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Sample records for improves protein identification

  1. Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

    PubMed Central

    Langó, Tamás; Róna, Gergely; Hunyadi-Gulyás, Éva; Turiák, Lilla; Varga, Julia; Dobson, László; Várady, György; Drahos, László; Vértessy, Beáta G.; Medzihradszky, Katalin F.; Szakács, Gergely; Tusnády, Gábor E.

    2017-01-01

    Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins. PMID:28211907

  2. Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins.

    PubMed

    Langó, Tamás; Róna, Gergely; Hunyadi-Gulyás, Éva; Turiák, Lilla; Varga, Julia; Dobson, László; Várady, György; Drahos, László; Vértessy, Beáta G; Medzihradszky, Katalin F; Szakács, Gergely; Tusnády, Gábor E

    2017-02-13

    Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins.

  3. Improvement of hydrophobic integral membrane protein identification by mild performic acid oxidation-assisted digestion.

    PubMed

    Cao, Rui; Liu, Yisong; Chen, Ping; Lv, Rong; Song, Qin; Sheng, Tingting; He, Quanyuan; Wang, Yin; Wang, Xianchun; Liang, Songping

    2010-12-15

    Integral membrane proteins (IMPs) are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of IMPs make them difficult to analyze. In proteomic analyses, hydrophobic peptides including transmembrane domains are often underrepresented, and this reduces the sequence coverage and reliability of the identified IMPs. Here we report a new strategy, mild performic acid oxidation treatment (mPAOT), for improvement of IMP identification. In the mPAOT strategy, the hydrophobicity of IMPs is significantly decreased by oxidizing their methionine and cysteine residues with performic acid, thereby improving the solubility and enzymolysis of these proteins. The application of the mPAOT strategy to the analysis of IMPs from human nasopharyngeal carcinoma CNE1 cell line demonstrated that many IMPs, including those with high hydrophobicity, could be reliably identified.

  4. Deglycosylation Step to Improve the Identification of Egg Proteins in Art Samples.

    PubMed

    Vinciguerra, Roberto; Galano, Eugenio; Vallone, Fabiana; Greco, Giovanna; Vergara, Alessandro; Bonaduce, Ilaria; Marino, Gennaro; Pucci, Pietro; Amoresano, Angela; Birolo, Leila

    2015-10-20

    A deglycosylation step using Peptide-N-Glycosidase F (PNGaseF) has been introduced in a standard proteomic protocol to more confidently identify egg based binders. The ingenuity of introducing a PNGaseF digestion was aimed at removing the molecular hindrance, made up by the heavily glycosylated egg proteins, before the protease(s) hydrolysis. This novelty in the protocol resulted in obtaining a significant increase of proteolytic egg peptides thus improving the quality and reliability of egg identification in artwork samples. The protocol has been set up on paint replicas and successfully tested on two historical samples of different origin.

  5. 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.

  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.

  7. IDSieve: Protein Identification Using Peptide pI Filtering of MS/MS Data for Improved Confidence in Identifications

    PubMed Central

    West, K.D.; Zhang, X.; Bundy, J.L.; Stephenson, J.L.; Cargile, B.J.; Bunger, M.K.; Garge, N.R.

    2011-01-01

    The main challenge of tandem mass spectrometry based proteomic analysis is to correctly match the tandem mass spectra produced to the correct peptides. However, the large number of protein sequences in a database increases the chances of a false positive identification for any given peptide match. Here we present an automated algorithm called IDSieve that utilizes target-decoy database search strategy in combination with pI filtering to allow greater confidence for peptide identifications. IDSieve considers the SEQUEST parameters Xcorr and äCn to assign statistical confidence (false discovery rates) to the peptide matches. The distribution of predicted pI values for peptide spectrum matches (PSMs) is considered separately for each immobilized pH gradient isoelectric focusing fraction, and matches with pI values within 1.5 times inter-quartile range (within pI range) are analyzed independently of matches outside the pI ranges. We tested the performance of IDSieve and Peptide/Protein Prophet on the SEQUEST outputs from 60 immobilized pH gradient isoelectric focusing fractions derived from mouse intestinal epithelial cell protein extracts. Our results demonstrated that IDSieve produced 1355 more peptide spectrum matches (or 330 more peptides) than Peptide Prophet using comparable false positive rate cutoffs. Therefore, combining pI filtering with the appropriate statistical significance measurements allows for a higher number of protein identifications without adversely affecting the false positive rate. We further tested the performance of pI filtering using ID Sieve when samples were prefractionated using either pH range 3.5–4.5 or 3–10, and either 24cm or 7cm IPG strips.

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-02-22

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

  11. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  14. A legume specific protein database (LegProt) improves the number of identified peptides, confidence scores and overall protein identification success rates for legume proteomics.

    PubMed

    Lei, Zhentian; Dai, Xinbin; Watson, Bonnie S; Zhao, Patrick X; Sumner, Lloyd W

    2011-07-01

    A legume specific protein database (LegProt) has been created containing sequences from seven legume species, i.e., Glycine max, Lotus japonicus, Medicago sativa, Medicago truncatula, Lupinusalbus, Phaseolus vulgaris, and Pisum sativum. The database consists of amino acid sequences translated from predicted gene models and 6-frame translations of tentative consensus (TC) sequences assembled from expressed sequence tags (ESTs) and singleton ESTs. This database was queried using mass spectral data for protein identification and identification success rates were compared to the NCBI nr database. Specifically, Mascot MS/MS ion searches of tandem nano-LC Q-TOFMS/MS mass spectral data showed that relative to the NCBI nr protein database, the LegProt database yielded a 54% increase in the average protein score (i.e., from NCBI nr 480 to LegProt 739) and a 50% increase in the average number of matched peptides (i.e., from NCBI nr 8 to LegProt 12). The overall identification success rate also increased from 88% (NCBI nr) to 93% (LegProt). Mascot peptide mass fingerprinting (PMF) searches of the LegProt database using MALDI-TOFMS data yielded a significant increase in the identification success rate from 19% (NCBI nr) to 34% (LegProt) while the average scores and average number of matched peptides showed insignificant changes. The results demonstrate that the LegProt database significantly increases legume protein identification success rates and the confidence levels compared to the commonly used NCBI nr. These improvements are primarily due to the presence of a large number of legume specific TC sequences in the LegProt database that were not found in NCBI nr. The LegProt database is freely available for download (http://bioinfo.noble.org/manuscript-support/legumedb) and will serve as a valuable resource for legume proteomics.

  15. IDENTIFICATION AND REMOVAL OF PROTEINS THAT CO-PURIFY WITH INFECTIOUS PRION PROTEIN IMPROVES THE ANALYSIS OF ITS SECONDARY STRUCTURE

    PubMed Central

    Moore, Roger A.; Timmes, Andrew; Wilmarth, Phillip A.; Safronetz, David; Priola, Suzette A.

    2013-01-01

    Prion diseases are neurodegenerative disorders associated with the accumulation of an abnormal isoform of the mammalian prion protein (PrP). Fourier transform infrared spectroscopy (FTIR) has previously been used to show that the conformation of aggregated, infectious PrP (PrPSc) varies between prion strains and these unique conformations may determine strain-specific disease phenotypes. However, the relative amounts of α-helix, β-sheet and other secondary structures have not always been consistent between studies suggesting that other proteins might be confounding the analysis of PrPSc secondary structure. We have used FTIR and tandem mass spectrometry to analyze enriched PrPSc from mouse and hamster prion strains both before and after the removal of protein contaminants that commonly co-purify with PrPSc. Our data show that non-PrP proteins do contribute to absorbances that have been associated with α-helical, loop, turn, and β-sheet structures attributed to PrPSc. The major contaminant, the α-helical protein ferritin, absorbs strongly at 1652cm−1 in the FTIR spectrum associated with PrPSc. However, even the removal of greater than 99% of the ferritin from PrPSc did not completely abolish absorbance at 1652cm−1. Our results show that contaminating proteins alter the FTIR spectrum attributed to PrPSc and suggest that the α-helical, loop/turn, and β-sheet secondary structure that remains following their removal are derived from PrPSc itself. PMID:21805638

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

    PubMed

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

    2012-08-01

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

  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. 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)).

  20. Millimeter radar improves target identification

    NASA Astrophysics Data System (ADS)

    McAulay, Alastair D.

    2011-06-01

    Recently developed millimeter wave radar has advantages for target identification over conventional microwave radar which typically use lower frequencies. We describe the pertinent features involved in the construction of the new millimeter wave radar, the pseudo-optical cavity source and the quasi-optical duplexer. The long wavelength relative to light allows the radar beam to penetrate through most weather because the wavelength is larger than the particle size for dust, drizzle rain, fog. Further the mm wave beam passes through an atmospheric transmission window that provides a dip in attenuation. The higher frequency than conventional radar provides higher Doppler frequencies, for example, than X-band radar. We show by simulation that small characteristic vibrations and slow turns of an aircraft become visible so that the Doppler signature improves identification. The higher frequency also reduces beam width, which increases transmit and receive antenna gains. For the same power the transmit beam extends to farther range and the increase in receive antenna gain increases signal to noise ratio for improved detection and identification. The narrower beam can also reduce clutter and reject other noise more readily. We show by simulation that the radar can be used at lower elevations over the sea than conventional radar.

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

    PubMed

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

    2015-01-01

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

  2. 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...

  3. Higher sequence coverage and improved confidence in the identification of cysteine-rich proteins from the wool cuticle using combined chemical and enzymatic digestion.

    PubMed

    Koehn, Henning; Clerens, Stefan; Deb-Choudhury, Santanu; Morton, James D; Dyer, Jolon M; Plowman, Jeffrey E

    2009-12-01

    Keratin-associated proteins (KAPs) are important constituents of the wool cuticle, comprised of the endo-, exocuticle and a-layers, which contribute significantly to the fibre's molecular and mechanical characteristics. Relatively little is known about the distribution of specific KAPs across these layers, and correct protein identification of individual KAPs is difficult due to extensive homology and identity among individual KAPs. We here present evidence that, by specifically exploiting the high-cysteine content of KAPs in the wool cuticle, using 2-nitro-5-thiocyanobenzoic acid (NTCB) cleavage in combination with tryptic digestion, a larger number of KAPs can be identified than with standard trypsin-only digests. A total of 27 KAPs were identified, six of which could only be identified using NTCB. Furthermore, NTCB-mediated cleavage of cuticle proteins generated unique peptides critical for unambiguous identification of two KAPs, as well as significantly increasing the overall sequence coverage of most identified KAPs. Interestingly, some of the peptides found to be unique to particular KAPs could only be found in either the exo- or endocuticle. We conclude that for the analysis of high sulphur proteomes, specific targeting of cysteine residues using chemical agents such as NTCB can provide critical information for unambiguous protein identification.

  4. Automated Protein Subfamily Identification and Classification

    PubMed Central

    Brown, Duncan P; Krishnamurthy, Nandini; Sjölander, Kimmen

    2007-01-01

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

  5. Data Analysis Strategies for Protein Modification Identification.

    PubMed

    Fu, Yan

    2016-01-01

    Mass spectrometry-based proteomics provides a powerful tool for large-scale analysis of protein modifications. Statistical and computational analysis of mass spectrometry data is a key step in protein modification identification. This chapter presents common and advanced data analysis strategies for modification identification, including variable modification search, unrestrictive approaches for modification discovery, false discovery rate estimation and control methods, and tools for modification site localization.

  6. 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.

  7. Improved system identification with Renormalization Group.

    PubMed

    Wang, Qing-Guo; Yu, Chao; Zhang, Yong

    2014-09-01

    This paper proposes an improved system identification method with Renormalization Group. Renormalization Group is applied to a fine data set to obtain a coarse data set. The least squares algorithm is performed on the coarse data set. The theoretical analysis under certain conditions shows that the parameter estimation error could be reduced. The proposed method is illustrated with examples.

  8. 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

  9. 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.

  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.

  11. Cleaning of raw peptide MS/MS spectra: improved protein identification following deconvolution of multiply charged peaks, isotope clusters, and removal of background noise.

    PubMed

    Mujezinovic, Nedim; Raidl, Günther; Hutchins, James R A; Peters, Jan-Michael; Mechtler, Karl; Eisenhaber, Frank

    2006-10-01

    The dominant ions in MS/MS spectra of peptides, which have been fragmented by low-energy CID, are often b-, y-ions and their derivatives resulting from the cleavage of the peptide bonds. However, MS/MS spectra typically contain many more peaks. These can result not only from isotope variants and multiply charged replicates of the peptide fragmentation products but also from unknown fragmentation pathways, sample-specific or systematic chemical contaminations or from noise generated by the electronic detection system. The presence of this background complicates spectrum interpretation. Besides dramatically prolonged computation time, it can lead to incorrect protein identification, especially in the case of de novo sequencing algorithms. Here, we present an algorithm for detection and transformation of multiply charged peaks into singly charged monoisotopic peaks, removal of heavy isotope replicates, and random noise. A quantitative criterion for the recognition of some noninterpretable spectra has been derived as a byproduct. The approach is based on numerical spectral analysis and signal detection methods. The algorithm has been implemented in a stand-alone computer program called MS Cleaner that can be obtained from the authors upon request.

  12. 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...

  13. 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

  14. Repeats identification using improved suffix trees.

    PubMed

    Huo, Hongwei; Wang, Xiaowu; Stojkovic, Vojislav

    2009-01-01

    The suffix tree data structure plays an important role in the efficient implementations of some querying algorithms. This paper presents the fast Rep(eats)Seeker algorithm for repeats identification based on the improvements of suffix tree construction. The leaf nodes and the branch nodes are numbered in different ways during the construction of a suffix tree and extra information is added to the branch nodes. The experimental results show that improvements reduce the running time of the RepSeeker algorithm without losing the accuracy. The experimental results coincide with the theoretical expectations.

  15. 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...

  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. Identification of ligands for bacterial sensor proteins.

    PubMed

    Fernández, Matilde; Morel, Bertrand; Corral-Lugo, Andrés; Rico-Jiménez, Miriam; Martín-Mora, David; López-Farfán, Diana; Reyes-Darias, José Antonio; Matilla, Miguel A; Ortega, Álvaro; Krell, Tino

    2016-02-01

    Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria.

  18. Digestion and depletion of abundant proteins improves proteomic coverage

    PubMed Central

    Fonslow, Bryan R.; Stein, Benjamin D.; Webb, Kristofor J.; Xu, Tao; Choi, Jeong; Park, Sung Kyu; Yates, John R.

    2012-01-01

    Two major challenges in proteomics are the large number of proteins and their broad dynamic range within the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances and observed greater than 3-fold improvement in low abundance human protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases, and proteomic pipelines. PMID:23160281

  19. Applications of graph theory in protein structure identification.

    PubMed

    Yan, Yan; Zhang, Shenggui; Wu, Fang-Xiang

    2011-10-14

    There is a growing interest in the identification of proteins on the proteome wide scale. Among different kinds of protein structure identification methods, graph-theoretic methods are very sharp ones. Due to their lower costs, higher effectiveness and many other advantages, they have drawn more and more researchers' attention nowadays. Specifically, graph-theoretic methods have been widely used in homology identification, side-chain cluster identification, peptide sequencing and so on. This paper reviews several methods in solving protein structure identification problems using graph theory. We mainly introduce classical methods and mathematical models including homology modeling based on clique finding, identification of side-chain clusters in protein structures upon graph spectrum, and de novo peptide sequencing via tandem mass spectrometry using the spectrum graph model. In addition, concluding remarks and future priorities of each method are given.

  20. Modified Protein Improves Vitiligo Symptoms in Mice

    MedlinePlus

    ... 2013 (historical) Modified Protein Improves Vitiligo Symptoms in Mice Altering a key protein involved in the development ... pigmentation loss associated with the skin disorder in mice, according to recent research funded by the NIH’s ...

  1. Identification of immunoreactive proteins of Mycobacterium avium subsp. paratuberculosis.

    PubMed

    Piras, Cristian; Soggiu, Alessio; Bonizzi, Luigi; Greco, Viviana; Ricchi, Matteo; Arrigoni, Norma; Bassols, Anna; Urbani, Andrea; Roncada, Paola

    2015-02-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the cause of a chronic enteritis of ruminants (bovine paratuberculosis (PTB)--Johne's disease) that is associated with enormous worldwide economic losses for the animal production. Diagnosis is based on observation of clinical signs, the detection of antibodies in milk or serum, or evaluation of bacterial culture from feces. The limit of these methods is that they are not able to detect the disease in the subclinical stage and are applicable only when the disease is already advanced. For this reason, the main purpose of this study is to use the MAP proteome to detect novel immunoreactive proteins that may be helpful for PTB diagnoses. 2DE and 2D immunoblotting of MAP proteins were performed using sera of control cattle and PTB-infected cattle in order to highlight the specific immunoreactive proteins. Among the assigned identifiers to immunoreactive spots it was found that most of them correspond to surface-located proteins while three of them have never been described before as antigens. The identification of these proteins improves scientific knowledge that could be useful for PTB diagnoses. The sequence of the identified protein can be used for the synthesis of immunoreactive peptides that could be screened for their immunoreaction against bovine sera infected with MAP. All MS data have been deposited in the ProteomeXchange consortium with identifier PXD001159 and DOI 10.6019/PXD001159.

  2. Identification of essential proteins based on ranking edge-weights in protein-protein interaction networks.

    PubMed

    Wang, Yan; Sun, Huiyan; Du, Wei; Blanzieri, Enrico; Viero, Gabriella; Xu, Ying; Liang, Yanchun

    2014-01-01

    Essential proteins are those that are indispensable to cellular survival and development. Existing methods for essential protein identification generally rely on knock-out experiments and/or the relative density of their interactions (edges) with other proteins in a Protein-Protein Interaction (PPI) network. Here, we present a computational method, called EW, to first rank protein-protein interactions in terms of their Edge Weights, and then identify sub-PPI-networks consisting of only the highly-ranked edges and predict their proteins as essential proteins. We have applied this method to publicly-available PPI data on Saccharomyces cerevisiae (Yeast) and Escherichia coli (E. coli) for essential protein identification, and demonstrated that EW achieves better performance than the state-of-the-art methods in terms of the precision-recall and Jackknife measures. The highly-ranked protein-protein interactions by our prediction tend to be biologically significant in both the Yeast and E. coli PPI networks. Further analyses on systematically perturbed Yeast and E. coli PPI networks through randomly deleting edges demonstrate that the proposed method is robust and the top-ranked edges tend to be more associated with known essential proteins than the lowly-ranked edges.

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

    PubMed

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

    2014-01-07

    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.

  4. 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

  5. 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/.

  6. Improving recombinant protein purification yield

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  7. 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,…

  8. Perspective: A Program to Improve Protein Biomarker Discovery for Cancer

    SciTech Connect

    Aebersold, Ruedi; Anderson, Leigh N.; Caprioli, Richard M.; Druker, Brian; Hartwell, L D.; Smith, Richard D.

    2005-06-01

    Biomarkers for cancer risk, early detection, prognosis, and therapeutic response promise to revolutionize cancer management. Protein biomarkers offer tremendous potential in this regard due to their great diversity and intimate involvement in physiology. An effective program to discover protein biomarkers using existing technology will require team science, an integrated informatics platform, identification and quantitation of candidate biomarkers in disease tissue, mouse models of disease, standardized reagents for analyzing candidate biomarkers in bodily fluids, and implementation of automation. Technology improvements for better fractionation of the proteome, selection of specific biomarkers from complex mixtures, and multiplexed assay of biomarkers would greatly enhance progress.

  9. Essential protein identification based on essential protein-protein interaction prediction by Integrated Edge Weights.

    PubMed

    Jiang, Yuexu; Wang, Yan; Pang, Wei; Chen, Liang; Sun, Huiyan; Liang, Yanchun; Blanzieri, Enrico

    2015-07-15

    Essential proteins play a crucial role in cellular survival and development process. Experimentally, essential proteins are identified by gene knockouts or RNA interference, which are expensive and often fatal to the target organisms. Regarding this, an alternative yet important approach to essential protein identification is through computational prediction. Existing computational methods predict essential proteins based on their relative densities in a protein-protein interaction (PPI) network. Degree, betweenness, and other appropriate criteria are often used to measure the relative density. However, no matter what criterion is used, a protein is actually ordered by the attributes of this protein per se. In this research, we presented a novel computational method, Integrated Edge Weights (IEW), to first rank protein-protein interactions by integrating their edge weights, and then identified sub PPI networks consisting of those highly-ranked edges, and finally regarded the nodes in these sub networks as essential proteins. We evaluated IEW on three model organisms: Saccharomyces cerevisiae (S. cerevisiae), Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegans). The experimental results showed that IEW achieved better performance than the state-of-the-art methods in terms of precision-recall and Jackknife measures. We had also demonstrated that IEW is a robust and effective method, which can retrieve biologically significant modules by its highly-ranked protein-protein interactions for S. cerevisiae, E. coli, and C. elegans. We believe that, with sufficient data provided, IEW can be used to any other organisms' essential protein identification. A website about IEW can be accessed from http://digbio.missouri.edu/IEW/index.html.

  10. Compositions and methods for improved protein production

    DOEpatents

    Bodie, Elizabeth A [San Carlos, CA; Kim, Steve [San Francisco, CA

    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.

  11. 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.

  12. 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

  13. Improvement of interfacial protein stability by CHAPS.

    PubMed

    Sah, Hongkee; Kim, Kil-Soo

    2006-04-01

    Emulsification of aqueous protein solutions in methylene chloride triggered the formation of water-insoluble aggregates at a water/methylene chloride interface. As a result, the amounts of beta-lactoglobulin and ovalbumin recovered in water were 36 and 44%, respectively. Addition of 5 mM: CHAPS in the aqueous phase raised the degree of beta-lactoglobulin recovery to 96%. Sodium taurocholate, however, failed to improve protein recovery. The stabilizing effect of CHAPS was also protein-specific and concentration-dependent: at >or=5 mM: , the surfactant caused unfolding of ovalbumin to make a water-soluble oligomer. CHAPS thus stabilizes proteins at an interface.

  14. 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.

  15. 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.

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

    DTIC Science & Technology

    2007-12-01

    subcellular localization can be found using the TargetP 105 localization predictor. Identification of possible cell membrane or cell wall...analyzed as vaccine candidates. Examples of this include membrane proteins that localized to subcellular organelle membranes, or proteins with...403-410. 105. Emanuelsson, O., H. Nielsen, S. Brunak, et al. 2000. Predicting subcellular localization of proteins based on their N-terminal amino

  17. 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.

  18. 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

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

    PubMed

    Tang, Haixu; Li, Sujun; Ye, Yuzhen

    2016-12-01

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

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

    PubMed Central

    Tang, Haixu; Li, Sujun; Ye, Yuzhen

    2016-01-01

    Metaproteomic studies adopt the common bottom-up proteomics approach to investigate the protein composition and the dynamics of protein expression in microbial communities. When matched metagenomic and/or metatranscriptomic data of the microbial communities are available, metaproteomic data analyses often employ a metagenome-guided approach, in which complete or fragmental protein-coding genes are first directly predicted from metagenomic (and/or metatranscriptomic) sequences or from their assemblies, and the resulting protein sequences are then used as the reference database for peptide/protein identification from MS/MS spectra. This approach is often limited because protein coding genes predicted from metagenomes are incomplete and fragmental. In this paper, we present a graph-centric approach to improving metagenome-guided peptide and protein identification in metaproteomics. Our method exploits the de Bruijn graph structure reported by metagenome assembly algorithms to generate a comprehensive database of protein sequences encoded in the community. We tested our method using several public metaproteomic datasets with matched metagenomic and metatranscriptomic sequencing data acquired from complex microbial communities in a biological wastewater treatment plant. The results showed that many more peptides and proteins can be identified when assembly graphs were utilized, improving the characterization of the proteins expressed in the microbial communities. The additional proteins we identified contribute to the characterization of important pathways such as those involved in degradation of chemical hazards. Our tools are released as open-source software on github at https://github.com/COL-IU/Graph2Pro. PMID:27918579

  1. 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…

  2. Improving bioorthogonal protein ubiquitylation by click reaction.

    PubMed

    Schneider, Daniel; Schneider, Tatjana; Rösner, Daniel; Scheffner, Martin; Marx, Andreas

    2013-06-15

    Posttranslational modification of proteins with ubiquitin (ubiquitylation) regulates numerous cellular processes. Besides functioning as a signal for proteasomal degradation, ubiquitylation has also non-proteolytic functions by altering the biochemical properties of the modified protein. To investigate the effect(s) of ubiquitylation on the properties of a protein, sufficient amounts of homogenously and well-defined ubiquitylated proteins are required. Here, we report on the elaboration of a method for the generation of high amounts of site-specifically mono-ubiquitylated proteins. Firstly, a one-step affinity purification scheme was developed for ubiquitin containing the unnatural amino acid azidohomoalanine at the C-terminal position. This ubiquitin was conjugated in a click reaction to recombinant DNA polymerase β, equipped with an alkyne function at a distinct position. Secondly, addition of defined amounts of SDS to the reaction significantly improved product formation. With these two technical improvements, we have developed a straight forward procedure for the efficient generation of site-specifically ubiquitylated proteins that can be used to study the effect(s) of ubiquitylation on the activities/properties of a protein.

  3. Identification Method in Software Process Improvement Areas

    NASA Astrophysics Data System (ADS)

    Hayashi, Akihiro; Kataoka, Nobuhiro

    With the prevalence of CMMI (Capability Maturity Model Integration) that is developed in the United States and the international standardization model of Software Process Assessment ISO/IEC 15504, SPI (Software Process Improvement) based on Software Process Assessment is gaining ground in Japan as well. One of the objectives in SPI is to prevent the occurrence of errors in the downstream process by thorough process management in the upstream process. In order to implement the SPI from such viewpoints, this paper suggests the method to analyze Problem Reports developed in the testing process of a project, to specify software bugs existent in leading processes and to identify the SPI in the upstream process. By means of Problem Reports that is capable of externalization and is used in testing processes, knowledge that has not been externalized will be specified as Defect Cluster. Furthermore, deliverables in each process will be identified from the properties of Defect Cluster to implement the SPI, which is of the essence, by identifying processes causing trouble. In the project that was the analytical target, there were 608 Problem Reports in its testing process. As a result of the analysis, it was found that by identifying processes in the upper process to improve with the techniques recommended in this paper, about 13% of cases causing trouble could be prevented from occurring, which will contribute to productivity improvement due to a decrease in the number of rework processes.

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

    PubMed

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

    2014-06-01

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

  6. 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.

  7. Identification of Trypanosome proteins in plasma from African sleeping sickness patients infected with T. b. rhodesiense.

    PubMed

    Eyford, Brett A; Ahmad, Rushdy; 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.

  8. Methods and Approaches to Mass Spectroscopy Based Protein Identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. 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.

  10. Identification of Protein Components of Yeast Telomerase

    DTIC Science & Technology

    2000-09-01

    for forming telomeres at sites with stretches of telomere- like DNA. The pifl mutants also exhibit increased loss and decreased recombination of...like DNA. The pifl mutants also exhibit increased loss 6 and decreased recombination of mitochondrial DNA and thus have a high fraction of...the fission yeast Schizosaccharomyces pombe that was predicted to encode a 805 amino acid protein. The S. pombe gene was called rphl+ (RRM3/PIF1

  11. 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.

  12. 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

  13. Microwave-assisted specific chemical digestion for rapid protein identification.

    PubMed

    Hua, Lin; Low, Teck Yew; Sze, Siu Kwan

    2006-01-01

    We have developed a rapid microwave-assisted protein digestion technique based on classic acid hydrolysis reaction with 2% formic acid solution. In this mild chemical environment, proteins are hydrolyzed to peptides, which can be directly analyzed by MALDI-MS or ESI-MS without prior sample purification. Dilute formic acid cleaves proteins specifically at the C-terminal of aspartyl (Asp) residues within 10 min of exposure to microwave irradiation. By adjusting the irradiation time, we found that the extent of protein fragmentation can be controlled, as shown by the single fragmentation of myoglobin at the C-terminal of any of the Asp residues. The efficacy and simplicity of this technique for protein identification are demonstrated by the peptide mass maps of in-gel digested myoglobin and BSA, as well as proteins isolated from Escherichia coli K12 cells.

  14. 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.

  15. 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.

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

    PubMed

    Negroni, Luc

    2007-01-01

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

  17. 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.

  18. 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.

  19. 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.

  20. Large-scale identification of adverse drug reaction-related proteins through a random walk model

    PubMed Central

    Chen, Xiaowen; Shi, Hongbo; Yang, Feng; Yang, Lei; Lv, Yingli; Wang, Shuyuan; Dai, Enyu; Sun, Dianjun; Jiang, Wei

    2016-01-01

    Adverse drug reactions (ADRs) are responsible for drug failure in clinical trials and affect life quality of patients. The identification of ADRs during the early phases of drug development is an important task. Therefore, predicting potential protein targets eliciting ADRs is essential for understanding the pathogenesis of ADRs. In this study, we proposed a computational algorithm,Integrated Network for Protein-ADR relations (INPADR), to infer potential protein-ADR relations based on an integrated network. First, the integrated network was constructed by connecting the protein-protein interaction network and the ADR similarity network using known protein-ADR relations. Then, candidate protein-ADR relations were further prioritized by performing a random walk with restart on this integrated network. Leave-one-out cross validation was used to evaluate the ability of the INPADR. An AUC of 0.8486 was obtained, which was a significant improvement compared to previous methods. We also applied the INPADR to two ADRs to evaluate its accuracy. The results suggested that the INPADR is capable of finding novel protein-ADR relations. This study provides new insight to our understanding of ADRs. The predicted ADR-related proteins will provide a reference for preclinical safety pharmacology studies and facilitate the identification of ADRs during the early phases of drug development. PMID:27805066

  1. A peptide-retrieval strategy enables significant improvement of quantitative performance without compromising confidence of identification.

    PubMed

    Tu, Chengjian; Shen, Shichen; Sheng, Quanhu; Shyr, Yu; Qu, Jun

    2017-01-30

    Reliable quantification of low-abundance proteins in complex proteomes is challenging largely owing to the limited number of spectra/peptides identified. In this study we developed a straightforward method to improve the quantitative accuracy and precision of proteins by strategically retrieving the less confident peptides that were previously filtered out using the standard target-decoy search strategy. The filtered-out MS/MS spectra matched to confidently-identified proteins were recovered, and the peptide-spectrum-match FDR were re-calculated and controlled at a confident level of FDR≤1%, while protein FDR maintained at ~1%. We evaluated the performance of this strategy in both spectral count- and ion current-based methods. >60% increase of total quantified spectra/peptides was respectively achieved for analyzing a spike-in sample set and a public dataset from CPTAC. Incorporating the peptide retrieval strategy significantly improved the quantitative accuracy and precision, especially for low-abundance proteins (e.g. one-hit proteins). Moreover, the capacity of confidently discovering significantly-altered proteins was also enhanced substantially, as demonstrated with two spike-in datasets. In summary, improved quantitative performance was achieved by this peptide recovery strategy without compromising confidence of protein identification, which can be readily implemented in a broad range of quantitative proteomics techniques including label-free or labeling approaches.

  2. Biochemical component identification by plasmonic improved whispering gallery mode optical resonance based sensor

    NASA Astrophysics Data System (ADS)

    Saetchnikov, Vladimir A.; Tcherniavskaia, Elina A.; Saetchnikov, Anton V.; Schweiger, Gustav; Ostendorf, Andreas

    2014-05-01

    Experimental data on detection and identification of variety of biochemical agents, such as proteins, microelements, antibiotic of different generation etc. in both single and multi component solutions under varied in wide range concentration analyzed on the light scattering parameters of whispering gallery mode optical resonance based sensor are represented. Multiplexing on parameters and components has been realized using developed fluidic sensor cell with fixed in adhesive layer dielectric microspheres and data processing. Biochemical component identification has been performed by developed network analysis techniques. Developed approach is demonstrated to be applicable both for single agent and for multi component biochemical analysis. Novel technique based on optical resonance on microring structures, plasmon resonance and identification tools has been developed. To improve a sensitivity of microring structures microspheres fixed by adhesive had been treated previously by gold nanoparticle solution. Another technique used thin film gold layers deposited on the substrate below adhesive. Both biomolecule and nanoparticle injections caused considerable changes of optical resonance spectra. Plasmonic gold layers under optimized thickness also improve parameters of optical resonance spectra. Biochemical component identification has been also performed by developed network analysis techniques both for single and for multi component solution. So advantages of plasmon enhancing optical microcavity resonance with multiparameter identification tools is used for development of a new platform for ultra sensitive label-free biomedical sensor.

  3. 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

  4. Identification and Characterization of β-Sitosterol Target Proteins

    PubMed Central

    Lomenick, Brett; Shi, Heping; Huang, Jing; Chen, Chuo

    2015-01-01

    β-Sitosterol is the most abundant plant sterol in the human diet. It is also the major component of several traditional medicines, including saw palmetto and devil’s claw. Although β-sitosterol is effective against enlarged prostate in human clinical trials and has anti-cancer and anti-inflammatory activities, the mechanisms of action are poorly understood. Here, we report the identification of two new binding proteins for β-sitosterol that may underlie its beneficial effects. PMID:25804720

  5. Unbiased identification of protein-bait interactions using biochemical enrichment and quantitative proteomics.

    PubMed

    Ong, Shao-En

    2010-03-01

    The use of recombinant proteins, antibodies, small molecules, or nucleic acids as affinity reagents is a simple yet powerful strategy to study the protein-bait interactions that drive biological processes. However, such experiments are often analyzed by Western blotting, limiting the ability to detect novel protein interactors. Unbiased protein identification by mass spectrometry (MS) extends these experiments beyond the study of pairwise interactions, allowing analyses of whole networks of protein-bait interactions. With the latest advances in MS, it is not uncommon to identify thousands of proteins from complex mixtures. Paradoxically, the improved sensitivity of proteomic analyses can make it more difficult to distinguish bait-specific interactions from the large background of identified proteins. In quantitative proteomics, MS signals from protein populations labeled with stable isotopes such as (13)C and (15)N can be identified and quantified relative to unlabeled counterparts. Using quantitative proteomics to compare biochemical enrichments with the bait of interest against those obtained with control baits allows sensitive detection and discrimination of specific protein-bait interactions among the large number of nonspecific interactions with beads. Ad hoc optimization of enrichment conditions is minimized, and mild purification conditions preserve secondary or high-order protein-protein interactions. The combination of biochemical enrichment and quantitative proteomics allows rapid characterization of molecular baits with their interacting proteins, providing tremendous insight into their biological mechanisms of action.

  6. DNA-Templated Aptamer Probe for Identification of Target Proteins.

    PubMed

    Bi, Wenjing; Bai, Xue; Gao, Fan; Lu, Congcong; Wang, Ye; Zhai, Guijin; Tian, Shanshan; Fan, Enguo; Zhang, Yukui; Zhang, Kai

    2017-04-04

    Using aptamers as molecular probes for biomarker discovery has attracted a great deal of attention in recent years. However, it is still a big challenge to accurately identify those protein markers that are targeted by aptamers under physiological conditions due to weak and noncovalent aptamer-protein interactions. Herein, we developed an aptamer based dual-probe using DNA-templated chemistry and photo-cross-linking technique for the identification of target proteins that are recognized by aptamers. In this system, the aptamer was modified by a single strand DNA as binding probe (BP), and another complementary DNA with a photoactive group and reporter group was modified as capture probe (CP). BP was first added to recruit the binding protein via aptamer recognition, and subsequently CP was added to let the cross-linker close to the target via DNA self-assembly, and then a covalent bond between CP and its binding protein was achieved via photo-cross-linking reaction. The captured protein can be detected or affinity enrichment using the tag, finally identified by MS. By use of lysozyme as a model substrate, we demonstrated that this multiple functionalized probe can be utilized for a successful labeling and enrichment of target protein even under a complicated and real environment. Thus, a novel method to precisely identify the aptamer-targeted proteins has been developed and it has a potential application for discovery of aptamer-based biomarkers.

  7. 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.

  8. Top-down protein identification using isotopic envelope fingerprinting.

    PubMed

    Xiao, Kaijie; Yu, Fan; Tian, Zhixin

    2017-01-30

    For top-down protein database search and identification from tandem mass spectra, our isotopic envelope fingerprinting search algorithm and ProteinGoggle search engine have demonstrated their strength of efficiently resolving heavily overlapping data as well separating non-ideal data with non-ideal isotopic envelopes from ideal ones with ideal isotopic envelopes. Here we report our updated ProteinGoggle 2.0 for intact protein database search with full-capacity. The indispensable updates include users' optional definition of dynamic post-translational modifications and static chemical labeling during database creation, comprehensive dissociation methods and ion series, as well as a Proteoform Score for each proteoform. ProteinGoggle has previously been benchmarked with both collision-based dissociation (CID, HCD) and electron-based dissociation (ETD) data of either intact proteins or intact proteomes. Here we report our further benchmarking of the new version of ProteinGoggle with publically available photon-based dissociation (UVPD) data (http://hdl.handle.net/2022/17316) of intact E. coli ribosomal proteins.

  9. Identification of the human testis protein phosphatase 1 interactome.

    PubMed

    Fardilha, Margarida; Esteves, Sara L C; Korrodi-Gregório, Luís; Vintém, Ana Paula; Domingues, Sara C; Rebelo, Sandra; Morrice, Nick; Cohen, Patricia T W; da Cruz e Silva, Odete A B; da Cruz e Silva, Edgar F

    2011-11-15

    Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins-PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26104144

  11. 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.

  12. 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.

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

    PubMed Central

    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.

    2015-01-01

    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 like 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. PMID:25092112

  14. Identification of Protein Interactions Involved in Cellular Signaling

    PubMed Central

    Westermarck, Jukka; Ivaska, Johanna; Corthals, Garry L.

    2013-01-01

    Protein-protein interactions drive biological processes. They are critical for all intra- and extracellular functions, and the technologies to analyze them are widely applied throughout the various fields of biological sciences. This study takes an in-depth view of some common principles of cellular regulation and provides a detailed account of approaches required to comprehensively map signaling protein-protein interactions in any particular cellular system or condition. We provide a critical review of the benefits and disadvantages of the yeast two-hybrid method and affinity purification coupled with mass spectrometric procedures for identification of signaling protein-protein interactions. In particular, we emphasize the quantitative and qualitative differences between tandem affinity and one-step purification (such as FLAG and Strep tag) methods. Although applicable to all types of interaction studies, a special section is devoted in this review to aspects that should be considered when attempting to identify signaling protein interactions that often are transient and weak by nature. Finally, we discuss shotgun and quantitative information that can be gleaned by MS-coupled methods for analysis of multiprotein complexes. PMID:23481661

  15. Distilling the essential features of a protein surface for improving protein-ligand docking, scoring, and virtual screening

    NASA Astrophysics Data System (ADS)

    Zavodszky, Maria I.; Sanschagrin, Paul C.; Kuhn, Leslie A.; Korde, Rajesh S.

    2002-12-01

    For the successful identification and docking of new ligands to a protein target by virtual screening, the essential features of the protein and ligand surfaces must be captured and distilled in an efficient representation. Since the running time for docking increases exponentially with the number of points representing the protein and each ligand candidate, it is important to place these points where the best interactions can be made between the protein and the ligand. This definition of favorable points of interaction can also guide protein structure-based ligand design, which typically focuses on which chemical groups provide the most energetically favorable contacts. In this paper, we present an alternative method of protein template and ligand interaction point design that identifies the most favorable points for making hydrophobic and hydrogen-bond interactions by using a knowledge base. The knowledge-based protein and ligand representations have been incorporated in version 2.0 of SLIDE and resulted in dockings closer to the crystal structure orientations when screening a set of 57 known thrombin and glutathione S-transferase (GST) ligands against the apo structures of these proteins. There was also improved scoring enrichment of the dockings, meaning better differentiation between the chemically diverse known ligands and a ˜15,000-molecule dataset of randomly-chosen small organic molecules. This approach for identifying the most important points of interaction between proteins and their ligands can equally well be used in other docking and design techniques. While much recent effort has focused on improving scoring functions for protein-ligand docking, our results indicate that improving the representation of the chemistry of proteins and their ligands is another avenue that can lead to significant improvements in the identification, docking, and scoring of ligands.

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

    PubMed

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

    2017-02-01

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

  17. 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

  18. Identification of potential protein markers of noble rot infected grapes.

    PubMed

    Lorenzini, Marilinda; Millioni, Renato; Franchin, Cinzia; Zapparoli, Giacomo; Arrigoni, Giorgio; Simonato, Barbara

    2015-07-15

    The evaluation of Botrytis cinerea as noble rot on withered grapes is of great importance to predict the wine sensory/organoleptic properties and to manage the winemaking process of Amarone, a passito dry red wine. This report describes the first proteomic analysis of grapes infected by noble rot under withering conditions to identify possible markers of fungal infection. 2-D gel electrophoresis revealed that protein profiles of infected and not infected grape samples are significantly different in terms of number of spots and relative abundance. Protein identification by MS analysis allowed to identify only in infected berries proteins of B. cinerea that represent potential markers of the presence of the fungus in the withered grapes.

  19. 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.

  20. Identification of hydrophobic proteins as biomarker candidates for colorectal cancer.

    PubMed

    Alvarez-Chaver, Paula; Rodríguez-Piñeiro, Ana M; Rodríguez-Berrocal, Francisco J; Martínez-Zorzano, Vicenta S; Páez de la Cadena, María

    2007-01-01

    Nowadays, colorectal cancer is one of the major causes of cancer death in Western countries. Due to the lack of biomarkers with clinical utility for this pathology, and considering that membrane and hydrophobic proteins have not been studied in depth, we performed a prefractionation of colorectal tissues prior to two-dimensional gel electrophoresis in order to identify hydrophobic proteins differentially expressed in colorectal cancer patients. Fractions enriched in hydrophobic proteins were obtained from healthy mucosa and tumor tissue by a specific extraction method based on temperature-dependent phase partitioning with Triton X-114. Proteins were separated by two-dimensional gel electrophoresis and gels were silver-stained, scanned and compared using the PDQuest software. Those spots presenting significantly different abundance were submitted to mass spectrometry for protein identification. Alterations in the expression of cytoskeletal proteins, including a decrease of vimentin and the absence of desmin, were found. We also detected alterations in antioxidant and transport proteins, chaperones, and in two isoforms of the calcium-binding protein S100A6. On the other hand, vimentin was chosen to corroborate the electrophoretic results by specific immunodetection. Most of the altered proteins have been related to cellular membranes, many of them to lipid rafts microdomains in the plasma membrane, and they have also been implicated in the control of cell proliferation, apoptosis, or metastasis. In conclusion, all the proteins found altered in colorectal tumor samples could be considered as candidates for future studies focused on their utility as markers for colorectal diagnosis and prognosis, or as targets for colorectal cancer therapy.

  1. Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

    PubMed Central

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

    2011-01-01

    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

  2. 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

  3. Proteomic analysis of secreted proteins from Arabidopsis thaliana seedlings: improved recovery following removal of phenolic compounds.

    PubMed

    Charmont, Stéphane; Jamet, Elisabeth; Pont-Lezica, Rafael; Canut, Hervé

    2005-02-01

    Arabidopsis thaliana seedlings grown in liquid culture were used to recover proteins secreted from the whole plant. The aim was to identify apoplastic proteins that may be lost during classical extraction procedures such as preparation of cell walls. The inclusion of polyvinyl-polypyrrolidone (PVPP) in the protocol of purification of secreted proteins allowed a more efficient identification of proteins after their separation by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analyses. Improvement of identification was 4-fold. It is related to an increased number of detectable peaks on mass spectra increasing the percentage of sequence coverage, and the identification confidence. The role of PVPP was to trap phenolic compounds and to prevent their unspecific interactions with proteins. These experiments resulted in the identification of 44 secreted proteins, of which 70% were not identified in previous cell wall proteomic studies. This may be due to specific gene regulation in seedlings and/or to a better access to apoplastic proteins not bound to cell walls.

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

    PubMed Central

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

    2009-01-01

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

  5. 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.

  6. 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

  7. Identification of lipid synthesis and secretion proteins in bovine milk.

    PubMed

    Lu, Jing; van Hooijdonk, Toon; Boeren, Sjef; Vervoort, Jacques; Hettinga, Kasper

    2014-02-01

    Lactation physiology is a process that is only partly understood. Proteomics techniques have shown to be useful to help advance the knowledge on lactation physiology in human and rodent species but have not been used as major tools for dairy cows, except for mastitis. In this paper, advanced non-targeted proteomics techniques (Filter aided sample preparation and NanoLC-Orbitrap-MS/MS) were applied to study the milk fat globule membrane and milk serum fraction, resulting in the identification of 246 proteins. Of these, 23 transporters and enzymes were related to lipid synthesis and secretion in mammary gland and their functions are discussed in detail. The identification of these intracellular transporters and enzymes in milk provides a possibility of using milk itself to study lipid synthesis and secretion pathways. This full-scale scan of milk proteins by using non-targeted proteomic analysis helps to reveal the important proteins involved in lipid synthesis and secretion for further examination in targeted studies.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Identification of proteins that modify cataract of mouse eye lens.

    PubMed

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

    2008-12-01

    The occurrence of a nuclear cataract in the eye lens due to disruption of the alpha3Cx46 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 PTMs 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 nonconservative 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.

  15. Identification of a fibronectin-binding protein from Staphylococcus epidermidis.

    PubMed

    Williams, Rachel J; Henderson, Brian; Sharp, Lindsay J; Nair, Sean P

    2002-12-01

    Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.

  16. In Silico identification of M. TB proteins with diagnostic potential

    PubMed Central

    2013-01-01

    TB, caused by Mycobacterium tuberculosis (MTB), is one of the major global infectious diseases. For the pandemic control, early diagnosis with sensitive and specific methods is fundamental. With the advent of bioinformatics’ tools, the identification of several proteins involved in the pathogenesis of TB (TB) has been possible. In the present work, the MTB genome was explored to look for molecules with possible antigenic properties for their evaluation as part of new generation diagnostic kits based on the release of cytokines. Seven proteins from the MTB proteome and some of their combinations suited the computational test and the results suggested their potential use for the diagnosis of infection in the following population groups: Cuba, Mexico, Malaysia and sub-Saharan Africa. Our predictions were performed using public bioinformatics tools plus three computer programs, developed by our group, to facilitate information retrieval and processing. PMID:23458073

  17. Identification of three novel mutations in hereditary protein S deficiency.

    PubMed

    Bustorff, T C; Freire, I; Gago, T; Crespo, F; David, D

    1997-01-01

    We report the application of single-stranded conformation polymorphism (SSCP) analysis to the screening of 15 functionally important Protein S (PS) gene (PS alpha) regions (4.243 Kb) in 6 unrelated families with PS deficiencies. Direct sequencing of the fragments with altered migration patterns led to the identification of the corresponding molecular alterations. A missense mutation, G to T transversion at codon Cys598, and two different alterations, leading either to allelic exclusion, or premature termination of the protein translation: a G to A transition at codon Trp465 and a 1 nt (T) insertion at codon 265, were identified. The 1 nt insertion was observed in three apparently unrelated families but with a common geographical origin and the mutated allele was undetectable in platelet mRNAs of affected individuals. Family analysis confirmed, in each case, a perfect cosegregation of the mutation with the PS deficiency. We conclude that these alterations represent the causative mutations.

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

    PubMed Central

    2012-01-01

    Background The regulation and function of mammalian RNAs has been increasingly appreciated to operate via RNA-protein interactions. With the recent discovery of thousands of novel human RNA molecules by high-throughput RNA sequencing, efficient methods to uncover RNA-protein interactions are urgently required. Existing methods to study proteins associated with a given RNA are laborious and require substantial amounts of cell-derived starting material. To overcome these limitations, we have developed a rapid and large-scale approach to characterize binding of in vitro transcribed labeled RNA to ~9,400 human recombinant proteins spotted on protein microarrays. Results We have optimized methodology to probe human protein microarrays with full-length RNA molecules and have identified 137 RNA-protein interactions specific for 10 coding and non-coding RNAs. Those proteins showed strong enrichment for common human RNA binding domains such as RRM, RBD, as well as K homology and CCCH type zinc finger motifs. Previously unknown RNA-protein interactions were discovered using this technique, and these interactions were biochemically verified between TP53 mRNA and Staufen1 protein as well as between HRAS mRNA and CNBP protein. Functional characterization of the interaction between Staufen 1 protein and TP53 mRNA revealed a novel role for Staufen 1 in preserving TP53 RNA stability. Conclusions Our approach demonstrates a scalable methodology, allowing rapid and efficient identification of novel human RNA-protein interactions using RNA hybridization to human protein microarrays. Biochemical validation of newly identified interactions between TP53-Stau1 and HRAS-CNBP using reciprocal pull-down experiments, both in vitro and in vivo, demonstrates the utility of this approach to study uncharacterized RNA-protein interactions. PMID:23157412

  19. PRIGSA: protein repeat identification by graph spectral analysis.

    PubMed

    Chakrabarty, Broto; Parekh, Nita

    2014-12-01

    Repetition of a structural motif within protein is associated with a wide range of structural and functional roles. In most cases the repeating units are well conserved at the structural level while at the sequence level, they are mostly undetectable suggesting the need for structure-based methods. Since most known methods require a training dataset, de novo approach is desirable. Here, we propose an efficient graph-based approach for detecting structural repeats in proteins. In a protein structure represented as a graph, interactions between inter- and intra-repeat units are well captured by the eigen spectra of adjacency matrix of the graph. These conserved interactions give rise to similar connections and a unique profile of the principal eigen spectra for each repeating unit. The efficacy of the approach is shown on eight repeat families annotated in UniProt, comprising of both solenoid and nonsolenoid repeats with varied secondary structure architecture and repeat lengths. The performance of the approach is also tested on other known benchmark datasets and the performance compared with two repeat identification methods. For a known repeat type, the algorithm also identifies the type of repeat present in the protein. A web tool implementing the algorithm is available at the URL http://bioinf.iiit.ac.in/PRIGSA/.

  20. 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.

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

    DOE PAGES

    Wang, Dong; Dasari, Surendra; Chambers, Matthew C.; ...

    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

  2. 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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23499924

  4. Charge State Coalescence During Electrospray Ionization Improves Peptide Identification by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Meyer, Jesse G.; A. Komives, Elizabeth

    2012-08-01

    We report the effects of supercharging reagents dimethyl sulphoxide (DMSO) and m-nitrobenzyl alcohol ( m-NBA) applied to untargeted peptide identification, with special emphasis on non-tryptic peptides. Peptides generated from a mixture of five standard proteins digested with trypsin, elastase, or pepsin were separated with nanoflow liquid chromatography using mobile phases modified with either 5 % DMSO or 0.1 % m-NBA. Eluting peptides were ionized by online electrospray and sequenced by both CID and ETD using data-dependent MS/MS. Statistically significant improvements in peptide identifications were observed with DMSO co-solvent. In order to understand this observation, we assessed the effects of supercharging reagents on the chromatographic separation and the electrospray quality. The increase in identifications was not due to supercharging, which was greater for the 0.1 % m-NBA co-solvent and not observed for the 5.0 % DMSO co-solvent. The improved MS/MS efficiency using the DMSO modified mobile phase appeared to result from charge state coalescence.

  5. 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.

  6. Identification of trichoplein, a novel keratin filament-binding protein.

    PubMed

    Nishizawa, Miwako; Izawa, Ichiro; Inoko, Akihito; Hayashi, Yuko; Nagata, Koh-ichi; Yokoyama, Tomoya; Usukura, Jiro; Inagaki, Masaki

    2005-03-01

    Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells.

  7. Identification of protein secretion systems in bacterial genomes.

    PubMed

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

    2016-03-16

    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 T6SS(iii) and T9SS were restricted to Bacteroidetes, and T6SS(ii) 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.

  8. Identification of Naegleria fowleri Proteins Linked to Primary Amebic Meningoencephalitis.

    PubMed

    Jamerson, Melissa; Schmoyer, Jacqueline A; Park, Jay; Marciano-Cabral, Francine; Cabral, Guy

    2017-01-12

    Naegleria fowleri (N. fowleri) causes Primary Amebic Meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). This opportunistic pathogen can exist in cyst, flagellate, or amebic forms, depending on environmental conditions. The amebic form can invade the brain following introduction into the nasal passages. When applied intranasally to a mouse model, cultured N. fowleri amebae exhibit low virulence. However, upon serial passage in mouse brain the amebae acquire a highly virulent state. In the present study, a proteomics approach was applied to the identification of N. fowleri ameba proteins whose expression was associated with the highly virulent state in mice. Mice were inoculated intranasally with axenically cultured or with mouse-passaged amebae. Examination by light and electron microscopy revealed no morphological differences. However, mouse-passaged amebae were more virulent in mice as indicated by exhibiting a two log10 titer decrease in median infective dose 50 (ID50). Scatter plot analysis of amebic lysates revealed a subset of proteins the expression of which was associated with highly virulent amebae. Tandem mass spectrometry indicated that this subset contained proteins that shared homology with those linked to cytoskeletal rearrangement and the invasion process. Invasion assays were performed in the presence of a select inhibitor to expand on the findings. The collective results suggest that N. fowleri gene products linked to cytoskeletal rearrangement and invasion may be candidate targets in the management of PAM.

  9. Identification of lipopolysaccharide-binding proteins in porcine milk

    PubMed Central

    Shahriar, Farshid; Gordon, John R.; Simko, Elemir

    2006-01-01

    Septicemia and endotoxemia initiated by bacterial lipopolysaccharide (LPS) are relatively common in suckling and weaned piglets. Maternal milk is a source of both nutrition and immune protection for piglets. Passive transfer of colostral antibodies is necessary for protection of neonatal piglets against diseases, but the concentration of immunoglobulins in milk rapidly declines during the 1st wk of lactation in all mammals. We hypothesized, therefore, that nonimmunoglobulin substances in milk contribute to the innate protection of neonates against septicemia during the suckling period. Using LPS-affinity chromatography for isolation of LPS-binding proteins and liquid chromatography–mass spectrometry for their identification, we identified in porcine milk the following proteins with LPS-binding capacity: lactoferrin, soluble CD14, serum amyloid A, α-S1 casein, β-casein, and κ-casein. For lactoferrin, α-S1 casein, and κ-casein, in vitro pepsin digestion did not inhibit LPS-binding activity, whereas combined digestion with pepsin and pancreatin abolished it. The biologic functions of these LPS-binding proteins and peptides were not determined. PMID:17042375

  10. 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

  11. 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.

  12. Leptospiral Outer Membrane Protein Microarray, a Novel Approach to Identification of Host Ligand-Binding Proteins

    PubMed Central

    Matsunaga, James; Haake, David A.

    2012-01-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens. PMID:22961849

  13. Algorithm Improvement Program Nuclide Identification Algorithm Scoring Criteria And Scoring Application - DNDO.

    SciTech Connect

    Enghauser, Michael

    2015-02-01

    The goal of the Domestic Nuclear Detection Office (DNDO) Algorithm Improvement Program (AIP) is to facilitate gamma-radiation detector nuclide identification algorithm development, improvement, and validation. Accordingly, scoring criteria have been developed to objectively assess the performance of nuclide identification algorithms. In addition, a Microsoft Excel spreadsheet application for automated nuclide identification scoring has been developed. This report provides an overview of the equations, nuclide weighting factors, nuclide equivalencies, and configuration weighting factors used by the application for scoring nuclide identification algorithm performance. Furthermore, this report presents a general overview of the nuclide identification algorithm scoring application including illustrative examples.

  14. 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).

  15. 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.

  16. Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining.

    PubMed

    Valdes, I; Pitarch, A; Gil, C; Bermúdez, A; Llorente, M; Nombela, C; Méndez, E

    2000-06-01

    The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.

  17. Web-based software for rapid "top-down" proteomic identification of protein biomarkers with implications for bacterial identification

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have developed web-based software for the rapid identification of protein biomarkers of bacterial microorganisms. Proteins from bacterial cell lysates were ionized by matrix-assisted laser desorption/ionization (MALDI), mass-isolated and fragmented using a time-of-flight/time-of-flight (TOF-TOF)...

  18. Identification of major rye secalins as coeliac immunoreactive proteins.

    PubMed

    Rocher, A; Calero, M; Soriano, F; Méndez, E

    1996-06-07

    Six distinct gamma- and omega-type secalins, together with two new low molecular mass glycoproteins, have been identified as the major coeliac immunoreactive proteins from a chloroform/methanol soluble extract from rye endosperm. These components were characterized by a combination of reverse-phase high-performance liquid chromatography, immunoblotting using a coeliac serum and microsequencing analysis. This allowed the identification of a group of secalins with different molecular masses according to their N-terminal amino-acid sequence: one omega-type secalin of 40 kDa (omega 1-40); three gamma-type secalins, one of 70 kDa (gamma-70) and two of 35 kDa (gamma-35); as well as two low molecular mass glycoproteins of 15 and 18 kDa, all exhibiting coeliac serum antigenicity. Moreover, four additional rye components, including two low molecular mass proteins, which did not react with coeliac sera, have also been identified. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the three main purified coeliac immunogenic secalins, gamma-70, gamma-35 and omega 1-40, indicated molecular masses of 71457, 32240 and 39117 Da, respectively. The omega 1-40 secalin displays a significant absorption in the visible region which could be related to its peculiar low capacity to bind both coeliac sera antibodies and Coomassie brilliant blue dye.

  19. 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.

  20. 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...

  1. A novel system identification technique for improved wearable hemodynamics assessment.

    PubMed

    Wiens, Andrew D; Inan, Omer T

    2015-05-01

    Recent advances have led to renewed interest in ballistocardiography (BCG), a noninvasive measure of the small movements of the body due to cardiovascular events. A broad range of platforms have been developed and verified for BCG measurement including beds, chairs, and weighing scales: while the body is coupled to such a platform, the cardiogenic movements are measured. Wearable BCG, measured with an accelerometer affixed to the body, may enable continuous, or more regular, monitoring during the day; however, the signals from such wearable BCGs represent local or distal accelerations of skin and tissue rather than the whole body. In this paper, we propose a novel method to reconstruct the BCG measured with a weighing scale (WS BCG) from a wearable sensor via a training step to remove these local effects. Preliminary validation of this method was performed with 15 subjects: the wearable sensor was placed at three locations on the surface of the body while WS BCG measurements were recorded simultaneously. A regularized system identification approach was used to reconstruct the WS BCG from the wearable BCG. Preliminary results suggest that the relationship between local and central disturbances is highly dependent on both the individual and the location where the accelerometer is placed on the body and that these differences can be resolved via calibration to accurately measure changes in cardiac output and contractility from a wearable sensor. Such measurements could be highly effective, for example, for improved monitoring of heart failure patients at home.

  2. Critical components required to improve deployable laboratory biological hazards identification

    NASA Astrophysics Data System (ADS)

    Niemeyer, Debra M.

    2004-08-01

    An ever-expanding global military mission necessitates quick and accurate identification of biological hazards, whether naturally occurring or man-made. Coupled with an ever-present threat of biological attack, an expanded U.S. presence in worn-torn locations like Southwest Asia presents unique public health challenges. We must heed modern day "lessons learned" from Operation Desert Shield and the Soviet Afghanistan Campaign and guard against rapid incapacitation of troop strength from endemic disease and biological attack. To minimize readiness impacts, field hygiene is enforced, and research on better medical countermeasures such as antibiotics and vaccines continues. However, there are no preventions or remedies for all military-relevant infectious diseases or biological agents. A deployable, streamlined, self-contained diagnostic and public health surveillance laboratory capability with a reach-back communication is critical to meeting global readiness challenges. Current deployable laboratory packages comprise primarily diagnostic or environmental sample testing capabilities. Discussion will focus on critical components needed to improve existing laboratory assets, and to facilitate deployment of small, specialized packages far forward. The ideal laboratory model described will become an essential tool for the Combatant or Incident Commander to maintain force projection in the expeditionary environment.

  3. Protein identification: the origins of peptide mass fingerprinting.

    PubMed

    Henzel, William J; Watanabe, Colin; Stults, John T

    2003-09-01

    Peptide mass fingerprinting (PMF) grew from a need for a faster, more efficient method to identify frequently observed proteins in electrophoresis gels. We describe the genesis of the idea in 1989, and show the first demonstration with fast atom bombardment mass spectrometry. Despite its promise, the method was seldom used until 1992, with the coming of significantly more sensitive commercial instrumentation based on MALDI-TOF-MS. We recount the evolution of the method and its dependence on a number of technical breakthroughs, both in mass spectrometry and in other areas. We show how it laid the foundation for high-throughput, high-sensitivity methods of protein analysis, now known as proteomics. We conclude with recommendations for further improvements, and speculation of the role of PMF in the future.

  4. 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.

  5. Spatially-directed protein identification from tissue sections by top-down LC-MS/MS with electron transfer dissociation.

    PubMed

    Schey, Kevin L; Anderson, David M; Rose, Kristie L

    2013-07-16

    MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for localizing both small molecules and intact proteins in a wide variety of tissue samples in both normal and diseased states. Identification of imaged signals in MALDI-IMS remains a bottleneck in the analysis and limits the interpretation of underlying biology of tissue specimens. In this work, spatially directed tissue microextraction of intact proteins followed by LC-MS/MS with electron transfer dissociation (ETD) was used to identify proteins from specific locations in three tissue types; ocular lens, brain, and kidney. Detection limits were such that a 1 μL extraction volume was sufficient to deliver proteins to the LC-MS/MS instrumentation with sufficient sensitivity to detect 50-100 proteins in a single experiment. Additionally, multiple modified proteins were identified; including truncated lens proteins that would be difficult to assign to an imaged mass using a bottom-up approach. Protein separation and identification are expected to improve with advances in intact protein fractionation/chromatography and advances in interpretation algorithms leading to increased depth of proteome coverage from distinct tissue locations.

  6. Spatially-Directed Protein Identification from Tissue Sections by Top-Down LC-MS/MS with Electron Transfer Dissociation

    PubMed Central

    Schey, Kevin L.; Anderson, David M.; Rose, Kristie L.

    2013-01-01

    MALDI imaging mass spectrometry (MALDI-IMS) has become a powerful tool for localizing both small molecules and intact proteins in a wide variety of tissue samples in both normal and diseased states. Identification of imaged signals in MALDI-IMS remains a bottleneck in the analysis and limits the interpretation of underlying biology of tissue specimens. In this work, spatially-directed tissue microextraction of intact proteins followed by LC-MS/MS with electron transfer dissociation (ETD) was used to identify proteins from specific locations in three tissue types; ocular lens, brain, and kidney. Detection limits were such that a 1 microliter extraction volume was sufficient to deliver proteins to the LC-MS/MS instrumentation with sufficient sensitivity to detect 50–100 proteins in a single experiment. Additionally, multiple modified proteins were identified; including truncated lens proteins that would be difficult to assign to an imaged mass using a bottom-up approach. Protein separation and identification are expected to improve with advances in intact protein fractionation/chromatography and advances in interpretation algorithms leading to increased depth of proteome coverage from distinct tissue locations. PMID:23718750

  7. A Novel System Identification Technique for Improved Wearable Hemodynamics Assessment

    PubMed Central

    Wiens, Andrew D.; Inan, Omer T.

    2015-01-01

    Recent advances have led to renewed interest in ballistocardiography (BCG), a non-invasive measure of the small reaction forces on the body from cardiovascular events. A broad range of platforms have been developed and verified for BCG measurement including beds, chairs, and weighing scales: while the body is coupled to such a platform, the cardiogenic movements of the center-of-mass (COM) are measured. Wearable BCG, measured with an accelerometer affixed to the body, may enable continuous, or more regular, monitoring during the day; however, the signals from such wearable BCGs represent local or distal accelerations of skin and tissue rather than the displacement of the body's COM. In this paper we propose a novel method to reconstruct the COM BCG from a wearable sensor via a training step to remove these local effects. Preliminary validation of this method was performed with fifteen subjects: the wearable sensor was placed at three locations on the surface of the body while COM BCG measurements were recorded simultaneously with a modified weighing scale. A regularized system identification approach was used to reconstruct the COM BCG from the wearable signal. Preliminary results suggest that the relationship between local and central forces is highly dependent on both the individual and the location where the wearable sensor is placed on the body and that these differences can be resolved via calibration to accurately measure changes in cardiac output and contractility from a wearable sensor. Such measurements could be highly effective, for example, for improved monitoring of heart failure patients at home. PMID:25561589

  8. Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry.

    PubMed

    Otto, A; Thiede, B; Müller, E C; Scheler, C; Wittmann-Liebold, B; Jungblut, P

    1996-10-01

    Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.

  9. 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.

  10. Identification of Central Nervous System Proteins in Human Blood Serum and Plasma.

    PubMed

    Miroshnichenko, Yu V; Petushkova, N A; Teryaeva, N B; Lisitsa, A V; Zgoda, V G; Belyaev, A Yu; Potapov, A A

    2015-11-01

    Mass-spectrometric identification of proteins in human blood plasma and serum was performed by comparing mass-spectra of fragmented peptides using Swiss-Prot and UniProtKB databases of amino acid sequences. After choosing the appropriate identification conditions we found that combination of spectrum search parameters are optimal for identification of CNS proteins. In the studied plasma and serum samples, 9 proteins involved into pathological processes in the nervous tissue were identified; 7 of them were identified in both plasma and serum.

  11. Effect of cleavage enzyme, search algorithm and decoy database on mass spectrometric identification of wheat gluten proteins.

    PubMed

    Vensel, William H; Dupont, Frances M; Sloane, Stacia; Altenbach, Susan B

    2011-07-01

    While tandem mass spectrometry (MS/MS) is routinely used to identify proteins from complex mixtures, certain types of proteins present unique challenges for MS/MS analyses. The major wheat gluten proteins, gliadins and glutenins, are particularly difficult to distinguish by MS/MS. Each of these groups contains many individual proteins with similar sequences that include repetitive motifs rich in proline and glutamine. These proteins have few cleavable tryptic sites, often resulting in only one or two tryptic peptides that may not provide sufficient information for identification. Additionally, there are less than 14,000 complete protein sequences from wheat in the current NCBInr release. In this paper, MS/MS methods were optimized for the identification of the wheat gluten proteins. Chymotrypsin and thermolysin as well as trypsin were used to digest the proteins and the collision energy was adjusted to improve fragmentation of chymotryptic and thermolytic peptides. Specialized databases were constructed that included protein sequences derived from contigs from several assemblies of wheat expressed sequence tags (ESTs), including contigs assembled from ESTs of the cultivar under study. Two different search algorithms were used to interrogate the database and the results were analyzed and displayed using a commercially available software package (Scaffold). We examined the effect of protein database content and size on the false discovery rate. We found that as database size increased above 30,000 sequences there was a decrease in the number of proteins identified. Also, the type of decoy database influenced the number of proteins identified. Using three enzymes, two search algorithms and a specialized database allowed us to greatly increase the number of detected peptides and distinguish proteins within each gluten protein group.

  12. RecRWR: a recursive random walk method for improved identification of diseases.

    PubMed

    Arrais, Joel Perdiz; Oliveira, José Luís

    2015-01-01

    High-throughput methods such as next-generation sequencing or DNA microarrays lack precision, as they return hundreds of genes for a single disease profile. Several computational methods applied to physical interaction of protein networks have been successfully used in identification of the best disease candidates for each expression profile. An open problem for these methods is the ability to combine and take advantage of the wealth of biomedical data publicly available. We propose an enhanced method to improve selection of the best disease targets for a multilayer biomedical network that integrates PPI data annotated with stable knowledge from OMIM diseases and GO biological processes. We present a comprehensive validation that demonstrates the advantage of the proposed approach, Recursive Random Walk with Restarts (RecRWR). The obtained results outline the superiority of the proposed approach, RecRWR, in identifying disease candidates, especially with high levels of biological noise and benefiting from all data available.

  13. 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—...

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

    PubMed

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

    2016-12-01

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

  15. Choosing an Optimal Database for Protein Identification from Tandem Mass Spectrometry Data.

    PubMed

    Kumar, Dhirendra; Yadav, Amit Kumar; Dash, Debasis

    2017-01-01

    Database searching is the preferred method for protein identification from digital spectra of mass to charge ratios (m/z) detected for protein samples through mass spectrometers. The search database is one of the major influencing factors in discovering proteins present in the sample and thus in deriving biological conclusions. In most cases the choice of search database is arbitrary. Here we describe common search databases used in proteomic studies and their impact on final list of identified proteins. We also elaborate upon factors like composition and size of the search database that can influence the protein identification process. In conclusion, we suggest that choice of the database depends on the type of inferences to be derived from proteomics data. However, making additional efforts to build a compact and concise database for a targeted question should generally be rewarding in achieving confident protein identifications.

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

    PubMed

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

    2008-12-01

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

  17. Comprehensive identification of proteins in Hodgkin lymphoma-derived Reed-Sternberg cells by LC-MS/MS.

    PubMed

    Wallentine, Jeremy C; Kim, Ki Kwon; Seiler, Charles E; Vaughn, Cecily P; Crockett, David K; Tripp, Sheryl R; Elenitoba-Johnson, Kojo S J; Lim, Megan S

    2007-11-01

    Mass spectrometry-based proteomics in conjunction with liquid chromatography and bioinformatics analysis provides a highly sensitive and high-throughput approach for the identification of proteins. Hodgkin lymphoma is a form of malignant lymphoma characterized by the proliferation of Reed-Sternberg cells and background reactive lymphocytes. Comprehensive analysis of proteins expressed and released by Reed-Sternberg cells would assist in the discovery of potential biomarkers and improve our understanding of its pathogenesis. The subcellular proteome of the three cellular compartments from L428 and KMH2 Hodgkin lymphoma-derived cell lines were fractionated, and analyzed by reverse-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry. Additionally, proteins released by Hodgkin lymphoma-derived L428 cells were extracted from serum-free culture media and analyzed. Peptide spectra were analyzed using TurboSEQUEST against the UniProt protein database (5.26.05; 188 712 entries). A subset of the identified proteins was validated by Western blot analysis, immunofluorescence microscopy and immunohistochemistry. A total of 1945 proteins were identified with 785 from the cytosolic fraction, 305 from the membrane fraction, 441 from the nuclear fraction and 414 released proteins using a minimum of two peptide identifications per protein and an error rate of <5.0%. Identification of proteins from diverse functional groups reflected the functional complexity of the Reed-Sternberg proteome. Proteins with previously reported oncogenic function in other cancers and from signaling pathways implicated in Hodgkin lymphoma were identified. Selected proteins without previously demonstrated expression in Hodgkin lymphoma were validated by Western blot analysis (B-RAF, Erb-B3), immunofluorescence microscopy (Axin1, Tenascin-X, Mucin-2) and immunohistochemistry using a tissue microarray (BRAF, PIM1). This study represents the first comprehensive inventory

  18. Proteogenomic Analysis Greatly Expands the Identification of Proteins Related to Reproduction in the Apogamous Fern Dryopteris affinis ssp. affinis

    PubMed Central

    Grossmann, Jonas; Fernández, Helena; Chaubey, Pururawa M.; Valdés, Ana E.; Gagliardini, Valeria; Cañal, María J.; Russo, Giancarlo; Grossniklaus, Ueli

    2017-01-01

    Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis. For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a “species-specific transcriptome database” (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non-model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models. PMID:28382042

  19. Identification of genes and proteins associated with anagen wool growth.

    PubMed

    Zhao, J; Liu, N; Liu, K; He, J; Yu, J; Bu, R; Cheng, M; De, W; Liu, J; Li, H

    2017-02-01

    Identifying genes of major effect for wool growth would offer strategies for improving the quality and increasing the yield of fine wool. In this study, we employed the Agilent Sheep Gene Expression Microarray and proteomic technology to investigate the gene expression patterns of body side skin (more wool growing) in Aohan fine wool sheep (a Chinese indigenous breed) in comparison with groin skin (no wool growing) at the anagen stage of the wool follicle. A microarray study revealed that 4772 probes were differentially expressed, including 2071 upregulated and 2701 downregulated probes, in the comparisons of body side skin vs. groin skin (S/G). The microarray results were verified by means of quantitative PCR. A total of 1099 probes were assigned to unique genes/transcripts. The number of distinct genes/transcripts (annotated) was 926, of which 352 were upregulated and 574 were downregulated. In S/G, 13 genes were upregulated by more than 10 fold, whereas 60 genes were downregulated by more than 10 fold. Further analysis revealed that the majority of the genes possibly related to the wool growth could be assigned to categories including regulation of cell division, intermediate filament, cytoskeletal part and growth factor activity. Several potential gene families may participate in hair growth regulation, including fibroblast growth factors, transforming growth factor-β, WNTs, insulin-like growth factor, vascular endothelial growth factors and so on. Proteomic analysis also revealed 196 differentially expressed protein points, of which 121 were identified as single protein points.

  20. 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.

  1. Improving the Use of Self-Generated Identification Codes

    ERIC Educational Resources Information Center

    Schnell, Rainer; Bachteler, Tobias; Reiher, Jorg

    2010-01-01

    In panel studies on sensitive topics, respondent-generated identification codes are often used to link records across surveys. However, usually a substantial number of cases are lost due to the codes. These losses may cause biased estimates. Using more components and linking the codes by the Levenshtein string distance function will reduce the…

  2. 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

  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. Single-step protease cleavage elution for identification of protein-protein interactions from GST pull-down and mass spectrometry.

    PubMed

    Luo, Lin; King, Nathan P; Yeo, Jeremy C; Jones, Alun; Stow, Jennifer L

    2014-01-01

    The study of protein-protein interactions is a major theme in biological disciplines. Pull-down or affinity-precipitation assays using GST fusion proteins have become one of the most common and valuable approaches to identify novel binding partners for proteins of interest (bait). Non-specific binding of prey proteins to the beads or to GST itself, however, inevitably complicates and impedes subsequent analysis of pull-down results. A variety of measures, each with inherent advantages and limitations, can minimise the extent of the background. This technical brief details and tests a modification of established GST pull-down protocols. By specifically eluting only the bait (minus the GST tag) and the associated non-specific binding proteins with a simple, single-step protease cleavage, a cleaner platform for downstream protein identification with MS is established. We present a proof of concept for this method, as evidenced by a GST pull-down/MS case study of the small guanosine triphosphatase (GTPase) Rab31 in which: (i) sensitivity was enhanced, (ii) a reduced level of background was observed, (iii) distinguishability of non-specific contaminant proteins from genuine binders was improved and (iv) a putative new protein-protein interaction was discovered. Our protease cleavage step is readily applicable to all further affinity tag pull-downs.

  5. 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...

  6. Protein identification with N and C-terminal sequence tags in proteome projects.

    PubMed

    Wilkins, M R; Gasteiger, E; Tonella, L; Ou, K; Tyler, M; Sanchez, J C; Gooley, A A; Walsh, B J; Bairoch, A; Appel, R D; Williams, K L; Hochstrasser, D F

    1998-05-08

    Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein

  7. 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.

  8. 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.

  9. Fast Photochemical Oxidation of Proteins Coupled to Multidimensional Protein Identification Technology (MudPIT): Expanding Footprinting Strategies to Complex Systems

    NASA Astrophysics Data System (ADS)

    Rinas, Aimee; Jones, Lisa M.

    2015-04-01

    Peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. Here we report the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Using this method led to a 37% increase in unique peptide identifications as well as a 70% increase in protein group identifications over one-dimensional data-dependent acquisition on the same samples. Furthermore, we demonstrate the combination of these methods as a means to investigate megadalton complexes.

  10. Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae.

    PubMed

    Kennedy-Darling, Julia; Guillen-Ahlers, Hector; Shortreed, Michael R; Scalf, Mark; Frey, Brian L; Kendziorski, Christina; Olivier, Michael; Gasch, Audrey P; Smith, Lloyd M

    2014-08-01

    DNA-protein interactions play critical roles in the control of genome expression and other fundamental processes. An essential element in understanding how these systems function is to identify their molecular components. We present here a novel strategy, Hybridization Capture of Chromatin Associated Proteins for Proteomics (HyCCAPP), to identify proteins that are interacting with any given region of the genome. This technology identifies and quantifies the proteins that are specifically interacting with a genomic region of interest by sequence-specific hybridization capture of the target region from in vivo cross-linked chromatin, followed by mass spectrometric identification and quantification of associated proteins. We demonstrate the utility of HyCCAPP by identifying proteins associated with three multicopy and one single-copy loci in yeast. In each case, a locus-specific pattern of target-associated proteins was revealed. The binding of previously unknown proteins was confirmed by ChIP in 11 of 17 cases. The identification of many previously known proteins at each locus provides strong support for the ability of HyCCAPP to correctly identify DNA-associated proteins in a sequence-specific manner, while the discovery of previously unknown proteins provides new biological insights into transcriptional and regulatory processes at the target locus.

  11. RPI-Bind: a structure-based method for accurate identification of RNA-protein binding sites.

    PubMed

    Luo, Jiesi; Liu, Liang; Venkateswaran, Suresh; Song, Qianqian; Zhou, Xiaobo

    2017-04-04

    RNA and protein interactions play crucial roles in multiple biological processes, while these interactions are significantly influenced by the structures and sequences of protein and RNA molecules. In this study, we first performed an analysis of RNA-protein interacting complexes, and identified interface properties of sequences and structures, which reveal the diverse nature of the binding sites. With the observations, we built a three-step prediction model, namely RPI-Bind, for the identification of RNA-protein binding regions using the sequences and structures of both proteins and RNAs. The three steps include 1) the prediction of RNA binding regions on protein, 2) the prediction of protein binding regions on RNA, and 3) the prediction of interacting regions on both RNA and protein simultaneously, with the results from steps 1) and 2). Compared with existing methods, most of which employ only sequences, our model significantly improves the prediction accuracy at each of the three steps. Especially, our model outperforms the catRAPID by >20% at the 3(rd) step. All of these results indicate the importance of structures in RNA-protein interactions, and suggest that the RPI-Bind model is a powerful theoretical framework for studying RNA-protein interactions.

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

    PubMed Central

    Barkla, Bronwyn J.

    2016-01-01

    Modern day agriculture practice is narrowing the genetic diversity in our food supply. This may compromise the ability to obtain high yield under extreme climactic conditions, threatening food security for a rapidly growing world population. To identify genetic diversity, tolerance mechanisms of cultivars, landraces and wild relatives of major crops can be identified and ultimately exploited for yield improvement. Quantitative proteomics allows for the identification of proteins that may contribute to tolerance mechanisms by directly comparing protein abundance under stress conditions between genotypes differing in their stress responses. In this review, a summary is provided of the data accumulated from quantitative proteomic comparisons of crop genotypes/cultivars which present different stress tolerance responses when exposed to various abiotic stress conditions, including drought, salinity, high/low temperature, nutrient deficiency and UV-B irradiation. This field of research aims to identify molecular features that can be developed as biomarkers for crop improvement, however without accurate phenotyping, careful experimental design, statistical robustness and appropriate biomarker validation and verification it will be challenging to deliver what is promised. PMID:28248236

  13. Functional module search in protein networks based on semantic similarity improves the analysis of proteomics data.

    PubMed

    Boyanova, Desislava; Nilla, Santosh; Klau, Gunnar W; Dandekar, Thomas; Müller, Tobias; Dittrich, Marcus

    2014-07-01

    The continuously evolving field of proteomics produces increasing amounts of data while improving the quality of protein identifications. Albeit quantitative measurements are becoming more popular, many proteomic studies are still based on non-quantitative methods for protein identification. These studies result in potentially large sets of identified proteins, where the biological interpretation of proteins can be challenging. Systems biology develops innovative network-based methods, which allow an integrated analysis of these data. Here we present a novel approach, which combines prior knowledge of protein-protein interactions (PPI) with proteomics data using functional similarity measurements of interacting proteins. This integrated network analysis exactly identifies network modules with a maximal consistent functional similarity reflecting biological processes of the investigated cells. We validated our approach on small (H9N2 virus-infected gastric cells) and large (blood constituents) proteomic data sets. Using this novel algorithm, we identified characteristic functional modules in virus-infected cells, comprising key signaling proteins (e.g. the stress-related kinase RAF1) and demonstrate that this method allows a module-based functional characterization of cell types. Analysis of a large proteome data set of blood constituents resulted in clear separation of blood cells according to their developmental origin. A detailed investigation of the T-cell proteome further illustrates how the algorithm partitions large networks into functional subnetworks each representing specific cellular functions. These results demonstrate that the integrated network approach not only allows a detailed analysis of proteome networks but also yields a functional decomposition of complex proteomic data sets and thereby provides deeper insights into the underlying cellular processes of the investigated system.

  14. MALDI imaging mass spectrometry of Pacific White Shrimp L. vannamei and identification of abdominal muscle proteins.

    PubMed

    Schey, Kevin L; Hachey, Amanda J; Rose, Kristie L; Grey, Angus C

    2016-06-01

    MALDI imaging mass spectrometry (IMS) has been applied to whole animal tissue sections of Pacific White Shrimp, Litopenaeus vannamei, in an effort to identify and spatially localize proteins in specific organ systems. Frozen shrimp were sectioned along the ventral-dorsal axis and methods were optimized for matrix application. In addition, tissue microextraction and homogenization was conducted followed by top-down LC-MS/MS analysis of intact proteins and searches of shrimp EST databases to identify imaged proteins. IMS images revealed organ system specific protein signals that highlighted the hepatopancreas, heart, nervous system, musculature, and cuticle. Top-down proteomics identification of abdominal muscle proteins revealed the sequence of the most abundant muscle protein that has no sequence homology to known proteins. Additional identifications of abdominal muscle proteins included titin, troponin-I, ubiquitin, as well as intact and multiple truncated forms of flightin; a protein known to function in high frequency contraction of insect wing muscles. The combined use of imaging mass spectrometry and top-down proteomics allowed for identification of novel proteins from the sparsely populated shrimp protein databases.

  15. Identification and characterization of a Dictyostelium discoideum ribosomal protein gene.

    PubMed Central

    Szymkowski, D E; Deering, R A

    1990-01-01

    We have identified a developmentally repressed large-subunit ribosomal protein gene of Dictyostelium discoideum based on sequence similarity to other ribosomal proteins. Protein rpl7 is homologous to large subunit ribosomal proteins from the rat and possibly to Mycoplasma capricolum and Escherichia coli, but is not similar to three sequenced ribosomal proteins in Dictyostelium. The rpl7 gene is present at one copy per genome, as are six other cloned Dictyostelium ribosomal proteins. Restriction fragment length polymorphisms exist for ribosomal protein genes rpl7, rp1024, and rp110 in strain HU182; most Dictyostelium ribosomal protein genes examined are linked no closer than 30-100 kb to each other in the genome. Dictyostelium ribosomal proteins are known to be developmentally regulated, and levels of rpl7 transcript gradually decrease during the 24-hour development cycle. This drop correlates with that of rp1024, indicating these and other ribosomal protein genes may be coordinately regulated. To determine the cellular location of the protein, we raised antibodies to an rpl7-derived branched synthetic peptide. These antibodies cross-reacted with one protein of the expected size in a ribosomal protein fraction of Dictyostelium, indicating that the product of gene rpl7 is localized in the ribosome. Images PMID:1975664

  16. 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.

  17. 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.

  18. 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.

  19. Improved method for predicting protein fold patterns with ensemble classifiers.

    PubMed

    Chen, W; Liu, X; Huang, Y; Jiang, Y; Zou, Q; Lin, C

    2012-01-27

    Protein folding is recognized as a critical problem in the field of biophysics in the 21st century. Predicting protein-folding patterns is challenging due to the complex structure of proteins. In an attempt to solve this problem, we employed ensemble classifiers to improve prediction accuracy. In our experiments, 188-dimensional features were extracted based on the composition and physical-chemical property of proteins and 20-dimensional features were selected using a coupled position-specific scoring matrix. Compared with traditional prediction methods, these methods were superior in terms of prediction accuracy. The 188-dimensional feature-based method achieved 71.2% accuracy in five cross-validations. The accuracy rose to 77% when we used a 20-dimensional feature vector. These methods were used on recent data, with 54.2% accuracy. Source codes and dataset, together with web server and software tools for prediction, are available at: http://datamining.xmu.edu.cn/main/~cwc/ProteinPredict.html.

  20. 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

    2013-04-09

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

  1. 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.

  2. 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

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

    PubMed

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

    2015-01-01

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

  4. 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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-03-08

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

  7. 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.

  8. 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.

  9. IDENTIFICATION OF PROTEIN FRACTIONS OF MILK COWS CASEIN COMPLEX.

    PubMed

    Iukalo, A V

    2015-01-01

    To date, dozens of biologically active peptides formed during proteolysis of casein fractions have been discovered. The use of these peptides is closely related to the necessity of their rapid identification. The aim of this work was the development of an electrophoresis system for rapid identification of individual fractions in serial studies and the separation of the milk casein complex. Considering the abnormal nature of the interaction of caseins with the sodium dodecyl sulfate and similar values of their molecular masses, the anode electrophoresis system in a homogeneous polyacrylamide gel was taken as a basis. Caseins, in this system, are separated according to their charge and located on the electrophoregram in accordance with the modern classification. Urea was used as a disaggregating agent in gel. It was shown that the use of Studier type apparatus for electrophoresis with changeable dimensions of electrophoretic chamber significantly reduces (to 45 min) the time for identification of casein fractions. This method may be useful for rapid identification of casein fractions, as well as for rapid analysis of natural milk and milk products.

  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.

  11. 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

  12. Identification and characterization of Euphorbia nivulia latex proteins.

    PubMed

    Badgujar, Shamkant B; Mahajan, Raghunath T

    2014-03-01

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

  13. Identification of a Protein for Prostate-Specific Infection

    DTIC Science & Technology

    2006-12-01

    P2) modified gp41 envelope protein was used to infect LNCaP cells in 24-well plates. The control vector that does not have gp41 -P2 envelope protein...on viral surface was also used to infect LNCaP cells as the control. Our results demonstrated that with the gp41 -P2 envelope protein on the surface...Insert sequences into lentiviral envelope protein gp41 As described in the annual report of last year, we have identified two peptides that can

  14. Improved protein refolding using hollow-fibre membrane dialysis.

    PubMed

    West, S M; Chaudhuri, J B; Howell, J A

    1998-03-05

    We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration membrane to refold bovine carbonic anhydrase, loaded into the lumen space, by removing the denaturant through controlled dialysis via the shell side space. When challenged with GdnHCl-denatured carbonic anhydrase, 70% of the loaded protein reptated through the membrane into the circulating dialysis buffer. Reptation occurred because the protein, in its fully unfolded configuration, was able to pass through the pores. The loss of carbonic anhydrase through the membrane was controlled by the dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min reduced the denaturant around the protein to a concentration that allowed the return of secondary structure, increasing the hydrodynamic radius, thus preventing protein transmission. Under these conditions a maximum of 42% of carbonic anhydrase was recovered (from a starting concentration of 5 mg/mL) with 94% activity. This is an improvement over refolding carbonic anhydrase by simple batch dilution, which gave a maximum reactivation of 85% with 35% soluble protein yield. The batch refolding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25 degrees C the temperature sensitivity was considerably reduced. In order to reduce the convection forces that give rise to aggregation and promote refolding the dialyzate was slowly heated from 4 to 25 degrees C. This slow, temperature-controlled refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membrane, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield.

  15. Large-scale identification of putative exported proteins in Candida albicans by genetic selection.

    PubMed

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

    2002-08-01

    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.

  16. 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

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

    PubMed Central

    Straub, Daniel

    2017-01-01

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

  18. Identification of urinary proteins potentially associated with diabetic kidney disease.

    PubMed

    Marikanty, R K; Gupta, M K; Cherukuvada, S V B; Kompella, S S S; Prayaga, A K; Konda, S; Polisetty, R V; Idris, M M; Rao, P V; Chandak, G R; Dakshinamurty, K V

    2016-01-01

    Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. Although several parameters are used to evaluate renal damage, in many instances, there is no pathological change until damage is already advanced. Mass spectrometry-based proteomics is a novel tool to identify newer diagnostic markers. To identify urinary proteins associated with renal complications in diabetes, we collected urine samples from 10 type 2 diabetes patients each with normoalbuminuria, micro- and macro-albuminuria and compared their urinary proteome with that of 10 healthy individuals. Urinary proteins were concentrated, depleted of albumin and five other abundant plasma proteins and in-gel trypsin digested after prefractionation on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The peptides were analyzed using a nanoflow reverse phase liquid chromatography system coupled to linear trap quadrupole-Orbitrap mass spectrometer. We identified large number of proteins in each group, of which many were exclusively present in individual patient groups. A total of 53 proteins were common in all patients but were absent in the controls. The majority of the proteins were functionally binding, biologically involved in metabolic processes, and showed enrichment of alternative complement and blood coagulation pathways. In addition to identifying reported proteins such as α2-HS-glycoprotein and Vitamin D binding protein, we detected novel proteins such as CD59, extracellular matrix protein 1 (ECM1), factor H, and myoglobin in the urine of macroalbuminuria patients. ECM1 and factor H are known to influence mesangial cell proliferation, and CD59 causes microvascular damage by influencing membrane attack complex deposition, suggestive their biological relevance to DN. Thus, we have developed a proteome database where various proteins exclusively present in the patients may be further investigated for their role as stage-specific markers and possible therapeutic targets.

  19. Identification of urinary proteins potentially associated with diabetic kidney disease

    PubMed Central

    Marikanty, R. K.; Gupta, M. K.; Cherukuvada, S. V. B.; Kompella, S. S. S; Prayaga, A. K.; Konda, S.; Polisetty, R. V.; Idris, M. M.; Rao, P. V.; Chandak, G. R.; Dakshinamurty, K. V.

    2016-01-01

    Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. Although several parameters are used to evaluate renal damage, in many instances, there is no pathological change until damage is already advanced. Mass spectrometry-based proteomics is a novel tool to identify newer diagnostic markers. To identify urinary proteins associated with renal complications in diabetes, we collected urine samples from 10 type 2 diabetes patients each with normoalbuminuria, micro- and macro-albuminuria and compared their urinary proteome with that of 10 healthy individuals. Urinary proteins were concentrated, depleted of albumin and five other abundant plasma proteins and in-gel trypsin digested after prefractionation on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The peptides were analyzed using a nanoflow reverse phase liquid chromatography system coupled to linear trap quadrupole-Orbitrap mass spectrometer. We identified large number of proteins in each group, of which many were exclusively present in individual patient groups. A total of 53 proteins were common in all patients but were absent in the controls. The majority of the proteins were functionally binding, biologically involved in metabolic processes, and showed enrichment of alternative complement and blood coagulation pathways. In addition to identifying reported proteins such as α2-HS-glycoprotein and Vitamin D binding protein, we detected novel proteins such as CD59, extracellular matrix protein 1 (ECM1), factor H, and myoglobin in the urine of macroalbuminuria patients. ECM1 and factor H are known to influence mesangial cell proliferation, and CD59 causes microvascular damage by influencing membrane attack complex deposition, suggestive their biological relevance to DN. Thus, we have developed a proteome database where various proteins exclusively present in the patients may be further investigated for their role as stage-specific markers and possible therapeutic targets. PMID

  20. 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.

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

    PubMed

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

    2012-03-01

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

  2. 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

  3. Identification of the Coated Vesicle Proteins That Bind Calmodulin

    DTIC Science & Technology

    1982-11-16

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS November 16, 1982 Pages 186-193 IDENTIFICATION OF THE COATED VESICLE...00/0 v80 C opyigh CI92byAaeicPe,.n.8 A#I rihts of reproduction in any form ,ese,’d. 186 Vol. 109, No. 1, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS allows...coated vesicles under 187 Vol. 109, No. 1, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ABC b e fgh i F

  4. 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

  5. Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

    PubMed Central

    Lennen, Rebecca M.; Politz, Mark G.; Kruziki, Max A.

    2013-01-01

    Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB+ strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts. PMID:23104810

  6. A proteogenomics approach integrating proteomics and ribosome profiling increases the efficiency of protein identification and enables the discovery of alternative translation start sites.

    PubMed

    Koch, Alexander; Gawron, Daria; Steyaert, Sandra; Ndah, Elvis; Crappé, Jeroen; De Keulenaer, Sarah; De Meester, Ellen; Ma, Ming; Shen, Ben; Gevaert, Kris; Van Criekinge, Wim; Van Damme, Petra; Menschaert, Gerben

    2014-12-01

    Next-generation transcriptome sequencing is increasingly integrated with MS to enhance MS-based protein and peptide identification. Recently, a breakthrough in transcriptome analysis was achieved with the development of ribosome profiling (ribo-seq). This technology is based on the deep sequencing of ribosome-protected mRNA fragments, thereby enabling the direct observation of in vivo protein synthesis at the transcript level. In order to explore the impact of a ribo-seq-derived protein sequence search space on MS/MS spectrum identification, we performed a comprehensive proteome study on a human cancer cell line, using both shotgun and N-terminal proteomics, next to ribosome profiling, which was used to delineate (alternative) translational reading frames. By including protein-level evidence of sample-specific genetic variation and alternative translation, this strategy improved the identification score of 69 proteins and identified 22 new proteins in the shotgun experiment. Furthermore, we discovered 18 new alternative translation start sites in the N-terminal proteomics data and observed a correlation between the quantitative measures of ribo-seq and shotgun proteomics with a Pearson correlation coefficient ranging from 0.483 to 0.664. Overall, this study demonstrated the benefits of ribosome profiling for MS-based protein and peptide identification and we believe this approach could develop into a common practice for next-generation proteomics.

  7. Identification of a novel protein complex containing annexin A4, rabphilin and synaptotagmin.

    PubMed

    Willshaw, Angela; Grant, Karen; Yan, Jun; Rockliffe, Nichola; Ambavarapu, Sailaja; Burdyga, Galina; Varro, Andrea; Fukuoka, Shin-Ichi; Gawler, Debra

    2004-02-13

    Rabphilin is a synaptic vesicle-associated protein proposed to play a role in regulating neurotransmitter release. Here we report the isolation and identification of a novel protein complex containing rabphilin, annexin A4 and synaptotagmin 1. We show that the rabphilin C2B domain interacts directly with the N-terminus of annexin A4 and mediates the co-complexing of these two proteins in PC12 cells. Analyzing the cellular localisation of these co-complexing proteins we find that annexin A4 is located on synaptic membranes and co-localises with rabphilin at the plasma membrane in PC12 cells. Given that rabphilin and synaptotagmin are synaptic vesicle proteins involved in neurotransmitter release, the identification of this complex suggests that annexin A4 may play a role in synaptic exocytosis.

  8. 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.

  9. 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.

  10. Linker engineering for fusion protein construction: Improvement and characterization of a GLP-1 fusion protein.

    PubMed

    Kong, Yuelin; Tong, Yue; Gao, Mingming; Chen, Chen; Gao, Xiangdong; Yao, Wenbing

    2016-01-01

    Protein engineering has been successfully applied in protein drug discovery. Using this technology, we previously have constructed a fusion protein by linking the globular domain of adiponectin to the C-terminus of a glucagon-like peptide-1 (GLP-1) analog. Herein, to further improve its bioactivity, we reconstructed this fusion protein by introducing linker peptides of different length and flexibility. The reconstructed fusion proteins were overexpressed in Escherichia coli and purified using nickel affinity chromatography. Their agonist activity towards receptors of GLP-1 and adiponectin were assessed in vitro by using luciferase assay and AMP-activated protein kinase (AMPK) immunoblotting, respectively. The effects of the selected fusion protein on glucose and lipid metabolism were evaluated in mice. The fusion protein reconstructed using a linker peptide of AMGPSSGAPGGGGS showed high potency in activating GLP-1 receptor and triggering AMPK phosphorylation via activating the adiponectin receptor. Remarkably, the optimized fusion protein was highly effective in lowering blood glucose and lipids in mice. Collectively, these findings demonstrate that the bioactivity of this GLP-1 fusion protein can be significantly promoted by linker engineering, and indicate that the optimized GLP-1 fusion protein is a promising lead structure for anti-diabetic drug discovery.

  11. Text Mining Improves Prediction of Protein Functional Sites

    PubMed Central

    Cohn, Judith D.; Ravikumar, Komandur E.

    2012-01-01

    We present an approach that integrates protein structure analysis and text mining for protein functional site prediction, called LEAP-FS (Literature Enhanced Automated Prediction of Functional Sites). The structure analysis was carried out using Dynamics Perturbation Analysis (DPA), which predicts functional sites at control points where interactions greatly perturb protein vibrations. The text mining extracts mentions of residues in the literature, and predicts that residues mentioned are functionally important. We assessed the significance of each of these methods by analyzing their performance in finding known functional sites (specifically, small-molecule binding sites and catalytic sites) in about 100,000 publicly available protein structures. The DPA predictions recapitulated many of the functional site annotations and preferentially recovered binding sites annotated as biologically relevant vs. those annotated as potentially spurious. The text-based predictions were also substantially supported by the functional site annotations: compared to other residues, residues mentioned in text were roughly six times more likely to be found in a functional site. The overlap of predictions with annotations improved when the text-based and structure-based methods agreed. Our analysis also yielded new high-quality predictions of many functional site residues that were not catalogued in the curated data sources we inspected. We conclude that both DPA and text mining independently provide valuable high-throughput protein functional site predictions, and that integrating the two methods using LEAP-FS further improves the quality of these predictions. PMID:22393388

  12. 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.

  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. High-throughput identification of protein localization dependency networks.

    PubMed

    Christen, Beat; Fero, Michael J; Hillson, Nathan J; Bowman, Grant; Hong, Sun-Hae; Shapiro, Lucy; McAdams, Harley H

    2010-03-09

    Bacterial cells are highly organized with many protein complexes and DNA loci dynamically positioned to distinct subcellular sites over the course of a cell cycle. Such dynamic protein localization is essential for polar organelle development, establishment of asymmetry, and chromosome replication during the Caulobacter crescentus cell cycle. We used a fluorescence microscopy screen optimized for high-throughput to find strains with anomalous temporal or spatial protein localization patterns in transposon-generated mutant libraries. Automated image acquisition and analysis allowed us to identify genes that affect the localization of two polar cell cycle histidine kinases, PleC and DivJ, and the pole-specific pili protein CpaE, each tagged with a different fluorescent marker in a single strain. Four metrics characterizing the observed localization patterns of each of the three labeled proteins were extracted for hundreds of cell images from each of 854 mapped mutant strains. Using cluster analysis of the resulting set of 12-element vectors for each of these strains, we identified 52 strains with mutations that affected the localization pattern of the three tagged proteins. This information, combined with quantitative localization data from epitasis experiments, also identified all previously known proteins affecting such localization. These studies provide insights into factors affecting the PleC/DivJ localization network and into regulatory links between the localization of the pili assembly protein CpaE and the kinase localization pathway. Our high-throughput screening methodology can be adapted readily to any sequenced bacterial species, opening the potential for databases of localization regulatory networks across species, and investigation of localization network phylogenies.

  15. Identification and Validation of PTEN Complex, Associated Proteins

    DTIC Science & Technology

    2005-11-01

    Rosalia construct was transcribed and translated using a wheat germ lysate transcription/translation system to generate an unphosphorylated protein...efficient using the wheat germ lysate transcription/translation, system the new antisera immunoprecipitated the protein as well as the C54 Ab, especially...pSGL-PTEN was in vitro translated in a Rabbit reticolocyte lysate system (A) or in a wheat germ system (B) in the presence of radioactively labeled

  16. Identification of immunoreactive proteins of Brucella melitensis by immunoproteomics.

    PubMed

    Zhao, Zhongpeng; Yan, Fang; Ji, Wenhui; Luo, Deyan; Liu, Xin; Xing, Li; Duan, Yueqiang; Yang, Penghui; Shi, Xiumin; Lu, Zhong; Wang, Xiliang

    2011-09-01

    Infection with Brucella causes brucellosis, a chronic disease in humans, which induces abortion and sterility in livestock. Among the different Brucella species, Brucella melitensis is considered the most virulent and is the predominant species associated with outbreaks in China. To date, no safe human vaccine is available against Brucella infection. The currently used live vaccines against Brucella in livestock induce antibodies that interfere with the diagnosis of field infection in vaccinated animals, which is harmful to eradication programs. However, there is as yet no complete profile of immunogenic proteins of B. melitensis. Towards the development of a safer, equally efficacious, and field infection-distinguishable vaccine, we used immunoproteomics to identify novel candidate immunogenic proteins from B. melitensis M5. Eighty-eight immunoreactive protein spots from B. melitensis M5 were identified by Western blotting and were assigned to sixty-one proteins by mass spectrometry, including many new immunoreactive proteins such as elongation factor G, F0F1 ATP synthase subunit beta, and OMP1. These provide many candidate immunoreactive proteins for vaccine development.

  17. Identification and characterization of the pseudorabies virus UL43 protein

    SciTech Connect

    Klupp, Barbara G.; Altenschmidt, Jan; Granzow, Harald; Fuchs, Walter; Mettenleiter, Thomas C. . E-mail: thomas.mettenleiter@fli.bund.de

    2005-04-10

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

  18. 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.

  19. 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.

  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. Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels

    PubMed Central

    Lee, Kimberly A.; Hammerle, Lisa P.; Andrews, Paul S.; Stokes, Matthew P.; Mustelin, Tomas; Silva, Jeffrey C.; Black, Roy A.; Doedens, John R.

    2011-01-01

    Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His6-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology. PMID:21987572

  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. Gas-phase concentration, purification, and identification of whole proteins from complex mixtures.

    PubMed

    Reid, Gavin E; Shang, Hao; Hogan, Jason M; Lee, Gil U; McLuckey, Scott A

    2002-06-26

    Five proteins present in a relatively complex mixture derived from a whole cell lysate fraction of E. coli have been concentrated, purified, and dissociated in the gas phase, using a quadrupole ion trap mass spectrometer. Concentration of intact protein ions was effected using gas-phase ion/ion proton-transfer reactions in conjunction with mass-to-charge dependent ion "parking" to accumulate protein ions initially dispersed over a range of charge states into a single lower charge state. Sequential ion isolation events interspersed with additional ion parking ion/ion reaction periods were used to "charge-state purify" the protein ion of interest. Five of the most abundant protein components present in the mixture were subjected to this concentration/purification procedure and then dissociated by collisional activation of their intact multiply charged precursor ions. Four of the five proteins were subsequently identified by matching the uninterpreted product ion spectra against a partially annotated protein sequence database, coupled with a novel scoring scheme weighted for the relative abundances of the experimentally observed product ions and the frequency of fragmentations occurring at preferential cleavage sites. The identification of these proteins illustrates the potential of this "top-down" protein identification approach to reduce the reliance on condensed-phase chemistries and extensive separations for complex protein mixture analysis.

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

    PubMed Central

    Laine, Elodie

    2016-01-01

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

  5. Odor Identification Screening Improves Diagnostic Classification in Incipient Alzheimer’s Disease

    PubMed Central

    Quarmley, Megan; Moberg, Paul J.; Mechanic-Hamilton, Dawn; Kabadi, Sushila; Arnold, Steven E.; Wolk, David A.; Roalf, David R.

    2017-01-01

    Background Measurements of olfaction may serve as useful biomarkers of incipient dementia. Here we examine the improvement in diagnostic accuracy of Alzheimer’s disease (AD) and mild cognitive impairment (MCI) when assessing both cognitive functioning and odor identification. Objective To determine the utility of odor identification as a supplementary screening test in incipient AD. Methods Sniffin’ Sticks Odor Identification Test (SS-OIT) and the Montreal Cognitive Assessment (MoCA) were administered in 262 AD, 174 MCI [150 amnestic (aMCI), and 24 non-amnestic (naMCI)], and 292 healthy older adults (HOA). Results Odor identification scores were higher in HOA relative to MCI or AD groups, and MCI outperformed AD. Odor identification scores were higher in aMCI single domain than aMCI multiple domain. Complementing MoCA scores with the SS-OIT significantly improved diagnostic accuracy of individuals with AD and MCI, including within MCI subgroups. Discussion Odor identification is a useful supplementary screening tool that provides additional information relevant for clinical categorization of AD and MCI, including those who are at highest risk to convert to AD. PMID:27886011

  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 giant Mimivirus protein functions using RNA interference.

    PubMed

    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.

  8. 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

  9. 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

  10. 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%.

  11. 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

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

    PubMed

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

    2010-06-01

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

  13. Identification of protein pheromones that promote aggressive behaviour.

    PubMed

    Chamero, Pablo; Marton, Tobias F; Logan, Darren W; Flanagan, Kelly; Cruz, Jason R; Saghatelian, Alan; Cravatt, Benjamin F; Stowers, Lisa

    2007-12-06

    Mice use pheromones, compounds emitted and detected by members of the same species, as cues to regulate social behaviours such as pup suckling, aggression and mating. Neurons that detect pheromones are thought to reside in at least two separate organs within the nasal cavity: the vomeronasal organ (VNO) and the main olfactory epithelium (MOE). Each pheromone ligand is thought to activate a dedicated subset of these sensory neurons. However, the nature of the pheromone cues and the identity of the responding neurons that regulate specific social behaviours are largely unknown. Here we show, by direct activation of sensory neurons and analysis of behaviour, that at least two chemically distinct ligands are sufficient to promote male-male aggression and stimulate VNO neurons. We have purified and analysed one of these classes of ligand and found its specific aggression-promoting activity to be dependent on the presence of the protein component of the major urinary protein (MUP) complex, which is known to comprise specialized lipocalin proteins bound to small organic molecules. Using calcium imaging of dissociated vomeronasal neurons (VNs), we have determined that the MUP protein activates a sensory neuron subfamily characterized by the expression of the G-protein Galpha(o) subunit (also known as Gnao) and Vmn2r putative pheromone receptors (V2Rs). Genomic analysis indicates species-specific co-expansions of MUPs and V2Rs, as would be expected among pheromone-signalling components. Finally, we show that the aggressive behaviour induced by the MUPs occurs exclusively through VNO neuronal circuits. Our results substantiate the idea of MUP proteins as pheromone ligands that mediate male-male aggression through the accessory olfactory neural pathway.

  14. Can computationally designed protein sequences improve secondary structure prediction?

    PubMed

    Bondugula, Rajkumar; Wallqvist, Anders; Lee, Michael S

    2011-05-01

    Computational sequence design methods are used to engineer proteins with desired properties such as increased thermal stability and novel function. In addition, these algorithms can be used to identify an envelope of sequences that may be compatible with a particular protein fold topology. In this regard, we hypothesized that sequence-property prediction, specifically secondary structure, could be significantly enhanced by using a large database of computationally designed sequences. We performed a large-scale test of this hypothesis with 6511 diverse protein domains and 50 designed sequences per domain. After analysis of the inherent accuracy of the designed sequences database, we realized that it was necessary to put constraints on what fraction of the native sequence should be allowed to change. With mutational constraints, accuracy was improved vs. no constraints, but the diversity of designed sequences, and hence effective size of the database, was moderately reduced. Overall, the best three-state prediction accuracy (Q(3)) that we achieved was nearly a percentage point improved over using a natural sequence database alone, well below the theoretical possibility for improvement of 8-10 percentage points. Furthermore, our nascent method was used to augment the state-of-the-art PSIPRED program by a percentage point.

  15. Improving Inpatients' Identification of Their Doctors: Use of FACE™ Cards

    PubMed Central

    Arora, Vineet M.; Schaninger, Caitlin; D'Arcy, Michael; Johnson, Julie K.; Humphrey, Holly J.; Woodruff, James N.; Meltzer, David

    2011-01-01

    Background Improving patients' ability to identify their inpatient physicians and understand their roles is vital to safe patient care. We designed picture cards to facilitate physician introductions. We assessed the effect of Feedback Care and Evaluation (FACE™) cards on patient: (1) ability to correctly identify their inpatient physicians, and (2) understanding of their roles. Methods In October 2006, team members introduced themselves with FACE™ cards, which included a photo and an explanation of their roles. During an inpatient interview research assistants asked patients to name their inpatient physicians and trainees, and rate their understanding of their physicians' roles. Results 1686 (80%) patients in the baseline period and 857 (67%) in the intervention period participated in the evaluation. With the FACE™ intervention, patients were significantly more likely to correctly identify at least one inpatient physician (attending, resident, or intern) [baseline 12.5% vs. intervention 21.1%; p<0.001]. Of the 181 patients who were able to correctly identify at least one inpatient physician in the intervention period, research assistants noted that 59% (n=107) had FACE™ cards visible in their rooms. Surprisingly, fewer patients rated their understanding of their physicians' roles as excellent or very good in the intervention period (45.6%) compared to the baseline period (55.3%) (p<0.001). Conclusions Although FACE™ cards improved patients' ability to identify their inpatient physicians, many patients still cannot identify their inpatient doctors. The FACE™ cards also served to highlight patients' misunderstanding of their physicians' roles. PMID:20043501

  16. Identification of a Protein for Prostate-Specific Infection

    DTIC Science & Technology

    2007-12-01

    tissue-specific manner. In the third year, we worked on using gp41 HIV glycoprotein to fuse with these two peptides in a lentiviral vector. The gp41 ...HIV gp41 protein, we could increase gene delivery by 30% to 70% into LNCaP prostate cancer cells (Fig. 3C). Although the the fusion of the...in Fig. 4. We use influenza HA protein instead of Sindbis E1 gp41 Vector genome C. C-terminus N-terminus C-terminus N-terminus Modified with our

  17. MALDI imaging and in situ identification of integral membrane proteins from rat brain tissue sections

    PubMed Central

    Nicklay, Joshua J.; Harris, Glenn A.; Schey, Kevin L.; Caprioli, Richard M.

    2013-01-01

    Transmembrane proteins are greatly underrepresented in data generated by imaging mass spectrometry (IMS) because of analytical challenges related to their size and solubility. Here we present the first example of MALDI IMS of two highly modified multi-transmembrane domain proteins, myelin proteolipid protein (PLP, 30 kDa) and DM-20 (26 kDa), from various regions of rat brain, namely the cerebrum, cerebellum, and medulla. We utilize a novel tissue pre-treatment aimed at transmembrane protein enrichment to show the in situ distribution of fatty acylation of these proteins, particularly of post-translational palmitoylation. Additionally, we demonstrate the utility of protease-encapsulated hydrogels for spatially localized on-tissue protein digestion and peptide extraction for subsequent direct coupling to LC-MS/MS for protein identification. PMID:23829295

  18. Leishmania infantum chagasi: a genome-based approach to identification of excreted/secreted proteins.

    PubMed

    DebRoy, Sruti; Keenan, Alexandra B; Ueno, Norikiyo; Jeronimo, Selma M B; Donelson, John E; Wilson, Mary E

    2010-12-01

    The parasitic protozoan, Leishmania, survives in harsh environments within its mammalian and sand fly hosts. Secreted proteins likely play critical roles in the parasite's interactions with its environment. As a preliminary identification of the spectrum of potential excreted/secreted (ES) proteins of Leishmania infantum chagasi (Lic), a causative agent of visceral leishmaniasis, we used standard algorithms to screen the annotated L. infantum genome for genes whose predicted protein products have an N-terminal signal peptide and lack transmembrane domains and membrane anchors. A suite of 181 candidate ES proteins were identified. These included several that were documented in the literature to be released by other Leishmania spp. Six candidate ES proteins were selected for further validation of their expression and release by different parasite stages. We found both amastigote-specific and promastigote-specific released proteins. The ES proteins of Lic are candidates for future studies of parasite virulence determinants and host protective immunity.

  19. Proteome study of the phloem sap of pumpkin using multidimensional protein identification technology.

    PubMed

    Cho, Won Kyong; Chen, Xiong-Yan; Rim, Yeonggil; Chu, Hyosub; Kim, Suwha; Kim, Seon-Won; Park, Zee-Yong; Kim, Jae-Yean

    2010-07-01

    The phloem is the major transport route for both small substances and large molecules, such as proteins and RNAs, from their sources to sink tissues. To investigate the proteins present in pumpkin phloem sap, proteome analysis using multidimensional protein identification technology was carried out. Pumpkin phloem peptides obtained by liquid chromatography/mass spectrometry/mass spectrometry were searched against pumpkin protein data derived from the National Center for Biotechnology Information. A total of 47 pumpkin phloem proteins were identified. The identified proteins mainly corresponded to enzymes involved in gibberellin biosynthesis, antioxidation processes, or defense mechanisms. Interestingly, seven enzymes required for gibberellin biosynthesis were identified for the first time by this proteomics approach. In summary, the new phloem proteins identified in this study provide strong evidence for stress and defense signaling and new insights regarding the role of gibberellin in the developmental programming of higher plants through the phloem.

  20. Identification and Characterization of Prostate Cancer Associated Protein Biomarkers Using High-Throughput Mass Spectrometry

    DTIC Science & Technology

    2007-12-01

    of PCa in cohort. AIM 3. Isolation and identification of the protein biomarkers. AIM 4. Development of MS-assisted immunoassay for PCa diagnostics... Immunoassays for PCa Diagnostics. We have previously identified biomarker protein/peptides that comprise disease- specific signature profiles...gray area), would be tested using large sample sets on MALDI and SELDI-based immunoassays using sample cohorts from Dr. Ian Thompson. Data

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

    PubMed Central

    FU, Si-Ling; LI, Jiang-Lin; CHEN, Jia; WANG, Qiu-Ting; LI, Jian-Jun; WANG, Xian-Chun

    2015-01-01

    The eggs of oviparous animals are storehouses of maternal proteins required for embryonic development. Identification and molecular characterization of such proteins will provide much insight into the regulation of embryonic development. We previously analyzed soluble proteins in the eggs of the black widow spider (Latrodectus tredecimguttatus), and report here on the extraction and mass spectrometric identification of the egg membrane proteins. Comparison of different lysis solutions indicated that the highest extraction of the membrane proteins was achieved with 3%-4% sodium laurate in 40 mmol/L Tris-HCl buffer containing 4% CHAPS and 2% DTT (pH 7.4). SDS-PAGE combined with nLC-MS/MS identified 39 proteins with membrane-localization annotation, including those with structural, catalytic, and regulatory activities. Nearly half of the identified membrane proteins were metabolic enzymes involved in various cellular processes, particularly energy metabolism and biosynthesis, suggesting that relevant metabolic processes were active during the embryonic development of the eggs. Several identified cell membrane proteins were involved in the special structure formation and function of the egg cell membranes. The present proteomic analysis of the egg membrane proteins provides new insight into the molecular mechanisms of spider embryonic development. PMID:26228476

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

    PubMed

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

    2014-01-01

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

  3. 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.

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

    PubMed Central

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

    2017-01-01

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

  5. Systematic identification of proteins that elicit drug side effects

    PubMed Central

    Kuhn, Michael; Al Banchaabouchi, Mumna; Campillos, Monica; Jensen, Lars Juhl; Gross, Cornelius; Gavin, Anne-Claude; Bork, Peer

    2013-01-01

    Side effect similarities of drugs have recently been employed to predict new drug targets, and networks of side effects and targets have been used to better understand the mechanism of action of drugs. Here, we report a large-scale analysis to systematically predict and characterize proteins that cause drug side effects. We integrated phenotypic data obtained during clinical trials with known drug–target relations to identify overrepresented protein–side effect combinations. Using independent data, we confirm that most of these overrepresentations point to proteins which, when perturbed, cause side effects. Of 1428 side effects studied, 732 were predicted to be predominantly caused by individual proteins, at least 137 of them backed by existing pharmacological or phenotypic data. We prove this concept in vivo by confirming our prediction that activation of the serotonin 7 receptor (HTR7) is responsible for hyperesthesia in mice, which, in turn, can be prevented by a drug that selectively inhibits HTR7. Taken together, we show that a large fraction of complex drug side effects are mediated by individual proteins and create a reference for such relations. PMID:23632385

  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/).

  7. Mass Spectrometric Identification of the Arginine and Lysine deficient Proline Rich Glutamine Rich Wheat Storage Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tandem mass spectrometry (MS/MS) of enzymatic digest has made possible identification of a wide variety of proteins and complex samples prepared by such techniques as RP-HPLC or 2-D gel electrophoresis. Success requires peptide fragmentation to be indicative of the peptide amino acid sequence. The f...

  8. 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...

  9. Identification of proteins suppressing the functions of oncogenic phosphatase of regenerating liver 1 and 3

    PubMed Central

    Lee, Ju-Dong; Jung, Haiyoung; Min, Sang-Hyun

    2016-01-01

    The phosphatase of regenerating liver (PRL) family, including PRL-1, PRL-2, and PRL-3, comprises protein tyrosine phosphatases whose deregulation is associated with the tumorigenesis and metastasis of many types of cancer. However, the underlying mechanism is poorly understood. In this study, aiming to increase understanding of the molecular mechanisms underlying the functions of PRL-1 and PRL-3, a yeast two-hybrid system was employed to screen for their interacting proteins. Alignment with the NCBI BLAST database revealed 12 interactive proteins: Synaptic nuclear envelope protein 2, emerin, mannose 6-phosphate receptor-binding protein 1, low-density lipoprotein receptor-related protein 10, Rab acceptor 1, tumor protein D52-like 2, selectin P ligand (SELPLG), guanylate binding protein 1, transmembrane and ubiquitin-like domain-containing 2, NADH:ubiquinone oxidoreductase subunit B8, syndecan 4 and FK506-binding protein 8 (FKBP8). These proteins are associated with cell proliferation, apoptosis, immune response, cell fate specification and metabolic process in biological process categories, and involved in various signaling pathways, including Alzheimer's disease, Parkinson's disease, Huntington's disease, hypertrophic cardiomyopathy and cell adhesion molecules. Interactions of PRL-1 with the prey proteins SELPLG and FKBP8 were confirmed by immunoprecipitation or immunostaining. Furthermore, SELPLG and FKBP8 suppressed PRL-1− or PRL-3-mediated p53 activity. Identification of the proteins interacting with PRL family proteins may provide valuable information to better understand the mechanism of PRL-mediated signal transduction in cancer and other diverse diseases. PMID:27882103

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

    PubMed

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

    2011-08-05

    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.

  11. Identification of two integral membrane proteins of Plasmodium falciparum.

    PubMed Central

    Smythe, J A; Coppel, R L; Brown, G V; Ramasamy, R; Kemp, D J; Anders, R F

    1988-01-01

    We describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage lambda gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a Mr 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a Mr 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with [3H]glucosamine and [3H]myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the Mr 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor. Images PMID:3293051

  12. Identification of leukemia cell surface proteins in clams

    SciTech Connect

    Reinisch, C.L.; Smolowitz, R.; Miosky, D. Marine Biological Lab., Woods Hole, MA )

    1988-09-01

    Soft-shell clams, Mya arenaria, develop leukemias which, in the advanced stages of disease, kill the host. The authors laboratory has developed an extensive panel of murine monoclonal antibodies to leukemia cells of Mya, and has used these powerful reagents to diagnose the disease with extreme accuracy. They have now ascertained that one membrane-associated protein of approximately 200kD is immunodominant. The function of this protein, regulation of its production and potential site of synthesis are being evaluated. Monoclonal antibodies have also permitted the exploration of the mechanism of leukemogensis. They have evaluated the specific staining pattern of one monoclonal antibody, and have concluded that at least one ontogenic source of leukemic cells may be connective tissue cells lining the sinusoids. Whether or not exposure to severely polluted sites such as New Bedford Harbor stimulates the export of immature hemocytes which then become transformed is at least one possibility amenable to testing using the monoclonal reagents.

  13. Identification of an additional protein involved in mannan biosynthesis

    PubMed Central

    Wang, Yan; Mortimer, Jennifer C; Davis, Jonathan; Dupree, Paul; Keegstra, Kenneth

    2013-01-01

    Galactomannans comprise a β-1,4-mannan backbone substituted with α-1,6-galactosyl residues. Genes encoding the enzymes that are primarily responsible for backbone synthesis and side-chain addition of galactomannans were previously identified and characterized. To identify additional genes involved in galactomannan biosynthesis, we previously performed deep EST profiling of fenugreek (Trigonella foenum-graecum L.) seed endosperm, which accumulates large quantities of galactomannans as a reserve carbohydrate during seed development. One of the candidate genes encodes a protein that is likely to be a glycosyltransferase. Because this protein is involved in mannan biosynthesis, we named it ‘mannan synthesis-related’ (MSR). Here, we report the characterization of a fenugreek MSR gene (TfMSR) and its two Arabidopsis homologs, AtMSR1 and AtMSR2. TfMSR was highly and specifically expressed in the endosperm. TfMSR, AtMSR1 and AtMSR2 proteins were all determined to be localized to the Golgi by fluorescence confocal microscopy. The level of mannosyl residues in stem glucomannans decreased by approximately 40% for Arabidopsis msr1 single T-DNA insertion mutants and by more than 50% for msr1 msr2 double mutants, but remained unchanged for msr2 single mutants. In addition, in vitro mannan synthase activity from the stems of msr1 single and msr1 msr2 double mutants also decreased. Expression of AtMSR1 or AtMSR2 in the msr1 msr2 double mutant completely or partially restored mannosyl levels. From these results, we conclude that the MSR protein is important for mannan biosynthesis, and offer some ideas about its role. PMID:22966747

  14. 34 CFR 200.37 - Notice of identification for improvement, corrective action, or restructuring.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ACADEMIC ACHIEVEMENT OF THE DISADVANTAGED Improving Basic Programs Operated by Local Educational Agencies..., and how the school compares in terms of academic achievement to other elementary and secondary schools... parents can become involved in addressing the academic issues that led to identification. (4)(i)...

  15. First identification of proteins involved in motility of Mycoplasma gallisepticum.

    PubMed

    Indikova, Ivana; Vronka, Martin; Szostak, Michael P

    2014-10-17

    Mycoplasma gallisepticum, the most pathogenic mycoplasma in poultry, is able to glide over solid surfaces. Although this gliding motility was first observed in 1968, no specific protein has yet been shown to be involved in gliding. We examined M. gallisepticum strains and clonal variants for motility and found that the cytadherence proteins GapA and CrmA were required for gliding. Loss of GapA or CrmA resulted in the loss of motility and hemadsorption and led to drastic changes in the characteristic flask-shape of the cells. To identify further genes involved in motility, a transposon mutant library of M. gallisepticum was generated and screened for motility-deficient mutants, using a screening assay based on colony morphology. Motility-deficient mutants had transposon insertions in gapA and the neighbouring downstream gene crmA. In addition, insertions were seen in gene mgc2, immediately upstream of gapA, in two motility-deficient mutants. In contrast to the GapA/CrmA mutants, the mgc2 motility mutants still possessed the ability to hemadsorb. Complementation of these mutants with a mgc2-hexahistidine fusion gene restored the motile phenotype. This is the first report assigning specific M. gallisepticum proteins to involvement in gliding motility.

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

    PubMed

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

    2010-08-13

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

  18. 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.

  19. Identification of membrane proteins mediating the interaction of human platelets

    PubMed Central

    Phillips, D; Jennings, L; Edwards, H

    1980-01-01

    Membrane glycoproteins that mediate platelet-platelet interactions were investigated by identifying those associated with the cytoskeletal structures from aggregated platelets. The cytoskeletal structures from washed platelets, thrombin-activated platelets (platelets incubated with thrombin in the presence of mM EDTA to prevent aggregation) and thrombin- aggregated platelets (platelets activated in the presence of mM Ca(++) were prepared by first treating platelet suspensions with 1 percent Triton X-100 and 5 mM EGTA and then isolating the insoluble residue by centrifugation. The readily identifiable structures in electron micrographs of the residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue from washed platelets had the shape and dimensions of actin filaments. Analysis of this residue by SDS gel electrophoresis showed that it consisted primarily of three proteins: actin (mol wt = 43,000), myosin (mol wt = 200,000) and a high molecular weight polypeptide (mol wt = 255,000) which had properties indentical to actin-binding protein (filamin). When platelets are activated with thrombin in the presence of EDTA to prevent aggregation, there was a marked increase in the amount of insoluble precipitate in the subsequent Triton extraction. Transmission electron microscopy showed that this residue not only contained the random array of actin filaments as seen above, but also organized structures from individual platelets which appeared as balls of electron-dense filamentous material approximately 1mum in diameter. SDS polyacrylamide gel analysis of the Triton residue of activated platelets showed that this preparation contained more actin, myosin and actin-binding protein than that from washed platelets plus polypeptides with mol wt of 56,000 and 90,000 and other minor polypeptides. Thus, thrombin activation appeared to increase polymerization of actin in association with other cytoskeletal proteins into structures that

  20. 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.

  1. Improving the quality of protein structures derived by NMR spectroscopy.

    PubMed

    Spronk, Christian A E M; Linge, Jens P; Hilbers, Cornelis W; Vuister, Geerten W

    2002-03-01

    Biomolecular structures provide the basis for many studies in several research areas such as homology modelling, structure-based drug design and functional genomics. It is an important prerequisite that the structure is reliable in terms of accurate description of the experimental data, and in terms of good quality of local- and overall geometry. Recent surveys indicate that structures solved by NMR-spectroscopy normally are of lower precision than high-resolution X-ray structures. Here, we present a refinement protocol that improves the quality of protein structures determined by NMR-spectroscopy to the level of those determined by high resolution X-ray crystallography in terms of local geometry. The protocol was tested on experimental data of the proteins IL4 and Ubiquitin and on simulated data of the protein Crambin. In almost all aspects, the protocol yielded better results in terms of accuracy and precision. Independent validation of the results for Ubiquitin, using residual dipolar couplings, indicates that the ensemble of NMR structure is substantially improved by the protocol.

  2. 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.

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

    PubMed

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

    2009-12-11

    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.

  5. 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

  6. 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

  7. Identification, nomenclature, and evolutionary relationships of mitogen-activated protein kinase (MAPK) genes in soybean.

    PubMed

    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.

  8. 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

  9. APoc: large-scale identification of similar protein pockets

    PubMed Central

    Gao, Mu; Skolnick, Jeffrey

    2013-01-01

    Motivation: Most proteins interact with small-molecule ligands such as metabolites or drug compounds. Over the past several decades, many of these interactions have been captured in high-resolution atomic structures. From a geometric point of view, most interaction sites for grasping these small-molecule ligands, as revealed in these structures, form concave shapes, or ‘pockets’, on the protein’s surface. An efficient method for comparing these pockets could greatly assist the classification of ligand-binding sites, prediction of protein molecular function and design of novel drug compounds. Results: We introduce a computational method, APoc (Alignment of Pockets), for the large-scale, sequence order-independent, structural comparison of protein pockets. A scoring function, the Pocket Similarity Score (PS-score), is derived to measure the level of similarity between pockets. Statistical models are used to estimate the significance of the PS-score based on millions of comparisons of randomly related pockets. APoc is a general robust method that may be applied to pockets identified by various approaches, such as ligand-binding sites as observed in experimental complex structures, or predicted pockets identified by a pocket-detection method. Finally, we curate large benchmark datasets to evaluate the performance of APoc and present interesting examples to demonstrate the usefulness of the method. We also demonstrate that APoc has better performance than the geometric hashing-based method SiteEngine. Availability and implementation: The APoc software package including the source code is freely available at http://cssb.biology.gatech.edu/APoc. Contact: skolnick@gatech.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:23335017

  10. X!TandemPipeline: A Tool to Manage Sequence Redundancy for Protein Inference and Phosphosite Identification.

    PubMed

    Langella, Olivier; Valot, Benoît; Balliau, Thierry; Blein-Nicolas, Mélisande; Bonhomme, Ludovic; Zivy, Michel

    2017-02-03

    X!TandemPipeline is a software designed to perform protein inference and to manage redundancy in the results of phosphosite identification by database search. It provides the minimal list of proteins or phosphosites that are present in a set of samples using grouping algorithms based on the principle of parsimony. Regarding proteins, a two-level classification is performed, where groups gather proteins sharing at least one peptide and subgroups gather proteins that are not distinguishable according to the identified peptides. Regarding phosphosites, an innovative approach based on the concept of phosphoisland is used to gather overlapping phosphopeptides. The graphical interface of X!TandemPipeline allows the users to launch X!tandem identification, to inspect spectra and to manually validate their assignment to peptides, to launch the grouping program, and to visualize elementary data as well as grouping and redundancy information. Identification results obtained from other search engines can also be processed. X!TandemPipeline results can be exported as ready-to-use tabulated files or as XML files that can be directly used by the PROTICdb database or by the MassChroQ quantification software. X!TandemPipeline runs fast, is easy to use, and can process hundreds of samples simultaneously. It is freely available under the GNU General Public License v3.0 at http://pappso.inra.fr/bioinfo/xtandempipeline/ .

  11. Improvement of a potential anthrax therapeutic by computational protein design.

    PubMed

    Wu, Sean J; Eiben, Christopher B; Carra, John H; Huang, Ivan; Zong, David; Liu, Peixian; Wu, Cindy T; Nivala, Jeff; Dunbar, Josef; Huber, Tomas; Senft, Jeffrey; Schokman, Rowena; Smith, Matthew D; Mills, Jeremy H; Friedlander, Arthur M; Baker, David; Siegel, Justin B

    2011-09-16

    Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

  12. Identification of Sequence Similarities among Isomerization Hotspots in Crystallin Proteins

    PubMed Central

    2017-01-01

    The eye lens crystallins represent an ideal target for studying the effects of aging on protein structure. Herein we examine separately the water-soluble (WS) and water-insoluble (WI) crystallin fractions and identify sites of isomerization and epimerization. Both collision-induced dissociation and radical-directed dissociation are needed for detection of these non-mass-shifting post-translational modifications. Isomerization levels differ significantly between the WS and the WI fractions from sheep, pig, and cow eye lenses. Residues that are most susceptible to isomerization are identified site-specifically and are found to reside in structurally disordered regions. However, isomerization in structured domains, although less common, often yields more dramatic effects on solubility. Numerous isomerization hotspots were also identified and occur in regions with aspartic acid and serine repeats. For example, 128KMEIVDDDVPSLW140 in βB3 crystallin contains three sequential aspartic acid residues and is isomerized heavily in the WI fractions, while it is not modified at all in the WS fractions. Potential causes for enhanced isomerization at sites with acidic residue repeats are presented. The importance of acidic residue repeats extends beyond the lens, as they are found in many other long-lived proteins associated with disease. PMID:28234481

  13. Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

    PubMed

    Han, Rui-Zhi; Xu, Guo-Chao; Dong, Jin-Jun; Ni, Ye

    2016-06-01

    Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

  14. 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

  15. 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

  16. The RIPper case: identification of RNA-binding protein targets by RNA immunoprecipitation.

    PubMed

    Köster, Tino; Haas, Meike; Staiger, Dorothee

    2014-01-01

    Control at the posttranscriptional level emerges as an important layer of regulation in the circadian timing system. RNA-binding proteins that specifically interact with cis-regulatory motifs within pre-mRNAs are key elements of this regulation. While the ability to interact with RNA in vitro has been demonstrated for numerous Arabidopsis RNA-binding proteins, a full understanding of posttranscriptional networks controlled by an RNA-binding protein requires the identification of its immediate in vivo targets. Here we describe differential RNA immunoprecipitation in transgenic Arabidopsis thaliana plants expressing RNA-binding protein variants epitope-tagged with green fluorescent protein. To control for RNAs that nonspecifically co-purify with the RNA-binding protein, transgenic plants are generated with a mutated version of the RNA-binding protein that is not capable of binding to its target RNAs. The RNA-binding protein variants are expressed under the control of their authentic promoter and cis-regulatory motifs. Incubation of the plants with formaldehyde in vivo cross-links the proteins to their RNA targets. A whole-cell extract is then prepared and subjected to immunoprecipitation with an antibody against the GFP tag and to mock precipitation with an antibody against the unrelated red fluorescent protein. The RNAs coprecipitating with the proteins are eluted from the immunoprecipitate and identified via reverse transcription-PCR.

  17. 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.

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

    PubMed Central

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

    2014-01-01

    Whole-cell matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid method for identification of microorganisms that is increasingly used in microbiology laboratories. This identification is based on the comparison of the tested isolate mass spectrum with reference databases. Using Neisseria meningitidis as a model organism, we showed that in one of the available databases, the Andromas database, 10 of the 13 species-specific biomarkers correspond to ribosomal proteins. Remarkably, one biomarker, ribosomal protein L32, was subject to inter-strain variability. The analysis of the ribosomal protein patterns of 100 isolates for which whole genome sequences were available, confirmed the presence of inter-strain variability in the molecular weight of 29 ribosomal proteins, thus establishing a correlation between the sequence type (ST) and/or clonal complex (CC) of each strain and its ribosomal protein pattern. Since the molecular weight of three of the variable ribosomal proteins (L30, L31 and L32) was included in the spectral window observed by MALDI-TOF MS in clinical microbiology, i.e., 3640–12000 m/z, we were able by analyzing the molecular weight of these three ribosomal proteins to classify each strain in one of six subgroups, each of these subgroups corresponding to specific STs and/or CCs. Their detection by MALDI-TOF allows therefore a quick typing of N. meningitidis isolates. PMID:23916798

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

    PubMed Central

    Zhang, Tao; Lu, Haojie; Li, Weijun; Hu, Ronggui; Chen, Zi

    2015-01-01

    The identification of arsenic direct-binding proteins is essential for determining the mechanism by which arsenic trioxide achieves its chemotherapeutic effects. At least two cysteines close together in the amino acid sequence are crucial to the binding of arsenic and essential to the identification of arsenic-binding proteins. In the present study, arsenic binding proteins were pulled down with streptavidin and identified using a liquid chromatograph-mass spectrometer (LC-MS/MS). More than 40 arsenic-binding proteins were separated, and redox-related proteins, glutathione S-transferase P1 (GSTP1), heat shock 70 kDa protein 9 (HSPA9) and pyruvate kinase M2 (PKM2), were further studied using binding assays in vitro. Notably, PKM2 has a high affinity for arsenic. In contrast to PKM2, GSTP1and HSPA9 did not combine with arsenic directly in vitro. These observations suggest that arsenic-mediated acute promyelocytic leukaemia (APL) suppressive effects involve PKM2. In summary, we identified several arsenic binding proteins in APL cells and investigated the therapeutic mechanisms of arsenic trioxide for APL. Further investigation into specific signal pathways by which PKM2 mediates APL developments may lead to a better understanding of arsenic effects on APL. PMID:26569224

  20. Identification of whey proteins in tradional Bulgarian yougurt.

    PubMed

    Ivanova, I; Antonova-Nikolova, S; Iliev, I

    2001-01-01

    Functional foods hold a great promise for future trends in human nutrition. Consumption of milk and milk products have a pronounced probiotic effects together with the expected modification of allergenic properties of milk due to the process of fermentation. The proteolytic system of lactic acid bacteria consists a cell wall bound proteinase and several intracellular peptidases, and can contribute to the liberation of bioactive peptides. Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional food. In our study we focused our attention on beta-lactoglobulin and alpha-lactalbumin in early stages of yogurt fermentation of traditional Bulgarian products. Biochemical techniques were used to measure the concentration of these two whey proteins during fermentation. At a result of the done study alteration in the concentration of beta-lactoglobulin and alpha-lactalbumin were detected. The studied proteolytic activity of the strains, used in the fermentation process confirmed the received results.

  1. Identification of bitter peptides in whey protein hydrolysate.

    PubMed

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

    2014-06-25

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

  2. 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

  3. Brief communication: multiplex X/Y-PCR improves sex identification in aDNA analysis.

    PubMed

    Schmidt, Diane; Hummel, Susanne; Herrmann, Bernd

    2003-08-01

    This study introduces a polymerase chain reaction (PCR)-based multiplex approach to improve the certainty of molecular sex identification on archaeological skeletal material. We coamplified amelogenin, two X-chromosomal short tandem repeats (STRs) (DXS6789 and DXS9898), and two Y-specific STRs (DYS391 and DYS392). The amplification results of this multiplex approach back each other up, and enable a reliable sex identification. This coamplification of X- and Y-specific markers in a multiplex assay combines the added advantage of positive identification of both female and male individuals with raising the validity of the diagnosis by obtaining multiple data simultaneously. This multiplex system was successfully applied to 3,000-year-old bone material.

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

    PubMed Central

    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 an 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. PMID:26539460

  5. 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

  6. PILOT_PROTEIN: Identification of unmodified and modified proteins via high-resolution mass spectrometry and mixed-integer linear optimization

    PubMed Central

    Baliban, Richard C.; DiMaggio, Peter A.; Plazas-Mayorca, Mariana D.; Garcia, Benjamin A.; Floudas, Christodoulos A.

    2012-01-01

    A novel protein identification framework, PILOT_PROTEIN, has been developed to construct a comprehensive list of all unmodified proteins that are present in a living sample. It uses the peptide identification results from the PILOT_SEQUEL algorithm to initially determine all unmodified proteins within the sample. Using a rigorous biclustering approach that groups incorrect peptide sequences with other homologous sequences, the number of false positives reported is minimized. A sequence tag procedure is then incorporated along with the untargeted PTM identification algorithm, PILOT_PTM, to determine a list of all modification types and sites for each protein. The unmodified protein identification algorithm, PILOT_PROTEIN, is compared to the methods SEQUEST, InsPecT, X!Tandem, VEMS, and ProteinProspector using both prepared protein samples and a more complex chromatin digest. The algorithm demonstrates superior protein identification accuracy with a lower false positive rate. All materials are freely available to the scientific community at http://pumpd.princeton.edu. PMID:22788846

  7. Identification of a GPER/GPR30 Antagonist with Improved Estrogen Receptor Counterselectivity

    PubMed Central

    Dennis, Megan K.; Field, Angela S.; Burai, Ritwik; Ramesh, Chinnasamy; Petrie, Whitney K.; Bologa, Cristian G.; Oprea, Tudor I.; Yamaguchi, Yuri; Hayashi, Shin-ichi; Sklar, S. Larry A.; Hathaway, Helen J.; Arterburn, Jeffrey B.; Prossnitz, Eric R.

    2011-01-01

    GPER/GPR30 is a seven-transmembrane G protein-coupled estrogen receptor that regulates many aspects of mammalian biology and physiology. We have previously described both a GPER-selective agonist G-1 and antagonist G15 based on a tetrahydro-3H-cyclopenta[c]quinoline scaffold. The antagonist lacks an ethanone moiety that likely forms important hydrogen bonds involved in receptor activation. Computational docking studies suggested that the lack of the ethanone substituent in G15 could minimize key steric conflicts, present in G-1, that limit binding within the ERα ligand binding pocket. In this report, we identify low-affinity cross-reactivity of the GPER antagonist G15 to the classical estrogen receptor ERα. To generate an antagonist with enhanced selectivity, we therefore synthesized an isosteric G-1 derivative, G36, containing an isopropyl moiety in place of the ethanone moiety. We demonstrate that G36 shows decreased binding and activation of ERα, while maintaining its antagonist profile towards GPER. G36 selectively inhibits estrogen-mediated activation of PI3K by GPER but not ERα. It also inhibits estrogen- and G-1-mediated calcium mobilization as well as ERK1/2 activation, with no effect on EGF-mediated ERK1/2 activation. Similar to G15, G36 inhibits estrogen- and G-1-stimulated proliferation of uterine epithelial cells in vivo. The identification of G36 as a GPER antagonist with improved ER counterselectivity represents a significant step towards the development of new highly selective therapeutics for cancer and other diseases. PMID:21782022

  8. 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.

  9. Improved identification of hordeins by cysteine alkylation with 2-bromoethylamine, SDS-PAGE and subsequent in-gel tryptic digestion.

    PubMed

    Rehulková, Helena; Marchetti-Deschmann, Martina; Pittenauer, Ernst; Allmaier, Günter; Rehulka, Pavel

    2009-11-01

    Proteomic-based description of varieties of barley (Hordeum vulgare L.) is a very important task especially in the food and brewing industry. This study is focused on major storage proteins in barley--hordeins--as a group of proteins soluble in alcohol/water mixtures that shows up significant changes in proteomic profiles for different varieties of barley. Unusual amino acid composition of hordeins with low numbers of lysine and arginine in combination with high content of proline and glutamine complicates their identification in a common proteomic workflow, because tryptic digestion produces just a few peptides amenable for successful mass spectrometric analysis. To increase the number of cleavage sites, in this work, cysteines in hordeins were chemically modified with 2-bromoethylamine (BEA) for their conversion into aminoethylcysteines. These mimic lysine residues and are recognized by trypsin as potential cleavage sites (if not followed by a proline residue) on the C-terminal side of the modified cysteine. Small extent of side reactions (towards histidine, N-terminus of the peptide, methionine, and also here the newly discovered reaction towards aminoethylcysteine) during modification with BEA could be observed after a longer period of reaction but this did not hinder the analysis when optimal conditions were used. Application of trypsin for in-gel digestion of hordeins, previously modified chemically with BEA, provided a higher number of short peptides and their subsequent mass spectrometric analysis resulted in an improved identification of hordeins. This approach can also be used for the analysis of other similar protein groups (e.g. gliadins in wheat) or other cysteines containing proteins having a low number of lysine and arginine residues in their primary structure.

  10. Proteome analysis of the penicillin producer Penicillium chrysogenum: characterization of protein changes during the industrial strain improvement.

    PubMed

    Jami, Mohammad-Saeid; Barreiro, Carlos; García-Estrada, Carlos; Martín, Juan-Francisco

    2010-06-01

    Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by "blue silver" colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin

  11. 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.

  12. Identification of lipids and lipid-binding proteins in phloem exudates from Arabidopsis thaliana.

    PubMed

    Guelette, Brandon S; Benning, Urs F; Hoffmann-Benning, Susanne

    2012-06-01

    The phloem plays a crucial role in assimilate and nutrient transport, pathogen response, and plant growth and development. Yet, few species have yielded pure phloem exudate and, if proteins need to be analysed, those species may not have sequenced genomes, making identification difficult. The enrichment of Arabidopsis thaliana phloem exudate in amounts large enough to allow for metabolite and protein analysis is described. Using this method, it was possible to identify 65 proteins present in the Arabidopsis phloem exudate. The majority of these proteins could be grouped by response to pathogens, stress, or hormones, carbon metabolism, protein interaction, modification, and turnover, and transcription factors. It was also possible to detect 11 proteins that play a role in lipid/fatty acid metabolism (aspartic protease, putative 3-β-hydroxysteroid dehydrogenase, UDP-sulphoquinovose synthase/SQD1, lipase, PIG-P-like protein: phosphatidylinositol-N-acetylglucosaminyltransferase), storage (glycine-rich protein), binding (annexin, lipid-associated family protein, GRP17/oleosin), and/or signalling (annexin, putative lipase, PIG-P-like protein). Along with putative lipid-binding proteins, several lipids and fatty acids could be identified. Only a few examples exist of lipids (jasmonic acid, oxylipins) or lipid-binding proteins (DIR1, acyl-CoA-binding protein) in the phloem. Finding hydrophobic compounds in an aqueous environment is not without precedence in biological systems: human blood contains a variety of lipids, many of which play a significant role in human health. In blood, lipids are transported while bound to proteins. The present findings of lipids and lipid-binding proteins in phloem exudates suggest that a similar long-distance lipid signalling exists in plants and may play an important role in plant growth and development.

  13. Differential extraction and enrichment of human sperm surface proteins in a proteome: identification of immunocontraceptive candidates.

    PubMed

    Shetty, J; Diekman, A B; Jayes, F C; Sherman, N E; Naaby-Hansen, S; Flickinger, C J; Herr, J C

    2001-08-01

    The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

  14. 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.

  15. Improving amino-acid identification, fit and C(alpha) prediction using the Simplex method in automated model building.

    PubMed

    Romo, Tod D; Sacchettini, James C; Ioerger, Thomas R

    2006-11-01

    Automated methods for protein model building in X-ray crystallography typically use a two-phased approach that involves first modeling the protein backbone followed by building in the side chains. The latter phase requires the identification of the amino-acid side-chain type as well as fitting of the side-chain model into the observed electron density. While mistakes in identification of individual side chains are common for a number of reasons, sequence alignment can sometimes be used to correct errors by mapping fragments into the true (expected) amino-acid sequence and exploiting contiguity constraints among neighbors. However, side chains cannot always be confidently aligned; this depends on having sufficient accuracy in the initial calls. The recognition of amino-acid side-chains based on the surrounding pattern of electron density, whether by features, density correlation or free atoms, can be sensitive to inaccuracies in the coordinates of the predicted backbone C(alpha) atoms to which they are anchored. By incorporating a Nelder-Mead Simplex search into the side-chain identification and model-building routines of TEXTAL, it is demonstrated that this form of residue-by-residue rigid-body real-space refinement (in which the C(alpha) itself is allowed to shift) can improve the initial accuracy of side-chain selection by over 25% on average (from 25% average identity to 32% on a test set of five representative proteins, without corrections by sequence alignment). This improvement in amino-acid selection accuracy in TEXTAL is often sufficient to bring the pairwise amino-acid identity of chains in the model out of the so-called ;twilight zone' for sequence-alignment methods. When coupled with sequence alignment, use of the Simplex search yielded improvements in side-chain accuracy on average by over 13 percentage points (from 64 to 77%) and up to 38 percentage points (from 40 to 78%) in one case compared with using sequence alignment alone.

  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.

    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.

  17. 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.

  18. 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

  19. Identification of albumin-binding proteins in capillary endothelial cells

    PubMed Central

    1988-01-01

    Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine. PMID:2839518

  20. 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.

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

    PubMed Central

    Wong, Wendy SW; Sainudiin, Raazesh; Nielsen, Rasmus

    2006-01-01

    Background Statistical methods for identifying positively selected sites in protein coding regions are one of the most commonly used tools in evolutionary bioinformatics. However, they have been limited by not taking the physiochemical properties of amino acids into account. Results We develop a new codon-based likelihood model for detecting site-specific selection pressures acting on specific physicochemical properties. Nonsynonymous substitutions are divided into substitutions that differ with respect to the physicochemical properties of interest, and those that do not. The substitution rates of these two types of changes, relative to the synonymous substitution rate, are then described by two parameters, γ and ω respectively. The new model allows us to perform likelihood ratio tests for positive selection acting on specific physicochemical properties of interest. The new method is first used to analyze simulated data and is shown to have good power and accuracy in detecting physicochemical selective pressure. We then re-analyze data from the class-I alleles of the human Major Histocompatibility Complex (MHC) and from the abalone sperm lysine. Conclusion Our new method allows a more flexible framework to identify selection pressure on particular physicochemical properties. PMID:16542458

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

    PubMed

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

    2007-10-01

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

  3. Improving the identification rate of endogenous peptides using electron transfer dissociation and collision-induced dissociation.

    PubMed

    Hayakawa, Eisuke; Menschaert, Gerben; De Bock, Pieter-Jan; Luyten, Walter; Gevaert, Kris; Baggerman, Geert; Schoofs, Liliane

    2013-12-06

    Tandem mass spectrometry (MS/MS) combined with bioinformatics tools have enabled fast and systematic protein identification based on peptide-to-spectrum matches. However, it remains challenging to obtain accurate identification of endogenous peptides, such as neuropeptides, peptide hormones, peptide pheromones, venom peptides, and antimicrobial peptides. Since these peptides are processed at sites that are difficult to predict reliably, the search of their MS/MS spectra in sequence databases needs to be done without any protease setting. In addition, many endogenous peptides carry various post-translational modifications, making it essential to take these into account in the database search. These characteristics of endogenous peptides result in a huge search space, frequently leading to poor confidence of the peptide characterizations in peptidomics studies. We have developed a new MS/MS spectrum search tool for highly accurate and confident identification of endogenous peptides by combining two different fragmentation methods. Our approach takes advantage of the combination of two independent fragmentation methods (collision-induced dissociation and electron transfer dissociation). Their peptide spectral matching is carried out separately in both methods, and the final score is built as a combination of the two separate scores. We demonstrate that this approach is very effective in discriminating correct peptide identifications from false hits. We applied this approach to a spectral data set of neuropeptides extracted from mouse pituitary tumor cells. Compared to conventional MS-based identification, i.e., using a single fragmentation method, our approach significantly increased the peptide identification rate. It proved also highly effective for scanning spectra against a very large search space, enabling more accurate genome-wide searches and searches including multiple potential post-translational modifications.

  4. 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.

  5. Improving CID, HCD, and ETD FT MS/MS degradome-peptidome identifications using high accuracy mass information

    SciTech Connect

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

    2011-11-07

    The peptidome (i.e. processed and degraded forms of proteins) of e.g. blood can potentially provide insights into disease processes, as well as a source of candidate biomarkers that are unobtainable using conventional bottom-up proteomics approaches. MS dissociation methods, including CID, HCD, and ETD, can each contribute distinct identifications using conventional peptide identification methods (Shen et al. J. Proteome Res. 2011), but such samples still pose significant analysis and informatics challenges. In this work, we explored a simple approach for better utilization of high accuracy fragment ion mass measurements provided e.g. by FT MS/MS and demonstrate significant improvements relative to conventional descriptive and probabilistic scores methods. For example, at the same FDR level we identified 20-40% more peptides than SEQUEST and Mascot scoring methods using high accuracy fragment ion information (e.g., <10 mass errors) from CID, HCD, and ETD spectra. Species identified covered >90% of all those identified from SEQUEST, Mascot, and MS-GF scoring methods. Additionally, we found that the merging the different fragment spectra provided >60% more species using the UStags method than achieved previously, and enabled >1000 peptidome components to be identified from a single human blood plasma sample with a 0.6% peptide-level FDR, and providing an improved basis for investigation of potentially disease-related peptidome components.

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

    PubMed

    Montowska, Magdalena; Pospiech, Edward

    2016-12-01

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

  7. Identification of long-lived proteins reveals exceptional stability of essential cellular structures

    PubMed Central

    Park, Sung Kyu; Harris, Michael S.; Ingolia, Nicholas T.; Yates, John R.; Hetzer, Martin W.

    2013-01-01

    Intracellular proteins with long lifespans have recently been linked to age-dependent defects, ranging from decreased fertility to the functional decline of neurons. Why long-lived proteins exist in metabolically active cellular environments and how they are maintained over time remains poorly understood. Here we provide a system-wide identification of proteins with exceptional lifespans in the rat brain. These proteins are inefficiently replenished despite being translated robustly throughout adulthood. Using nucleoporins as a paradigm for long-term protein persistence, we found that nuclear pore complexes (NPCs) are maintained over a cell’s life through slow but finite exchange of even its most stable subcomplexes. This maintenance is limited, however, as some nucleoporin levels decrease during aging, providing a rationale for the previously observed age-dependent deterioration of NPC function. Our identification of a long-lived proteome reveals cellular components that are at increased risk for damage accumulation, linking long-term protein persistence to the cellular aging process. PMID:23993091

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

    PubMed Central

    Pospiech, Edward

    2016-01-01

    Summary New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey) and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages). Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed after 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation. PMID:28115907

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

    PubMed

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

    2011-03-04

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

  10. 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

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

    PubMed

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

    2017-02-01

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

  12. Analysis of proteins with the 'hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

    PubMed Central

    Pidugu, Lakshmi S; Maity, Koustav; Ramaswamy, Karthikeyan; Surolia, Namita; Suguna, Kaza

    2009-01-01

    Background The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects. PMID:19473548

  13. An Improved Fast Photochemical Oxidation of Proteins (FPOP) Platform for Protein Therapeutics

    NASA Astrophysics Data System (ADS)

    Zhang, Ying; Rempel, Don L.; Zhang, Hao; Gross, Michael L.

    2015-03-01

    Unlike small-molecule drugs, the size and dynamics of protein therapeutics challenge existing methods for assessing their high order structures (HOS). To extend fast photochemical oxidation of proteins (FPOP) to protein therapeutics, we modified its platform by introducing a mixing step prior to laser irradiation to minimize unwanted H2O2-induced oxidation. This improvement plus standardizing each step yield better reproducibility as determined by a fitting process whereby we used a non-FPOP spectrum as a template to report the unmodified level. We also tested different buffer systems for this modified FPOP platform with cytochrome c. The outcome is a standard oxidation profile that can be compared between different laboratories and regulatory agencies that wish to adopt FPOP for quality control purposes.

  14. An improved fast photochemical oxidation of proteins (FPOP) platform for protein therapeutics.

    PubMed

    Zhang, Ying; Rempel, Don L; Zhang, Hao; Gross, Michael L

    2015-03-01

    Unlike small-molecule drugs, the size and dynamics of protein therapeutics challenge existing methods for assessing their high order structures (HOS). To extend fast photochemical oxidation of proteins (FPOP) to protein therapeutics, we modified its platform by introducing a mixing step prior to laser irradiation to minimize unwanted H(2)O(2)-induced oxidation. This improvement plus standardizing each step yield better reproducibility as determined by a fitting process whereby we used a non-FPOP spectrum as a template to report the unmodified level. We also tested different buffer systems for this modified FPOP platform with cytochrome c. The outcome is a standard oxidation profile that can be compared between different laboratories and regulatory agencies that wish to adopt FPOP for quality control purposes.

  15. 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

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

    PubMed

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

    2007-09-04

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

  17. 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.

  18. Creating an antibacterial with in vivo efficacy: synthesis and characterization of potent inhibitors of the bacterial cell division protein FtsZ with improved pharmaceutical properties.

    PubMed

    Haydon, David J; Bennett, James M; Brown, David; Collins, Ian; Galbraith, Greta; Lancett, Paul; Macdonald, Rebecca; Stokes, Neil R; Chauhan, Pramod K; Sutariya, Jignesh K; Nayal, Narendra; Srivastava, Anil; Beanland, Joy; Hall, Robin; Henstock, Vincent; Noula, Caterina; Rockley, Chris; Czaplewski, Lloyd

    2010-05-27

    3-Methoxybenzamide (1) is a weak inhibitor of the essential bacterial cell division protein FtsZ. Alkyl derivatives of 1 are potent antistaphylococcal compounds with suboptimal drug-like properties. Exploration of the structure-activity relationships of analogues of these inhibitors led to the identification of potent antistaphylococcal compounds with improved pharmaceutical properties.

  19. Extracting features from protein sequences to improve deep extreme learning machine for protein fold recognition.

    PubMed

    Ibrahim, Wisam; Abadeh, Mohammad Saniee

    2017-03-27

    Protein fold recognition is an important problem in bioinformatics to predict three-dimensional structure of a protein. One of the most challenging tasks in protein fold recognition problem is the extraction of efficient features from the amino-acid sequences to obtain better classifiers. In this paper, we have proposed six descriptors to extract features from protein sequences. These descriptors are applied in the first stage of a three-stage framework PCA-DELM-LDA to extract feature vectors from the amino-acid sequences. Principal Component Analysis PCA has been implemented to reduce the number of extracted features. The extracted feature vectors have been used with original features to improve the performance of the Deep Extreme Learning Machine DELM in the second stage. Four new features have been extracted from the second stage and used in the third stage by Linear Discriminant Analysis LDA to classify the instances into 27 folds. The proposed framework is implemented on the independent and combined feature sets in SCOP datasets. The experimental results show that extracted feature vectors in the first stage could improve the performance of DELM in extracting new useful features in second stage.

  20. 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

  1. Identification, N-terminal region sequencing and similarity analysis of differentially expressed proteins in Paracoccidioides brasiliensis.

    PubMed

    Cunha, A F; Sousa, M V; Silva, S P; Jesuíno, R S; Soares, C M; Felipe, M S

    1999-04-01

    Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America. This human pathogen is a dimorphic fungus existing as mycelium (26 degrees C) and in infected tissues as a yeast form (36 degrees C). The in vitro differentiation process is reversible and dependent on temperature shift. In the present study, the total proteins from both forms of P. brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis. Differentially expressed proteins were identified. Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions. The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program. The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum. This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P. brasiliensis.

  2. 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

  3. Detection of outlier residues for improving interface prediction in protein heterocomplexes.

    PubMed

    Chen, Peng; Wong, Limsoon; Li, Jinyan

    2012-01-01

    Sequence-based understanding and identification of protein binding interfaces is a challenging research topic due to the complexity in protein systems and the imbalanced distribution between interface and noninterface residues. This paper presents an outlier detection idea to address the redundancy problem in protein interaction data. The cleaned training data are then used for improving the prediction performance. We use three novel measures to describe the extent a residue is considered as an outlier in comparison to the other residues: the distance of a residue instance from the center instance of all residue instances of the same class label (Dist), the probability of the class label of the residue instance (PCL), and the importance of within-class and between-class (IWB) residue instances. Outlier scores are computed by integrating the three factors; instances with a sufficiently large score are treated as outliers and removed. The data sets without outliers are taken as input for a support vector machine (SVM) ensemble. The proposed SVM ensemble trained on input data without outliers performs better than that with outliers. Our method is also more accurate than many literature methods on benchmark data sets. From our empirical studies, we found that some outlier interface residues are truly near to noninterface regions, and some outlier noninterface residues are close to interface regions.

  4. Identification and Characterization of a Novel Secreted Immunoglobulin Binding Protein from Group A Streptococcus

    PubMed Central

    Fagan, Peter K.; Reinscheid, Dieter; Gottschalk, Birgit; Chhatwal, Gursharan S.

    2001-01-01

    Immunoglobulin binding proteins are one of several pathogenicity factors which have been associated with invasive disease caused by group A streptococci. The surface-bound M and M-like proteins of Streptococcus pyogenes are the most characterized of these immunoglobulin binding proteins, and in most cases they bind only a single antibody class. Here we report the identification of a novel non-M-type secreted protein, designated SibA (for secreted immunoglobulin binding protein from group A streptococcus), which binds all immunoglobulin G (IgG) subclasses, the Fc and Fab fragments, and also IgA and IgM. SibA has no significant sequence homology to any M-related proteins, is not found in the vir regulon, and contains none of the characteristic M-protein regions, such as the A or C repeats. Like M proteins, however, SibA does have relatively high levels of alanine, lysine, glutamic acid, leucine, and glycine. SibA and M proteins also share an alpha-helical N-terminal secondary structure which has been previously implicated in immunoglobulin binding in M proteins. Evidence presented here indicates that this is also the case for SibA. SibA also has regions of local similarity with other coiled-coil proteins such as Listeria monocytogenes P45 autolysin, human myosin heavy chain, macrogolgin, and Schistoma mansoni paramyosin, some of which are of potential significance since cross-reactive antibodies between myosin proteins and M proteins have been implicated in the development of the autoimmune sequelae of streptococcal disease. PMID:11447160

  5. Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

    PubMed Central

    Chen, Fangyu; Jiang, Liangrong; Zheng, Jingsheng; Huang, Rongyu; Wang, Houcong; Hong, Zonglie; Huang, Yumin

    2014-01-01

    Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development. PMID:24699514

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

    PubMed

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

    2012-01-01

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

  7. Redox Proteomics Identification of Oxidatively Modified Myocardial Proteins in Human Heart Failure: Implications for Protein Function

    PubMed Central

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

    2012-01-01

    Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n = 14) and non-failing human hearts (n = 13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF. PMID:22606238

  8. Identification of a Bacteria-Specific Binding Protein from the Sequenced Bacterial Genome.

    PubMed

    Kong, Minsuk; Ryu, Sangryeol

    2016-01-01

    Novel and specific recognition elements are of central importance in the development of a pathogen detection method. Here, we describe a simple method for identifying the cell-wall binding domain (CBD) from a sequenced bacterial genome employing homology search for phage lysin genes. A putative CBD (CPF369_CBD) was identified from a genome of Clostridium perfringens type strain ATCC 13124, and its function was studied with the CBDGFP fusion protein recombinantly expressed in Escherichia coli. Fluorescence microscopy showed the specific binding of the fusion protein to C. perfringens cells, which demonstrates the potential of this method for the identification of novel bioprobes for specific detection of pathogenic bacteria.

  9. 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.

  10. 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.

  11. Identification of lysine-acetylated mitochondrial proteins and their acetylation sites.

    PubMed

    Hartl, Markus; König, Ann-Christine; Finkemeier, Iris

    2015-01-01

    The (ε)N-acetylation of lysine side chains is a highly conserved posttranslational modification of both prokaryotic and eukaryotic proteins. Lysine acetylation not only occurs on histones in the nucleus but also on many mitochondrial proteins in plants and animals. As the transfer of the acetyl group to lysine eliminates its positive charge, lysine acetylation can affect the biological function of proteins. This chapter describes two methods for the identification of lysine-acetylated proteins in plant mitochondria using an anti-acetyllysine antibody. We describe the Western blot analysis of a two-dimensional blue native-polyacrylamide gel electrophoresis with an anti-acetyllysine antibody as well as the immuno-enrichment of lysine-acetylated peptides followed by liquid chromatography-tandem mass spectrometry data acquisition and analysis.

  12. A method for the identification of proteins secreted by lactic acid bacteria grown in complex media.

    PubMed

    Sánchez, Borja; Chaignepain, Sthéphane; Schmitter, Jean-Marie; Urdaci, María C

    2009-06-01

    Lactic acid bacteria (LAB) are known for their special nutritional requirements, being usually cultured in complex media to achieve optimal growth. In this paper, a protocol based on trichloroacetic acid precipitation of peptides and proteins is presented. The method has been tested on four probiotic LAB strains grown in De Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the culture of such bacteria. This protocol allowed the detection of 19 proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being successfully identified by tandem MS. Thereafter, the 10 were found to be secreted or surface associated by bioinformatic means. In conclusion, this work supplies a method for the identification of proteins secreted by LAB, allowing discrimination between the proteins present in the MRS and those produced by probiotic LAB.

  13. 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.

  14. Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins.

    PubMed

    Pham, Victoria C; Henzel, William J; Lill, Jennie R

    2005-11-01

    A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins.

  15. 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.

  16. A photometric mode identification method, including an improved non-adiabatic treatment of the atmosphere

    NASA Astrophysics Data System (ADS)

    Dupret, M.-A.; De Ridder, J.; De Cat, P.; Aerts, C.; Scuflaire, R.; Noels, A.; Thoul, A.

    2003-02-01

    We present an improved version of the method of photometric mode identification of Heynderickx et al. (\\cite{hey}). Our new version is based on the inclusion of precise non-adiabatic eigenfunctions determined in the outer stellar atmosphere according to the formalism recently proposed by Dupret et al. (\\cite{dup}). Our improved photometric mode identification technique is therefore no longer dependent on ad hoc parameters for the non-adiabatic effects. It contains the complete physical conditions of the outer atmosphere of the star, provided that rotation does not play a key role. We apply our method to the two slowly pulsating B stars HD 74560 and HD 138764 and to the beta Cephei star EN (16) Lac. Besides identifying the degree l of the pulsating stars, our method is also a tool for improving the knowledge of stellar interiors and atmospheres, by imposing constraints on parameters such as the metallicity and the mixing-length parameter alpha (a procedure we label non-adiabatic asteroseismology). The non-adiabatic eigenfunctions needed for the mode identification are available upon request from the authors.

  17. Improving the Quality of Protein Crystals Using Stirring Crystallization

    NASA Astrophysics Data System (ADS)

    Adachi, Hiroaki; Matsumura, Hiroyoshi; Niino, Ai; Takano, Kazufumi; Kinoshita, Takayoshi; Warizaya, Masaichi; Inoue, Tsuyoshi; Mori, Yusuke; Sasaki, Takatomo

    2004-04-01

    Recent reports state that a high magnetic field improves the crystal quality of bovine adenosine deaminase (ADA) with an inhibitor [Kinoshita et al.: Acta Cryst. D59 (2003) 1333]. In this paper, we examine the effect of stirring solution on ADA crystallization using a vapor-diffusion technique with rotary and figure-eight motion shakers. The probability of obtaining high-quality crystals is increased with stirring in a figure-eight pattern. Furthermore, rotary stirring greatly increased the probability of obtaining high-quality crystals, however, nucleation time was also increased. The crystal structure with the inhibitor was determined at a high resolution using a crystal obtained from a stirred solution. These results indicate that stirring with simple equipment is as useful as the high magnetic field technique for protein crystallization.

  18. An Improved Protein Surface Extraction Method Using Rotating Cylinder Probe.

    PubMed

    Singh, Kalpana; Lahiri, Tapobrata

    2017-03-01

    For extraction of information on binding sites of a protein, the commonly known geometry-based methods utilize the corresponding PDB file to extract its surface as a first step. Finally, the surface is used to find the binding site atoms. As shown in this paper work, since none of the mostly used surface extraction methods can retrieve a sizeable percentage of the binding site atoms, the scope of development of a better method remains. In this direction, this paper presents a new benchmarking criteria based on utilization of binding site information to compare performance of these surface extraction methods. Also, a new surface extraction method is introduced based on the use of a rotating cylinder probe adapting from the work of Weisel et al. (Chem Cent J 1:7-23, 2007. doi: 10.1186/1752-153X-1-7 ). The result of the new method shows a significant improvement of performance in comparison to the existing methods.

  19. Protein haptenation by amoxicillin: high resolution mass spectrometry analysis and identification of target proteins in serum.

    PubMed

    Ariza, Adriana; Garzon, Davide; Abánades, Daniel R; de los Ríos, Vivian; Vistoli, Giulio; Torres, María J; Carini, Marina; Aldini, Giancarlo; Pérez-Sala, Dolores

    2012-12-21

    Allergy towards wide spectrum antibiotics such as amoxicillin (AX) is a major health problem. Protein haptenation by covalent conjugation of AX is considered a key process for the allergic response. However, the nature of the proteins involved has not been completely elucidated. Human serum albumin (HSA) is the most abundant protein in plasma and is considered a major target for haptenation by drugs, including β-lactam antibiotics. Here we report a procedure for immunological detection of AX-protein adducts with antibodies recognizing the lateral chain of the AX molecule. With this approach we detected human serum proteins modified by AX in vitro and identified HSA, transferrin and immunoglobulins heavy and light chains as prominent AX-modified proteins. Since HSA was the major AX target, we characterized AX-HSA interaction using high resolution LTQ orbitrap MS. At 0.5mg/mL AX, we detected one main AX-HSA adduct involving residues Lys 190, 199 or 541, whereas higher AX concentrations elicited a more extensive modification. In molecular modeling studies Lys190 and Lys 199 were found the most reactive residues towards AX, with surrounding residues favoring adduct formation. These findings provide novel tools and insight for the study of protein haptenation and the mechanisms involved in AX-elicited allergic reactions.

  20. PPIcons: identification of protein-protein interaction sites in selected organisms.

    PubMed

    Sriwastava, Brijesh K; Basu, Subhadip; Maulik, Ujjwal; Plewczynski, Dariusz

    2013-09-01

    The physico-chemical properties of interaction interfaces have a crucial role in characterization of protein-protein interactions (PPI). In silico prediction of participating amino acids helps to identify interface residues for further experimental verification using mutational analysis, or inhibition studies by screening library of ligands against given protein. Given the unbound structure of a protein and the fact that it forms a complex with another known protein, the objective of this work is to identify the residues that are involved in the interaction. We attempt to predict interaction sites in protein complexes using local composition of amino acids together with their physico-chemical characteristics. The local sequence segments (LSS) are dissected from the protein sequences using a sliding window of 21 amino acids. The list of LSSs is passed to the support vector machine (SVM) predictor, which identifies interacting residue pairs considering their inter-atom distances. We have analyzed three different model organisms of Escherichia coli, Saccharomyces Cerevisiae and Homo sapiens, where the numbers of considered hetero-complexes are equal to 40, 123 and 33 respectively. Moreover, the unified multi-organism PPI meta-predictor is also developed under the current work by combining the training databases of above organisms. The PPIcons interface residues prediction method is measured by the area under ROC curve (AUC) equal to 0.82, 0.75, 0.72 and 0.76 for the aforementioned organisms and the meta-predictor respectively.

  1. Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins

    PubMed Central

    1979-01-01

    To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS- polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface. PMID:479301

  2. Identification of the protein components of protein-bound polysaccharide (PSK) that interact with NKL cells.

    PubMed

    Jiménez, Eva; Garcia-Lora, Angel; Martinez, Marisol; Garrido, Federico

    2005-04-01

    We identified the protein components of a protein-bound polysaccharide (PSK) that are responsible for the biological function of this immunomodulator in its interaction with NKL cells, an NK-derived cell line previously known to be activated by this extract, obtained from the basidiomycete Coriolus versiocolor. In addition, we show that PSK protein interacts with NKL cells through a different receptor from that used by IL-2. This was deduced from the different molecular weights of the PSK/NKL and IL-2/NKL receptor complexes. We show that PSK is composed of a highly glycosylated 12-kDa protein. Protein-bound polysaccharide interacts in vitro with an NKL receptor of approximately 48 kDa, whereas IL-2 shows a similar interaction with NKL receptor proteins of approximately 64 and 75 kDa. Our results may explain why PSK and IL-2 use completely different intracellular routes for their biological activities in NKL cells-i.e., regulating different PKC isozymes, mitogen-activated protein kinases, and nuclear transcription factors.

  3. Computational Identification and Comparative Analysis of Secreted and Transmembrane Proteins in Six Burkholderia Species

    PubMed Central

    Nguyen, Thao Thi; Lee, Hyun-Hee; Park, Jungwook; Park, Inmyoung; Seo, Young-Su

    2017-01-01

    As a step towards discovering novel pathogenesis-related proteins, we performed a genome scale computational identification and characterization of secreted and transmembrane (TM) proteins, which are mainly responsible for bacteria-host interactions and interactions with other bacteria, in the genomes of six representative Burkholderia species. The species comprised plant pathogens (B. glumae BGR1, B. gladioli BSR3), human pathogens (B. pseudomallei K96243, B. cepacia LO6), and plant-growth promoting endophytes (Burkholderia sp. KJ006, B. phytofirmans PsJN). The proportions of putative classically secreted proteins (CSPs) and TM proteins among the species were relatively high, up to approximately 20%. Lower proportions of putative type 3 non-classically secreted proteins (T3NCSPs) (~10%) and unclassified non-classically secreted proteins (NCSPs) (~5%) were observed. The numbers of TM proteins among the three clusters (plant pathogens, human pathogens, and endophytes) were different, while the distribution of these proteins according to the number of TM domains was conserved in which TM proteins possessing 1, 2, 4, or 12 TM domains were the dominant groups in all species. In addition, we observed conservation in the protein size distribution of the secreted protein groups among the species. There were species-specific differences in the functional characteristics of these proteins in the various groups of CSPs, T3NCSPs, and unclassified NCSPs. Furthermore, we assigned the complete sets of the conserved and unique NCSP candidates of the collected Burkholderia species using sequence similarity searching. This study could provide new insights into the relationship among plant-pathogenic, human-pathogenic, and endophytic bacteria. PMID:28381962

  4. Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins1[OPEN

    PubMed Central

    Tsai, Allen Yi-Lun; Kunieda, Tadashi; Rogalski, Jason; Foster, Leonard J.; Ellis, Brian E.

    2017-01-01

    Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features. PMID:28003327

  5. Identification of Late Embryogenesis Abundant (LEA) protein putative interactors using phage display.

    PubMed

    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.

  6. pH fractionation and identification of proteins: comparing column chromatofocusing versus liquid isoelectric focusing techniques.

    PubMed

    Gunther, Nereus W; Paul, Moushumi; Nuñez, Alberto; Liu, Yanhong

    2012-06-01

    In proteomic investigations, a number of different separation techniques can be applied to fractionate whole cell proteomes into more manageable fractions for subsequent analysis. In this work, utilizing HPLC and mass spectrometry for protein identification, two different fractionation methods were compared and contrasted to determine the potential of each method for the simple and reproducible fractionation of a bacterial proteome. Column-based chromatofocusing and liquid-based isoelectric focusing both utilized pH gradients to produce similar results in terms of the numbers of proteins successfully identified (402 and 378 proteins) and the consistency of proteins identified from one experiment to the next (<10% change). However, there was limited overlap in the protein sets with <50% of the proteins identified as common between the sets of proteins identified by the different systems. In addition to the numbers of proteins identified and consistency of those identified, the reduced monetary costs of experimentation and increased assay flexibility produced by using isoelectric focusing was considered in order to adopt a system best suited for comparative proteomic projects.

  7. 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.

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

    PubMed

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

    2014-06-01

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

  9. Identification of Obscure yet Conserved Actin-Associated Proteins in Giardia lamblia

    PubMed Central

    Nayeri, Arash; Xu, Jennifer W.; Krtková, Jana; Cande, W. Zacheus

    2014-01-01

    Consistent with its proposed status as an early branching eukaryote, Giardia has the most divergent actin of any eukaryote and lacks core actin regulators. Although conserved actin-binding proteins are missing from Giardia, its actin is utilized similarly to that of other eukaryotes and functions in core cellular processes such as cellular organization, endocytosis, and cytokinesis. We set out to identify actin-binding proteins in Giardia using affinity purification coupled with mass spectroscopy (multidimensional protein identification technology [MudPIT]) and have identified >80 putative actin-binding proteins. Several of these have homology to conserved proteins known to complex with actin for functions in the nucleus and flagella. We validated localization and interaction for seven of these proteins, including 14-3-3, a known cytoskeletal regulator with a controversial relationship to actin. Our results indicate that although Giardia lacks canonical actin-binding proteins, there is a conserved set of actin-interacting proteins that are evolutionarily indispensable and perhaps represent some of the earliest functions of the actin cytoskeleton. PMID:24728194

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

    PubMed

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

    2017-02-07

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

  11. Weight and see: loading working memory improves incidental identification of irrelevant faces.

    PubMed

    Carmel, David; Fairnie, Jake; Lavie, Nilli

    2012-01-01

    Are task-irrelevant stimuli processed to a level enabling individual identification? This question is central both for perceptual processing models and for applied settings (e.g., eye-witness testimony). Lavie's load theory proposes that working memory actively maintains attentional prioritization of relevant over irrelevant information. Loading working memory thus impairs attentional prioritization, leading to increased processing of task-irrelevant stimuli. Previous research has shown that increased working memory load leads to greater interference effects from response-competing distractors. Here we test the novel prediction that increased processing of irrelevant stimuli under high working memory load should lead to a greater likelihood of incidental identification of entirely irrelevant stimuli. To test this, we asked participants to perform a word-categorization task while ignoring task-irrelevant images. The categorization task was performed during the retention interval of a working memory task with either low or high load (defined by memory set size). Following the final experimental trial, a surprise question assessed incidental identification of the irrelevant image. Loading working memory was found to improve identification of task-irrelevant faces, but not of building stimuli (shown in a separate experiment to be less distracting). These findings suggest that working memory plays a critical role in determining whether distracting stimuli will be subsequently identified.

  12. Identification of Cellular Proteins that Interact with Human Cytomegalovirus Immediate-Early Protein 1 by Protein Array Assay

    PubMed Central

    Puerta Martínez, Francisco; Tang, Qiyi

    2013-01-01

    Human cytomegalovirus (HCMV) gene expression during infection is characterized as a sequential process including immediate-early (IE), early (E), and late (L)-stage gene expression. The most abundantly expressed gene at the IE stage of infection is the major IE (MIE) gene that produces IE1 and IE2. IE1 has been the focus of study because it is an important protein, not only for viral gene expression but also for viral replication. It is believed that IE1 plays important roles in viral gene regulation by interacting with cellular proteins. In the current study, we performed protein array assays and identified 83 cellular proteins that interact with IE1. Among them, seven are RNA-binding proteins that are important in RNA processing; more than half are nuclear proteins that are involved in gene regulations. Tumorigenesis-related proteins are also found to interact with IE1, implying that the role of IE1 in tumorigenesis might need to be reevaluated. Unexpectedly, cytoplasmic proteins, such as Golgi autoantigen and GGA1 (both related to the Golgi trafficking protein), are also found to be associated with IE1. We also employed a coimmunoprecipitation assay to test the interactions of IE1 and some of the proteins identified in the protein array assays and confirmed that the results from the protein array assays are reliable. Many of the proteins identified by the protein array assay have not been previously reported. Therefore, the functions of the IE1-protein interactions need to be further explored in the future. PMID:24385082

  13. Improving the Identification of Neonatal Encephalopathy: Utility of a Web-Based Video Tool.

    PubMed

    Ivy, Autumn S; Clark, Catherine L; Bahm, Sarah M; Meurs, Krisa P Van; Wusthoff, Courtney J

    2017-04-01

    Objective This study tested the effectiveness of a video teaching tool in improving identification and classification of encephalopathy in infants. Study Design We developed an innovative video teaching tool to help clinicians improve their skills in interpreting the neonatal neurological examination for grading encephalopathy. Pediatric residents were shown 1-minute video clips demonstrating exam findings in normal neonates and neonates with various degrees of encephalopathy. Findings from five domains were demonstrated: spontaneous activity, level of alertness, posture/tone, reflexes, and autonomic responses. After each clip, subjects were asked to identify whether the exam finding was normal or consistent with mild, moderate, or severe abnormality. Subjects were then directed to a web-based teaching toolkit, containing a compilation of videos demonstrating normal and abnormal findings on the neonatal neurological examination. Immediately after training, subjects underwent posttesting, again identifying exam findings as normal, mild, moderate, or severe abnormality. Results Residents improved in their overall ability to identify and classify neonatal encephalopathy after viewing the teaching tool. In particular, the identification of abnormal spontaneous activity, reflexes, and autonomic responses were most improved. Conclusion This pretest/posttest evaluation of an educational tool demonstrates that after viewing our toolkit, pediatric residents were able to improve their overall ability to detect neonatal encephalopathy.

  14. Identification of G protein-coupled receptor signaling pathway proteins in marine diatoms using comparative genomics

    PubMed Central

    2013-01-01

    Background The G protein-coupled receptor (GPCR) signaling pathway plays an essential role in signal transmission and response to external stimuli in mammalian cells. Protein components of this pathway have been characterized in plants and simpler eukaryotes such as yeast, but their presence and role in unicellular photosynthetic eukaryotes have not been determined. We use a comparative genomics approach using whole genome sequences and gene expression libraries of four diatoms (Pseudo-nitzschia multiseries, Thalassiosira pseudonana, Phaeodactylum tricornutum and Fragilariopsis cylindrus) to search for evidence of GPCR signaling pathway proteins that share sequence conservation to known GPCR pathway proteins. Results The majority of the core components of GPCR signaling were well conserved in all four diatoms, with protein sequence similarity to GPCRs, human G protein α- and β-subunits and downstream effectors. There was evidence for the Gγ-subunit and thus a full heterotrimeric G protein only in T. pseudonana. Phylogenetic analysis of putative diatom GPCRs indicated similarity but deep divergence to the class C GPCRs, with branches basal to the GABAB receptor subfamily. The extracellular and intracellular regions of these putative diatom GPCR sequences exhibited large variation in sequence length, and seven of these sequences contained the necessary ligand binding domain for class C GPCR activation. Transcriptional data indicated that a number of the putative GPCR sequences are expressed in diatoms under various stress conditions in culture, and that many of the GPCR-activated signaling proteins, including the G protein, are also expressed. Conclusions The presence of sequences in all four diatoms that code for the proteins required for a functional mammalian GPCR pathway highlights the highly conserved nature of this pathway and suggests a complex signaling machinery related to environmental perception and response in these unicellular organisms. The lack of

  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. An improved plant leaf protein extraction method for high resolution two-dimensional polyacrylamide gel electrophoresis and comparative proteomics.

    PubMed

    Alam, I; Sharmin, Sa; Kim, K-H; Kim, Y-G; Lee, Jj; Lee, B-H

    2013-02-01

    We report here a simple and universally applicable protocol for extracting high quality proteins from plant leaf tissues. The protocol provides improved resolution and reproducibility of two-dimensional polyacrylamide gel electrophoresis (2-DE) and reduces the time required to analyze samples. Partitioning rubisco by polyethylene glycol (PEG) fractionation provides clearer detection of low-abundance proteins. Co-extraction of interfering substances increases the sample conductivity, which results in poor electrophoretic separation. Re-extraction of PEG-fractionated samples with phenol effectively eliminated interfering substances, which results in optimal conductivity during separation in the first dimension of the isoelectric focusing. Smooth focusing reduces analysis time and provides superior resolution in 2-DE gels. Incubating the samples at -80° C instead of -20° C reduced protein precipitation time to 2-3 h. Removal of nonprotein contaminants and the use of sonication increased protein solubility without additional reagents. These changes enabled loading and separation of maximum amounts of proteins, which permitted improved protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). An immunological approach revealed that little or no ribulose-1, 5-bisphosphte bisphosphate carboxylase oxygenase was present in the PEG supernatant. In addition, low-abundance proteins, such as myelocytomatosis transcription factor (MYC) and alpha subunit of heterotrimeric guanine nucleotide-binding protein complex (Gα), were detected only in the modified PEG supernatant and not in the total protein. These results suggest that our protocol produced high quality proteins and made many low-abundant proteins available for proteomic analysis. The successful application of this protocol for analyzing the leaf proteomes of soybean, Miscanthus sinensis, barley, Chinese cabbage, peanut and tea (Camellia sinensis) suggests

  17. Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis.

    PubMed

    Cao, Heping; Deterding, Leesa J; Venable, John D; Kennington, Elizabeth A; Yates, John R; Tomer, Kenneth B; Blackshear, Perry J

    2006-02-15

    Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption-ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)-tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines.

  18. Identification of the anti-inflammatory protein tristetraprolin as a hyperphosphorylated protein by mass spectrometry and site-directed mutagenesis

    PubMed Central

    Cao, Heping; Deterding, Leesa J.; Venable, John D.; Kennington, Elizabeth A.; Yates, John R.; Tomer, Kenneth B.; Blackshear, Perry J.

    2005-01-01

    Tristetraprolin (TTP) is a zinc-finger protein that binds to AREs (AU-rich elements) within certain mRNAs and causes destabilization of those mRNAs. Mice deficient in TTP develop a profound inflammatory syndrome with erosive arthritis, autoimmunity and myeloid hyperplasia. Previous studies showed that TTP is phosphorylated extensively in intact cells. However, limited information is available about the identities of these phosphorylation sites. We investigated the phosphorylation sites in human TTP from transfected HEK-293 cells by MS and site-directed mutagenesis. A number of phosphorylation sites including Ser66, Ser88, Thr92, Ser169, Ser186, Ser197, Ser218, Ser228, Ser276 and Ser296 were identified by MS analyses using MALDI (matrix-assisted laser-desorption–ionization)-MS, MALDI-tandem MS, LC (liquid chromatography)–tandem MS and multidimensional protein identification technology. Mutations of Ser197, Ser218 and Ser228 to alanine in the human protein significantly increased TTP's gel mobility (likely to be stoichiometric), whereas mutations at the other sites had little effect on its gel mobility. Dephosphorylation and in vivo labelling studies showed that mutant proteins containing multiple mutations were still phosphorylated, and all were able to bind to RNA probes containing AREs. Confocal microscopy showed a similar cytosolic localization of TTP among the various proteins. Ser197, Ser218 and Ser228 are predicted by motif scanning to be potential sites for protein kinase A, glycogen synthase kinase-3 and extracellular-signal-regulated kinase 1 (both Ser218 and Ser228) respectively. The present study has identified multiple phosphorylation sites in the anti-inflammatory protein TTP in mammalian cells and should provide the molecular basis for further studies on the function and regulation of TTP in controlling pro-inflammatory cytokines. PMID:16262601

  19. 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.

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

    PubMed

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

    2016-03-01

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

  1. Identification of Bovine Sperm Acrosomal Proteins that Interact with a 32kDa Acrosomal Matrix Protein

    PubMed Central

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

    2016-01-01

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

  2. 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...

  3. Identification of AKAP79 as a Protein Phosphatase 1 catalytic binding protein

    PubMed Central

    Le, Andrew. V.; Tavalin, Steven. J.

    2011-01-01

    The ubiquitously expressed and highly promiscuous Protein Phosphatase 1 (PP1) regulates many cellular processes. Targeting PP1 to specific locations within the cell allows for the regulation of PP1 by conferring substrate specificity. In the present study, we identified AKAP79 as a novel PP1 regulatory subunit. Immunoprecipitaiton of the AKAP from rat brain extract found that the PP1 catalytic subunit co-purified with the anchoring protein. This is a direct interaction, demonstrated by pulldown experiments using purified proteins. Interestingly, the addition of AKAP79 to purified PP1 catalytic subunit decreased phosphatase activity with an IC50 of 811±0.56 nM of the anchoring protein. Analysis of AKAP79 identified a PP1 binding site that conformed to a consensus PP1 binding motif (FxxR/KxR/K) in the first 44 amino acids of the anchoring protein. This was confirmed when a peptide mimicking this region of AKAP79 was able to bind PP1 by both pulldown assay and Surface Plasmon Resonance. However, PP1 was still able to bind to AKAP79 upon deletion of this region, suggestion additional sites of contact between the anchoring protein and the phosphatase. Importantly, this consensus PP1 binding motif was found not to be responsible for PP1 inhibition, but rather enhanced phosphatase activity, as deletion of this domain resulted in an increased inhibition of PP1 activity. Instead, a second interaction domain localized to residues 150–250 of AKAP79 was required for the inhibition of PP1. However, the inhibitory actions of AKAP79 on PP1 are substrate dependent, as the anchoring protein did not inhibit PP1 dephosphorylation of phospho-PSD-95, a substrate found in AKAP79 complexes in the brain. These combined observations suggest that AKAP79 acts as a PP1 regulatory subunit that can direct PP1 activity towards specific targets in the AKAP79 complex. PMID:21561082

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

    PubMed

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

    2011-02-25

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

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

    PubMed

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

    2015-01-01

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

  6. An improved method for identification of small non-coding RNAs in bacteria using support vector machine

    PubMed Central

    Barman, Ranjan Kumar; Mukhopadhyay, Anirban; Das, Santasabuj

    2017-01-01

    Bacterial small non-coding RNAs (sRNAs) are not translated into proteins, but act as functional RNAs. They are involved in diverse biological processes like virulence, stress response and quorum sensing. Several high-throughput techniques have enabled identification of sRNAs in bacteria, but experimental detection remains a challenge and grossly incomplete for most species. Thus, there is a need to develop computational tools to predict bacterial sRNAs. Here, we propose a computational method to identify sRNAs in bacteria using support vector machine (SVM) classifier. The primary sequence and secondary structure features of experimentally-validated sRNAs of Salmonella Typhimurium LT2 (SLT2) was used to build the optimal SVM model. We found that a tri-nucleotide composition feature of sRNAs achieved an accuracy of 88.35% for SLT2. We validated the SVM model also on the experimentally-detected sRNAs of E. coli and Salmonella Typhi. The proposed model had robustly attained an accuracy of 81.25% and 88.82% for E. coli K-12 and S. Typhi Ty2, respectively. We confirmed that this method significantly improved the identification of sRNAs in bacteria. Furthermore, we used a sliding window-based method and identified sRNAs from complete genomes of SLT2, S. Typhi Ty2 and E. coli K-12 with sensitivities of 89.09%, 83.33% and 67.39%, respectively. PMID:28383059

  7. 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.

  8. Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins.

    PubMed

    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.

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

    PubMed

    Quintero, Jael; Figueroa, Diana Carolina; Barcelo, Rafael; Breci, Linda; Astiazaran-Garcia, Humberto; Rascon, Lucila; Robles-Zepeda, Ramon; Garibay-Escobar, Adriana; Velazquez-Contreras, Enrique; Avila, Gloria Leon; Hernandez-Hernandez, Jose Manuel; Velazquez, Carlos

    2013-08-01

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

  10. 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

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

    PubMed

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

    2000-08-01

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

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

    PubMed

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

    1999-04-01

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

  13. Protein architecture of avian reovirus S1133 and identification of the cell attachment protein.

    PubMed Central

    Martínez-Costas, J; Grande, A; Varela, R; García-Martínez, C; Benavente, J

    1997-01-01

    There are a number of discrepancies in the literature regarding the protein composition of the avian reoviruses. The present study demonstrates that avian reovirus S1133 contains at least 10 proteins (lambdaA, lambdaB, lambdaC, muA, muB, muBC, muBN, sigmaA, sigmaB, and sigmaC). Polypeptides muB, muBC, muBN, sigmaB, and sigmaC are components of the outer capsid layer of the virus, while lambdaA, lambdaB, muA, and sigmaA are core polypeptides. Protein lambdaC is a component of both layers, extending from the inner core to the outer capsid. The minor outer-capsid polypeptide sigmaC is shown to be the cell attachment protein, since it is the only viral polypeptide present in extracts of S1133-infected cells that binds specifically to chicken embryo fibroblasts; furthermore, its binding to avian cells was competitively inhibited by S1133 reovirions but not by mammalian reovirions. Our results also show that sigmaC is an oligomeric protein both in the virion and free in the cytoplasm, and preliminary results suggest that the multimer is made up of three monomeric units. PMID:8985323

  14. Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS.

    PubMed

    Ziegler, Dominik; Pothier, Joël F; Ardley, Julie; Fossou, Romain Kouakou; Pflüger, Valentin; de Meyer, Sofie; Vogel, Guido; Tonolla, Mauro; Howieson, John; Reeve, Wayne; Perret, Xavier

    2015-07-01

    Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.

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

    SciTech Connect

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

    2007-02-01

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

  16. 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.

  17. 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.

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

    PubMed

    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 the

  19. Improving protein-protein interaction prediction using evolutionary information from low-quality MSAs

    PubMed Central

    Várnai, Csilla; Burkoff, Nikolas S.; Wild, David L.

    2017-01-01

    Evolutionary information stored in multiple sequence alignments (MSAs) has been used to identify the interaction interface of protein complexes, by measuring either co-conservation or co-mutation of amino acid residues across the interface. Recently, maximum entropy related correlated mutation measures (CMMs) such as direct information, decoupling direct from indirect interactions, have been developed to identify residue pairs interacting across the protein complex interface. These studies have focussed on carefully selected protein complexes with large, good-quality MSAs. In this work, we study protein complexes with a more typical MSA consisting of fewer than 400 sequences, using a set of 79 intramolecular protein complexes. Using a maximum entropy based CMM at the residue level, we develop an interface level CMM score to be used in re-ranking docking decoys. We demonstrate that our interface level CMM score compares favourably to the complementarity trace score, an evolutionary information-based score measuring co-conservation, when combined with the number of interface residues, a knowledge-based potential and the variability score of individual amino acid sites. We also demonstrate, that, since co-mutation and co-complementarity in the MSA contain orthogonal information, the best prediction performance using evolutionary information can be achieved by combining the co-mutation information of the CMM with co-conservation information of a complementarity trace score, predicting a near-native structure as the top prediction for 41% of the dataset. The method presented is not restricted to small MSAs, and will likely improve interface prediction also for complexes with large and good-quality MSAs. PMID:28166227

  20. Protein-protein interaction and molecular dynamics analysis for identification of novel inhibitors in Burkholderia cepacia GG4.

    PubMed

    Gupta, Money; Chauhan, Rashi; Prasad, Yamuna; Wadhwa, Gulshan; Jain, Chakresh Kumar

    2016-12-01

    The lack of complete treatments and appearance of multiple drug-resistance strains of Burkholderia cepacia complex (Bcc) are causing an increased risk of lung infections in cystic fibrosis patients. Bcc infection is a big risk to human health and demands an urgent need to identify new therapeutics against these bacteria. Network biology has emerged as one of the prospective hope in identifying novel drug targets and hits. We have applied protein-protein interaction methodology to identify new drug-target candidates (orthologs) in Burkhloderia cepacia GG4, which is an important strain for studying the quorum-sensing phenomena. An evolutionary based ortholog mapping approach has been applied for generating the large scale protein-protein interactions in B. Cepacia. As a case study, one of the identified drug targets; GEM_3202, a NH (3)-dependent NAD synthetase protein has been studied and the potential ligand molecules were screened using the ZINC database. The three dimensional structure (NH (3)-dependent NAD synthetase protein) has been predicted from MODELLERv9.11 tool using multiple PDB templates such as 3DPI, 2PZ8 and 1NSY with sequence identity of 76%, 50% and 50% respectively. The structure has been validated with Ramachandaran plot having 100% residues of NadE in allowed region and overall quality factor of 81.75 using ERRAT tool. High throughput screening and Vina resulted in two potential hits against NadE such as ZINC83103551 and ZINC38008121. These molecules showed lowest binding energy of -5.7kcalmol(-1) and high stability in the binding pockets during molecular dynamics simulation analysis. The similar approach for target identification could be applied for clinical strains of other pathogenic microbes.

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

    PubMed

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

    2017-01-01

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

  2. Protein Identification Via Surface-Induced Dissociation in an FT-ICR Mass Spectrometer and a Patchwork Sequencing Approach

    SciTech Connect

    Fernandez, Facundo; Wysocki, Vicki H.; Futrell, Jean H.; Laskin, Julia

    2006-05-01

    Surface-induced dissociation (SID) and collision-induced dissociation (CID) are ion activation techniques based on energetic collisions with a surface or gas molecules, respectively. One noticeable difference between CID and SID is that SID does not require a collision gas for ion activation and is therefore directly compatible with the high vacuum requirement of Fourier Transform Ion Cyclotron Resonance mass spectrometers (FT-ICR MS). Eliminating the introduction of collision gas into the ICR cell for collisional activation dramatically shortens the acquisition time for MS/MS experiments, suggesting that SID could be utilized for high-throughput MS/MS studies in FT-ICR MS. We demonstrate for the first time the utility of SID combined with FT-ICR MS for protein identification. Tryptic digests of standard proteins were analyzed using a hybrid 6-Tesla FT-ICR MS with SID and CID capabilities. SID spectra of mass-selected singly and doubly charged peptides were obtained using a diamond-coated target mounted at the rear trapping plate of the ICR cell. The broad internal energy distribution deposited into the precursor ion following collision with the diamond surface allowed a variety of fragmentation channels to be accessed by SID. Composition and sequence qualifiers produced by SID of tryptic peptides were used to improve the statistical significance of database searches. Protein identification MASCOT scores obtained using SID were comparable or better than scores obtained using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) –the conventional ion activation technique in FT-ICR MS.

  3. Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that el...

  4. Microbial Protein-Antigenome Determination (MAD) Technology: A Proteomics-Based Strategy for Rapid Identification of Microbial Targets of Host Humoral Immune Responses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunogenic, pathogen-specific proteins have excellent potential for development of novel management modalities. Here, we describe an innovative application of proteomics called Microbial protein-Antigenome Determination (MAD) Technology for rapid identification of native microbial proteins that eli...

  5. 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

  6. Identification of the major functional proteins of prokaryotic lipid droplets[S

    PubMed Central

    Ding, Yunfeng; Yang, Li; Zhang, Shuyan; Wang, Yang; Du, Yalan; Pu, Jing; Peng, Gong; Chen, Yong; Zhang, Huina; Yu, Jinhai; Hang, Haiying; Wu, Peng; Yang, Fuquan; Yang, Hongyuan; Steinbüchel, Alexander; Liu, Pingsheng

    2012-01-01

    Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly efficient in converting biomass to TAG. We obtained highly purified LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identified, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. The structure predicted for ro02104 resembles that of apolipoproteins, the structural proteins of plasma lipoproteins in mammals. Deletion of ro02104 resulted in the formation of supersized LDs, indicating that ro02104 plays a critical role in cellular LD dynamics. The putative α helix of the ro02104 LD-targeting domain (amino acids 83–146) is also similar to that of apolipoproteins. We report the identification of 228 proteins in the proteome of prokaryotic LDs, identify a putative structural protein of this organelle, and suggest that apolipoproteins may have an evolutionarily conserved role in the storage and trafficking of neutral lipids. PMID:22180631

  7. Proteomic analysis of urine exosomes by multidimensional protein identification technology (MudPIT).

    PubMed

    Wang, Zhen; Hill, Salisha; Luther, James M; Hachey, David L; Schey, Kevin L

    2012-01-01

    Exosomes are membrane vesicles that are secreted by cells upon fusion of multivesicular bodies with the plasma membrane. Exosomal proteomics has emerged as a powerful approach to understand the molecular composition of exosomes and has potential to accelerate biomarker discovery. Different proteomic analysis methods have been previously employed to establish several exosome protein databases. In this study, TFE solution-phase digestion was compared with in-gel digestion and found to yield similar results. Proteomic analysis of urinary exosomes was performed by multidimensional protein identification technology (MudPIT) after TFE digestion. Nearly, 3280 proteins were identified from nine human urine samples with 31% overlap among nine samples. Gene ontology (GO) analysis, coupled with detection of all of the members of ESCRT machinery complex, supports the multivesicular origin of these particles. These results significantly expand the existing database of urinary exosome proteins. Our results also indicate that more than 1000 proteins can be detected from exosomes prepared from as little as 25 mL of urine. This study provides the largest set of proteins present in human urinary exosome proteomes, provides a valuable reference for future studies, and provides methods that can be applied to exosomal proteomic analysis from other tissue sources.

  8. Identification of eight point mutations in protein S deficiency type I--analysis of 15 pedigrees.

    PubMed

    Gómez, E; Poort, S R; Bertina, R M; Reitsma, P H

    1995-05-01

    We described molecular genetic studies of 15 patients with protein S deficiency type I (i.e. reduced total protein S antigen). All the exons of the PROS 1 gene were analyzed both by PCR and direct sequencing in all 15 probands. This analysis led to the identification of point mutations affecting eight individuals. One of these mutations (codon-25, insertion of T) has been described previously in a Dutch pedigree. The other mutations are novel and all are located in exons that code for the protein S domain that is homologous to the steroid hormone binding globulins. They include two amino acid replacements (one individual with 340 Gly--> Val, and two individuals with 467 Val --> Gly), and four frameshift mutations due to either one bp deletions (in codon 261 deletion of T and in codon 267 deletion of G) or insertions (in codon 565 insertion T and after codon 578 insertions of C). Studies performed in six families (totalling 43 subjects) showed cosegregation of the genetic abnormality with reduced plasma protein S levels, and provided genetic evidence for a heterozygous protein S deficiency in 25 of them. The yield of mutations in this study (53%) confirms that the percentage of protein S deficient cases in which a point mutation is found remains low.

  9. 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.

  10. 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.

  11. XSTREAM: A practical algorithm for identification and architecture modeling of tandem repeats in protein sequences

    PubMed Central

    Newman, Aaron M; Cooper, James B

    2007-01-01

    Background Biological sequence repeats arranged in tandem patterns are widespread in DNA and proteins. While many software tools have been designed to detect DNA tandem repeats (TRs), useful algorithms for identifying protein TRs with varied levels of degeneracy are still needed. Results To address limitations of current repeat identification methods, and to provide an efficient and flexible algorithm for the detection and analysis of TRs in protein sequences, we designed and implemented a new computational method called XSTREAM. Running time tests confirm the practicality of XSTREAM for analyses of multi-genome datasets. Each of the key capabilities of XSTREAM (e.g., merging, nesting, long-period detection, and TR architecture modeling) are demonstrated using anecdotal examples, and the utility of XSTREAM for identifying TR proteins was validated using data from a recently published paper. Conclusion We show that XSTREAM is a practical and valuable tool for TR detection in protein and nucleotide sequences at the multi-genome scale, and an effective tool for modeling TR domains with diverse architectures and varied levels of degeneracy. Because of these useful features, XSTREAM has significant potential for the discovery of naturally-evolved modular proteins with applications for engineering novel biostructural and biomimetic materials, and identifying new vaccine and diagnostic targets. PMID:17931424

  12. 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.

  13. Biotinylated Quercetin as an Intrinsic Photoaffinity Proteomics Probe for the Identification of Quercetin Target Proteins

    PubMed Central

    Wang, Rongsheng E.; Hunt, Clayton R.; Chen, Jiawei; Taylor, John-Stephen

    2012-01-01

    Quercetin is a flavonoid natural product that is found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying all potential targets of quercetin. We describe an O7- biotinylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing quercetin binding proteins, which can then be identified by LC MS/MS. BioQ was shown to inhibit heat induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor. BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV-irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin antibody. Analysis of the protein bands by trypsinization and LC MS/MS led to the identification of heat shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quercetin. Most of the proteins identified have also been previously suggested to be potential anticancer targets, suggesting that quercetin's antitumor activity may be due to its ability to inhibit multiple target proteins. PMID:21798748

  14. 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.

  15. An improved PSO algorithm for parameter identification of nonlinear dynamic hysteretic models

    NASA Astrophysics Data System (ADS)

    Zhang, Junhao; Xia, Pinqi

    2017-02-01

    The nonlinear dynamic hysteretic models used in nonlinear dynamic analysis contain generally lots of model parameters which need to be identified accurately and effectively. The accuracy and effectiveness of identification depend generally on the complexity of model, number of model parameters and proximity of initial values of the parameters. The particle swarm optimization (PSO) algorithm has the random searching ability and has been widely applied to the parameter identification in the nonlinear dynamic hysteretic models. However, the PSO algorithm may get trapped in the local optimum and appear the premature convergence not to obtain the real optimum results. In this paper, an improved PSO algorithm for identifying parameters of nonlinear dynamic hysteretic models has been presented by defining a fitness function for hysteretic model. The improved PSO algorithm can enhance the global searching ability and avoid to appear the premature convergence of the conventional PSO algorithm, and has been applied to identify the parameters of two nonlinear dynamic hysteretic models which are the Leishman-Beddoes (LB) dynamic stall model of rotor blade and the anelastic displacement fields (ADF) model of elastomeric damper which can be used as the lead-lag damper in rotor. The accuracy and effectiveness of the improved PSO algorithm for identifying parameters of the LB model and the ADF model are validated by comparing the identified results with test results. The investigations have indicated that in order to reduce the influence of randomness caused by using the PSO algorithm on the accuracy of identified parameters, it is an effective method to increase the number of repeated identifications.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Improved short adjacent repeat identification using three evolutionary Monte Carlo schemes.

    PubMed

    Xu, Jin; Li, Qiwei; Li, Victor O K; Li, Shuo-Yen Robert; Fan, Xiaodan

    2013-01-01

    This paper employs three Evolutionary Monte Carlo (EMC) schemes to solve the Short Adjacent Repeat Identification Problem (SARIP), which aims to identify the common repeat units shared by multiple sequences. The three EMC schemes, i.e., Random Exchange (RE), Best Exchange (BE), and crossover are implemented on a parallel platform. The simulation results show that compared with the conventional Markov Chain Monte Carlo (MCMC) algorithm, all three EMC schemes can not only shorten the computation time via speeding up the convergence but also improve the solution quality in difficult cases. Moreover, we observe that the performances of different EMC schemes depend on the degeneracy degree of the motif pattern.

  1. Leucine supplementation chronically improves muscle protein synthesis in older adults consuming the RDA for protein

    PubMed Central

    Casperson, Shanon L.; Sheffield-Moore, Melinda; Hewlings, Susan J.; Paddon-Jones, Douglas

    2013-01-01

    SUMMARY Background & aim Protein-energy supplementation is routinely employed to combat muscle loss. However, success is often compromised by increased satiety, poor palatability, high costs and low compliance. Methods For 2-weeks we supplemented meals of older individuals with leucine (4 g/meal; 3 meals/day; days 2–14). Metabolic studies were performed prior to (Day 1) and following (Day 15) supplementation. Leucine was not provided on metabolic study days. Venous blood and vastus lateralis muscle biopsies were obtained during a primed constant infusion of L-[ring-13C6] phenylalanine. Mixed muscle fractional synthesis rate (FSR), body composition and markers of nutrient signaling (mTOR, 4E-BP1 and p70S6K1 phosphorylation) were measured before and after a low protein/carbohydrate simulated meal. Results The meal modestly increased FSR on Day 1 (postabsorptive: 0.063 ± 0.004 vs. postprandial: 0.075 ± 0.006%/h; p = 0.03), however, two weeks of leucine supplementation increased postabsorptive FSR (p = 0.004) and the response to the meal (p = 0.01) (postabsorptive: 0.074 ± 0.007 vs. postprandial: 0.10 ± 0.007%/h). Changes in FSR were mirrored by increased phosphorylation of mTOR, 4E-BP1 and p70S6K1 (p ≤ 0.1). No change in fat free mass was observed (p > 0.05). Conclusions In older adults, leucine supplementation may improve muscle protein synthesis in response to lower protein meals. PMID:22357161

  2. Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae.

    PubMed

    Ferreira-Camargo, Livia S; Tran, Miller; Beld, Joris; Burkart, Michael D; Mayfield, Stephen P

    2015-12-01

    Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly, little has been done to determine which processes serve as rate-limiting steps for protein accumulation. In other expression systems, as Escherichia coli, Chinese hamster ovary cells, and insect cells, recombinant protein accumulation can be hampered by cell's inability to fold the target polypeptide into the native state, resulting in aggregation and degradation. To determine if chloroplasts' ability to oxidize proteins that require disulfide bonds into a stable conformation is a rate-limiting step of protein accumulation, three recombinant strains, each expressing a different recombinant protein, were analyzed. These recombinant proteins included fluorescent GFP, a reporter containing no disulfide bonds; Gaussia princeps luciferase, a luminescent reporter containing disulfide bonds; and an immunotoxin, an antibody-fusion protein containing disulfide bonds. Each strain was analyzed for its ability to accumulate proteins when supplemented with selenocystamine, a small molecule capable of catalyzing the formation of disulfide bonds. Selenocystamine supplementation led to an increase in luciferase and immunotoxin but not GFP accumulation. These results demonstrated that selenocystamine can increase the accumulation of proteins containing disulfide bonds and suggests that a rate-limiting step in chloroplast protein accumulation is the disulfide bonds formation in recombinant proteins native structure.

  3. Identification of protein-protein interactions of the occlusion-derived virus-associated proteins of Helicoverpa armigera nucleopolyhedrovirus.

    PubMed

    Peng, Ke; Wu, Minzhi; Deng, Fei; Song, Jingjiao; Dong, Chunsheng; Wang, Hualin; Hu, Zhihong

    2010-03-01

    The purpose of this study was to identify protein-protein interactions among the components of the occlusion-derived virus (ODV) of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), a group II alphabaculovirus in the family Baculoviridae. To achieve this, 39 selected genes of potential ODV structural proteins were cloned and expressed in the Gal4 yeast two-hybrid (Y2H) system. The direct-cross Y2H assays identified 22 interactions comprising 13 binary interactions [HA9-ODV-EC43, ODV-E56-38K, ODV-E56-PIF3, LEF3-helicase, LEF3-alkaline nuclease (AN), GP41-38K, GP41-HA90, 38K-PIF3, 38K-PIF2, VP80-HA100, ODV-E66-PIF3, ODV-E66-PIF2 and PIF3-PIF2] and nine self-associations (IE1, HA44, LEF3, HA66, GP41, CG30, 38K, PIF3 and P24). Five of these interactions - LEF3-helicase and LEF3-AN, and the self-associations of IE1, LEF3 and 38K - have been reported previously in Autographa californica multiple nucleopolyhedrovirus. As HA44 and HA100 were two newly identified ODV proteins of group II viruses, their interactions were further confirmed. The self-association of HA44 was verified with a His pull-down assay and the interaction of VP80-HA100 was confirmed by a co-immunoprecipitation assay. A summary of the protein-protein interactions of baculoviruses reported so far, comprising 68 interactions with 45 viral proteins and five host proteins, is presented, which will facilitate our understanding of the molecular mechanisms of baculovirus infection.

  4. 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

  5. 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.

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

    PubMed

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

    2004-12-01

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

  7. Identification of in vivo-induced bacterial protein antigens during calf infection with Chlamydia psittaci.

    PubMed

    Kästner, Julia; Saluz, Hans Peter; Hänel, Frank

    2015-05-01

    Chlamydia (C.) psittaci, the causative agent of ornithosis, is an obligate intracellular pathogen with a unique developmental cycle and a high potential for zoonotic transmission. Various mammalian hosts, such as cattle, horse, sheep and man that are in close contact with contaminated birds can get infected (referred to as psittacosis). Since little is known about long-term sequelae of chronic disease and the molecular mechanisms of chlamydial pathogenesis, a key step in understanding the in vivo situation is the identification of C. psittaci infection-associated proteins. For this, we investigated sera of infected calves. Using the immunoscreening approach In Vivo Induced Antigen Technology (IVIAT) including all relevant controls, we focused on C. psittaci proteins, which are induced in vivo during infection. Sera were pooled, extensively adsorbed against in vitro antigens to eliminate false positive results, and used to screen an inducible C. psittaci 02DC15 genomic expression library. Screening and control experiments revealed 19 immunogenic proteins, which are expressed during infection. They are involved in transport and oxidative stress response, heme and folate biosynthesis, DNA replication, recombination and repair, cell envelope, bacterial secretion systems and hypothetical proteins of so far unknown functions. Some of the proteins found may be considered as diagnostic markers or as candidates for the development of vaccines.

  8. Experience with a mouse intranasal test for the predictive identification of respiratory sensitization potential of proteins.

    PubMed

    Blaikie, L; Basketter, D A

    1999-08-01

    The predictive identification of respiratory allergenic potential is an important primary step in the safety evaluation of (novel) proteins, such as the enzymes used in a range of consumer laundry products. In the past this has been achieved by assessing the relative ability of proteins to give rise to the formation of anaphylactic antibody in the guinea pig. Recently, an alternative model has been proposed which assesses the formation of specific IgG1 antibody in a mouse intranasal test (MINT), the assumption being that specific IgG1 antibody is a surrogate for anaphylactic antibody in the mouse. This procedure has undergone successful initial intralaboratory and interlaboratory assessment. In the present work, the MINT has been evaluated in a more thorough intralaboratory study using eight enzymes plus ovalbumin. While the data generated with a reference enzyme protein, Alcalase, showed good reproducibility, results with the remaining eight proteins led to estimates of their relative antigenic or sensitization potential several of which were at variance from those derived from the guinea pig/ human experience. In consequence, it is concluded that the MINT requires substantial further investigation before it can be adopted as a model for the assessment of the relative ability of proteins to behave as respiratory allergens.

  9. 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.

  10. Identification of proteins associated with ion homeostasis and salt tolerance in barley.

    PubMed

    Wu, Dezhi; Shen, Qiufang; Qiu, Long; Han, Yong; Ye, Linzheng; Jabeen, Zahra; Shu, Qingyao; Zhang, Guoping

    2014-06-01

    Identification and characterization of proteins involved in salt tolerance are imperative for revealing its genetic mechanisms. In this study, ionic and proteomic responses of a Tibetan wild barley XZ16 and a well-known salt-tolerant barley cv. CM72 were analyzed using inductively coupled plasma-optical emission spectrometer, 2DE, and MALDI-TOF/TOF MS techniques to determine salt-induced differences in element and protein profiles between the two genotypes. In total, 41 differentially expressed proteins were identified in roots and leaves, and they were associated with ion homeostasis, cell redox homeostasis, metabolic process, and photosynthesis. Under salinity stress, calmodulin, Na/K transporters, and H(+) -ATPases were involved in establishment of ion homeostasis for barley plants. Moreover, ribulose-1,5-bisphosphate carboxylase/oxygenase activase and oxygen-evolving enhancer proteins were significantly upregulated under salinity stress, indicating the great impact of salinity on photosynthesis. In comparison with CM72, XZ16 had greater relative dry weight and lower Na accumulation in the shoots under salinity stress. A higher expression of HvNHX1 in the roots, and some specific proteins responsible for ion homeostasis and cell redox homeostasis, was also found in XZ16 exposed to salt stress. The current results showed that Tibetan wild barley XZ16 and cultivated barley cultivar CM72 differ in the mechanism of salt tolerance.

  11. 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.

  12. 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

  13. Improved pulse transit time estimation by system identification analysis of proximal and distal arterial waveforms.

    PubMed

    Xu, Da; Ryan, Kathy L; Rickards, Caroline A; Zhang, Guanqun; Convertino, Victor A; Mukkamala, Ramakrishna

    2011-10-01

    We investigated the system identification approach for potentially improved estimation of pulse transit time (PTT), a popular arterial stiffness marker. In this approach, proximal and distal arterial waveforms are measured and respectively regarded as the input and output of a system. Next, the system impulse response is identified from all samples of the measured input and output. Finally, the time delay of the impulse response is detected as the PTT estimate. Unlike conventional foot-to-foot detection techniques, this approach is designed to provide an artifact robust estimate of the true PTT in the absence of wave reflection. The approach is also applicable to arbitrary types of arterial waveforms. We specifically applied a parametric system identification technique to noninvasive impedance cardiography (ICG) and peripheral arterial blood pressure waveforms from 15 humans subjected to lower-body negative pressure. We assessed the technique through the correlation coefficient (r) between its 1/PTT estimates and measured diastolic pressure (DP) per subject and the root mean squared error (RMSE) of the DP predicted from these estimates and measured DP. The technique achieved average r and RMSE values of 0.81 ± 0.16 and 4.3 ± 1.3 mmHg. For comparison, the corresponding values were 0.59 ± 0.37 (P < 0.05) and 5.9 ± 2.5 (P < 0.01) mmHg for the conventional technique applied to the same waveforms and 0.28 ± 0.40 (P < 0.001) and 7.2 ± 1.8 (P < 0.001) mmHg for the conventional technique with the ECG waveform substituted for the ICG waveform. These results demonstrate, perhaps for the first time, that the system identification approach can indeed improve PTT estimation.

  14. The utility of including pathology reports in improving the computational identification of patients

    PubMed Central

    Chen, Wei; Huang, Yungui; Boyle, Brendan; Lin, Simon

    2016-01-01

    Background: Celiac disease (CD) is a common autoimmune disorder. Efficient identification of patients may improve chronic management of the disease. Prior studies have shown searching International Classification of Diseases-9 (ICD-9) codes alone is inaccurate for identifying patients with CD. In this study, we developed automated classification algorithms leveraging pathology reports and other clinical data in Electronic Health Records (EHRs) to refine the subset population preselected using ICD-9 code (579.0). Materials and Methods: EHRs were searched for established ICD-9 code (579.0) suggesting CD, based on which an initial identification of cases was obtained. In addition, laboratory results for tissue transglutaminse were extracted. Using natural language processing we analyzed pathology reports from upper endoscopy. Twelve machine learning classifiers using different combinations of variables related to ICD-9 CD status, laboratory result status, and pathology reports were experimented to find the best possible CD classifier. Ten-fold cross-validation was used to assess the results. Results: A total of 1498 patient records were used including 363 confirmed cases and 1135 false positive cases that served as controls. Logistic model based on both clinical and pathology report features produced the best results: Kappa of 0.78, F1 of 0.92, and area under the curve (AUC) of 0.94, whereas in contrast using ICD-9 only generated poor results: Kappa of 0.28, F1 of 0.75, and AUC of 0.63. Conclusion: Our automated classification system presented an efficient and reliable way to improve the performance of CD patient identification. PMID:27994938

  15. 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

  16. 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

  17. 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.

  18. 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 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.

  19. 3D pharmacophoric similarity improves multi adverse drug event identification in pharmacovigilance.

    PubMed

    Vilar, Santiago; Tatonetti, Nicholas P; Hripcsak, George

    2015-03-06

    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.

  20. Incorporating conditional random fields and active learning to improve sentiment identification.

    PubMed

    Zhang, Kunpeng; Xie, Yusheng; Yang, Yi; Sun, Aaron; Liu, Hengchang; Choudhary, Alok

    2014-10-01

    Many machine learning, statistical, and computational linguistic methods have been developed to identify sentiment of sentences in documents, yielding promising results. However, most of state-of-the-art methods focus on individual sentences and ignore the impact of context on the meaning of a sentence. In this paper, we propose a method based on conditional random fields to incorporate sentence structure and context information in addition to syntactic information for improving sentiment identification. We also investigate how human interaction affects the accuracy of sentiment labeling using limited training data. We propose and evaluate two different active learning strategies for labeling sentiment data. Our experiments with the proposed approach demonstrate a 5%-15% improvement in accuracy on Amazon customer reviews compared to existing supervised learning and rule-based methods.

  1. Protein sequence classification with improved extreme learning machine algorithms.

    PubMed

    Cao, Jiuwen; Xiong, Lianglin

    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.

  2. Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

  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. 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

  5. Identification of protein interaction antagonists using the repressed transactivator two-hybrid system.

    PubMed

    Joshi, Phalgun B; Hirst, Martin; Malcolm, Tom; Parent, Jennifer; Mitchell, David; Lund, Karen; Sadowski, Ivan

    2007-05-01

    The repressed transactivator (RTA) yeast two-hybrid system was developed to enable genetic identification of interactions with transcriptional activator proteins. We have devised modifications of this system that enable its use in screening for inhibitors of protein interactions from small molecule compound libraries. We show that inhibition of protein interactions can be measured by monitoring growth in selective medium containing 3-aminotriazole (3-AT) and using this assay have identified inhibitors of four independent protein interactions in screens with a 23,000 small molecule compound library. Compounds found to inhibit one of the tested interactions between FKBP12 and the transforming growth factor beta receptor (TGFbeta-R) were validated in vivo and found to inhibit calcineurin-dependent signaling in T cells. One of these compounds was also found to cause elevated basal expression of a TGFbeta-R/SMAD-dependent reporter gene. These results demonstrate the capability of the RTA small molecule screening assay for discovery of potentially novel therapeutic compounds.

  6. 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.

  7. 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).

  8. 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.

  9. Identification of cooperative folding units in a set of native proteins.

    PubMed Central

    Wallqvist, A.; Smythers, G. W.; Covell, D. G.

    1997-01-01

    Cooperative unfolding penalties are calculated by statistically evaluating an ensemble of denatured states derived from native structures. The ensemble of denatured states is determined by dividing the native protein into short contiguous segments and defining all possible combinations of native, i.e., interacting, and non-native, i.e., non-interacting, segments. We use a novel knowledge-based scoring function, derived from a set of non-homologous proteins in the Protein Data Bank, to describe the interactions among residues. This procedure is used for the structural identification of cooperative folding cores for four globular proteins: bovine pancreatic trypsin inhibitor, horse heart cytochrome c, French bean plastocyanin, and staphylococcal nuclease. The theoretical folding units are shown to correspond to regions that exhibit enhanced stability against denaturation as determined from experimental hydrogen exchange protection factors. Using a sequence similarity score for related sequences, we show that, in addition to residues necessary for enzymatic function, those amino acids comprising structurally important folding cores are also preferentially conserved during evolution. This implies that the identified folding cores may be part of an array of fundamental structural folding units. PMID:9260276

  10. A Comprehensive Approach to Identification of Surface-Exposed, Outer Membrane-Spanning Proteins of Leptospira interrogans

    PubMed Central

    Pinne, Marija; Haake, David A.

    2009-01-01

    Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via fresh water and colonization of the renal tubules of their reservoir hosts or infection of accidental hosts, including humans. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in virulence mechanisms of pathogens and the adaptation to various environmental conditions, including those of the mammalian host. Little is known about the surface-exposed OMPs in Leptospira, particularly those with outer membrane-spanning domains. Herein, we describe a comprehensive strategy for identification and characterization of leptospiral transmembrane OMPs. The genomic sequence of L. interrogans serovar Copenhageni strain Fiocruz L1–130 allowed us to employ the β-barrel prediction programs, PRED-TMBB and TMBETA-NET, to identify potential transmembrane OMPs. Several complementary methods were used to characterize four novel OMPs, designated OmpL36, OmpL37, OmpL47 and OmpL54. In addition to surface immunofluorescence and surface biotinylation, we describe surface proteolysis of intact leptospires as an improved method for determining the surface exposure of leptospiral proteins. Membrane integration was confirmed using techniques for removal of peripheral membrane proteins. We also demonstrate deficiencies in the Triton X-114 fractionation method for assessing the outer membrane localization of transmembrane OMPs. Our results establish a broadly applicable strategy for the elucidation of novel surface-exposed outer membrane-spanning proteins of Leptospira, an essential step in the discovery of potential virulence factors, diagnostic antigens and vaccine candidates. PMID:19562037

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

    PubMed

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

    2014-02-01

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

  12. Protein identification from dried nipple aspirate fluid on Guthrie cards using mass spectrometry

    PubMed Central

    DELMONICO, LUCAS; AREIAS, VIVIAN RABELLO; PINTO, RODRIGO CÉSAR; MATOS, CINTIA DA SILVA; ROSA, MARCO FELIPE FRANCO; DE AZEVEDO, CAROLINA MARIA; ALVES, GILDA

    2015-01-01

    Nipple aspirate fluid (NAF) requires investigation as a potential source of biomarkers for early diagnosis or risk assessment in breast cancer and other breast disorders. The present study demonstrated that proteins were easily extracted from dried NAF spots on Guthrie cards and were suitable for mass spectrometry analysis. NAF was obtained from 80 women, collected on Guthrie cards, between 2007 and 2010. The NAF-proteins were extracted from the card by incubating the card in water. These proteins were then quantified and separated using one-dimensional, 12% SDS-PAGE, gel electrophoresis and on high-resolution gradient gels at different concentrations (4–12, 8–16 and 4–20%). The bands with the most abundant proteins were excised from the gradient gels and the proteins were identified by liquid chromatography quadrupole time of flight. Immunoglobulins, Zn-α2-glicoprotein, apoliprotein D and prolactin inducible protein were among those identified. The NAF-Guthrie card collection method has not been applied previously, however, NAF proteins have been identified using other collecting techniques, confirming the feasibility of the NAF Guthrie card collection method for analyzing the proteins within NAF. The NAF-Guthrie card collecting method has multiple advantages, including being inexpensive, non-invasive, reliable and painless, and the cards can be stored at room temperature. Examining NAF may assist in identifying individuals at a higher risk of breast cancer and in improving patient prognosis. PMID:25760982

  13. Shape of Key Malaria Protein Could Help Improve Vaccine Efficacy

    MedlinePlus

    ... new findings show the importance of understanding the biology behind this conformational change, possibly due to a ... Nucleotide Polymorphism Phylogenetics & Ontology Proteomics & Protein Analysis Systems Biology Data Portals Software Applications BCBB Mobyle Interface Designer ( ...

  14. Beyond Grand Rounds: A Comprehensive and Sequential Intervention to Improve Identification of Delirium

    PubMed Central

    Ramaswamy, Ravishankar; Dix, Edward F.; Drew, Janet E.; Diamond, James J.; Inouye, Sharon K.; Roehl, Barbara J. O.

    2011-01-01

    Purpose of the Study: Delirium is a widespread concern for hospitalized seniors, yet is often unrecognized. A comprehensive and sequential intervention (CSI) aiming to effect change in clinician behavior by improving knowledge about delirium was tested. Design and Methods: A 2-day CSI program that consisted of progressive 4-part didactic series, including evidence-based reviews of delirium recognition, prevention, and management, interspersed with interactive small group sessions and practical case conferences was conceptualized in consultation with a leading expert on delirium. Pretest and posttest instruments were designed to test the attendees on their knowledge and confidence around delirium identification. Results: An average of 71 people attended each didactic session. Among all responses, 50 pretests and posttests were matched based on numeric coding (6 MD/DOs, 34 RNs, and 10 others). Mean pretest and posttest scores were 7.9 and 10.8 points, respectively (maximum: 17), showing a positive change in knowledge scores after the intervention (2.9 points, p < .001). Improvement in knowledge scores was higher in the cohort attending 2 or more lectures (3.8 points, p < .001) compared with those attending only 1 lecture (1.3 points, p < .12). Confidence in identifying patients with delirium increased by 28% (p < .001), and self-assessed capacity to correctly administer the Confusion Assessment Method increased by 36% (p < .001). Implications: A novel CSI increased clinician knowledge about delirium identification and management and improved confidence and self-assessed capacity to identify delirium in the hospitalized elderly patients. This strategy, which incorporates multiple reinforcing modes of education, may ultimately be more effective in influencing clinician behavior when compared with traditional grand rounds. PMID:20855818

  15. 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

  16. Improvement of antibody immobilization using hyperbranched polymer and protein A.

    PubMed

    Shen, Guangyu; Cai, Chenbo; Wang, Kun; Lu, Jilin

    2011-02-01

    For the construction of a well-defined antibody surface, protein A was used as a binding material to immobilize antibodies onto gold-derivatized transducers. The traditional method tends to assemble protein A directly onto the gold-derivatized transducers. In this paper, we tried to indirectly bind protein A onto sensors through hyperbranched polymer (HBP) which was synthesized from p-phenylenediamine and trimesic acid. The three-dimensional structure of HBP and the characteristics including orientation control and biocompatibility of protein A led to highly efficient immunoreactions and enhanced detection system performance. With this strategy, cysteamine monolayer was first assembled onto Au electrodes associated with the piezoelectric quartz crystal; secondly, the cysteamine-modified gold electrode was further modified by the activated HBP; thirdly, protein A was immobilized onto the HBP film; and finally, antibodies were immobilized onto the surface of protein A film for detecting the corresponding antigen. The quartz crystal microbalance immunosensor thus fabricated was applied to detect hepatitis B surface antigen in solutions that ranged from 0.71 to 300 μg mL(-1). The detection limit was estimated to be 0.53 μg mL(-1). The immunosensor holds good selectivity, sensitivity, and repeatability.

  17. Improvement of the technique of identification of electrons and positrons with use of electromagnetic calorimeter of the CLAS detector

    SciTech Connect

    Gevorgyan, N. E.; Dashyan, N. B.; Paremuzyan, R. G.; Stepanyan, S. G.

    2010-01-01

    We study the dependence of the sensitivity of response of the electromagnetic calorimeter of CLAS plant on the momenta of electrons and positrons. We made calculation of this dependence and elaborated a method for its employment in identification of e- and e+. We have shown that the new method of selection of e- and e+ improves the quality of identification by about 10%. We used the experimental data obtained with the plant CLAS of linear accelerator at Jefferson laboratory (USA).

  18. Utilization of advanced clutter suppression algorithms for improved standoff detection and identification of radionuclide threats

    NASA Astrophysics Data System (ADS)

    Cosofret, Bogdan R.; Shokhirev, Kirill; Mulhall, Phil; Payne, David; Harris, Bernard

    2014-05-01

    Technology development efforts seek to increase the capability of detection systems in low Signal-to-Noise regimes encountered in both portal and urban detection applications. We have recently demonstrated significant performance enhancement in existing Advanced Spectroscopic Portals (ASP), Standoff Radiation Detection Systems (SORDS) and handheld isotope identifiers through the use of new advanced detection and identification algorithms. The Poisson Clutter Split (PCS) algorithm is a novel approach for radiological background estimation that improves the detection and discrimination capability of medium resolution detectors. The algorithm processes energy spectra and performs clutter suppression, yielding de-noised gamma-ray spectra that enable significant enhancements in detection and identification of low activity threats with spectral target recognition algorithms. The performance is achievable at the short integration times (0.5 - 1 second) necessary for operation in a high throughput and dynamic environment. PCS has been integrated with ASP, SORDS and RIID units and evaluated in field trials. We present a quantitative analysis of algorithm performance against data collected by a range of systems in several cluttered environments (urban and containerized) with embedded check sources. We show that the algorithm achieves a high probability of detection/identification with low false alarm rates under low SNR regimes. For example, utilizing only 4 out of 12 NaI detectors currently available within an ASP unit, PCS processing demonstrated Pd,ID > 90% at a CFAR (Constant False Alarm Rate) of 1 in 1000 occupancies against weak activity (7 - 8μCi) and shielded sources traveling through the portal at 30 mph. This vehicle speed is a factor of 6 higher than was previously possible and results in significant increase in system throughput and overall performance.

  19. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to multidimensional protein identification technology.

    PubMed

    Gonzalez-Begne, Mireya; Lu, Bingwen; Liao, Lujian; Xu, Tao; Bedi, Gurrinder; Melvin, James E; Yates, John R

    2011-11-04

    In-depth analysis of the salivary proteome is fundamental to understanding the functions of salivary proteins in the oral cavity and to reveal disease biomarkers involved in different pathophysiological conditions, with the ultimate goal of improving patient diagnosis and prognosis. Submandibular and sublingual glands contribute saliva rich in glycoproteins to the total saliva output, making them valuable sources for glycoproteomic analysis. Lectin-affinity chromatography coupled to mass spectrometry-based shotgun proteomics was used to explore the submandibular/sublingual (SM/SL) saliva glycoproteome. A total of 262 N- and O-linked glycoproteins were identified by multidimensional protein identification technology (MudPIT). Only 38 were previously described in SM and SL salivas from the human salivary N-linked glycoproteome, while 224 were unique. Further comparison analysis with SM/SL saliva of the human saliva proteome, revealed 125 glycoproteins not formerly reported in this secretion. KEGG pathway analyses demonstrated that many of these glycoproteins are involved in processes such as complement and coagulation cascades, cell communication, glycosphingolipid biosynthesis neo-lactoseries, O-glycan biosynthesis, glycan structures-biosynthesis 2, starch and sucrose metabolism, peptidoglycan biosynthesis or others pathways. In summary, lectin-affinity chromatography coupled to MudPIT mass spectrometry identified many novel glycoproteins in SM/SL saliva. These new additions to the salivary proteome may prove to be a critical step for providing reliable biomarkers in the diagnosis of a myriad of oral and systemic diseases.

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

    PubMed

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

    2017-03-01

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

  1. Identification and characterization of functional homologs of nitrogenase cofactor biosynthesis protein NifB from methanogens

    PubMed Central

    Fay, Aaron W.; Wiig, Jared A.; Lee, Chi Chung; Hu, Yilin

    2015-01-01

    Nitrogenase biosynthesis protein NifB catalyzes the radical S-adenosyl-L-methionine (SAM)-dependent insertion of carbide into the M cluster, the cofactor of the molybdenum nitrogenase from Azotobacter vinelandii. Here, we report the identification and characterization of two naturally “truncated” homologs of NifB from Methanosarcina acetivorans (NifBMa) and Methanobacterium thermoautotrophicum (NifBMt), which contain a SAM-binding domain at the N terminus but lack a domain toward the C terminus that shares homology with NifX, an accessory protein in M cluster biosynthesis. NifBMa and NifBMt are monomeric proteins containing a SAM-binding [Fe4S4] cluster (designated the SAM cluster) and a [Fe4S4]-like cluster pair (designated the K cluster) that can be processed into an [Fe8S9] precursor to the M cluster (designated the L cluster). Further, the K clusters in NifBMa and NifBMt can be converted to L clusters upon addition of SAM, which corresponds to their ability to heterologously donate L clusters to the biosynthetic machinery of A. vinelandii for further maturation into the M clusters. Perhaps even more excitingly, NifBMa and NifBMt can catalyze the removal of methyl group from SAM and the abstraction of hydrogen from this methyl group by 5′-deoxyadenosyl radical that initiates the radical-based incorporation of methyl-derived carbide into the M cluster. The successful identification of NifBMa and NifBMt as functional homologs of NifB not only enabled classification of a new subset of radical SAM methyltransferases that specialize in complex metallocluster assembly, but also provided a new tool for further characterization of the distinctive, NifB-catalyzed methyl transfer and conversion to an iron-bound carbide. PMID:26627238

  2. Application of proteomics to investigate stress-induced proteins for improvement in crop protection.

    PubMed

    Afroz, Amber; Ali, Ghulam Muhammad; Mir, Asif; Komatsu, Setsuko

    2011-05-01

    Proteomics has contributed to defining the specific functions of genes and proteins involved in plant-pathogen interactions. Proteomic studies have led to the identification of many pathogenicity and defense-related genes and proteins expressed during phytopathogen infections, resulting in the collection of an enormous amount of data. However, the molecular basis of plant-pathogen interactions remains an intensely active area of investigation. In this review, the role of differential analysis of proteins expressed during fungal, bacterial, and viral infection is discussed, as well as the role of JA and SA in the production of stress related proteins. Resistance acquired upon induction of stress related proteins in intact plant leaves is mediated by potentiation of pathogens via signal elicitors. Stress related genes extensively used in biotechnology had been cited. Stress related proteins identified must be followed through for studying the molecular mechanism for plant defense against pathogens.

  3. Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer*S⃞

    PubMed Central

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

    2009-01-01

    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 are associated with tamoxifen resistance is a first step toward 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 nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on ∼5,500 pooled tumor cells (corresponding to ∼550 ng of 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 reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with ≥2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy

  4. Hydrolysis by Alcalase Improves Hypoallergenic Properties of Goat Milk Protein

    PubMed Central

    Yun, Sung-Seob; Lee, Won-Jae; Kim, Jin-Wook; Ha, Ho-Kyung; Yoo, Michelle

    2016-01-01

    Goat milk is highly nutritious and is consumed in many countries, but the development of functional foods from goat milk has been slow compared to that for other types of milk. The aim of this study was to develop a goat milk protein hydrolysate (GMPH) with enhanced digestibility and better hypoallergenic properties in comparison with other protein sources such as ovalbumin and soy protein. Goat milk protein was digested with four commercial food-grade proteases (separately) under various conditions to achieve the best hydrolysis of αs -casein and β-lactoglobulin. It was shown that treatment with alcalase (0.4%, 60℃ for 30 min) effectively degraded these two proteins, as determined by SDS-PAGE, measurement of nonprotein nitrogen content, and reverse-phase high-performance liquid chromatography. Hydrolysis with alcalase resulted in a significant decrease in β-lactoglobulin concentration (almost to nil) and a ~40% reduction in the level of αs-casein. Quantification of histamine and TNF-α released from HMC-1 cells (human mast cell line) showed that the GMPH did not induce an allergic response when compared to the control. Hence, the GMPH may be useful for development of novel foods for infants, the elderly, and convalescent patients, to replace cow milk. PMID:27621693

  5. Identification of chromosome regions controlling seed storage proteins of narrow-leafed lupin (Lupinus angustifolius).

    PubMed

    Li, Xin; Islam, Shahidul; Yang, Huaan; Ma, Wujun; Yan, Guijun

    2013-05-01

    Narrow-leafed lupin (Lupinus angustifolius L.) is a valuable legume crop for animal feed and human health food because of its high proteins content. However, the genetics of seed storage proteins is unclear, limiting further improvement of protein quantity and quality. In this study, matrix-assisted laser desorption/ionization time of flight mass spectrometry was used for the first time to analyze lupin seed storage proteins and the spectra generated was treated as markers to investigate the chromosome locations controlling seed storage proteins in the narrow-leafed lupin. In a recombinant inbred line population of 89 individuals, 48 polymorphic protein peaks were identified and seven of which were successfully mapped onto four existing linkage groups: two on NLL-04, three on NLL-05, one on NLL-07 and one on NLL-14, with LOD values ranging from 2.6 to 7.7 confirming a significant linkage. Most protein-based markers showed distorted segregation and were failed to be integrated into the reference map. Among them, 31 were grouped into six clusters and the other ten were totally unlinked. This study provides a significant clue to study the comparative genomics/proteomics among legumes as well as for protein marker-assisted breeding. The distribution pattern of genes controlling seed storage protein revealed in this study probably exists universally among legumes or even all plants and animals. Whether genes controlling seed storage protein share the same gene expression pattern controlling other enzymes and what is the mechanism behind it are the questions which remain to be answered in the future.

  6. Large-scale identification of human protein function using topological features of interaction network

    PubMed Central

    Li, Zhanchao; Liu, Zhiqing; Zhong, Wenqian; Huang, Menghua; Wu, Na; Xie, Yun; Dai, Zong; Zou, Xiaoyong

    2016-01-01

    The annotation of protein function is a vital step to elucidate the essence of life at a molecular level, and it is also meritorious in biomedical and pharmaceutical industry. Developments of sequencing technology result in constant expansion of the gap between the number of the known sequences and their functions. Therefore, it is indispensable to develop a computational method for the annotation of protein function. Herein, a novel method is proposed to identify protein function based on the weighted human protein-protein interaction network and graph theory. The network topology features with local and global information are presented to characterise proteins. The minimum redundancy maximum relevance algorithm is used to select 227 optimized feature subsets and support vector machine technique is utilized to build the prediction models. The performance of current method is assessed through 10-fold cross-validation test, and the range of accuracies is from 67.63% to 100%. Comparing with other annotation methods, the proposed way possesses a 50% improvement in the predictive accuracy. Generally, such network topology features provide insights into the relationship between protein functions and network architectures. The source code of Matlab is freely available on request from the authors. PMID:27849060

  7. Large-scale identification of human protein function using topological features of interaction network

    NASA Astrophysics Data System (ADS)

    Li, Zhanchao; Liu, Zhiqing; Zhong, Wenqian; Huang, Menghua; Wu, Na; Xie, Yun; Dai, Zong; Zou, Xiaoyong

    2016-11-01

    The annotation of protein function is a vital step to elucidate the essence of life at a molecular level, and it is also meritorious in biomedical and pharmaceutical industry. Developments of sequencing technology result in constant expansion of the gap between the number of the known sequences and their functions. Therefore, it is indispensable to develop a computational method for the annotation of protein function. Herein, a novel method is proposed to identify protein function based on the weighted human protein-protein interaction network and graph theory. The network topology features with local and global information are presented to characterise proteins. The minimum redundancy maximum relevance algorithm is used to select 227 optimized feature subsets and support vector machine technique is utilized to build the prediction models. The performance of current method is assessed through 10-fold cross-validation test, and the range of accuracies is from 67.63% to 100%. Comparing with other annotation methods, the proposed way possesses a 50% improvement in the predictive accuracy. Generally, such network topology features provide insights into the relationship between protein functions and network architectures. The source code of Matlab is freely available on request from the authors.

  8. Improved solubility and emulsification of wet-milled corn germ protein recovered by ultrafiltration-diafiltration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study evaluated ultrafiltration-diafiltration (UFDF) as a means to improve the extractability of wet-milled corn germ protein and determined its effects on the functional properties of the recovered protein product. Wet germ (WG) and finished germ (FG) proteins (Pr) were extracted by using 0.1M...

  9. Leveraging ancestry to improve causal variant identification in exome sequencing for monogenic disorders.

    PubMed

    Brown, Robert; Lee, Hane; Eskin, Ascia; Kichaev, Gleb; Lohmueller, Kirk E; Reversade, Bruno; Nelson, Stanley F; Pasaniuc, Bogdan

    2016-01-01

    Recent breakthroughs in exome-sequencing technology have made possible the identification of many causal variants of monogenic disorders. Although extremely powerful when closely related individuals (eg, child and parents) are simultaneously sequenced, sequencing of a single case is often unsuccessful due to the large number of variants that need to be followed up for functional validation. Many approaches filter out common variants above a given frequency threshold (eg, 1%), and then prioritize the remaining variants according to their functional, structural and conservation properties. Here we present methods that leverage the genetic structure across different populations to improve filtering performance while accounting for the finite sample size of the reference panels. We show that leveraging genetic structure reduces the number of variants that need to be followed up by 16% in simulations and by up to 38% in empirical data of 20 exomes from individuals with monogenic disorders for which the causal variants are known.

  10. Use of system identification techniques for improving airframe finite element models using test data

    NASA Technical Reports Server (NTRS)

    Hanagud, Sathya V.; Zhou, Weiyu; Craig, James I.; Weston, Neil J.

    1991-01-01

    A method for using system identification techniques to improve airframe finite element models was developed and demonstrated. The method uses linear sensitivity matrices to relate changes in selected physical parameters to changes in total system matrices. The values for these physical parameters were determined using constrained optimization with singular value decomposition. The method was confirmed using both simple and complex finite element models for which pseudo-experimental data was synthesized directly from the finite element model. The method was then applied to a real airframe model which incorporated all the complexities and details of a large finite element model and for which extensive test data was available. The method was shown to work, and the differences between the identified model and the measured results were considered satisfactory.

  11. Use of system identification techniques for improving airframe finite element models using test data

    NASA Technical Reports Server (NTRS)

    Hanagud, Sathya V.; Zhou, Weiyu; Craig, James I.; Weston, Neil J.

    1993-01-01

    A method for using system identification techniques to improve airframe finite element models using test data was developed and demonstrated. The method uses linear sensitivity matrices to relate changes in selected physical parameters to changes in the total system matrices. The values for these physical parameters were determined using constrained optimization with singular value decomposition. The method was confirmed using both simple and complex finite element models for which pseudo-experimental data was synthesized directly from the finite element model. The method was then applied to a real airframe model which incorporated all of the complexities and details of a large finite element model and for which extensive test data was available. The method was shown to work, and the differences between the identified model and the measured results were considered satisfactory.

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

    PubMed Central

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

    2016-01-01

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

  13. Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands

    PubMed Central

    Ning, Fangkun; Wang, Chao; Berry, Karin Zemski; Kandasamy, Pitchaimani; Liu, Haolin; Murphy, Robert C.; Voelker, Dennis R.; Nho, Chu Won; Pan, Choel-Ho; Dai, Shaodong; Niu, Liwen; Chu, Hong-Wei; Zhang, Gongyi

    2014-01-01

    The short palate, lung and nasal epithelial clone 1 (SPLUNC1) protein is a member of the palate, lung, and nasal epithelium clone (PLUNC) family, also known as bactericidal/permeability-increasing (BPI) fold-containing protein, family A, member 1 (BPIFA1). SPLUNC1 is an abundant protein in human airways, but its function remains poorly understood. The lipid ligands of SPLUNC1 as well as other PLUNC family members are largely unknown, although some reports provide evidence that lipopolysaccharide (LPS) could be a lipid ligand. Unlike previous hypotheses, we found significant structural differences between SPLUNC1 and BPI. Recombinant SPLUNC1 produced in HEK 293 cells harbored several molecular species of sphingomyelin and phosphatidylcholine as its ligands. Significantly, in vitro lipid-binding studies failed to demonstrate interactions between SPLUNC1 and LPS, lipoteichoic acid, or polymyxin B. Instead, one of the major and most important pulmonary surfactant phospholipids, dipalmitoylphosphatidylcholine (DPPC), bound to SPLUNC1 with high affinity and specificity. We found that SPLUNC1 could be the first protein receptor for DPPC. These discoveries provide insight into the specific determinants governing the interaction between SPLUNC1 and lipids and also shed light on novel functions that SPLUNC1 and other PLUNC family members perform in host defense.—Ning, F., Wang, C., Berry, K. Z., Kandasamy, P., Liu, H., Murphy, R. C., Voelker, D. R., Nho, C. W., Pan, C.-H., Dai, S., Niu, L., Chu, H.-W., Zhang, G. Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands. PMID:25223608

  14. Identification of multiple salicylic acid-binding proteins using two high throughput screens

    PubMed Central

    Manohar, Murli; Tian, Miaoying; Moreau, Magali; Park, Sang-Wook; Choi, Hyong Woo; Fei, Zhangjun; Friso, Giulia; Asif, Muhammed; Manosalva, Patricia; von Dahl, Caroline C.; Shi, Kai; Ma, Shisong; Dinesh-Kumar, Savithramma P.; O'Doherty, Inish; Schroeder, Frank C.; van Wijk, Klass J.; Klessig, Daniel F.

    2014-01-01

    Salicylic acid (SA) is an important hormone involved in many diverse plant processes, including floral induction, stomatal closure, seed germination, adventitious root initiation, and thermogenesis. It also plays critical functions during responses to abiotic and biotic stresses. The role(s) of SA in signaling disease resistance is by far the best studied process, although it is still only partially understood. To obtain insights into how SA carries out its varied functions, particularly in activating disease resistance, two new high throughput screens were developed to identify novel SA-binding proteins (SABPs). The first utilized crosslinking of the photo-reactive SA analog 4-AzidoSA (4AzSA) to proteins in an Arabidopsis leaf extract, followed by immuno-selection with anti-SA antibodies and then mass spectroscopy-based identification. The second utilized photo-affinity crosslinking of 4AzSA to proteins on a protein microarray (PMA) followed by detection with anti-SA antibodies. To determine whether the candidate SABPs (cSABPs) obtained from these screens were true SABPs, recombinantly-produced proteins were generated and tested for SA-inhibitable crosslinking to 4AzSA, which was monitored by immuno-blot analysis, SA-inhibitable binding of the SA derivative 3-aminoethylSA (3AESA), which was detected by a surface plasmon resonance (SPR) assay, or SA-inhibitable binding of [3H]SA, which was detected by size exclusion chromatography. Based on our criteria that true SABPs must exhibit SA-binding activity in at least two of these assays, nine new SABPs are identified here; nine others were previously reported. Approximately 80 cSABPs await further assessment. In addition, the conflicting reports on whether NPR1 is an SABP were addressed by showing that it bound SA in all three of the above assays. PMID:25628632

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

    PubMed

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

    2007-09-01

    Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast-like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up-regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin-like gene. The 2138-bp full-length S. aurata osteopontin-like cDNA was shown to encode a 374 amino-acid