine framework to predict protein interactions. DomainSVM method utilizes evidence of multiple interacting domains to predict a protein interaction. It outperforms existing methods of PPI prediction by achieving very high explanation ratios, precision, specificity, sensitivity and F-measure values in a 10 fold cross-validation study conducted on the positive and negative PPIs in yeast. A Functional comparison study using GO annotations on the positive and the negative test sets is presented in addition to discussing novel PPI predictions in Salmonella Typhimurium.

  1. Improving the Understanding of Pathogenesis of Human Papillomavirus 16 via Mapping Protein-Protein Interaction Network

    PubMed Central

    Dong, Yongcheng; Kuang, Qifan; Dai, Xu; Li, Rong; Wu, Yiming; Leng, Weijia; Li, Yizhou; Li, Menglong

    2015-01-01

    The human papillomavirus 16 (HPV16) has high risk to lead various cancers and afflictions, especially, the cervical cancer. Therefore, investigating the pathogenesis of HPV16 is very important for public health. Protein-protein interaction (PPI) network between HPV16 and human was used as a measure to improve our understanding of its pathogenesis. By adopting sequence and topological features, a support vector machine (SVM) model was built to predict new interactions between HPV16 and human proteins. All interactions were comprehensively investigated and analyzed. The analysis indicated that HPV16 enlarged its scope of influence by interacting with human proteins as much as possible. These interactions alter a broad array of cell cycle progression. Furthermore, not only was HPV16 highly prone to interact with hub proteins and bottleneck proteins, but also it could effectively affect a breadth of signaling pathways. In addition, we found that the HPV16 evolved into high carcinogenicity on the condition that its own reproduction had been ensured. Meanwhile, this work will contribute to providing potential new targets for antiviral therapeutics and help experimental research in the future. PMID:25961044

  2. An Improved Algorithm of Congruent Matching Cells (CMC) Method for Firearm Evidence Identifications

    PubMed Central

    Tong, Mingsi; Song, John; Chu, Wei

    2015-01-01

    The Congruent Matching Cells (CMC) method was invented at the National Institute of Standards and Technology (NIST) for firearm evidence identifications. The CMC method divides the measured image of a surface area, such as a breech face impression from a fired cartridge case, into small correlation cells and uses four identification parameters to identify correlated cell pairs originating from the same firearm. The CMC method was validated by identification tests using both 3D topography images and optical images captured from breech face impressions of 40 cartridge cases fired from a pistol with 10 consecutively manufactured slides. In this paper, we discuss the processing of the cell correlations and propose an improved algorithm of the CMC method which takes advantage of the cell correlations at a common initial phase angle and combines the forward and backward correlations to improve the identification capability. The improved algorithm is tested by 780 pairwise correlations using the same optical images and 3D topography images as the initial validation. PMID:26958441

  3. An Improved Algorithm of Congruent Matching Cells (CMC) Method for Firearm Evidence Identifications.

    PubMed

    Tong, Mingsi; Song, John; Chu, Wei

    2015-01-01

    The Congruent Matching Cells (CMC) method was invented at the National Institute of Standards and Technology (NIST) for firearm evidence identifications. The CMC method divides the measured image of a surface area, such as a breech face impression from a fired cartridge case, into small correlation cells and uses four identification parameters to identify correlated cell pairs originating from the same firearm. The CMC method was validated by identification tests using both 3D topography images and optical images captured from breech face impressions of 40 cartridge cases fired from a pistol with 10 consecutively manufactured slides. In this paper, we discuss the processing of the cell correlations and propose an improved algorithm of the CMC method which takes advantage of the cell correlations at a common initial phase angle and combines the forward and backward correlations to improve the identification capability. The improved algorithm is tested by 780 pairwise correlations using the same optical images and 3D topography images as the initial validation. PMID:26958441

  4. Improved "optical highlighter" probes derived from discosoma red fluorescent protein.

    PubMed

    Robinson, Lisbeth C; Marchant, Jonathan S

    2005-02-01

    The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda < 760 nm) femtosecond irradiation--a phenomenon that underpins the application of DsRed as an "optical highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.

  5. Relaxation of backbone bond geometry improves protein energy landscape modeling.

    PubMed

    Conway, Patrick; Tyka, Michael D; DiMaio, Frank; Konerding, David E; Baker, David

    2014-01-01

    A key issue in macromolecular structure modeling is the granularity of the molecular representation. A fine-grained representation can approximate the actual structure more accurately, but may require many more degrees of freedom than a coarse-grained representation and hence make conformational search more challenging. We investigate this tradeoff between the accuracy and the size of protein conformational search space for two frequently used representations: one with fixed bond angles and lengths and one that has full flexibility. We performed large-scale explorations of the energy landscapes of 82 protein domains under each model, and find that the introduction of bond angle flexibility significantly increases the average energy gap between native and non-native structures. We also find that incorporating bonded geometry flexibility improves low resolution X-ray crystallographic refinement. These results suggest that backbone bond angle relaxation makes an important contribution to native structure energetics, that current energy functions are sufficiently accurate to capture the energetic gain associated with subtle deformations from chain ideality, and more speculatively, that backbone geometry distortions occur late in protein folding to optimize packing in the native state.

  6. Improving NMR Protein Structure Quality by Rosetta Refinement: a Molecular Replacement Study

    SciTech Connect

    Ramelot, T.; Raman, S; Kuzin, A; Hunt, J; Baker, D; Kennedy, M

    2009-01-01

    The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in

  7. Improving NMR Protein Structure Quality by Rosetta Refinement: A Molecular Replacement Study

    SciTech Connect

    Ramelot, Theresa A.; Raman, Srivatsan; Kuzin, Alexander P.; Xiao, Rong; Ma, LiChung; Acton, Thomas; Hunt, John F.; Montelione, Gaetano; Baker, David; Kennedy, Michael A.

    2009-04-01

    The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259–264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results

  8. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    SciTech Connect

    Blackburn, W.D.; Tallant, E.A.; Wallace, R.W.

    1986-05-01

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca/sup 2 +/. One mechanism by which Ca/sup 2 +/ may trigger neutrophil activation is through Ca/sup 2 +//calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca/sup 2 +//CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and /sup 125/I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound /sup 125/I-CaM in a Ca/sup 2 +/-dependent manner. One predominant region of /sup 125/I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca/sup 2 +/-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 ..mu..g/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca/sup 2 +/-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca/sup 2 +//calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase.

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

    PubMed

    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.

  10. Identification of active ingredients in Wuzhuyu decoction improving migraine in mice by spectral efficiency association.

    PubMed

    Pan, Xueqiang; Wang, Manyuan; Wu, Yanchuan; Lu, Xuran; Shang, Yawen; Xu, Yongsong; Zhai, Yongsong; Li, Jing; Li, Zhaoxia; Gong, Muxin

    2015-07-01

    Wuzhuyu decoction is a traditional Chinese medicine used for the effective treatment of migraines, termed 'Jueyin headache', in China. However, there have been few investigations to clarify the composition of Wuzhuyu decoction for the treatment of migraines. In the present study, 10 types of Wuzhuyu decoction were analyzed by chromatograms. 5-hydroxytryptamine (5-HT)-depletion mouse models of migraine were prepared by subcutaneous injection of reserpine and placement of autologous blood clots in the cerebral cortex. The levels of 5-HT, noradrenaline (NE), dopamine (DA), nitric oxide (NO) and nitric oxide synthase (NOS) in the brain tissues and sera of the mice were determined. The ingredients and pharmacodynamic indices of the Wuzhuyu decoctions were analyzed using spectral efficiency association by partial least squares regression. The levels of 5-HT, NE and DA in the mouse brain tissues were reduced to 337.785 ± 84.504, 171.173 ± 65.172 and 242.075 ± 158.621 mg/g brain tissue, respectively. The level of NO in the brain tissues increased to 0.425 ± 0.184 µmol/g protein and the activities of NOS in the brain tissues and sera increased to 0.719 ± 0.477 U/mg and 50.688 ± 8.132 U/ml, respectively. Regarding the ingredients of the Wuzhuyu decoction, those with significant regression coefficients were ginsenoside-Rg1, Re, Rb1, rutaevine (Rv), limonin (Li), evodiamine (Ev), rutaecarpine (Ru) and substance X (awaiting identification). Rg1, Re, Rb1, Rv, Li, Ev, Ru and X in the Wuzhuyu decoction were observed to yield the pharmacological effects, whereas Rb1, Rv and Ev were important in index improvement.

  11. Improving protein coreference resolution by simple semantic classification

    PubMed Central

    2012-01-01

    Background Current research has shown that major difficulties in event extraction for the biomedical domain are traceable to coreference. Therefore, coreference resolution is believed to be useful for improving event extraction. To address coreference resolution in molecular biology literature, the Protein Coreference (COREF) task was arranged in the BioNLP Shared Task (BioNLP-ST, hereafter) 2011, as a supporting task. However, the shared task results indicated that transferring coreference resolution methods developed for other domains to the biological domain was not a straight-forward task, due to the domain differences in the coreference phenomena. Results We analyzed the contribution of domain-specific information, including the information that indicates the protein type, in a rule-based protein coreference resolution system. In particular, the domain-specific information is encoded into semantic classification modules for which the output is used in different components of the coreference resolution. We compared our system with the top four systems in the BioNLP-ST 2011; surprisingly, we found that the minimal configuration had outperformed the best system in the BioNLP-ST 2011. Analysis of the experimental results revealed that semantic classification, using protein information, has contributed to an increase in performance by 2.3% on the test data, and 4.0% on the development data, in F-score. Conclusions The use of domain-specific information in semantic classification is important for effective coreference resolution. Since it is difficult to transfer domain-specific information across different domains, we need to continue seek for methods to utilize such information in coreference resolution. PMID:23157272

  12. Comprehensive identification of novel proteins and N-glycosylation sites in royal jelly

    PubMed Central

    2014-01-01

    Background Royal jelly (RJ) is a proteinaceous secretion produced from the hypopharyngeal and mandibular glands of nurse bees. It plays vital roles in honeybee biology and in the improvement of human health. However, some proteins remain unknown in RJ, and mapping N-glycosylation modification sites on RJ proteins demands further investigation. We used two different liquid chromatography-tandem mass spectrometry techniques, complementary N-glycopeptide enrichment strategies, and bioinformatic approaches to gain a better understanding of novel and glycosylated proteins in RJ. Results A total of 25 N-glycosylated proteins, carrying 53 N-glycosylation sites, were identified in RJ proteins, of which 42 N-linked glycosylation sites were mapped as novel on RJ proteins. Most of the glycosylated proteins were related to metabolic activities and health improvement. The 13 newly identified proteins were also mainly associated with metabolic processes and health improvement activities. Conclusion Our in-depth, large-scale mapping of novel glycosylation sites represents a crucial step toward systematically revealing the functionality of N-glycosylated RJ proteins, and is potentially useful for producing a protein with desirable pharmacokinetic and biological activity using a genetic engineering approach. The newly-identified proteins significantly extend the proteome coverage of RJ. These findings contribute vital and new knowledge to our understanding of the innate biochemical nature of RJ at both the proteome and glycoproteome levels. PMID:24529077

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

    PubMed Central

    Dumpala, Pradeep R.; Peterson, Brian C.; Lawrence, Mark L.; Karsi, Attila

    2015-01-01

    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. PMID:26168192

  14. Identification of Key Proteins in Human Epithelial Cells Responding to Bystander Signals From Irradiated Trout Skin

    PubMed Central

    Smith, Richard; Wang, Jiaxi; Seymour, Colin; Mothersill, Carmel; Howe, Orla

    2015-01-01

    Radiation-induced bystander signaling has been found to occur in live rainbow trout fish (Oncorhynchus mykiss). This article reports identification of key proteomic changes in a bystander reporter cell line (HaCaT) grown in low-dose irradiated tissue-conditioned media (ITCM) from rainbow trout fish. In vitro explant cultures were generated from the skin of fish previously exposed to low doses (0.1 and 0.5 Gy) of X-ray radiation in vivo. The ITCM was harvested from all donor explant cultures and placed on recipient HaCaT cells to observe any change in protein expression caused by the bystander signals. Proteomic methods using 2-dimensional (2D) gel electrophoresis and mass spectroscopy were employed to screen for novel proteins expressed. The proteomic changes measured in HaCaT cells receiving the ITCM revealed that exposure to 0.5 Gy induced an upregulation of annexin A2 and cingulin and a downregulation of Rho-GDI2, F-actin-capping protein subunit beta, microtubule-associated protein RP/EB family member, and 14-3-3 proteins. The 0.1 Gy dose also induced a downregulation of Rho-GDI2, hMMS19, F-actin-capping protein subunit beta, and microtubule-associated protein RP/EB family member proteins. The proteins reported may influence apoptotic signaling, as the results were suggestive of an induction of cell communication, repair mechanisms, and dysregulation of growth signals. PMID:26673684

  15. [Studies on protein-based identification method of genetically modified capsicum].

    PubMed

    Liu, Jianjun; Deng, Pingjian; Fang, Shisong; Zhao, Jin

    2003-03-01

    The detection system based on protein is a method to evaluate the safety of genetically modified foods (GMF). Using cecropin BD gene in capsicum, a detecting method was set up. It is a system of evaluating the real expressive condition and safety of the foreign target protein of GMF. In this studies, with the preformative technic method, a satisfactory results by making use of hemolymph of immunized pupae of Antheraea pernyi as standard experimental material was achieved, comparing with the realities of the goal protein expressive condition of cecropin D gene in capsicum. The detecting steps were as following: the goal protein from material was extracted roughly, then with CM-Sepharose-FF ion-exchange chromatography twice, the goal protein was purified moderately. The purified product was identified by detecting the anti-bacterial activity, electrophoresis, biological auto-photography of the goal protein and MADDI-TOF mass spectrum. The results showed that the expressive foreign target protein in transgenic capsicum was in accordance with standard protein in the physical and chemical property, anti-bacterial activity and molecular weight. It indicated that expression of the target gene in capsicum is real, it corresponded to expected value. The separation, purification and identification methods of cecropin D were established in the study. By means of the comparative experiments about anti-bacterial activity and molecular weight of anti-bacterial peptide(ABP) from GM-capsicum and hemolymph of immunized pupae of Antheraea pernyi, the identification method of target protein from GM-capsicum was set up. The method is easy to be operated, fast and feasible.

  16. A Novel Spectral Library Workflow to Enhance Protein Identifications

    PubMed Central

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

    2013-01-01

    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. PMID:23391412

  17. An intelligent fault identification method of rolling bearings based on LSSVM optimized by improved PSO

    NASA Astrophysics Data System (ADS)

    Xu, Hongbo; Chen, Guohua

    2013-02-01

    This paper presents an intelligent fault identification method of rolling bearings based on least squares support vector machine optimized by improved particle swarm optimization (IPSO-LSSVM). The method adopts a modified PSO algorithm to optimize the parameters of LSSVM, and then the optimized model could be established to identify the different fault patterns of rolling bearings. Firstly, original fault vibration signals are decomposed into some stationary intrinsic mode functions (IMFs) by empirical mode decomposition (EMD) method and the energy feature indexes extraction based on IMF energy entropy is analyzed in detail. Secondly, the extracted energy indexes serve as the fault feature vectors to be input to the IPSO-LSSVM classifier for identifying different fault patterns. Finally, a case study on rolling bearing fault identification demonstrates that the method can effectively enhance identification accuracy and convergence rate.

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed

    Kashima, Yuri; Kanematsu, Satoshi; Asai, Saori; Kusada, Mio; Watanabe, Suzuyo; Kawashima, Takuji; Nakamura, Tadashi; Shimada, Masaya; Goto, Tsuyoshi; Nagaoka, Satoshi

    2014-01-01

    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.

  20. Identification of a Novel Hypocholesterolemic Protein, Major Royal Jelly Protein 1, Derived from Royal Jelly

    PubMed Central

    Asai, Saori; Kusada, Mio; Watanabe, Suzuyo; Kawashima, Takuji; Nakamura, Tadashi; Shimada, Masaya; Goto, Tsuyoshi; Nagaoka, Satoshi

    2014-01-01

    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. PMID:25144734

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

    PubMed Central

    Krehenbrink, Martin; Downie, J Allan

    2008-01-01

    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 the genes encoding the

  2. Support Vector Machines for Improved Peptide Identification from Tandem Mass Spectrometry Database Search

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.

    2009-05-06

    Accurate identification of peptides is a current challenge in mass spectrometry (MS) based proteomics. The standard approach uses a search routine to compare tandem mass spectra to a database of peptides associated with the target organism. These database search routines yield multiple metrics associated with the quality of the mapping of the experimental spectrum to the theoretical spectrum of a peptide. The structure of these results make separating correct from false identifications difficult and has created a false identification problem. Statistical confidence scores are an approach to battle this false positive problem that has led to significant improvements in peptide identification. We have shown that machine learning, specifically support vector machine (SVM), is an effective approach to separating true peptide identifications from false ones. The SVM-based peptide statistical scoring method transforms a peptide into a vector representation based on database search metrics to train and validate the SVM. In practice, following the database search routine, a peptides is denoted in its vector representation and the SVM generates a single statistical score that is then used to classify presence or absence in the sample

  3. Identification of Transmembrane Protein 88 (TMEM88) as a Dishevelled-binding Protein*

    PubMed Central

    Lee, Ho-Jin; Finkelstein, David; Li, Xiaofeng; Wu, Dianqing; Shi, De-Li; Zheng, Jie J.

    2010-01-01

    Wnt signaling pathways are involved in embryonic development and adult tissue maintenance and have been implicated in tumorigenesis. Dishevelled (Dvl/Dsh) protein is one of key components in Wnt signaling and plays essential roles in regulating these pathways through protein-protein interactions. Identifying and characterizing Dvl-binding proteins are key steps toward understanding biological functions. Given that the tripeptide VWV (Val-Trp-Val) binds to the PDZ domain of Dvl, we searched publically available databases to identify proteins containing the VWV motif at the C terminus that could be novel Dvl-binding partners. On the basis of the cellular localization and expression patterns of the candidates, we selected for further study the TMEM88 (target protein transmembrane 88), a two-transmembrane-type protein. The interaction between the PDZ domain of Dvl and the C-terminal tail of TMEM88 was confirmed by using NMR and fluorescence spectroscopy. Furthermore, in HEK293 cells, TMEM88 attenuated the Wnt/β-catenin signaling induced by Wnt-1 ligand in a dose-dependent manner, and TMEM88 knockdown by RNAi increased Wnt activity. In Xenopus, TMEM88 protein is sublocalized at the cell membrane and inhibits Wnt signaling induced by Xdsh but not β-catenin. In addition, TMEM88 protein inhibits the formation of a secondary axis normally induced by Xdsh. The findings suggest that TMEM88 plays a role in regulating Wnt signaling. Indeed, analysis of microarray data revealed that the expression of the Tmem88 gene was strongly correlated with that of Wnt signaling-related genes in embryonic mouse intestines. Together, we propose that TMEM88 associates with Dvl proteins and regulates Wnt signaling in a context-dependent manner. PMID:21044957

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

    PubMed

    Manna, Sam; Brewster, Jessica; Barth, Christian

    2013-01-01

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

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

    PubMed

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

    2013-10-24

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

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

    SciTech Connect

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

    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.

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

    PubMed

    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.

  8. Identification of Signaling Protein Complexes by Parallel Affinity Precipitation Coupled with Mass Spectrometry

    PubMed Central

    Lu, Heng; Lin, Qishan; Zhao, Jihe

    2014-01-01

    Protein–protein interactions play a pivotal role in both inter- and intra-cellular signaling. Identification of signaling protein complexes can thus shed important new insights into cell communications. We developed a parallel affinity precipitation protocol to overcome the disadvantages of the tandem affinity purification procedure, such as the potential disruption of target protein conformation, subcellular localization or function by epitope tags, the potential need of large amounts of cell culture or generation of stable cell lines, and relatively long duration the two-step precipitation takes. This new simplified assay of protein interaction is quick, economic and specific. This paper describes the details in the design and method of the assay. PMID:24839392

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

    SciTech Connect

    Umar, Arzu; Kang, Hyuk; Timmermans, A. M.; Look, Maxime P.; Meijer-van Gelder, M. E.; den Bakker, Michael A.; Jaitly, Navdeep; Martens, John W.; Luider, Theo M.; Foekens, John A.; Pasa-Tolic, Ljiljana

    2009-06-01

    Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC coupled with FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells (corresponding to ~550 ng protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n=24 and n=27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag (AMT) reference databases.

  10. LC-MSMS identification of Arabidopsis thaliana heat-stable seed proteins: enriching for LEA-type proteins by acid treatment.

    PubMed

    Oliveira, E; Amara, I; Bellido, D; Odena, M A; Domínguez, E; Pagès, M; Goday, A

    2007-11-01

    Protein identification in systems containing very highly abundant proteins is not always efficient and usually requires previous enrichment or fractionation steps in order to uncover minor proteins. In plant seeds, identification of late embryogenesis abundant (LEA) proteins is often masked by the presence of the large family of storage proteins. LEA-proteins are predicted to play a role in plant stress tolerance. They are highly hydrophilic proteins, generally heat-stable, and correlate with dehydration in seeds or vegetative tissues. In the present work, we analyze the protein composition of heat-stable Arabidopsis thaliana seed extracts after treatment with trichloroacetic acid (TCA). The composition of the proteins that precipitate and those that remain in solution in 3% TCA was analyzed by two different approaches: 1D SDS-PAGE coupled to LC-ESI-MSMS analysis and a gel-free protocol associated with LC-MALDI-MSMS. Our results indicate that treating total heat-soluble extracts with 3% TCA is an effective procedure to remove storage proteins by selective precipitation and this fractionation step provides a soluble fraction highly enriched in Lea-type proteins. The analysis and determination of protein identities in this acid-soluble fraction by MS technology is a suitable system for large-scale identification of Lea-proteins present in seeds.

  11. Improving the identification and management of chronic kidney disease in primary care: lessons from a staged improvement collaborative

    PubMed Central

    Harvey, Gill; Oliver, Kathryn; Humphreys, John; Rothwell, Katy; Hegarty, Janet

    2015-01-01

    Quality problem Undiagnosed chronic kidney disease (CKD) contributes to a high cost and care burden in secondary care. Uptake of evidence-based guidelines in primary care is inconsistent, resulting in variation in the detection and management of CKD. Initial assessment Routinely collected general practice data in one UK region suggested a CKD prevalence of 4.1%, compared with an estimated national prevalence of 8.5%. Of patients on CKD registers, ∼30% were estimated to have suboptimal management according to Public Health Observatory analyses. Choice of solution An evidence-based framework for implementation was developed. This informed the design of an improvement collaborative to work with a sample of 30 general practices. Implementation A two-phase collaborative was implemented between September 2009 and March 2012. Key elements of the intervention included learning events, improvement targets, Plan-Do-Study-Act cycles, benchmarking of audit data, facilitator support and staff time reimbursement. Evaluation Outcomes were evaluated against two indicators: number of patients with CKD on practice registers; percentage of patients achieving evidence-based blood pressure (BP) targets, as a marker for CKD care. In Phase 1, recorded prevalence of CKD in collaborative practices increased ∼2-fold more than that in comparator local practices; in Phase 2, this increased to 4-fold, indicating improved case identification. Management of BP according to guideline recommendations also improved. Lessons learned An improvement collaborative with tailored facilitation support appears to promote the uptake of evidence-based guidance on the identification and management of CKD in primary care. A controlled evaluation study is needed to rigorously evaluate the impact of this promising improvement intervention. PMID:25525148

  12. Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

    PubMed

    Lallemand, Jérôme; Bouché, Frédéric; Desiron, Carole; Stautemas, Jennifer; de Lemos Esteves, Frédéric; Périlleux, Claire; Tocquin, Pierre

    2015-01-01

    Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

  13. Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

    PubMed

    Lallemand, Jérôme; Bouché, Frédéric; Desiron, Carole; Stautemas, Jennifer; de Lemos Esteves, Frédéric; Périlleux, Claire; Tocquin, Pierre

    2015-01-01

    Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing. PMID:25705212

  14. Extracellular peptidase hunting for improvement of protein production in plant cells and roots

    PubMed Central

    Lallemand, Jérôme; Bouché, Frédéric; Desiron, Carole; Stautemas, Jennifer; de Lemos Esteves, Frédéric; Périlleux, Claire; Tocquin, Pierre

    2015-01-01

    Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing. PMID:25705212

  15. Ultrasensitive isolation, identification and quantification of DNA–protein adducts by ELISA-based RADAR assay

    PubMed Central

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-01-01

    Enzymes that form transient DNA–protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA–protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA–protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA–protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA–protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA–protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1–DNA and Top2a–DNA adducts in human cells, and gyrase–DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  16. Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

    PubMed

    Eliahoo, Elad; Litovco, Phyana; Ben Yosef, Ron; Bendalak, Keren; Ziv, Tamar; Manor, Haim

    2014-04-01

    Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹⁵N and ¹⁴N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.

  17. Identification of Late Embryogenesis Abundant (LEA) Protein Putative Interactors Using Phage Display

    PubMed Central

    Kushwaha, Rekha; Lloyd, Taylor D.; Schäfermeyer, Kim R.; Kumar, Santosh; Downie, Allan Bruce

    2012-01-01

    Arabidopsis thaliana seeds without functional SEED MATURATION PROTEIN1 (SMP1), a boiling soluble protein predicted to be of intrinsic disorder, presumed to be a LATE EMBRYOGENESIS ABUNDANT (LEA) family protein based on sequence homology, do not enter secondary dormancy after 3 days at 40 °C. We hypothesized that SMP1 may protect a heat labile protein involved in the promotion of secondary dormancy. Recombinant SMP1 and GmPM28, its soybean (Glycine max), LEA4 homologue, protected the labile GLUCOSE-6-PHOSPHATE DEHYROGENASE enzyme from heat stress, as did a known protectant, Bovine Serum Albumin, whether the LEA protein was in solution or attached to the bottom of microtiter plates. Maintenance of a biological function for both recombinant LEA proteins when immobilized encouraged a biopanning approach to screen for potential protein interactors. Phage display with two Arabidopsis seed, T7 phage, cDNA libraries, normalized for transcripts present in the mature, dehydrated, 12-, 24-, or 36-h imbibed seeds, were used in biopans against recombinant SMP1 and GmPM28. Phage titer increased considerably over four rounds of biopanning for both LEA proteins, but not for BSA, at both 25 and at 41 °C, regardless of the library used. The prevalence of multiple, independent clones encoding portions of specific proteins repeatedly retrieved from different libraries, temperatures and baits, provides evidence suggesting these LEA proteins are discriminating which proteins they protect, a novel finding. The identification of putative LEA-interacting proteins provides targets for reverse genetic approaches to further dissect the induction of secondary dormancy in seeds in response to heat stress. PMID:22837651

  18. Sequence signatures and the probabilistic identification of proteins in the Myc-Max-Mad network.

    PubMed

    Atchley, William R; Fernandes, Andrew D

    2005-05-01

    Accurate identification of specific groups of proteins by their amino acid sequence is an important goal in genome research. Here we combine information theory with fuzzy logic search procedures to identify sequence signatures or predictive motifs for members of the Myc-Max-Mad transcription factor network. Myc is a well known oncoprotein, and this family is involved in cell proliferation, apoptosis, and differentiation. We describe a small set of amino acid sites from the N-terminal portion of the basic helix-loop-helix (bHLH) domain that provide very accurate sequence signatures for the Myc-Max-Mad transcription factor network and three of its member proteins. A predictive motif involving 28 contiguous bHLH sequence elements found 337 network proteins in the GenBank NR database with no mismatches or misidentifications. This motif also identifies at least one previously unknown fungal protein with strong affinity to the Myc-Max-Mad network. Another motif found 96% of known Myc protein sequences with only a single mismatch, including sequences from genomes previously not thought to contain Myc proteins. The predictive motif for Myc is very similar to the ancestral sequence for the Myc group estimated from phylogenetic analyses. Based on available crystal structure studies, this motif is discussed in terms of its functional consequences. Our results provide insight into evolutionary diversification of DNA binding and dimerization in a well characterized family of regulatory proteins and provide a method of identifying signature motifs in protein families.

  19. Bidimensional tandem mass spectrometry for selective identification of nitration sites in proteins.

    PubMed

    Amoresano, Angela; Chiappetta, Giovanni; Pucci, Piero; D'Ischia, Marco; Marino, Gennaro

    2007-03-01

    Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures. PMID:17243771

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

    PubMed

    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.

  1. Identification of Proteins Whose Interaction with Na+,K+-ATPase Is Triggered by Ouabain.

    PubMed

    Akimova, O A; Kapilevich, L V; Orlov, S N; Lopina, O D

    2016-09-01

    Prolonged exposure of different epithelial cells (canine renal epithelial cells (MDCK), vascular endothelial cells from porcine aorta (PAEC), human umbilical vein endothelial cells (HUVEC), cervical adenocarcinoma (HeLa), as well as epithelial cells from colon carcinoma (Caco-2)) with ouabain or with other cardiotonic steroids was shown earlier to result in the death of these cells. Intermediates in the cell death signal cascade remain unknown. In the present study, we used proteomics methods for identification of proteins whose interaction with Na+,K+-ATPase is triggered by ouabain. After exposure of Caco-2 human colorectal adenocarcinoma cells with 3 µM of ouabain for 3 h, the protein interacting in complex with Na+,K+-ATPase was coimmunoprecipitated using antibodies against the enzyme α1-subunit. Proteins of coimmunoprecipitates were separated by 2D electrophoresis in polyacrylamide gel. A number of proteins in the coimmunoprecipitates with molecular masses of 71-74, 46, 40-43, 38, and 33-35 kDa was revealed whose binding to Na+,K+-ATPase was activated by ouabain. Analyses conducted by mass spectroscopy allowed us to identify some of them, including seven signal proteins from superfamilies of glucocorticoid receptors, serine/threonine protein kinases, and protein phosphatases 2C, Src-, and Rho-GTPases. The possible participation of these proteins in activation of cell signaling terminated by cell death is discussed. PMID:27682173

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

    PubMed

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

    2016-01-14

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

  3. The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression.

    PubMed

    Sheffield, Jeanne; Taylor, Nigel; Fauquet, Claude; Chen, Sixue

    2006-03-01

    Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots.

  4. Identification of novel γ-secretase-associated proteins in detergent-resistant membranes from brain.

    PubMed

    Hur, Ji-Yeun; Teranishi, Yasuhiro; Kihara, Takahiro; Yamamoto, Natsuko Goto; Inoue, Mitsuhiro; Hosia, Waltteri; Hashimoto, Masakazu; Winblad, Bengt; Frykman, Susanne; Tjernberg, Lars O

    2012-04-01

    In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease. PMID:22315232

  5. Identification of Novel γ-Secretase-associated Proteins in Detergent-resistant Membranes from Brain*

    PubMed Central

    Hur, Ji-Yeun; Teranishi, Yasuhiro; Kihara, Takahiro; Yamamoto, Natsuko Goto; Inoue, Mitsuhiro; Hosia, Waltteri; Hashimoto, Masakazu; Winblad, Bengt; Frykman, Susanne; Tjernberg, Lars O.

    2012-01-01

    In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease. PMID:22315232

  6. Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

    PubMed

    Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

    2001-08-01

    A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

  7. Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis.

    PubMed

    Agallou, Maria; Athanasiou, Evita; Samiotaki, Martina; Panayotou, George; Karagouni, Evdokia

    2016-01-01

    Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests. PMID:26906226

  8. Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis

    PubMed Central

    Samiotaki, Martina; Panayotou, George; Karagouni, Evdokia

    2016-01-01

    Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests. PMID:26906226

  9. Strategies and computational tools for improving randomized protein libraries.

    PubMed

    Patrick, Wayne M; Firth, Andrew E

    2005-10-01

    In the last decade, directed evolution has become a routine approach for engineering proteins with novel or altered properties. Concurrently, a trend away from purely 'blind' randomization strategies and towards more 'semi-rational' approaches has also become apparent. In this review, we discuss ways in which structural information and predictive computational tools are playing an increasingly important role in guiding the design of randomized libraries: web servers such as ConSurf-HSSP and SCHEMA allow the prediction of sites to target for producing functional variants, while algorithms such as GLUE, PEDEL and DRIVeR are useful for estimating library completeness and diversity. In addition, we review recent methodological developments that facilitate the construction of unbiased libraries, which are inherently more diverse than biased libraries and therefore more likely to yield improved variants.

  10. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

    PubMed

    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean.

  11. The importance of peptide detectability for protein identification, quantification, and experiment design in MS/MS proteomics

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Tang, Haixu

    2010-01-01

    Peptide detectability is defined as the probability that a peptide is identified in an LC-MS/MS experiment and has been useful in providing solutions to protein inference and label-free quantification. Previously, predictors for peptide detectability trained on standard or complex samples were proposed. Although the models trained on complex samples may benefit from the large training data sets, it is unclear to what extent they are affected by the unequal abundances of identified proteins. To address this challenge and improve detectability prediction, we present a new algorithm for the iterative learning of peptide detectability from complex mixtures. We provide evidence that the new method approximates detectability with useful accuracy and, based on its design, can be used to interpret the outcome of other learning strategies. We studied the properties of peptides from the bacterium Deinococcus radiodurans and found that at standard quantities, its tryptic peptides can be roughly classified as either detectable or undetectable, with a relatively small fraction having medium detectability. We extend the concept of detectability from peptides to proteins and apply the model to predict the behavior of a replicate LC-MS/MS experiment from a single analysis. Finally, our study summarizes a theoretical framework for peptide/protein identification and label-free quantification. PMID:21067214

  12. Identification of canine helper T-cell epitopes from the fusion protein of canine distemper virus

    PubMed Central

    Ghosh, Souravi; Walker, John; Jackson, David C

    2001-01-01

    The fusion protein of canine distemper virus (CDV-F), a 662 amino-acid envelope protein, was used as the target molecule for identification of canine T helper (Th) epitopes. A library of 94 peptides, each 17 residues in length overlapping by 10 residues and covering the entire sequence of CDV-F, was screened using a lymphocyte proliferation assay with peripheral blood mononuclear cells (PBMC) obtained from dogs inoculated with canine distemper virus (CDV) vaccine. Initially we observed low and inconsistent proliferation of PBMC in response to these peptides, even when using cells obtained from dogs that had received multiple doses of CDV. Subsequently, the use of expanded cell populations derived by in vitro stimulation of canine PBMC with pools of peptides allowed the identification of a number of putative canine Th-epitopes within the protein sequence of CDV-F. There were two major clusters of Th-epitopes identified close to the cleavage site of the F0 fusion protein, while some others were scattered in both the F1 and F2 fragments of the protein. Some of these peptides, in particular peptide 35 (p35), were stimulatory in dogs of different breeds and ages. The identification of such promiscuous canine Th-epitopes encouraged us to assemble p35 in tandem with luteinising hormone releasing hormone (LHRH) a 10 amino-acid residue synthetic peptide representing a B-cell epitope which alone induces no antibody in dogs. The totally synthetic immunogen was able to induce the production of very high titres of antibodies against LHRH in all dogs tested. These results indicate that p35 could be an ideal candidate for use as a Th-epitope for use in outbred dogs. PMID:11576221

  13. Identification of protein adduction using mass spectrometry: Protein adducts as biomarkers and predictors of toxicity mechanisms.

    PubMed

    Yang, Xiangkun; Bartlett, Michael G

    2016-03-15

    The determination of protein-xenobiotic adducts using mass spectrometry is an emerging area which allows detailed understanding of the underlying mechanisms involved in toxicity. These approaches can also be used to reveal potential biomarkers of exposure or toxic response. The following review covers studies of protein adducts resulting from exposure to a wide variety of xenobiotics including organophosphates, polycyclic aromatic hydrocarbons, acetaminophen, alkylating agents and other related compounds. PMID:26842586

  14. Analysis and identification of toxin targets by topological properties in protein-protein interaction network.

    PubMed

    Yang, Lei; Wang, Jizhe; Wang, Huiping; Lv, Yingli; Zuo, Yongchun; Jiang, Wei

    2014-05-21

    Proteins do not exert their function in isolation of one another, but interact together in protein-protein interaction (PPI) networks. Analysis of topological properties of proteins in the PPI network is very helpful to understand the function of proteins. However, until recently, no one has ever undertaken to investigate toxin targets by topological properties. In this study, for the first time, 12 topological properties are used to investigate the characteristics of toxin targets in the PPI network. Most of the topological properties are found to be statistically discriminative between toxin targets and other proteins, and toxin targets tend to play more important roles in the PPI network. In addition, based on the topological properties and the sequence information, support vector machine (SVM) is used to predict toxin targets. The results obtained by the jackknife test and 10-fold cross validation are encouraging, indicating that SVM is a useful tool for predicting toxin targets, or at least can play complementary roles in relevant areas.

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

    SciTech Connect

    Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique . E-mail: vkruys@ulb.ac.be

    2006-04-28

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

  16. Identification of a cDNA encoding a novel small secretory protein, neurosecretory protein GL, in the chicken hypothalamic infundibulum.

    PubMed

    Ukena, Kazuyoshi; Iwakoshi-Ukena, Eiko; Taniuchi, Shusuke; Bessho, Yuki; Maejima, Sho; Masuda, Keiko; Shikano, Kenshiro; Kondo, Kunihiro; Furumitsu, Megumi; Tachibana, Tetsuya

    2014-03-28

    To find novel neuropeptide and/or peptide hormone precursors in the avian brain, we performed a cDNA subtractive screen of the chicken hypothalamic infundibulum, which contains one of the feeding and neuroendocrine centers. After sequencing 596 clones, we identified a novel cDNA encoding a previously unknown protein. The deduced precursor protein consisted of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. This small protein was flanked at the N-terminus by a signal peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated specific expression of the NPGL precursor mRNA in the hypothalamic infundibulum. Furthermore, the mRNA levels in the hypothalamic infundibulum increased during post-hatching development. In situ hybridization analysis showed that the cells containing the NPGL precursor mRNA were localized in the medial mammillary nucleus and infundibular nucleus within the hypothalamic infundibulum of 8- and 15-day-old chicks. Subcutaneous infusion of NPGL in chicks increased body weight gain without affecting food intake. To our knowledge, this is the first report to describe the identification and localization of the NPGL precursor mRNA and the function of its translated product in animals. Our findings indicate that NPGL may participate in the growth process in chicks.

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

    NASA Technical Reports Server (NTRS)

    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.

  18. Identification and Characterisation of Simiate, a Novel Protein Linked to the Fragile X Syndrome

    PubMed Central

    Derlig, Kristin; Gießl, Andreas; Brandstätter, Johann Helmut; Enz, Ralf; Dahlhaus, Regina

    2013-01-01

    A strict regulation of protein expression during developmental stages and in response to environmental signals is essential to every cell and organism. Recent research has shown that the mammalian brain is particularly sensitive to alterations in expression patterns of specific proteins and cognitive deficits as well as autistic behaviours have been linked to dysregulated protein expression. An intellectual disability characterised by changes in the expression of a variety of proteins is the fragile X syndrome. Due to the loss of a single mRNA binding protein, the Fragile X Mental Retardation Protein FMRP, vast misregulation of the mRNA metabolism is taking place in the disease. Here, we present the identification and characterisation of a novel protein named Simiate, whose mRNA contains several FMRP recognition motifs and associates with FMRP upon co-precipitation. Sequence analysis revealed that the protein evolved app. 1.7 billion years ago when eukaryotes developed. Applying antibodies generated against Simiate, the protein is detected in a variety of tissues, including the mammalian brain. On the subcellular level, Simiate localises to somata and nuclear speckles. We show that Simiate and nuclear speckles experience specific alterations in FMR1-/- mice. An antibody-based block of endogenous Simiate revealed that the protein is essential for cell survival. These findings suggest not only an important role for Simiate in gene transcription and/or RNA splicing, but also provide evidence for a function of nuclear speckles in the fragile X syndrome. Indeed, transcription and splicing are two fundamental mechanisms to control protein expression, that underlie not only synaptic plasticity and memory formation, but are also affected in several diseases associated with mental disabilities. PMID:24349419

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

    PubMed

    Cheng, Yiming; Perocchi, Fabiana

    2015-07-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Membrane protein isolation and identification by covalent binding for proteome research.

    PubMed

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, Xiangmin

    2015-11-01

    A novel method to achieve highly efficient identification of membrane proteins (MPs) has been developed based on a covalent binding (CB) strategy. For this purpose, magnetic nanoparticles coated with a PEG layer were synthesized. The PEG chain end was functionalized to form the PEG-tresyl group, which is an octopus-like long arm to capture the free amino groups of MPs. The long arm could be used to bind proteins in a high concentration of the SDS medium. Then, the SDS and interfering substances were completely depleted by washing. The CB proteins could form a molecular monolayer on the surface of the nanoparticles in the denatured state, which was significantly favorable for the proteolysis of MPs. Therefore, isolation with CB and highly efficient digestion resulted in a larger scale of MPs. The method has been verified by a proteome identification of mouse liver samples. A total of 2946 MPs were identified in an MP fraction. A total of 1505 proteins were characterized as integral MPs, and 735 MPs were identified beyond the largest database summarized by PeptideAtlas. This approach has great potential for membrane proteome research. PMID:26376103

  2. Identification of repetitive units in protein structures with ReUPred.

    PubMed

    Hirsh, Layla; Piovesan, Damiano; Paladin, Lisanna; Tosatto, Silvio C E

    2016-06-01

    Over the last decade, numerous studies have demonstrated the fundamental importance of tandem repeat (TR) proteins in many biological processes. A plethora of new repeat structures have also been solved. The recently published RepeatsDB provides information on TR proteins. However, a detailed structural characterization of repetitive elements is largely missing, as repeat unit annotation is manually curated and currently covers only 3 % of the bona fide TR proteins. Repeat Protein Unit Predictor (ReUPred) is a novel method for the fast automatic prediction of repeat units and repeat classification using an extensive Structure Repeat Unit Library (SRUL) derived from RepeatsDB. ReUPred uses an iterative structural search against the SRUL to find repetitive units. On a test set of solenoid proteins, ReUPred is able to correctly detect 92 % of the proteins. Unlike previous methods, it is also able to correctly classify solenoid repeats in 89 % of cases. It also outperforms two recent state-of-the-art methods for the repeat unit identification problem. The accurate prediction of repeat units increases the number of annotated repeat units by an order of magnitude compared to the sequence-based Pfam classification. ReUPred is implemented in Python for Linux and freely available from the URL: http://protein.bio.unipd.it/reupred/ .

  3. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

    PubMed Central

    Saritas-Yildirim, Banu; Pliner, Hannah A.; Ochoa, Angelica; Silva, Elena M.

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development. PMID:26327321

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

    PubMed Central

    Debarba, João Antonio; Monteiro, Karina Mariante; Moura, Hercules; Barr, John R.; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

    2015-01-01

    Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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. PMID:26393918

  5. SCNProDB: A database for the identification of soybean cyst nematode proteins

    PubMed Central

    Natarajan, Savithiry; Tavakolan, Mona; Alkharouf, Nadim W; Matthews, Benjamin F

    2014-01-01

    Soybean cyst nematode (Heterodera glycines, SCN) is the most destructive pathogen of soybean around the world. Crop rotation and resistant cultivars are used to mitigate the damage of SCN, but these approaches are not completely successful because of the varied SCN populations. Thus, the limitations of these practices with soybean dictate investigation of other avenues of protection of soybean against SCN, perhaps through genetically engineering of broad resistance to SCN. For better understanding of the consequences of genetic manipulation, elucidation of SCN protein composition at the subunit level is necessary. We have conducted studies to determine the composition of SCN proteins using a proteomics approach in our laboratory using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate SCN proteins and to characterize the proteins further using mass spectrometry. Our analysis resulted in the identification of several hundred proteins. In this investigation, we developed a web based database (SCNProDB) containing protein information obtained from our previous published studies. This database will be useful to scientists who wish to develop SCN resistant soybean varieties through genetic manipulation and breeding efforts. The database is freely accessible from: http://bioinformatics.towson.edu/Soybean_SCN_proteins_2D_Gel_DB/Gel1.aspx PMID:25097385

  6. Identification of repetitive units in protein structures with ReUPred.

    PubMed

    Hirsh, Layla; Piovesan, Damiano; Paladin, Lisanna; Tosatto, Silvio C E

    2016-06-01

    Over the last decade, numerous studies have demonstrated the fundamental importance of tandem repeat (TR) proteins in many biological processes. A plethora of new repeat structures have also been solved. The recently published RepeatsDB provides information on TR proteins. However, a detailed structural characterization of repetitive elements is largely missing, as repeat unit annotation is manually curated and currently covers only 3 % of the bona fide TR proteins. Repeat Protein Unit Predictor (ReUPred) is a novel method for the fast automatic prediction of repeat units and repeat classification using an extensive Structure Repeat Unit Library (SRUL) derived from RepeatsDB. ReUPred uses an iterative structural search against the SRUL to find repetitive units. On a test set of solenoid proteins, ReUPred is able to correctly detect 92 % of the proteins. Unlike previous methods, it is also able to correctly classify solenoid repeats in 89 % of cases. It also outperforms two recent state-of-the-art methods for the repeat unit identification problem. The accurate prediction of repeat units increases the number of annotated repeat units by an order of magnitude compared to the sequence-based Pfam classification. ReUPred is implemented in Python for Linux and freely available from the URL: http://protein.bio.unipd.it/reupred/ . PMID:26898549

  7. Identification of DNA-binding and protein-binding proteins using enhanced graph wavelet features.

    PubMed

    Zhu, Yuan; Zhou, Weiqiang; Dai, Dao-Qing; Yan, Hong

    2013-01-01

    Interactions between biomolecules play an essential role in various biological processes. For predicting DNA-binding or protein-binding proteins, many machine-learning-based techniques have used various types of features to represent the interface of the complexes, but they only deal with the properties of a single atom in the interface and do not take into account the information of neighborhood atoms directly. This paper proposes a new feature representation method for biomolecular interfaces based on the theory of graph wavelet. The enhanced graph wavelet features (EGWF) provides an effective way to characterize interface feature through adding physicochemical features and exploiting a graph wavelet formulation. Particularly, graph wavelet condenses the information around the center atom, and thus enhances the discrimination of features of biomolecule binding proteins in the feature space. Experiment results show that EGWF performs effectively for predicting DNA-binding and protein-binding proteins in terms of Matthew's correlation coefficient (MCC) score and the area value under the receiver operating characteristic curve (AUC). PMID:24334394

  8. Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection

    PubMed Central

    Zhou, Fangbin; Xu, Xindong; Wu, Sijia; Cui, Xiaobing; Fan, Lin; Pan, Weiqing

    2015-01-01

    The lack of effective and accurate diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. The current serodiagnostics for TB are inadequate mainly due to lack of TB-specific antigens with highly accurate diagnosis. In the current study, we aimed to identify novel diagnostic antigens using glutathione S-transferase (GST)-fusion protein technique. We determined the reactivity of these recombinant proteins arrayed in solution and on GSH-immobilized microplates with TB patient sera. Of 409 TB proteins produced, ninety-two yielded seropositive reactions, fourteen including eight novel proteins showed strong immunoreactivity. Further, six were selected and constructed as a multiple-antigen combination set through analysis of various combinations. A comparative study of the multiple-antigen combination set and a commercially available kit revealed that the combination set showed 66.3% (95% CI 60.5–71.8) sensitivity, which was significantly higher than that of the commercial kit [31.6% (95% CI 26.3–37.3)]. The specificity of both methods was similar at 89.6% (95% CI 83.3–95.4) and 90.6% (95% CI 83.0–95.6), respectively. This study provides a set of novel diagnostic protein markers with great potential for the development of novel diagnostic tools for active TB. PMID:26481294

  9. Identification of arabidopsis proteins that interact with the cauliflower mosaic virus (CaMV) movement protein.

    PubMed

    Huang, Z; Andrianov, V M; Han, Y; Howell, S H

    2001-11-01

    Gene I of cauliflower mosaic virus (CaMV) encodes a protein that is required for virus movement. The CaMV movement protein (MP) was used in a yeast 2-hybrid system to screen an Arabidopsis cDNA library for cDNAs encoding MP-interacting (MPI) proteins. Three different clones were found encoding proteins (MPI1, -2 and -7) that interact with the N-terminal third of the CaMV MP. The interaction in the 2-hybrid system between MPI7 and CaMV MP mutants correlated with the infectivity of the mutants. A non-infectious MP mutant, ER2A, with two amino acid changes in the N-terminal third of the MP failed to interact with MPI7, while an infectious second-site mutant, that differed from ER2A by only a single amino acid change, interacted in the 2-hybrid system. MPI7 is encoded by a member of a large, but diverse gene family in Arabidopsis. MPI7 is related in sequence, size and hydropathy profile to mammalian proteins (such as rat PRA1) described as a rab acceptor. The gene encoding MPI7 is expressed widely is Arabidopsis plants. and in transgenic plants the MPI7:GFP fusion protein is localized in the cytoplasm, concentrated in punctate spots. In protoplasts transfected with CFP:MP and MPI7:YFP, CFP:MP colocalized to some of the sites where MPI7:YFP is expressed. At these sites, fluorescence resonance energy transfer (FRET) between fluorophores was observed indicating an interaction in planta between the CaMV MP and MPI7.

  10. Improved Computation of Protein-Protein Relative Binding Energies with the Nwat-MMGBSA Method.

    PubMed

    Maffucci, Irene; Contini, Alessandro

    2016-09-26

    A MMGBSA variant (here referred to as Nwat-MMGBSA), based on the inclusion of a certain number of explicit water molecules (Nwat) during the calculations, has been tested on a set of 20 protein-protein complexes, using the correlation between predicted and experimental binding energy as the evaluation metric. Besides the Nwat parameter, the effect of the force field, the molecular dynamics simulation length, and the implicit solvent model used in the MMGBSA analysis have been also evaluated. We found that considering 30 interfacial water molecules improved the correlation between predicted and experimental binding energies by up to 30%, compared to the standard approach. Moreover, the correlation resulted in being rather sensitive to the force field and, to a minor extent, to the implicit solvent model and to the length of the MD simulation. PMID:27500550

  11. An Improved Identification Method of Magnetizing Inrush Current Phenomena in Distribution System

    NASA Astrophysics Data System (ADS)

    Maeda, Tatsuhiko; Naitoh, Tadashi; Toyama, Atsushi; Takeda, Keiki

    In this paper, the authors propose an improved identification method of the magnetizing inrush current phenomena. A first improvement is the development of determinant method about the transformer connection, which gives a calculation method of magnetic flux, using with theoretical pattern of inrush current ratio. And then, since the Aitken's Δ2-process, which uses the estimation of the saturation on/off time, has many extrema, it is difficult to determinate the saturation on/off time. Therefore, using the pattern of derivative current, a new determinant method of the saturation on/off time is developed.

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

    PubMed Central

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

    2012-01-01

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

  13. Identification and characterization of a surface-exposed, 66-kilodalton protein from Borrelia burgdorferi.

    PubMed Central

    Probert, W S; Allsup, K M; LeFebvre, R B

    1995-01-01

    The surface-exposed antigens of Borrelia burgdorferi represent important targets for the development of a protective immune response. We have identified a proteinase K-accessible, 66-kDa protein from B. burgdorferi and have demonstrated that at least a portion of this protein is surface exposed. The 66-kDa protein was purified by sequential extraction of spirochetes with butanol and Triton X-114 followed by preparative gel electrophoresis. Polyclonal antibodies developed against the purified 66-kDa protein were Borrelia spp. specific, whereas a monoclonal antibody, Route 66, displayed a genospecies-specific pattern of recognition for the 66-kDa protein. N-terminal amino acid sequence was obtained from an internal fragment, a truncated version, and the full-length form of the 66-kDa protein. A search of protein and gene databases for homologous sequences yielded a match with the predicted amino acid sequence from a segment of B. burgdorferi chromosomal DNA (P. A. Rosa, D. Hogan, and T. G. Schwan, J. Clin. Microbiol. 29:524-532, 1991). The construction of primers based on this DNA sequence and the N-terminal amino acid sequence allowed the amplification and cloning of the 66-kDa-protein gene. The identity of the cloned gene was verified by the recognition of the expressed gene product by Route 66. Pulsed-field gel electrophoresis and Southern blot analysis were performed to confirm the chromosomal location of the 66-kDa-protein gene. This study describes the identification and cloning of the first chromosomally encoded, surface-exposed protein from B. burgdoferi. PMID:7729905

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

    PubMed

    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.

  15. Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

    PubMed Central

    Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Bhargava, Kalpana

    2014-01-01

    Background Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. Methods In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. Results Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. Conclusion/Significance This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers. PMID:24842778

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

    PubMed Central

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

    2014-01-01

    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. PMID:25351924

  17. Identification of Asp isomerization in proteins by ¹⁸O labeling and tandem mass spectrometry.

    PubMed

    Zhang, Jennifer; Katta, Viswanatham

    2012-01-01

    Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) via succinimide intermediate is a common route of degradation for proteins that can affect their structural integrity. As Asp/isoAsp is isobaric in mass, it is difficult to identify the site of modification by LC-MS/MS peptide mapping. Here, we describe an approach to label the Asp residue involved in isomerization at the protein level by hydrolyzing the succinimide intermediate in H₂¹⁸O. Tryptic digestion of this labeled protein will result in peptides containing the site of isomerization being 2 Da heavier than the ¹⁶O-containing counterparts, due to ¹⁸O incorporation during the hydrolysis process. Comparison of tandem mass spectra of isomerized peptides with and without ¹⁸O incorporation allows easy identification of the Asp residue involved. This method proved to be especially useful in identifying the sites when isomerization occurs in Asp-Asp motifs.

  18. Phytochemical-mediated Protein Expression Profiling and the Potential Applications in Therapeutic Drug Target Identifications.

    PubMed

    Wong, Fai-Chu; Tan, Siok-Thing; Chai, Tsun-Thai

    2016-07-29

    Many phytochemicals derived from edible medicinal plants have been investigated intensively for their various bioactivities. However, the detailed mechanism and their corresponding molecular targets frequently remain elusive. In this review, we present a summary of the research works done on phytochemical-mediated molecular targets, identified via proteomic approach. Concurrently, we also highlighted some pharmaceutical drugs which could be traced back to their origins in phytochemicals. For ease of presentation, these identified protein targets were categorized into two important healthcare-related fields, namely anti-bacterial and anti-cancer research. Through this review, we hope to highlight the usefulness of comparative proteomic as a powerful tool in phytochemical-mediated protein target identifications. Likewise, we wish to inspire further investigations on some of these protein targets identified over the last few years. With contributions from all researchers, the accumulative efforts could eventually lead to the discovery of some target-specific, low-toxicity therapeutic agents.

  19. Systematic Identification of Novel Protein Domain Families Associated with Nuclear Functions

    PubMed Central

    Doerks, Tobias; Copley, Richard R.; Schultz, Jörg; Ponting, Chris P.; Bork, Peer

    2002-01-01

    A systematic computational analysis of protein sequences containing known nuclear domains led to the identification of 28 novel domain families. This represents a 26% increase in the starting set of 107 known nuclear domain families used for the analysis. Most of the novel domains are present in all major eukaryotic lineages, but 3 are species specific. For about 500 of the 1200 proteins that contain these new domains, nuclear localization could be inferred, and for 700, additional features could be predicted. For example, we identified a new domain, likely to have a role downstream of the unfolded protein response; a nematode-specific signalling domain; and a widespread domain, likely to be a noncatalytic homolog of ubiquitin-conjugating enzymes. PMID:11779830

  20. Improving Information Technology Adoption and Implementation Through the Identification of Appropriate Benefits: Creating IMPROVE-IT

    PubMed Central

    Sittig, Dean F

    2007-01-01

    This paper describes the objectives of a collaborative initiative that attempts to provide the evidence that increased information technology (IT) capabilities, availability, and use lead directly to improved clinical quality, safety, and effectiveness within the inpatient hospital setting. This collaborative network has defined specific measurement indicators in an attempt to examine the existence, timing, and level of improvements in health outcomes that can be derived from IT investment. These indicators are in three areas: (1) IT costs (which includes both initial and ongoing investment), (2) IT infusion (ie, system availability, adoption, and deployment), and (3) health performance (eg, clinical efficacy, efficiency, quality, and effectiveness). Herein, we outline the theoretical framework, the methodology employed to create the metrics, and the benefits that can be obtained. PMID:17513287

  1. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  2. Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)*

    PubMed Central

    Champasa, Kanokwan; Longwell, Scott A.; Eldridge, Aimee M.; Stemmler, Elizabeth A.; Dube, Danielle H.

    2013-01-01

    Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the

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

    SciTech Connect

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    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.

  6. Identification of angiotensin-converting enzyme inhibitory proteins from mycelium of Pleurotus pulmonarius (oyster mushroom).

    PubMed

    Ibadallah, Badjie Xietaqieuallah; Abdullah, Noorlidah; Shuib, Adawiyah Suriza

    2015-01-01

    Pleurotus pulmonarius (grey oyster mushroom) has been acknowledged as a recuperative agent for many diseases in addition to its recognition as a nutritious provision. We performed a study on P. pulmonarius mycelium for an antihypertensive effect via the angiotensin-converting enzyme inhibitory activity. The preliminary assay on the mycelial water extract demonstrated that the angiotensin-converting enzyme inhibitory activity had an IC50 value of 720 µg/mL. Further protein purifications via ammonium sulphate precipitation and RP-HPLC resulted in 60× stronger angiotensin-converting enzyme inhibitory activity than that of the mycelial water extract (IC50 = 12 µg/mL). Protein identification and characterisation by MALDI-TOF/TOF, later corroborated by LC-MS/MS, indicated three proteins that are responsible for the blood pressure lowering effects via different mechanisms: serine proteinase inhibitor-like protein, nitrite reductase-like protein, and DEAD/DEAH box RNA helicase-like protein. PMID:25590365

  7. Quantification, 2DE analysis and identification of enriched glycosylated proteins from mouse muscles: Difficulties and alternatives.

    PubMed

    de Fátima MenegociEugênio, Patrícia; Assunção, Nilson Antonio; Sciandra, Francesca; Aquino, Adriano; Brancaccio, Andrea; Carrilho, Emanuel

    2016-01-01

    One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a pre-purified sample using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study; the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method, however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie Blue R-250 dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign 12 new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue.

  8. Screening and identification of the differential proteins in kidney with complete unilateral ureteral obstruction

    PubMed Central

    Zhao, Qi; Yang, Yi; Wang, Chang-Lin; Hou, Ying; Chen, Hui

    2015-01-01

    Obstructive nephropathy is a major cause of renal failure, particularly in infants and children, and indications for therapeutic intervention remain highly controversial. There is a great need for the development of new methods to monitor patients, and the biomarker research field is a promising approach for this purpose to be used as prognostic tools for early disease detection and the choice of the optimal treatment and monitoring. Here, we presented our comparative proteomics study of rat kidney with complete unilateral ureteral obstruction (CUUO). Proteins from the groups of CUUO and corresponding sham rat kidney tissues were subjected to 2-D gel electrophoresis, and then protein identification by mass spectrometry. We identified 39 proteins with differential expression between kidney tissues from sham operated group and those with CUUO. These identified proteins were reported to be involved in cell apoptosis, energy metabolism and injuries of mitochondrion and oxidative stress, and so on. We confirmed 3 identified proteins by immunoblot analysis and immunofluorescence staining and assessed their mRNA levels in renal tissues. Our results demonstrate protein alterations that reflect the pathological situation of the obstructed kidneys, which may help understand the relationship between oxidative stress and obstructive nephropathy. PMID:26045767

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

    PubMed

    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.

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

    PubMed

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

    2015-02-01

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

  11. The Effect of Peptide Identification Search Algorithms on MS2-Based Label-Free Protein Quantification

    PubMed Central

    Degroeve, Sven; Staes, An; De Bock, Pieter-Jan

    2012-01-01

    Abstract Several approaches exist for the quantification of proteins in complex samples processed by liquid chromatography-mass spectrometry followed by fragmentation analysis (MS2). One of these approaches is label-free MS2-based quantification, which takes advantage of the information computed from MS2 spectrum observations to estimate the abundance of a protein in a sample. As a first step in this approach, fragmentation spectra are typically matched to the peptides that generated them by a search algorithm. Because different search algorithms identify overlapping but non-identical sets of peptides, here we investigate whether these differences in peptide identification have an impact on the quantification of the proteins in the sample. We therefore evaluated the effect of using different search algorithms by examining the reproducibility of protein quantification in technical repeat measurements of the same sample. From our results, it is clear that a search engine effect does exist for MS2-based label-free protein quantification methods. As a general conclusion, it is recommended to address the overall possibility of search engine-induced bias in the protein quantification results of label-free MS2-based methods by performing the analysis with two or more distinct search engines. PMID:22804230

  12. Diesel engine noise source identification based on EEMD, coherent power spectrum analysis and improved AHP

    NASA Astrophysics Data System (ADS)

    Zhang, Junhong; Wang, Jian; Lin, Jiewei; Bi, Fengrong; Guo, Qian; Chen, Kongwu; Ma, Liang

    2015-09-01

    As the essential foundation of noise reduction, many noise source identification methods have been developed and applied to engineering practice. To identify the noise source in the board-band frequency of different engine parts at various typical speeds, this paper presents an integrated noise source identification method based on the ensemble empirical mode decomposition (EEMD), the coherent power spectrum analysis, and the improved analytic hierarchy process (AHP). The measured noise is decomposed into several IMFs with physical meaning, which ensures the coherence analysis of the IMFs and the vibration signals are meaningful. An improved AHP is developed by introducing an objective weighting function to replace the traditional subjective evaluation, which makes the results no longer dependent on the subject performances and provides a better consistency in the meantime. The proposed noise identification model is applied to identifying a diesel engine surface radiated noise. As a result, the frequency-dependent contributions of different engine parts to different test points at different speeds are obtained, and an overall weight order is obtained as oil pan  >  left body  >  valve chamber cover  >  gear chamber casing  >  right body  >  flywheel housing, which provides an effectual guidance for the noise reduction.

  13. Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment

    PubMed Central

    Dasari, Surendra; Chambers, Matthew C.; Martinez, Misti A.; Carpenter, Kristin L.; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo J.; Tabb, David L.

    2012-01-01

    Spectral libraries have emerged as a viable alternative to protein sequence databases for peptide identification. These libraries contain previously detected peptide sequences and their corresponding tandem mass spectra (MS/MS). Search engines can then identify peptides by comparing experimental MS/MS scans to those in the library. Many of these algorithms employ the dot product score for measuring the quality of a spectrum-spectrum match (SSM). This scoring system does not offer a clear statistical interpretation and ignores fragment ion m/z discrepancies in the scoring. We developed a new spectral library search engine, Pepitome, which employs statistical systems for scoring SSMs. Pepitome outperformed the leading library search tool, SpectraST, when analyzing data sets acquired on three different mass spectrometry platforms. We characterized the reliability of spectral library searches by confirming shotgun proteomics identifications through RNA-Seq data. Applying spectral library and database searches on the same sample revealed their complementary nature. Pepitome identifications enabled the automation of quality analysis and quality control (QA/QC) for shotgun proteomics data acquisition pipelines. PMID:22217208

  14. l2 Multiple Kernel Fuzzy SVM-Based Data Fusion for Improving Peptide Identification.

    PubMed

    Jian, Ling; Xia, Zhonghang; Niu, Xinnan; Liang, Xijun; Samir, Parimal; Link, Andrew J

    2016-01-01

    SEQUEST is a database-searching engine, which calculates the correlation score between observed spectrum and theoretical spectrum deduced from protein sequences stored in a flat text file, even though it is not a relational and object-oriental repository. Nevertheless, the SEQUEST score functions fail to discriminate between true and false PSMs accurately. Some approaches, such as PeptideProphet and Percolator, have been proposed to address the task of distinguishing true and false PSMs. However, most of these methods employ time-consuming learning algorithms to validate peptide assignments [1] . In this paper, we propose a fast algorithm for validating peptide identification by incorporating heterogeneous information from SEQUEST scores and peptide digested knowledge. To automate the peptide identification process and incorporate additional information, we employ l2 multiple kernel learning (MKL) to implement the current peptide identification task. Results on experimental datasets indicate that compared with state-of-the-art methods, i.e., PeptideProphet and Percolator, our data fusing strategy has comparable performance but reduces the running time significantly. PMID:26394437

  15. Identification of ligand-binding pockets in proteins using residue preference methods.

    PubMed

    Qiu, Zhijun; Wang, Xicheng

    2009-01-01

    Identification of ligand-binding pockets in proteins is pivotal to protein function definition and drug discovery. In this study, we focus on determining the binding pockets in proteins for potential ligands without any a priori knowledge. Three methods based upon residue preference concept are proposed to predict ligand-binding pockets, where we deal with three types of residue preference (residue based, atom based and atom-contact-pair based preference), respectively. Two test sets were chosen to examine the proposed methods. Two different identification rules (named Top1 and Top2) are used to detect ligand-binding pockets. The results show that the atom-contact-pair method has good accuracy and high efficiency, better than the other two methods. By means of preference analysis for amino acids and atom-contact-pairs, we find that Gly and atom-contact-pairs on aromatic residues appear at ligand-binding pockets more frequently. The former favors pocket flexibility, and the latter shows that aggregate hydrophobic surface may play an important role in complex formation.

  16. Redox proteomics: Methods for the identification and enrichment of redox-modified proteins and their applications.

    PubMed

    Lennicke, Claudia; Rahn, Jette; Heimer, Nadine; Lichtenfels, Rudolf; Wessjohann, Ludger A; Seliger, Barbara

    2016-01-01

    PTMs are defined as covalent additions to functional groups of amino acid residues in proteins like phosphorylation, glycosylation, S-nitrosylation, acetylation, methylation, lipidation, SUMOylation as well as oxidation. Oxidation of proteins has been characterized as a double-edged sword. While oxidative modifications, in particular of cysteine residues, are widely involved in the regulation of cellular homeostasis, oxidative stress resulting in the oxidation of biomolecules along with the disruption of their biological functions can be associated with the development of diseases, such as cancer, diabetes, and neurodegenerative diseases, respectively. This is also the case for advanced glycation end products, which result from chemical reactions of keto compounds such as oxidized sugars with proteins. The role of oxidative modifications under physiological and pathophysiological conditions remains largely unknown. Recently, novel technologies have been established that allow the enrichment, identification, and characterization of specific oxidative PTMs (oxPTMs). This is essential to develop strategies to prevent and treat diseases that are associated with oxidative stress. Therefore this review will focus on (i) the methods and technologies, which are currently applied for the detection, identification, and quantification of oxPTMs including the design of high throughput approaches and (ii) the analyses of oxPTMs related to physiological and pathological conditions.

  17. Proteomic identification of FHL1 as the protein mutated in human reducing body myopathy

    PubMed Central

    Schessl, Joachim; Zou, Yaqun; McGrath, Meagan J.; Cowling, Belinda S.; Maiti, Baijayanta; Chin, Steven S.; Sewry, Caroline; Battini, Roberta; Hu, Ying; Cottle, Denny L.; Rosenblatt, Michael; Spruce, Lynn; Ganguly, Arupa; Kirschner, Janbernd; Judkins, Alexander R.; Golden, Jeffrey A.; Goebel, Hans-Hilmar; Muntoni, Francesco; Flanigan, Kevin M.; Mitchell, Christina A.; Bönnemann, Carsten G.

    2008-01-01

    Reducing body myopathy (RBM) is a rare disorder causing progressive muscular weakness characterized by aggresome-like inclusions in the myofibrils. Identification of genes responsible for RBM by traditional genetic approaches has been impossible due to the frequently sporadic occurrence in affected patients and small family sizes. As an alternative approach to gene identification, we used laser microdissection of intracytoplasmic inclusions identified in patient muscle biopsies, followed by nanoflow liquid chromatography–tandem mass spectrometry and proteomic analysis. The most prominent component of the inclusions was the Xq26.3-encoded four and a half LIM domain 1 (FHL1) protein, expressed predominantly in skeletal but also in cardiac muscle. Mutational analysis identified 4 FHL1 mutations in 2 sporadic unrelated females and in 2 families with severely affected boys and less-affected mothers. Transfection of kidney COS-7 and skeletal muscle C2C12 cells with mutant FHL1 induced the formation of aggresome-like inclusions that incorporated both mutant and wild-type FHL1 and trapped other proteins in a dominant-negative manner. Thus, a novel laser microdissection/proteomics approach has helped identify both inherited and de novo mutations in FHL1, thereby defining a new X-linked protein aggregation disorder of muscle. PMID:18274675

  18. Identification of ligand-binding pockets in proteins using residue preference methods.

    PubMed

    Qiu, Zhijun; Wang, Xicheng

    2009-01-01

    Identification of ligand-binding pockets in proteins is pivotal to protein function definition and drug discovery. In this study, we focus on determining the binding pockets in proteins for potential ligands without any a priori knowledge. Three methods based upon residue preference concept are proposed to predict ligand-binding pockets, where we deal with three types of residue preference (residue based, atom based and atom-contact-pair based preference), respectively. Two test sets were chosen to examine the proposed methods. Two different identification rules (named Top1 and Top2) are used to detect ligand-binding pockets. The results show that the atom-contact-pair method has good accuracy and high efficiency, better than the other two methods. By means of preference analysis for amino acids and atom-contact-pairs, we find that Gly and atom-contact-pairs on aromatic residues appear at ligand-binding pockets more frequently. The former favors pocket flexibility, and the latter shows that aggregate hydrophobic surface may play an important role in complex formation. PMID:19689426

  19. Immunochemical Methods Applied to Art-Historical Materials: Identification and Localization of Proteins by ELISA and IFM.

    PubMed

    Cartechini, Laura; Palmieri, Melissa; Vagnini, Manuela; Pitzurra, Lucia

    2016-02-01

    Despite the large diffusion of natural organic substances in art-historical materials, their characterization presents many challenges due to the chemical complexity and instability with respect to degradation processes. Among natural products, proteins have been largely used in the past as binders but also as adhesives or additives in coating layers. Nevertheless, biological identification of proteins in art-historical objects is one of the most recent achievements obtained in heritage science thanks to the development of specifically tailored bio-analytical strategies. In the context of this active emerging discipline, immunological methods stand out for sensitivity, specificity and versatility for both protein recognition and localization in micro-samples. Furthermore, the growing use of immunological techniques for advanced diagnostics and clinical applications ensures continuous improvement in their analytical performance. Considering such, this review provides an overview of the most recent applications of enzyme linked immunosorbent assay and immunofluorescence microscopy techniques in the field of heritage materials. Specifically, the main strengths and potentials of the two techniques as well as their limits and drawbacks are presented and discussed herein. PMID:27572988

  20. Phytic acid reduction in soy protein improves zinc bioavailability

    SciTech Connect

    Zhou, J.R.; Wong, M.S.; Burns, R.A.; Erdman, J.W. Jr. Mead Johnson Research Center, Evansville, IN )

    1991-03-15

    The objective of this study was to confirm previous studies that have suggested that reduction of phytic acid in soy improved zinc bioavailability (BV). Two commercially-produced soybean isolates containing either a normal phytic acid level or a reduced level were formulated into diets so as to provide 6 or 9 ppm zinc. Control diets were egg white protein-based and contained 3, 6 or 9 ppm zinc from zinc carbonate. Weanling male rats were fed these diets for 21 days and food intake and weight gain monitored. Slope ratio analysis of total tibia zinc content compared to total zinc intake revealed that zinc BV from reduced phytic acid soy isolate-containing diets was indistinguishable from control egg white diets. In contrast, zinc BV from normal soy isolate diets was significantly reduced compared to reduced phytic acid and control diets. These results coupled with other results indicate that phytic acid is the inhibitory factor in soybean products that results in reduced zinc BV.

  1. Heterodimeric protein complex identification by naïve Bayes classifiers

    PubMed Central

    2013-01-01

    Background Protein complexes are basic cellular entities that carry out the functions of their components. It can be found that in databases of protein complexes of yeast like CYC2008, the major type of known protein complexes is heterodimeric complexes. Although a number of methods for trying to predict sets of proteins that form arbitrary types of protein complexes simultaneously have been proposed, it can be found that they often fail to predict heterodimeric complexes. Results In this paper, we have designed several features characterizing heterodimeric protein complexes based on genomic data sets, and proposed a supervised-learning method for the prediction of heterodimeric protein complexes. This method learns the parameters of the features, which are embedded in the naïve Bayes classifier. The log-likelihood ratio derived from the naïve Bayes classifier with the parameter values obtained by maximum likelihood estimation gives the score of a given pair of proteins to predict whether the pair is a heterodimeric complex or not. A five-fold cross-validation shows good performance on yeast. The trained classifiers also show higher predictability than various existing algorithms on yeast data sets with approximate and exact matching criteria. Conclusions Heterodimeric protein complex prediction is a rather harder problem than heteromeric protein complex prediction because heterodimeric protein complex is topologically simpler. However, it turns out that by designing features specialized for heterodimeric protein complexes, predictability of them can be improved. Thus, the design of more sophisticate features for heterodimeric protein complexes as well as the accumulation of more accurate and useful genome-wide data sets will lead to higher predictability of heterodimeric protein complexes. Our tool can be downloaded from http://imi.kyushu-u.ac.jp/~om/. PMID:24299017

  2. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    DOEpatents

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

  3. Characterization of quinoa seed proteome combining different protein precipitation techniques: Improvement of knowledge of nonmodel plant proteomics.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Piovesana, Susy; Stampachiacchiere, Serena; Ventura, Salvatore; Zenezini Chiozzi, Riccardo; Laganà, Aldo

    2015-03-01

    A shotgun proteomics approach was used to characterize the quinoa seed proteome. To obtain comprehensive proteomic data from quinoa seeds three different precipitation procedures were employed: MeOH/CHCl3 /double-distilled H2 O, acetone either alone or with trichloroacetic acid; the isolated proteins were then in-solution digested and the resulting peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. However, since quinoa is a nonmodel plant species, only a few protein sequences are included in the most widely known protein sequence databases. To improve the data reliability a UniProt subdatabase, containing only proteins of Caryophillales order, was used. A total of 352 proteins were identified and evaluated both from a qualitative and quantitative point of view. This combined approach is certainly useful to increase the final number of identifications, but no particular class of proteins was extracted and identified in spite of the different chemistries and the different precipitation protocols. However, with respect to the other two procedures, from the relative quantitative analysis, based on the number of spectral counts, the trichloroacetic acid/acetone protocol was the best procedure for sample handling and quantitative protein extraction. This study could pave the way to further high-throughput studies on Chenopodium Quinoa.

  4. Characterization of quinoa seed proteome combining different protein precipitation techniques: Improvement of knowledge of nonmodel plant proteomics.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Piovesana, Susy; Stampachiacchiere, Serena; Ventura, Salvatore; Zenezini Chiozzi, Riccardo; Laganà, Aldo

    2015-03-01

    A shotgun proteomics approach was used to characterize the quinoa seed proteome. To obtain comprehensive proteomic data from quinoa seeds three different precipitation procedures were employed: MeOH/CHCl3 /double-distilled H2 O, acetone either alone or with trichloroacetic acid; the isolated proteins were then in-solution digested and the resulting peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. However, since quinoa is a nonmodel plant species, only a few protein sequences are included in the most widely known protein sequence databases. To improve the data reliability a UniProt subdatabase, containing only proteins of Caryophillales order, was used. A total of 352 proteins were identified and evaluated both from a qualitative and quantitative point of view. This combined approach is certainly useful to increase the final number of identifications, but no particular class of proteins was extracted and identified in spite of the different chemistries and the different precipitation protocols. However, with respect to the other two procedures, from the relative quantitative analysis, based on the number of spectral counts, the trichloroacetic acid/acetone protocol was the best procedure for sample handling and quantitative protein extraction. This study could pave the way to further high-throughput studies on Chenopodium Quinoa. PMID:25580831

  5. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  6. Identification of two bvg-repressed surface proteins of Bordetella pertussis.

    PubMed Central

    Stenson, T H; Peppler, M S

    1995-01-01

    Bordetella pertussis, the etiological agent of whooping cough, has the ability to modulate its phenotype in response to environmental conditions by using the BvgAS sensory transduction system which is encoded by the vir locus (now known as bvg). The BvgAS system is part of a large family of two-component sensory transduction systems which are common to a number of pathogenic bacteria. Although much is known about the proteins which exist in the B. pertussis virulent (X-mode or phase I) phenotype, relatively little is known about the proteins produced in the avirulent (C-mode or phase III) phenotype. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing techniques to demonstrate the existence of at least 22 vir-repressed molecules which are increased in the avirulent phenotype. In addition, a series of monoclonal antibodies which are specific for the surface of avirulent B. pertussis were developed. Using immunological and protein techniques, we characterized two of these antigens as surface-exposed proteins. One of these antigens is expressed only in B. pertussis but not in the related species B. parapertussis and B. bronchiseptica. The other antigen is also present in B. parapertussis and B. bronchiseptica but is expressed at lower levels which are not regulated by bvg. The identification and characterization of vir-repressed proteins (and the genes which encode and regulate them) may help elucidate a physiological role for modulation of this obligate human pathogen. PMID:7558280

  7. Identification and analysis of copine/BONZAI proteins among evolutionarily diverse plant species.

    PubMed

    Zou, Baohong; Hong, Xuexue; Ding, Yuan; Wang, Xiang; Liu, He; Hua, Jian

    2016-08-01

    Copines are evolutionarily conserved calcium-dependent membrane-binding proteins with potentially critical biological functions. In plants, the function of these proteins has not been analyzed except for in Arabidopsis thaliana where they play critical roles in development and disease resistance. To facilitate functional studies of copine proteins in crop plants, genome-wide identification, curation, and phylogeny analysis of copines in 16 selected plant species were conducted. All the identified 32 plant copines have conserved features of the two C2 domains (C2A and C2B) and the von Willebrand factor A (vWA) domain. Different from animal and protozoa copines, plant copines have glycine at the second residue potentially acquiring a unique protein myristoylation modification. Phylogenetic analysis suggests that copine was present as one copy when evolving from green algae to basal flowering plants, and duplicated before the divergence of monocots and dicots. In addition, gene expression and protein localization study of rice copines suggests both conserved and different properties of copines in dicots and monocots. This study will contribute to uncovering the role of copine genes in different plant species. PMID:27484220

  8. Rice transglutaminase gene: Identification, protein expression, functionality, light dependence and specific cell location.

    PubMed

    Campos, N; Castañón, S; Urreta, I; Santos, M; Torné, J M

    2013-05-01

    Transglutaminases (TGases), that catalyze post-translational modification of proteins, are scarcely known in plants. As part of a project to characterize transglutaminase genes in new plant species, the identification and characterization of a TGase in rice is presented. Using differential primers, a cDNA (tgo) of 1767bp from genomic rice DNA amplification was obtained. The primers were designed from the rice DNA sequence relatively homologous to the gene encoding active maize chloroplast TGase. Amino acid sequence of the deduced rice TGase protein (TGO) indicated that it contains the enzyme catalytic triad (Cys-His-Asp), three repeats, myristoylation domains and a leucine zipper motif. The TGO recombinant protein was characterized, showing specific activity regulation, and indicating that tgo encoded for an authentic TGase. Substrate preference and Ca(2+) dependent activity were also detected. In the rice plant TGO protein was immunolocalized in the grana chloroplasts, in protein vesicles near them, and in the bulliform cells. Immunoblot analyses, tgo mRNA expression, and TGase activity indicated that TGO expression in rice was light dependent and regulated by the illumination period. This work increases significantly our plant TGase understanding. Its functional role in rice, which is a good model system for C3 plants, is discussed.

  9. Identification of major Toxoneuron nigriceps venom proteins using an integrated transcriptomic/proteomic approach.

    PubMed

    Laurino, Simona; Grossi, Gerarda; Pucci, Pietro; Flagiello, Angela; Bufo, Sabino Aurelio; Bianco, Giuliana; Salvia, Rosanna; Vinson, S Bradleigh; Vogel, Heiko; Falabella, Patrizia

    2016-09-01

    Endoparasitoids in the order Hymenoptera are natural enemies of several herbivorous insect pest species. During oviposition they inject a mixture of factors, which include venom, into the host, ensuring the successful parasitism and the development of their progeny. Although these parasitoid factors are known to be responsible for host manipulation, such as immune system suppression, little is known about both identity and function of the majority of their venom components. To identify the major proteins of Toxoneuron nigriceps (Hymenoptera: Braconidae) venom, we used an integrated transcriptomic and proteomic approach. The tandem-mass spectrometric (LC-MS/MS) data combined with T. nigriceps venom gland transcriptome used as a reference database resulted in the identification of a total of thirty one different proteins. While some of the identified proteins have been described in venom from several parasitoids, others were identified for the first time. Among the identified proteins, hydrolases constituted the most abundant family followed by transferases, oxidoreductases, ligases, lyases and isomerases. The hydrolases identified in the T. nigriceps venom glands included proteases, peptidases and glycosidases, reported as common components of venom from several parasitoid species. Taken together, the identified proteins included factors that could potentially inhibit the host immune system, manipulate host physiological processes and host development, as well as provide nutrients to the parasitoid progeny, degrading host tissues by specific hydrolytic enzymes. The venom decoding provides us with information about the identity of candidate venom factors which could contribute to the success of parasitism, together with other maternal and embryonic factors. PMID:27388778

  10. Bio-guided identification of proteins for the diagnosis of cysticercosis in swine.

    PubMed

    Nativel, Priscilla; Rahantamalala, Anjanirina; Ramiandrisoa, Sitraka; Rasoamampianinaa, Virginie; Duchateau, Magalie; Chamot-Rooke, Julia; Guebey, Remy; Rasamoelina-Andriamanivo, Harentsoaniaina; Jambou, Ronan

    2016-04-15

    Neurocysticercosis (NCC) is one of the most prevalent parasitic infection of the brain and the most common cause of seizures in adults in tropical countries. Cysticercosis is caused by larvae of Taenia solium, a human tapeworm. Pig or humans are infected by ingestion of eggs in food contaminated by human feces. Diagnosis and treatment of pigs is a pillar of the control of the disease in a country. However current diagnostic tests are based on ELISA and/or Western blot using native antigens needing laboratory facilities not available in rural areas. Development of a pen side diagnostic test for swines, makes sense. Immunochromatographic test should be adapted for this purpose. To design it we started a bio-guided identification of new proteins in cysticercus fluid. Proteins were analyzed using ion exchange chromatography and 2D separation and were selected by Western blot analysis using sera from infected/non infected pigs. Spots from the Coomassie-stained gel corresponding to these proteins were then analyzed by mass spectroscopy and proteins were identified using a bank of Expressed Sequence Tags (EST) of T. solium. Eighteen new proteins of interest were identified and nine were selected for further development. PMID:26995717

  11. Identification and analysis of copine/BONZAI proteins among evolutionarily diverse plant species.

    PubMed

    Zou, Baohong; Hong, Xuexue; Ding, Yuan; Wang, Xiang; Liu, He; Hua, Jian

    2016-08-01

    Copines are evolutionarily conserved calcium-dependent membrane-binding proteins with potentially critical biological functions. In plants, the function of these proteins has not been analyzed except for in Arabidopsis thaliana where they play critical roles in development and disease resistance. To facilitate functional studies of copine proteins in crop plants, genome-wide identification, curation, and phylogeny analysis of copines in 16 selected plant species were conducted. All the identified 32 plant copines have conserved features of the two C2 domains (C2A and C2B) and the von Willebrand factor A (vWA) domain. Different from animal and protozoa copines, plant copines have glycine at the second residue potentially acquiring a unique protein myristoylation modification. Phylogenetic analysis suggests that copine was present as one copy when evolving from green algae to basal flowering plants, and duplicated before the divergence of monocots and dicots. In addition, gene expression and protein localization study of rice copines suggests both conserved and different properties of copines in dicots and monocots. This study will contribute to uncovering the role of copine genes in different plant species.

  12. Identification of proteins from prunus persica that interact with peach latent mosaic viroid.

    PubMed

    Dubé, Audrey; Bisaillon, Martin; Perreault, Jean-Pierre

    2009-12-01

    Peach latent mosaic viroid (PLMVd) is a small, single-stranded, circular RNA pathogen that infects Prunus persica trees. As with all other known viroids, the PLMVd genome does not encode any proteins. Consequently, it must interact with host cellular factors in order to ensure its life cycle. With the objective of identifying cellular proteins that interact with PLMVd, Northwestern hybridizations were performed using partially purified peach leaf extracts. Mass spectrometric analysis of the detected RNA-protein complexes led to the identification of six putative RNA-binding proteins. One of these was found to be elongation factor 1-alpha (eEF1A), and because of its known involvement in the replication and translation of various RNA viruses, further characterizations were performed. Initially, the existence of this interaction received support from an experiment that immunoprecipitated the eEF1A from a crude extract of infected peach leaves, coupled with reverse transcription-PCR detection of the PLMVd. Subsequently, eEF1A interaction with PLMVd strands of both polarities was confirmed in vitro by electrophoresis mobility shift assays, fluorescence spectroscopy, and the prediction of an altered PLMVd RNase mapping profile in the presence of the protein. The potential contribution of eEF1A to the molecular biology of PLMVd, including for viroid replication, is discussed.

  13. HotSpot Wizard: a web server for identification of hot spots in protein engineering.

    PubMed

    Pavelka, Antonin; Chovancova, Eva; Damborsky, Jiri

    2009-07-01

    HotSpot Wizard is a web server for automatic identification of 'hot spots' for engineering of substrate specificity, activity or enantioselectivity of enzymes and for annotation of protein structures. The web server implements the protein engineering protocol, which targets evolutionarily variable amino acid positions located in the active site or lining the access tunnels. The 'hot spots' for mutagenesis are selected through the integration of structural, functional and evolutionary information obtained from: (i) the databases RCSB PDB, UniProt, PDBSWS, Catalytic Site Atlas and nr NCBI and (ii) the tools CASTp, CAVER, BLAST, CD-HIT, MUSCLE and Rate4Site. The protein structure and e-mail address are the only obligatory inputs for the calculation. In the output, HotSpot Wizard lists annotated residues ordered by estimated mutability. The results of the analysis are mapped on the enzyme structure and visualized in the web browser using Jmol. The HotSpot Wizard server should be useful for protein engineers interested in exploring the structure of their favourite protein and for the design of mutations in site-directed mutagenesis and focused directed evolution experiments. HotSpot Wizard is available at http://loschmidt.chemi.muni.cz/hotspotwizard/.

  14. Imbalance in chemical space: How to facilitate the identification of protein-protein interaction inhibitors.

    PubMed

    Kuenemann, Mélaine A; Labbé, Céline M; Cerdan, Adrien H; Sperandio, Olivier

    2016-01-01

    Protein-protein interactions (PPIs) play vital roles in life and provide new opportunities for therapeutic interventions. In this large data analysis, 3,300 inhibitors of PPIs (iPPIs) were compared to 17 reference datasets of collectively ~566,000 compounds (including natural compounds, existing drugs, active compounds on conventional targets, etc.) using a chemoinformatics approach. Using this procedure, we showed that comparable classes of PPI targets can be formed using either the similarity of their ligands or the shared properties of their binding cavities, constituting a proof-of-concept that not only can binding pockets be used to group PPI targets, but that these pockets certainly condition the properties of their corresponding ligands. These results demonstrate that matching regions in both chemical space and target space can be found. Such identified classes of targets could lead to the design of PPI-class-specific chemical libraries and therefore facilitate the development of iPPIs to the stage of drug candidates. PMID:27034268

  15. Imbalance in chemical space: How to facilitate the identification of protein-protein interaction inhibitors

    NASA Astrophysics Data System (ADS)

    Kuenemann, Mélaine A.; Labbé, Céline M.; Cerdan, Adrien H.; Sperandio, Olivier

    2016-04-01

    Protein-protein interactions (PPIs) play vital roles in life and provide new opportunities for therapeutic interventions. In this large data analysis, 3,300 inhibitors of PPIs (iPPIs) were compared to 17 reference datasets of collectively ~566,000 compounds (including natural compounds, existing drugs, active compounds on conventional targets, etc.) using a chemoinformatics approach. Using this procedure, we showed that comparable classes of PPI targets can be formed using either the similarity of their ligands or the shared properties of their binding cavities, constituting a proof-of-concept that not only can binding pockets be used to group PPI targets, but that these pockets certainly condition the properties of their corresponding ligands. These results demonstrate that matching regions in both chemical space and target space can be found. Such identified classes of targets could lead to the design of PPI-class-specific chemical libraries and therefore facilitate the development of iPPIs to the stage of drug candidates.

  16. Imbalance in chemical space: How to facilitate the identification of protein-protein interaction inhibitors

    PubMed Central

    Kuenemann, Mélaine A.; Labbé, Céline M.; Cerdan, Adrien H.; Sperandio, Olivier

    2016-01-01

    Protein-protein interactions (PPIs) play vital roles in life and provide new opportunities for therapeutic interventions. In this large data analysis, 3,300 inhibitors of PPIs (iPPIs) were compared to 17 reference datasets of collectively ~566,000 compounds (including natural compounds, existing drugs, active compounds on conventional targets, etc.) using a chemoinformatics approach. Using this procedure, we showed that comparable classes of PPI targets can be formed using either the similarity of their ligands or the shared properties of their binding cavities, constituting a proof-of-concept that not only can binding pockets be used to group PPI targets, but that these pockets certainly condition the properties of their corresponding ligands. These results demonstrate that matching regions in both chemical space and target space can be found. Such identified classes of targets could lead to the design of PPI-class-specific chemical libraries and therefore facilitate the development of iPPIs to the stage of drug candidates. PMID:27034268

  17. Identification of protein partners in mycobacteria using a single-step affinity purification method.

    PubMed

    Płociński, Przemysław; Laubitz, Daniel; Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein-protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein-protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  18. A label-free differential quantitative mass spectrometry method for the characterization and identification of protein changes during citrus fruit development

    PubMed Central

    2010-01-01

    Background Citrus is one of the most important and widely grown commodity fruit crops. In this study a label-free LC-MS/MS based shot-gun proteomics approach was taken to explore three main stages of citrus fruit development. These approaches were used to identify and evaluate changes occurring in juice sac cells in various metabolic pathways affecting citrus fruit development and quality. Results Protein changes in citrus juice sac cells were identified and quantified using label-free shotgun methodologies. Two alternative methods, differential mass-spectrometry (dMS) and spectral counting (SC) were used to analyze protein changes occurring during earlier and late stages of fruit development. Both methods were compared in order to develop a proteomics workflow that could be used in a non-model plant lacking a sequenced genome. In order to resolve the bioinformatics limitations of EST databases from species that lack a full sequenced genome, we established iCitrus. iCitrus is a comprehensive sequence database created by merging three major sources of sequences (HarvEST:citrus, NCBI/citrus/unigenes, NCBI/citrus/proteins) and improving the annotation of existing unigenes. iCitrus provided a useful bioinformatics tool for the high-throughput identification of citrus proteins. We have identified approximately 1500 citrus proteins expressed in fruit juice sac cells and quantified the changes of their expression during fruit development. Our results showed that both dMS and SC provided significant information on protein changes, with dMS providing a higher accuracy. Conclusion Our data supports the notion of the complementary use of dMS and SC for label-free comparative proteomics, broadening the identification spectrum and strengthening the identification of trends in protein expression changes during the particular processes being compared. PMID:21162737

  19. Identification and baculovirus expression of the VP4 protein of the human group B rotavirus ADRV.

    PubMed Central

    Mackow, E R; Werner-Eckert, R; Fay, M E; Tao, H; Chen, G

    1993-01-01

    A complete cDNA copy of the fourth RNA segment of the human group B rotavirus adult diarrheal rotavirus (ADRV) has been cloned into lambda phage and excised into plasmid pSK Bluescript. Gene segment 4 contains 2,303 bases and encodes one long open reading frame beginning at base 16 and terminating at base 2263. The encoded protein contains 749 amino acids, with a calculated molecular mass of 84.4 kDa and a pI of 6.1. Gene 4 cDNA was inserted into a recombinant baculovirus via homologous recombination. The gene 4 polypeptide migrates at 84 kDa when expressed either by a recombinant baculovirus or in vitro in a rabbit reticulocyte lysate. The gene 4 protein is immunoprecipitable by hyperimmune serum to ADRV, human ADRV convalescent-phase serum, a porcine group B rotavirus infection serum, and a monoclonal antibody made to ADRV virion. Guinea pig hyperimmune serum to the baculovirus-expressed ADRV VP4 protein recognizes virus and immunoprecipitates an 84-kDa protein from in vitro translations of total ADRV mRNA. In addition, the gene 4-encoded protein shares significant amino acid identity and similarity with the group A rotavirus VP4 protein. This information, together with our previous identification of an 84-kDa protein present on iodinated intact virion but not EDTA-treated ADRV, suggests that gene 4 encodes the VP4 protein equivalent present on the outer capsid of ADRV. The ADRV VP4 protein is also 58% identical to the IDIR rat group B rotavirus gene segment 3 protein. The substantial differences between these two group B VP4 proteins suggests that they are distantly related and likely to define two different group B rotavirus VP4 serotypes. The baculovirus-expressed VP4 protein should be useful for developing serotyping reagents and tests for human and animal group B rotaviruses as well as for addressing the role of VP4 in ADRV neutralization. Images PMID:8386274

  20. Improving protein array performance: focus on washing and storage conditions.

    PubMed

    Nath, Nidhi; Hurst, Robin; Hook, Brad; Meisenheimer, Poncho; Zhao, Kate Q; Nassif, Nadine; Bulleit, Robert F; Storts, Douglas R

    2008-10-01

    For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.

  1. Identification of Protein-Protein Interactions via a Novel Matrix-Based Sequence Representation Model with Amino Acid Contact Information.

    PubMed

    Ding, Yijie; Tang, Jijun; Guo, Fei

    2016-09-24

    Identification of protein-protein interactions (PPIs) is a difficult and important problem in biology. Since experimental methods for predicting PPIs are both expensive and time-consuming, many computational methods have been developed to predict PPIs and interaction networks, which can be used to complement experimental approaches. However, these methods have limitations to overcome. They need a large number of homology proteins or literature to be applied in their method. In this paper, we propose a novel matrix-based protein sequence representation approach to predict PPIs, using an ensemble learning method for classification. We construct the matrix of Amino Acid Contact (AAC), based on the statistical analysis of residue-pairing frequencies in a database of 6323 protein-protein complexes. We first represent the protein sequence as a Substitution Matrix Representation (SMR) matrix. Then, the feature vector is extracted by applying algorithms of Histogram of Oriented Gradient (HOG) and Singular Value Decomposition (SVD) on the SMR matrix. Finally, we feed the feature vector into a Random Forest (RF) for judging interaction pairs and non-interaction pairs. Our method is applied to several PPI datasets to evaluate its performance. On the S . c e r e v i s i a e dataset, our method achieves 94 . 83 % accuracy and 92 . 40 % sensitivity. Compared with existing methods, and the accuracy of our method is increased by 0 . 11 percentage points. On the H . p y l o r i dataset, our method achieves 89 . 06 % accuracy and 88 . 15 % sensitivity, the accuracy of our method is increased by 0 . 76 % . On the H u m a n PPI dataset, our method achieves 97 . 60 % accuracy and 96 . 37 % sensitivity, and the accuracy of our method is increased by 1 . 30 % . In addition, we test our method on a very important PPI network, and it achieves 92 . 71 % accuracy. In the Wnt-related network, the accuracy of our method is increased by 16 . 67 % . The source code and all datasets are available

  2. Identification of Protein-Protein Interactions via a Novel Matrix-Based Sequence Representation Model with Amino Acid Contact Information.

    PubMed

    Ding, Yijie; Tang, Jijun; Guo, Fei

    2016-01-01

    Identification of protein-protein interactions (PPIs) is a difficult and important problem in biology. Since experimental methods for predicting PPIs are both expensive and time-consuming, many computational methods have been developed to predict PPIs and interaction networks, which can be used to complement experimental approaches. However, these methods have limitations to overcome. They need a large number of homology proteins or literature to be applied in their method. In this paper, we propose a novel matrix-based protein sequence representation approach to predict PPIs, using an ensemble learning method for classification. We construct the matrix of Amino Acid Contact (AAC), based on the statistical analysis of residue-pairing frequencies in a database of 6323 protein-protein complexes. We first represent the protein sequence as a Substitution Matrix Representation (SMR) matrix. Then, the feature vector is extracted by applying algorithms of Histogram of Oriented Gradient (HOG) and Singular Value Decomposition (SVD) on the SMR matrix. Finally, we feed the feature vector into a Random Forest (RF) for judging interaction pairs and non-interaction pairs. Our method is applied to several PPI datasets to evaluate its performance. On the S . c e r e v i s i a e dataset, our method achieves 94 . 83 % accuracy and 92 . 40 % sensitivity. Compared with existing methods, and the accuracy of our method is increased by 0 . 11 percentage points. On the H . p y l o r i dataset, our method achieves 89 . 06 % accuracy and 88 . 15 % sensitivity, the accuracy of our method is increased by 0 . 76 % . On the H u m a n PPI dataset, our method achieves 97 . 60 % accuracy and 96 . 37 % sensitivity, and the accuracy of our method is increased by 1 . 30 % . In addition, we test our method on a very important PPI network, and it achieves 92 . 71 % accuracy. In the Wnt-related network, the accuracy of our method is increased by 16 . 67 % . The source code and all datasets are available

  3. Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS.

    PubMed

    Janovská, Sylva; Pávková, Ivona; Hubálek, Martin; Lenco, Juraj; Macela, Ales; Stulík, Jirí

    2007-02-15

    Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins. PMID:17241671

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

    PubMed

    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.

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

    SciTech Connect

    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.

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

    PubMed Central

    Gorfe, Alemayehu A.

    2015-01-01

    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. PMID:26506102

  7. Identification of cytochrome P450 2C2 protein complexes in mouse liver.

    PubMed

    Li, Bin; Yau, Peter; Kemper, Byron

    2011-08-01

    Interactions of microsomal cytochromes P450 (CYPs) with other proteins in the microsomal membrane are important for their function. In addition to their redox partners, CYPs have been reported to interact with other proteins not directly involved in their enzymatic function. In this study, proteins were identified that interact with CYP2C2 in vivo in mouse liver. Flag-tagged CYP2C2 was expressed exogenously in mouse liver and was affinity purified, along with associated proteins which were identified by MS and confirmed by Western blotting. Over 20 proteins reproducibly copurified with CYP2C2. The heterogeneous sedimentation velocity of CYP2C2 and associated proteins by centrifugation in sucrose gradients and sequential immunoprecipitation analysis were consistent with multiple CYP2C2 complexes of differing composition. The abundance of CYPs and other drug metabolizing enzymes and NAD/NADP requiring enzymes associated with CYP2C2 suggest that complexes of these proteins may improve enzymatic efficiency or facilitate sequential metabolic steps. Chaperones, which may be important for maintaining CYP function, and reticulons, endoplasmic reticulum proteins that shape the morphology of the endoplasmic reticulum and are potential endoplasmic reticulum retention proteins for CYPs, were also associated with CYP2C2.

  8. Characterization and identification of protein O-GlcNAcylation sites with substrate specificity

    PubMed Central

    2014-01-01

    Background Protein O-GlcNAcylation, involving the attachment of single N-acetylglucosamine (GlcNAc) to the hydroxyl group of serine or threonine residues. Elucidation of O-GlcNAcylation sites on proteins is required in order to decipher its crucial roles in regulating cellular processes and aid in drug design. With an increasing number of O-GlcNAcylation sites identified by mass spectrometry (MS)-based proteomics, several methods have been proposed for the computational identification of O-GlcNAcylation sites. However, no development that focuses on the investigation of O-GlcNAcylated substrate motifs has existed. Thus, we were motivated to design a new method for the identification of protein O-GlcNAcylation sites with the consideration of substrate site specificity. Results In this study, 375 experimentally verified O-GlcNAcylation sites were collected from dbOGAP, which is an integrated resource for protein O-GlcNAcylation. Due to the difficulty in characterizing the substrate motifs by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. To construct the predictive models learned from the identified substrate motifs, we adopted Support Vector Machines (SVMs). A five-fold cross validation was used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 0.76, 0.80, and 0.78, respectively. Additionally, an independent testing set, which was really blind to the training data of predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (0.94) and outperform three other O-GlcNAcylation site prediction tools. Conclusion This work proposed a computational method to identify informative substrate motifs for O-GlcNAcylation sites. The evaluation of cross validation and independent testing indicated that the identified motifs were effective in the identification of O-GlcNAcylation sites. A case study demonstrated that the

  9. Improving protein–protein interactions prediction accuracy using protein evolutionary information and relevance vector machine model

    PubMed Central

    An, Ji‐Yong; Meng, Fan‐Rong; Chen, Xing; Yan, Gui‐Ying; Hu, Ji‐Pu

    2016-01-01

    Abstract Predicting protein–protein interactions (PPIs) is a challenging task and essential to construct the protein interaction networks, which is important for facilitating our understanding of the mechanisms of biological systems. Although a number of high‐throughput technologies have been proposed to predict PPIs, there are unavoidable shortcomings, including high cost, time intensity, and inherently high false positive rates. For these reasons, many computational methods have been proposed for predicting PPIs. However, the problem is still far from being solved. In this article, we propose a novel computational method called RVM‐BiGP that combines the relevance vector machine (RVM) model and Bi‐gram Probabilities (BiGP) for PPIs detection from protein sequences. The major improvement includes (1) Protein sequences are represented using the Bi‐gram probabilities (BiGP) feature representation on a Position Specific Scoring Matrix (PSSM), in which the protein evolutionary information is contained; (2) For reducing the influence of noise, the Principal Component Analysis (PCA) method is used to reduce the dimension of BiGP vector; (3) The powerful and robust Relevance Vector Machine (RVM) algorithm is used for classification. Five‐fold cross‐validation experiments executed on yeast and Helicobacter pylori datasets, which achieved very high accuracies of 94.57 and 90.57%, respectively. Experimental results are significantly better than previous methods. To further evaluate the proposed method, we compare it with the state‐of‐the‐art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM‐BiGP method is significantly better than the SVM‐based method. In addition, we achieved 97.15% accuracy on imbalance yeast dataset, which is higher than that of balance yeast dataset. The promising experimental results show the efficiency and robust of the proposed method, which can be an automatic

  10. In silico identification of gene amplification targets for improvement of lycopene production.

    PubMed

    Choi, Hyung Seok; Lee, Sang Yup; Kim, Tae Yong; Woo, Han Min

    2010-05-01

    The identification of genes to be deleted or amplified is an essential step in metabolic engineering for strain improvement toward the enhanced production of desired bioproducts. In the past, several methods based on flux analysis of genome-scale metabolic models have been developed for identifying gene targets for deletion. Genome-wide identification of gene targets for amplification, on the other hand, has been rather difficult. Here, we report a strategy called flux scanning based on enforced objective flux (FSEOF) to identify gene amplification targets. FSEOF scans all the metabolic fluxes in the metabolic model and selects fluxes that increase when the flux toward product formation is enforced as an additional constraint during flux analysis. This strategy was successfully employed for the identification of gene amplification targets for the enhanced production of the red-colored antioxidant lycopene. Additional metabolic engineering based on gene knockout simulation resulted in further synergistic enhancement of lycopene production. Thus, FSEOF can be used as a general strategy for selecting genome-wide gene amplification targets in silico.

  11. A numerical study of sensory-guided multiple views for improved object identification

    NASA Astrophysics Data System (ADS)

    Blakeslee, B. A.; Zelnio, E. G.; Koditschek, D. E.

    2014-06-01

    We explore the potential on-line adjustment of sensory controls for improved object identification and discrimination in the context of a simulated high resolution camera system carried onboard a maneuverable robotic platform that can actively choose its observational position and pose. Our early numerical studies suggest the significant efficacy and enhanced performance achieved by even very simple feedback-driven iteration of the view in contrast to identification from a fixed pose, uninformed by any active adaptation. Specifically, we contrast the discriminative performance of the same conventional classification system when informed by: a random glance at a vehicle; two random glances at a vehicle; or a random glance followed by a guided second look. After each glance, edge detection algorithms isolate the most salient features of the image and template matching is performed through the use of the Hausdor↵ distance, comparing the simulated sensed images with reference images of the vehicles. We present initial simulation statistics that overwhelmingly favor the third scenario. We conclude with a sketch of our near-future steps in this study that will entail: the incorporation of more sophisticated image processing and template matching algorithms; more complex discrimination tasks such as distinguishing between two similar vehicles or vehicles in motion; more realistic models of the observers mobility including platform dynamics and eventually environmental constraints; and expanding the sensing task beyond the identification of a specified object selected from a pre-defined library of alternatives.

  12. Improved x-ray detection and particle identification with avalanche photodiodes

    SciTech Connect

    Diepold, Marc Franke, Beatrice; Götzfried, Johannes; Hänsch, Theodor W.; Krauth, Julian J.; Mulhauser, Françoise; Nebel, Tobias; Pohl, Randolf; Fernandes, Luis M. P.; Amaro, Fernando D.; Gouvea, Andrea L.; Monteiro, Cristina M. B.; Santos, Joaquim M. F. dos; Machado, Jorge; Amaro, Pedro; Santos, José Paulo; and others

    2015-05-15

    Avalanche photodiodes are commonly used as detectors for low energy x-rays. In this work, we report on a fitting technique used to account for different detector responses resulting from photoabsorption in the various avalanche photodiode layers. The use of this technique results in an improvement of the energy resolution at 8.2 keV by up to a factor of 2 and corrects the timing information by up to 25 ns to account for space dependent electron drift time. In addition, this waveform analysis is used for particle identification, e.g., to distinguish between x-rays and MeV electrons in our experiment.

  13. Protein Sequence Classification with Improved Extreme Learning Machine Algorithms

    PubMed Central

    2014-01-01

    Precisely classifying a protein sequence from a large biological protein sequences database plays an important role for developing competitive pharmacological products. Comparing the unseen sequence with all the identified protein sequences and returning the category index with the highest similarity scored protein, conventional methods are usually time-consuming. Therefore, it is urgent and necessary to build an efficient protein sequence classification system. In this paper, we study the performance of protein sequence classification using SLFNs. The recent efficient extreme learning machine (ELM) and its invariants are utilized as the training algorithms. The optimal pruned ELM is first employed for protein sequence classification in this paper. To further enhance the performance, the ensemble based SLFNs structure is constructed where multiple SLFNs with the same number of hidden nodes and the same activation function are used as ensembles. For each ensemble, the same training algorithm is adopted. The final category index is derived using the majority voting method. Two approaches, namely, the basic ELM and the OP-ELM, are adopted for the ensemble based SLFNs. The performance is analyzed and compared with several existing methods using datasets obtained from the Protein Information Resource center. The experimental results show the priority of the proposed algorithms. PMID:24795876

  14. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38).

    PubMed Central

    Luzio, J P; Brake, B; Banting, G; Howell, K E; Braghetta, P; Stanley, K K

    1990-01-01

    Organelle-specific integral membrane proteins were identified by a novel strategy which gives rise to monospecific antibodies to these proteins as well as to the cDNA clones encoding them. A cDNA expression library was screened with a polyclonal antiserum raised against Triton X-114-extracted organelle proteins and clones were then grouped using antibodies affinity-purified on individual fusion proteins. The identification, molecular cloning and sequencing are described of a type 1 membrane protein (TGN38) which is located specifically in the trans-Golgi network. Images Fig. 1. Fig. 3. PMID:2204342

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

    PubMed

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. PMID:23894077

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

    SciTech Connect

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

    1995-12-31

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

  17. Extraction, purification and identification of antifreeze proteins from cold acclimated malting barley (Hordeum vulgare L.).

    PubMed

    Ding, Xiangli; Zhang, Hui; Chen, Haiying; Wang, Li; Qian, Haifeng; Qi, Xiguang

    2015-05-15

    Antifreeze proteins from cold-acclimated malting barley were extracted by infiltration-centrifugation. The infiltration time was optimised, and its extraction effect was evaluated. The effect of cold acclimation on the accumulation of barley antifreeze proteins (BaAFPs) was assessed by comparing the thermal hysteresis activities (THA) of proteins extracted from both cold acclimated and non-cold acclimated barley grain. Ultra-filtration, ammonium precipitation and column chromatography were used successively to purify the BaAFPs, and MALDI-TOF-MS/MS was used for protein identification. The results showed that infiltration-centrifugation was more targeted than the traditional method, and 10h was the optimal infiltration time. THA was observed only after cold acclimation implied that AFPs only began to accumulate after cold acclimation. After purification, BaAFP-I was obtained at an electrophoresis level and its THA was 1.04°C (18.0 mg ml(-1)). The mass fingerprinting and sequencing results indicated the homology of the BaAFP-I to alpha-amylase inhibitor BDAI-1 (Hordeum vulgare).

  18. Identification and isolation of a hyperphosphorylated, conformationally changed intermediate of human protein tau expressed in yeast.

    PubMed

    Vandebroek, Tom; Vanhelmont, Thomas; Terwel, Dick; Borghgraef, Peter; Lemaire, Katleen; Snauwaert, Johan; Wera, Stefaan; Van Leuven, Fred; Winderickx, Joris

    2005-08-30

    Hyperphosphorylation and aggregation of protein tau are typical for neurodegenerative tauopathies, including Alzheimer's disease (AD). We demonstrate here that human tau expressed in yeast acquired pathological phosphoepitopes, assumed a pathological conformation, and formed aggregates. These processes were modulated by yeast kinases Mds1 and Pho85, orthologues of GSK-3beta and cdk5, respectively. Surprisingly, inactivation of Pho85 increased phosphorylation of tau-4R, concomitant with increased conformational change defined by antibody MC1 and a 40-fold increase in aggregation. Soluble protein tau, purified from yeast lacking PHO85, spontaneously and rapidly formed tau filaments in vitro. Further fractionation of tau by anion-exchange chromatography yielded a hyperphosphorylated monomeric subfraction, termed hP-tau/MC1, with slow electrophoretic mobility and enriched with all major epitopes, including MC1. Isolated hP-tau/MC1 vastly accelerated in vitro aggregation of wild-type tau-4R, demonstrating its functional capacity to initiate aggregation, as well as its structural stability. Combined, this novel yeast model recapitulates hyperphosphorylation, conformation, and aggregation of protein tau, provides insight in molecular changes crucial in tauopathies, offers a source for isolation of modified protein tau, and has potential for identification of modulating compounds and genes.

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

    PubMed Central

    Liu, Pei; Zhang, Huoming; Yu, Boying; Xiong, Liming; Xia, Yiji

    2015-01-01

    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. PMID:25720653

  20. Identification of Protein Partners in Mycobacteria Using a Single-Step Affinity Purification Method

    PubMed Central

    Cysewski, Dominik; Stoduś, Krystian; Kowalska, Katarzyna; Dziembowski, Andrzej

    2014-01-01

    Tuberculosis is a leading cause of death in developing countries. Efforts are being made to both prevent its spread and improve curability rates. Understanding the biology of the bacteria causing the disease, Mycobacterium tuberculosis (M. tuberculosis), is thus vital. We have implemented improved screening methods for protein–protein interactions based on affinity purification followed by high-resolution mass spectrometry. This method can be efficiently applied to both medium- and high-throughput studies aiming to characterize protein–protein interaction networks of tubercle bacilli. Of the 4 tested epitopes FLAG, enhanced green fluorescent protein (eGFP), protein A and haemagglutinin, the eGFP tag was found to be most useful on account of its easily monitored expression and its ability to function as a simultaneous tool for subcellular localization studies. It presents a relatively low background with cost-effective purification. RNA polymerase subunit A (RpoA) was used as a model for investigation of a large protein complex. When used as bait, it co-purified with all remaining RNA polymerase core subunits as well as many accessory proteins. The amount of RpoA strongly correlated with the amount of quantification peptide used as part of the tagging system in this study (SH), making it applicable for semi-quantification studies. Interactions between the components of the RpoA-eGFP protein complex were further confirmed using protein cross-linking. Dynamic changes in the composition of protein complexes under induction of UV damage were observed when UvrA-eGFP expressing cells treated with UV light were used to co-purify UvrA interaction partners. PMID:24664103

  1. Improving Protein Fold Recognition by Deep Learning Networks

    PubMed Central

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-01-01

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold. PMID:26634993

  2. Improving Protein Fold Recognition by Deep Learning Networks.

    PubMed

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-12-04

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl's benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

  3. Improving Protein Fold Recognition by Deep Learning Networks

    NASA Astrophysics Data System (ADS)

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-12-01

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold.

  4. Improving Protein Fold Recognition by Deep Learning Networks.

    PubMed

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-01-01

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl's benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold. PMID:26634993

  5. 3D Pharmacophoric Similarity improves Multi Adverse Drug Event Identification in Pharmacovigilance

    PubMed Central

    Vilar, Santiago; Tatonetti, Nicholas P.; Hripcsak, George

    2015-01-01

    Adverse drugs events (ADEs) detection constitutes a considerable concern in patient safety and public health care. For this reason, it is important to develop methods that improve ADE signal detection in pharmacovigilance databases. Our objective is to apply 3D pharmacophoric similarity models to enhance ADE recognition in Offsides, a pharmacovigilance resource with drug-ADE associations extracted from the FDA Adverse Event Reporting System (FAERS). We developed a multi-ADE predictor implementing 3D drug similarity based on a pharmacophoric approach, with an ADE reference standard extracted from the SIDER database. The results showed that the application of our 3D multi-type ADE predictor to the pharmacovigilance data in Offsides improved ADE identification and generated enriched sets of drug-ADE signals. The global ROC curve for the Offsides ADE candidates ranked with the 3D similarity score showed an area of 0.7. The 3D predictor also allows the identification of the most similar drug that causes the ADE under study, which could provide hypotheses about mechanisms of action and ADE etiology. Our method is useful in drug development, screening potential adverse effects in experimental drugs, and in drug safety, applicable to the evaluation of ADE signals selected through pharmacovigilance data mining. PMID:25744369

  6. 3D Pharmacophoric Similarity improves Multi Adverse Drug Event Identification in Pharmacovigilance

    NASA Astrophysics Data System (ADS)

    Vilar, Santiago; Tatonetti, Nicholas P.; Hripcsak, George

    2015-03-01

    Adverse drugs events (ADEs) detection constitutes a considerable concern in patient safety and public health care. For this reason, it is important to develop methods that improve ADE signal detection in pharmacovigilance databases. Our objective is to apply 3D pharmacophoric similarity models to enhance ADE recognition in Offsides, a pharmacovigilance resource with drug-ADE associations extracted from the FDA Adverse Event Reporting System (FAERS). We developed a multi-ADE predictor implementing 3D drug similarity based on a pharmacophoric approach, with an ADE reference standard extracted from the SIDER database. The results showed that the application of our 3D multi-type ADE predictor to the pharmacovigilance data in Offsides improved ADE identification and generated enriched sets of drug-ADE signals. The global ROC curve for the Offsides ADE candidates ranked with the 3D similarity score showed an area of 0.7. The 3D predictor also allows the identification of the most similar drug that causes the ADE under study, which could provide hypotheses about mechanisms of action