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Sample records for improves protein identification

  1. On slide chemical modification as a means to improve confidence in protein identifications made by peptide mass finger prints

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The identification of proteins from large protein databases such as NCBInr using peptide mass finger prints (PMFs) obtained on a MALDI-TOF mass spectrometer continues to be a challenge. A strategy that can be used to improve confidence in these identifications is to carry out some form of chemical ...

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

    PubMed

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

    2008-08-01

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

  3. 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.

  4. Improved protein identification using automated high mass measurement accuracy MALDI FT-ICR MS peptide mass fingerprinting

    NASA Astrophysics Data System (ADS)

    Horn, David M.; Peters, Eric C.; Klock, Heath; Meyers, Andrew; Brock, Ansgar

    2004-11-01

    A comparison between automated peptide mass fingerprinting systems using MALDI-TOF and MALDI FT-ICR MS is presented using 86 overexpressed proteins from Thermotoga maritima. The high mass measurement accuracy of FT-ICR MS greatly reduces the probability of an incorrect assignment of a protein in peptide mass fingerprinting by significantly decreasing the score and peptide sequence coverage of the highest ranked random protein match from the database. This improved mass accuracy led to the identification of all 86 proteins with the FT-ICR data versus 84 proteins using the TOF data against the T. maritima database. The beneficial effect of mass accuracy becomes much more evident with the addition of variable modifications and an increase in the size of the database used in the search. A search of the same data against the T. maritima database with the addition of a variable modification resulted in 77 identifications using MALDI-TOF and 84 identifications using MALDI FT-ICR MS. When searching the NCBInr database, the FT-ICR based system identified 82 of 86 proteins while the TOF based system could only identify 73. The MALDI FT-ICR based system has the further advantage of producing fewer unassigned masses in each peptide mass fingerprint, resulting in greatly reduced sequence coverage and score for the highest ranked random match and improving confidence in the correctly assigned top scoring protein. Finally, the use of rms error as a measure for instrumental mass accuracy is discussed.

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

    PubMed

    Marshall, Skye

    2016-04-01

    Methods of identifying malnutrition in the rehabilitation setting require further examination so that patient outcomes may be improved. The purpose of this narrative review was to: (1) examine the defining characteristics of malnutrition, starvation, sarcopenia and cachexia; (2) review the validity of nutrition screening tools and nutrition assessment tools in the rehabilitation setting; and (3) determine the prevalence of malnutrition in the rehabilitation setting by geographical region and method of diagnosis. A narrative review was conducted drawing upon international literature. Starvation represents one form of malnutrition. Inadequate energy and protein intake are the critical factor in the aetiology of malnutrition, which is distinct from sarcopenia and cachexia. Eight nutrition screening tools and two nutrition assessment tools have been evaluated for criterion validity in the rehabilitation setting, and consideration must be given to the resources of the facility and the patient group in order to select the appropriate tool. The prevalence of malnutrition in the rehabilitation setting ranges from 14-65% worldwide with the highest prevalence reported in rural, European and Australian settings. Malnutrition is highly prevalent in the rehabilitation setting, and consideration must be given to the patient group when determining the most appropriate method of identification so that resources may be used efficaciously and the chance of misdiagnosis minimised. PMID:26921933

  6. RBRIdent: An algorithm for improved identification of RNA-binding residues in proteins from primary sequences.

    PubMed

    Xiong, Dapeng; Zeng, Jianyang; Gong, Haipeng

    2015-06-01

    Rapid and correct identification of RNA-binding residues based on the protein primary sequences is of great importance. In most prevalent machine-learning-based identification methods; however, either some features are inefficiently represented, or the redundancy between features is not effectively removed. Both problems may weaken the performance of a classifier system and raise its computational complexity. Here, we addressed the above problems and developed a better classifier (RBRIdent) to identify the RNA-binding residues. In an independent benchmark test, RBRIdent achieved an accuracy of 76.79%, Matthews correlation coefficient of 0.3819 and F-measure of 75.58%, remarkably outperforming all prevalent methods. These results suggest the necessity of proper feature description and the essential role of feature selection in this project. All source data and codes are freely available at http://166.111.152.91/RBRIdent. PMID:25846271

  7. 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

  8. Susceptibility to denaturation of caseins in milk samples for improving protein conformational study and their identification.

    PubMed

    Pepe, Giacomo; Tenore, Gian Carlo; Conte, Carmine; Russo, Mariateresa; Manfra, Michele; Campiglia, Pietro

    2013-01-01

    Caseins are phosphoproteins kept together by non-covalent interactions to form a highly stabilised dispersion in milk. This study intended to evaluate the different effects of denaturing solvents and solutions on caseins in order to optimise the chromatographic resolution for a better identification of individual casein fractions. The caseins were obtained from bovine skimmed milks by precipitation at pH 4.3, and the proteins were dissolved in water and three different solutions. The casein separation was performed by reversed-phase high-performance liquid chromatography. Each casein was identified by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry. The best result was achieved by treating the caseins with solution D. PMID:22978643

  9. Enhancements to the Rosetta Energy Function Enable Improved Identification of Small Molecules that Inhibit Protein-Protein Interactions

    PubMed Central

    Karanicolas, John

    2015-01-01

    Protein-protein interactions are among today’s most exciting and promising targets for therapeutic intervention. To date, identifying small-molecules that selectively disrupt these interactions has proven particularly challenging for virtual screening tools, since these have typically been optimized to perform well on more “traditional” drug discovery targets. Here, we test the performance of the Rosetta energy function for identifying compounds that inhibit protein interactions, when these active compounds have been hidden amongst pools of “decoys.” Through this virtual screening benchmark, we gauge the effect of two recent enhancements to the functional form of the Rosetta energy function: the new “Talaris” update and the “pwSHO” solvation model. Finally, we conclude by developing and validating a new weight set that maximizes Rosetta’s ability to pick out the active compounds in this test set. Looking collectively over the course of these enhancements, we find a marked improvement in Rosetta’s ability to identify small-molecule inhibitors of protein-protein interactions. PMID:26484863

  10. RT-SVR+q: A Strategy for Post-Mascot Analysis Using Retention Time and q Value Metric to Improve Peptide and Protein Identifications

    PubMed Central

    Cao, Weifeng; Ma, Di; Kapur, Arvinder; Patankar, Manish S; Ma, Yadi; Li, Lingjun

    2011-01-01

    Shotgun proteomics commonly utilizes database search like Mascot to identify proteins from tandem MS/MS spectra. False discovery rate (FDR) is often used to assess the confidence of peptide identifications. However, a widely accepted FDR of 1% sacrifices the sensitivity of peptide identification while improving the accuracy. This article details a machine learning approach combining retention time based support vector regressor (RT-SVR) with q value based statistical analysis to improve peptide and protein identifications with high sensitivity and accuracy. The use of confident peptide identifications as training examples and careful feature selection ensures high R values (>0.900) for all models. The application of RT-SVR model on Mascot results (p=0.10) increases the sensitivity of peptide identifications. q value, as a function of deviation between predicted and experimental RTs(Δ RT), is used to assess the significance of peptide identifications. We demonstrate that the peptide and protein identifications increase by up to 89.4% and 83.5%, respectively, for a specified q value of 0.01 when applying the method to proteomic analysis of the natural killer leukemia cell line (NKL). This study establishes an effective methodology and provides a platform for profiling confident proteomes in more relevant species as well as a future investigation of accurate protein quantification. PMID:21888997

  11. Improving protein identification from peptide mass fingerprinting through a parameterized multi-level scoring algorithm and an optimized peak detection.

    PubMed

    Gras, R; Müller, M; Gasteiger, E; Gay, S; Binz, P A; Bienvenut, W; Hoogland, C; Sanchez, J C; Bairoch, A; Hochstrasser, D F; Appel, R D

    1999-12-01

    We have developed a new algorithm to identify proteins by means of peptide mass fingerprinting. Starting from the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) spectra and environmental data such as species, isoelectric point and molecular weight, as well as chemical modifications or number of missed cleavages of a protein, the program performs a fully automated identification of the protein. The first step is a peak detection algorithm, which allows precise and fast determination of peptide masses, even if the peaks are of low intensity or they overlap. In the second step the masses and environmental data are used by the identification algorithm to search in protein sequence databases (SWISS-PROT and/or TrEMBL) for protein entries that match the input data. Consequently, a list of candidate proteins is selected from the database, and a score calculation provides a ranking according to the quality of the match. To define the most discriminating scoring calculation we analyzed the respective role of each parameter in two directions. The first one is based on filtering and exploratory effects, while the second direction focuses on the levels where the parameters intervene in the identification process. Thus, according to our analysis, all input parameters contribute to the score, however with different weights. Since it is difficult to estimate the weights in advance, they have been computed with a generic algorithm, using a training set of 91 protein spectra with their environmental data. We tested the resulting scoring calculation on a test set of ten proteins and compared the identification results with those of other peptide mass fingerprinting programs. PMID:10612280

  12. Combination of acid labile detergent and C18 Empore™ disks for improved identification and sequence coverage of in-gel digested proteins.

    PubMed

    Koehn, Henning; Lau, Benjamin; Clerens, Stefan; Plowman, Jeffrey E; Dyer, Jolon M; Ramli, Umi Salamah; Deb-Choudhury, Santanu

    2011-04-01

    A protocol for improved extraction of peptides from in-gel protein digests, using a combination of the acid labile surfactant, sodium deoxycholate (SDC) and C18 Empore™ membranes, is presented. This approach results in better mass spectrum quality, higher numbers of identified peptide peaks and improved identification scores compared to standard tryptic digestion protocols, or protocols using only SDC or only C18 Empore™ disks. The advantages of the new protocol are demonstrated for two different types of samples: Merino wool intermediate filament proteins and Elaeis guineensis (oil palm) mesocarp proteins. PMID:21327873

  13. 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

  14. Protein identification and Peptide expression resolver: harmonizing protein identification with protein expression data.

    PubMed

    Kearney, Paul; Butler, Heather; Eng, Kevin; Hugo, Patrice

    2008-01-01

    Proteomic discovery platforms generate both peptide expression information and protein identification information. Peptide expression data are used to determine which peptides are differentially expressed between study cohorts, and then these peptides are targeted for protein identification. In this paper, we demonstrate that peptide expression information is also a powerful tool for enhancing confidence in protein identification results. Specifically, we evaluate the following hypothesis: tryptic peptides originating from the same protein have similar expression profiles across samples in the discovery study. Evidence supporting this hypothesis is provided. This hypothesis is integrated into a protein identification tool, PIPER (Protein Identification and Peptide Expression Resolver), that reduces erroneous protein identifications below 5%. PIPER's utility is illustrated by application to a 72-sample biomarker discovery study where it is demonstrated that false positive protein identifications can be reduced below 5%. Consequently, it is recommended that PIPER methodology be incorporated into proteomic studies where both protein expression and identification data are collected. PMID:18062667

  15. 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

  16. 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

  17. BuildSummary: using a group-based approach to improve the sensitivity of peptide/protein identification in shotgun proteomics.

    PubMed

    Sheng, Quanhu; Dai, Jie; Wu, Yibo; Tang, Haixu; Zeng, Rong

    2012-03-01

    The target-decoy database search strategy is widely accepted as a standard method for estimating the false discovery rate (FDR) of peptide identification, based on which peptide-spectrum matches (PSMs) from the target database are filtered. To improve the sensitivity of protein identification given a fixed accuracy (frequently defined by a protein FDR threshold), a postprocessing procedure is often used that integrates results from different peptide search engines that had assayed the same data set. In this work, we show that PSMs that are grouped by the precursor charge, the number of missed internal cleavage sites, the modification state, and the numbers of protease termini and that the proteins grouped by their unique peptide count should be filtered separately according to the given FDR. We also develop an iterative procedure to filter the PSMs and proteins simultaneously, according to the given FDR. Finally, we present a general framework to integrate the results from different peptide search engines using the same FDR threshold. Our method was tested with several shotgun proteomics data sets that were acquired by multiple LC/MS instruments from two different biological samples. The results showed a satisfactory performance. We implemented the method in a user-friendly software package called BuildSummary, which can be downloaded for free from http://www.proteomics.ac.cn/software/proteomicstools/index.htm as part of the software suite ProteomicsTools. PMID:22217156

  18. 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)).

  19. 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...

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

    SciTech Connect

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

    2008-03-15

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

  1. Combining Isoelectric Point-Based Fractionation, Liquid Chromatography and Mass Spectrometry to Improve Peptide Detection and Protein Identification

    PubMed Central

    Cologna, Stephanie M.; Russell, William K.; Lim, Peniel J.; Vigh, Gyula; Russell, David H.

    2010-01-01

    The off-line coupling of an isoelectric trapping device termed membrane separated wells for isoelectric focusing and trapping (MSWIFT) to mass spectrometry-based proteomic studies is described. The MSWIFT is a high capacity, high-throughput, mass spectrometry compatible, isoelectric trapping device that provides isoelectric point (pI) based separations of complex mixtures of peptides. In MSWIFT, separation and analyte trapping are achieved by migrating the peptide ions through membranes having fixed pH values until the peptide pI is bracketed by the pH values of adjacent membranes. The pH values of the membranes can be tuned, thus affording a high degree of experimental flexibility. Specific advantages of using MSWIFT for sample pre-fractionation include: (i) small sample volumes (~200 μl), (ii) customized membranes over a large pH range, (iii) flexibility in the number of desired fractions, (iv) membrane compatibility with a variety of solvents systems and (v) resulting fractions do not require sample cleanup prior to MS analysis. Here, we demonstrate the utility of MSWIFT for mass spectrometry-based detection of peptides in improving dynamic range and the reduction of ion suppression effects for high-throughput separations of tryptic peptides. PMID:20537905

  2. Improved method for protein complex detection using bottleneck proteins

    PubMed Central

    2013-01-01

    Background Detecting protein complexes is one of essential and fundamental tasks in understanding various biological functions or processes. Therefore accurate identification of protein complexes is indispensable. Methods For more accurate detection of protein complexes, we propose an algorithm which detects dense protein sub-networks of which proteins share closely located bottleneck proteins. The proposed algorithm is capable of finding protein complexes which allow overlapping with each other. Results We applied our algorithm to several PPI (Protein-Protein Interaction) networks of Saccharomyces cerevisiae and Homo sapiens, and validated our results using public databases of protein complexes. The prediction accuracy was even more improved over our previous work which used also bottleneck information of the PPI network, but showed limitation when predicting small-sized protein complex detection. Conclusions Our algorithm resulted in overlapping protein complexes with significantly improved F1 score over existing algorithms. This result comes from high recall due to effective network search, as well as high precision due to proper use of bottleneck information during the network search. PMID:23566214

  3. 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.

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

    PubMed

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

    2009-05-31

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

  5. Direct Maximization of Protein Identifications from Tandem Mass Spectra*

    PubMed Central

    Spivak, Marina; Weston, Jason; Tomazela, Daniela; MacCoss, Michael J.; Noble, William Stafford

    2012-01-01

    The goal of many shotgun proteomics experiments is to determine the protein complement of a complex biological mixture. For many mixtures, most methodological approaches fall significantly short of this goal. Existing solutions to this problem typically subdivide the task into two stages: first identifying a collection of peptides with a low false discovery rate and then inferring from the peptides a corresponding set of proteins. In contrast, we formulate the protein identification problem as a single optimization problem, which we solve using machine learning methods. This approach is motivated by the observation that the peptide and protein level tasks are cooperative, and the solution to each can be improved by using information about the solution to the other. The resulting algorithm directly controls the relevant error rate, can incorporate a wide variety of evidence and, for complex samples, provides 18–34% more protein identifications than the current state of the art approaches. PMID:22052992

  6. Identification of extracellularly phosphorylated membrane proteins.

    PubMed

    Burghoff, Sandra; Willberg, Wibke; Schrader, Jürgen

    2015-10-01

    Ecto-protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC-MS/MS. A "false-positive" strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G-protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface-exposed peptides when extracellular adenosine-5'-triphosphate is elevated during purinergic signalling. PMID:26152529

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

    PubMed

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

    2015-01-01

    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

  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. An improved PCR method for gender identification of eagles.

    PubMed

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

    2008-06-01

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

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

    PubMed Central

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

    2009-01-01

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

  11. Comprehensive Identification of Proteins from MALDI Imaging*

    PubMed Central

    Maier, Stefan K.; Hahne, Hannes; Gholami, Amin Moghaddas; Balluff, Benjamin; Meding, Stephan; Schoene, Cédrik; Walch, Axel K.; Kuster, Bernhard

    2013-01-01

    Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a powerful tool for the visualization of proteins in tissues and has demonstrated considerable diagnostic and prognostic value. One main challenge is that the molecular identity of such potential biomarkers mostly remains unknown. We introduce a generic method that removes this issue by systematically identifying the proteins embedded in the MALDI matrix using a combination of bottom-up and top-down proteomics. The analyses of ten human tissues lead to the identification of 1400 abundant and soluble proteins constituting the set of proteins detectable by MALDI IMS including >90% of all IMS biomarkers reported in the literature. Top-down analysis of the matrix proteome identified 124 mostly N- and C-terminally fragmented proteins indicating considerable protein processing activity in tissues. All protein identification data from this study as well as the IMS literature has been deposited into MaTisse, a new publically available database, which we anticipate will become a valuable resource for the IMS community. PMID:23782541

  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. Insect Seminal Fluid Proteins: Identification and Function

    PubMed Central

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

    2014-01-01

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

  14. Proteomics technologies for the global identification and quantification of proteins.

    PubMed

    Brewis, Ian A; Brennan, P

    2010-01-01

    This review provides an introduction for the nonspecialist to proteomics and in particular the major approaches available for global protein identification and quantification. Proteomics technologies offer considerable opportunities for improved biological understanding and biomarker discovery. The central platform for proteomics is tandem mass spectrometry (MS) but a number of other technologies, resources, and expertise are absolutely required to perform meaningful experiments. These include protein separation science (and protein biochemistry in general), genomics, and bioinformatics. There are a range of workflows available for protein (or peptide) separation prior to tandem MS and subsequent bioinformatics analysis to achieve protein identifications. The predominant approaches are 2D electrophoresis (2DE) and subsequent MS, liquid chromatography-MS (LC-MS), and GeLC-MS. Beyond protein identification, there are a number of well-established options available for protein quantification. Difference gel electrophoresis (DIGE) following 2DE is one option but MS-based methods (most commonly iTRAQ-Isobaric Tags for Relative and Absolute Quantification or SILAC-Stable Isotope Labeling by Amino Acids) are now the preferred options. Sample preparation is critical to performing good experiments and subcellular fractionation can additionally provide protein localization information compared with whole cell lysates. Differential detergent solubilization is another valid option. With biological fluids, it is possible to remove the most abundant proteins by immunodepletion. Sample enrichment is also used extensively in certain analyses and most commonly in phosphoproteomics with the initial purification of phosphopeptides. Proteomics produces considerable datasets and resources to facilitate the necessary extended analysis of this data are improving all the time. Beyond the opportunities afforded by proteomics there are definite challenges to achieving full proteomic coverage

  15. 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. PMID:26511375

  16. Identification of Topping Responsive Proteins in Tobacco Roots

    PubMed Central

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

    2016-01-01

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

  17. Identification of Topping Responsive Proteins in Tobacco Roots.

    PubMed

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

    2016-01-01

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

  18. Applications of graph theory in protein structure identification.

    PubMed

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

    2011-01-01

    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. PMID:22165974

  19. 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. PMID:25268881

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

    PubMed Central

    Liu, Bin; Wang, Shanyi; Wang, Xiaolong

    2015-01-01

    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/. PMID:26482832

  1. 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/.

  2. Byonic: Advanced Peptide and Protein Identification Software

    PubMed Central

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

    2013-01-01

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

  3. Protein identification problem from a Bayesian point of view

    PubMed Central

    Li, Yong Fuga; Arnold, Randy J.; Radivojac, Predrag

    2012-01-01

    We present a generic Bayesian framework for the peptide and protein identification in proteomics, and provide a unified interpretation for the database searching and the de novo peptide sequencing approaches that are used in peptide identification. We describe several probabilistic graphical models and a variety of prior distributions that can be incorporated into the Bayesian framework to model different types of prior information, such as the known protein sequences, the known protein abundances, the peptide precursor masses, the estimated peptide retention time and the peptide detectabilities. Various applications of the Bayesian framework are discussed theoretically, including its application to the identification of peptides containing mutations and post-translational modifications. PMID:24761189

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

    PubMed Central

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

    2015-01-01

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

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

    ERIC Educational Resources Information Center

    Wilson, Karl A.; Tan-Wilson, Anna

    2013-01-01

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

  6. 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. PMID:25892709

  7. 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...

  8. Improving Protein Expression Prediction Using Extra Features and Ensemble Averaging

    PubMed Central

    Fernandes, Armando; Vinga, Susana

    2016-01-01

    The article focus is the improvement of machine learning models capable of predicting protein expression levels based on their codon encoding. Support vector regression (SVR) and partial least squares (PLS) were used to create the models. SVR yields predictions that surpass those of PLS. It is shown that it is possible to improve the models predictive ability by using two more input features, codon identification number and codon count, besides the already used codon bias and minimum free energy. In addition, applying ensemble averaging to the SVR or PLS models also improves the results even further. The present work motivates the test of different ensembles and features with the aim of improving the prediction models whose correlation coefficients are still far from perfect. These results are relevant for the optimization of codon usage and enhancement of protein expression levels in synthetic biology problems. PMID:26934190

  9. Engineering Cells to Improve Protein Expression

    PubMed Central

    Xiao, Su; Shiloach, Joseph; Betenbaugh, Michael J.

    2014-01-01

    Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes. PMID:24704806

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

    PubMed

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

    2005-01-01

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

  11. Compositions and methods for improved protein production

    DOEpatents

    Bodie, Elizabeth A.; Kim, Steve

    2012-07-10

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

  12. 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.

  13. 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

  14. Computational Methods for Protein Identification from Mass Spectrometry Data

    PubMed Central

    McHugh, Leo; Arthur, Jonathan W

    2008-01-01

    Protein identification using mass spectrometry is an indispensable computational tool in the life sciences. A dramatic increase in the use of proteomic strategies to understand the biology of living systems generates an ongoing need for more effective, efficient, and accurate computational methods for protein identification. A wide range of computational methods, each with various implementations, are available to complement different proteomic approaches. A solid knowledge of the range of algorithms available and, more critically, the accuracy and effectiveness of these techniques is essential to ensure as many of the proteins as possible, within any particular experiment, are correctly identified. Here, we undertake a systematic review of the currently available methods and algorithms for interpreting, managing, and analyzing biological data associated with protein identification. We summarize the advances in computational solutions as they have responded to corresponding advances in mass spectrometry hardware. The evolution of scoring algorithms and metrics for automated protein identification are also discussed with a focus on the relative performance of different techniques. We also consider the relative advantages and limitations of different techniques in particular biological contexts. Finally, we present our perspective on future developments in the area of computational protein identification by considering the most recent literature on new and promising approaches to the problem as well as identifying areas yet to be explored and the potential application of methods from other areas of computational biology. PMID:18463710

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

    PubMed

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

    2011-03-01

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

  16. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

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

  17. 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.

  18. Genetically modified proteins: functional improvement and chimeragenesis

    PubMed Central

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

    2015-01-01

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

  19. Identification of four plastid-localized protein kinases.

    PubMed

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

    2016-06-01

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

  20. 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…

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

    SciTech Connect

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

    2005-04-11

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

  2. 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.

  3. Identification of kinetically hot residues in proteins.

    PubMed Central

    Demirel, M. C.; Atilgan, A. R.; Jernigan, R. L.; Erman, B.; Bahar, I.

    1998-01-01

    A number of recent studies called attention to the presence of kinetically important residues underlying the formation and stabilization of folding nuclei in proteins, and to the possible existence of a correlation between conserved residues and those participating in the folding nuclei. Here, we use the Gaussian network model (GNM), which recently proved useful in describing the dynamic characteristics of proteins for identifying the kinetically hot residues in folded structures. These are the residues involved in the highest frequency fluctuations near the native state coordinates. Their high frequency is a manifestation of the steepness of the energy landscape near their native state positions. The theory is applied to a series of proteins whose kinetically important residues have been extensively explored: chymotrypsin inhibitor 2, cytochrome c, and related C2 proteins. Most of the residues previously pointed out to underlie the folding process of these proteins, and to be critically important for the stabilization of the tertiary fold, are correctly identified, indicating a correlation between the kinetic hot spots and the early forming structural elements in proteins. Additionally, a strong correlation between kinetically hot residues and loci of conserved residues is observed. Finally, residues that may be important for the stability of the tertiary structure of CheY are proposed. PMID:9865946

  4. BioID Identification of Lamin-Associated Proteins.

    PubMed

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

    2016-01-01

    A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The identification of proteins associated with lamins is likely to provide fundamental insight into these mechanisms. BioID (proximity-dependent biotin identification) is a unique and powerful method for identifying protein-protein and proximity-based interactions in living cells. BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID-fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry. BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. BioID is applicable to insoluble proteins such as lamins that are often refractory to study by other methods and can identify weak and/or transient interactions. We discuss the use of BioID to elucidate novel lamin-interacting proteins and its applications in a broad range of biological systems, and provide detailed protocols to guide new applications. PMID:26778550

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-02-01

    Protein citrullination is a post-translational modification of arginine that is catalyzed by the Protein Arginine Deiminase (PAD) family of enzymes. Aberrantly increased citrullination is associated with a host of inflammatory diseases and cancer and PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis, lupus, atherosclerosis, and ulcerative colitis. In rheumatoid arthritis, citrullinated proteins serve as key antigens for rheumatoid arthritis-associated autoantibodies. These data suggest that citrullinated proteins may serve more generally as biomarkers of specific disease states, however, the identification of citrullinated residues remains challenging due to the small 1Da mass change that occurs upon citrullination. Herein, we highlight the available techniques to identify citrullinated proteins/residues focusing on advanced MS techniques as well as chemical derivatization strategies that are currently being employed to identify citrullinated proteins as well as the specific residues modified within those proteins. PMID:26517730

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2011-12-01

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

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

    PubMed

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

    2010-08-01

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

  11. Identification of essential proteins based on edge clustering coefficient.

    PubMed

    Wang, Jianxin; Li, Min; Wang, Huan; Pan, Yi

    2012-01-01

    Identification of essential proteins is key to understanding the minimal requirements for cellular life and important for drug design. The rapid increase of available protein-protein interaction (PPI) data has made it possible to detect protein essentiality on network level. A series of centrality measures have been proposed to discover essential proteins based on network topology. However, most of them tended to focus only on the location of single protein, but ignored the relevance between interactions and protein essentiality. In this paper, a new centrality measure for identifying essential proteins based on edge clustering coefficient, named as NC, is proposed. Different from previous centrality measures, NC considers both the centrality of a node and the relationship between it and its neighbors. For each interaction in the network, we calculate its edge clustering coefficient. A node’s essentiality is determined by the sum of the edge clustering coefficients of interactions connecting it and its neighbors. The new centrality measure NC takes into account the modular nature of protein essentiality. NC is applied to three different types of yeast protein-protein interaction networks, which are obtained from the DIP database, the MIPS database and the BioGRID database, respectively. The experimental results on the three different networks show that the number of essential proteins discovered by NC universally exceeds that discovered by the six other centrality measures: DC, BC, CC, SC, EC, and IC. Moreover, the essential proteins discovered by NC show significant cluster effect. PMID:22084147

  12. Identification of Chemical-Adducted Proteins in Urine by Multi-dimensional Protein Identification Technology (LC/LC–MS/MS)

    PubMed Central

    Labenski, Matthew T.; Fisher, Ashley A.; Monks, Terrence J.; Lau, Serrine S.

    2014-01-01

    Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein adducts (Kleiner et al., Chem Res Toxicol 11:1283–1290, 1998), multi-dimensional protein identification technology (MuDPIT) has been utilized. MuDPIT was first described by Link et al. as a new technique useful for protein identification from a complex mixture of proteins (Link et al., Nat Biotechnol 17:676–682, 1999). MuDPIT utilizes two different HPLC columns to further enhance peptide separation, increasing the number of peptide hits and protein coverage. The technology is extremely useful for proteomes, such as the urine proteome, samples from immunoprecipitations, and 1D gel bands resolved from a tissue homogenate or lysate. In particular, MuDPIT has enhanced the field of adduct hunting for adducted peptides, since it is more capable of identifying lesser abundant peptides, such as those that are adducted, than the more standard LC–MS/MS. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response they elicit. PMID:20972764

  13. 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...

  14. Method for Rapid Protein Identification in a Large Database

    PubMed Central

    Zhang, Wenli; Zhao, Xiaofang

    2013-01-01

    Protein identification is an integral part of proteomics research. The available tools to identify proteins in tandem mass spectrometry experiments are not optimized to face current challenges in terms of identification scale and speed owing to the exponential growth of the protein database and the accelerated generation of mass spectrometry data, as well as the demand for nonspecific digestion and post-modifications in complex-sample identification. As a result, a rapid method is required to mitigate such complexity and computation challenges. This paper thus aims to present an open method to prevent enzyme and modification specificity on a large database. This paper designed and developed a distributed program to facilitate application to computer resources. With this optimization, nearly linear speedup and real-time support are achieved on a large database with nonspecific digestion, thus enabling testing with two classical large protein databases in a 20-blade cluster. This work aids in the discovery of more significant biological results, such as modification sites, and enables the identification of more complex samples, such as metaproteomics samples. PMID:24000323

  15. 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.

  16. 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.

  17. Bioinformatics pipeline for functional identification and characterization of proteins

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

  18. Tuber borchii fruit body: 2-dimensional profile and protein identification.

    PubMed

    Pierleoni, Raffaella; Buffalini, Michele; Vallorani, Luciana; Guidi, Chiara; Zeppa, Sabrina; Sacconi, Cinzia; Pucci, Piero; Amoresano, Angela; Casbarra, Annarita; Stocchi, Vilberto

    2004-04-01

    The formation of the fruit body represents the final phase of the ectomycorrhizal fungus T. borchii life cycle. Very little is known concerning the molecular and biochemical processes involved in the fructification phase. 2-DE maps of unripe and ripe ascocarps revealed different protein expression levels and the comparison of the electropherograms led to the identification of specific proteins for each developmental phase. Associating micropreparative 2-DE to microchemical approaches, such as N-terminal sequencing and 2-D gel-electrophoresis mass-spectrometry, proteins playing pivotal roles in truffle physiology were identified. PMID:15081280

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

    PubMed

    Wang, Fuqiang; Wang, Ling; Xu, Zhiyang; Liang, Gaolin

    2013-01-01

    Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders. PMID:23638173

  20. Novel identification of expressed genes and functional classification of hypothetical proteins from Neisseria meningitidis serogroup A.

    PubMed

    Bernardini, Giulia; Arena, Simona; Braconi, Daniela; Scaloni, Andrea; Santucci, Annalisa

    2007-09-01

    To implement the 2-DE database of serogroup A Neisseria meningitidis (MenA) and improve its potential of investigation in bacterial biology, cell extracts were separated by tricine-SDS-PAGE and 131 novel proteins were identified by microLC-ESI-IT-MS/MS. These identifications extended to 404, the number of MenA gene expression products characterized at the proteome level, approximately covering 20% of the total ORFs predicted from genome sequence. This technical approach was particularly useful in ascertaining expression of ribosomal as well as hypothetical proteins. Particular attention was paid to functional characterization of hypothetical proteins by means of software analyses and database searches. PMID:17849410

  1. Improved Stochastic Subspace System Identification for Structural Health Monitoring

    NASA Astrophysics Data System (ADS)

    Chang, Chia-Ming; Loh, Chin-Hsiung

    2015-07-01

    Structural health monitoring acquires structural information through numerous sensor measurements. Vibrational measurement data render the dynamic characteristics of structures to be extracted, in particular of the modal properties such as natural frequencies, damping, and mode shapes. The stochastic subspace system identification has been recognized as a power tool which can present a structure in the modal coordinates. To obtain qualitative identified data, this tool needs to spend computational expense on a large set of measurements. In study, a stochastic system identification framework is proposed to improve the efficiency and quality of the conventional stochastic subspace system identification. This framework includes 1) measured signal processing, 2) efficient space projection, 3) system order selection, and 4) modal property derivation. The measured signal processing employs the singular spectrum analysis algorithm to lower the noise components as well as to present a data set in a reduced dimension. The subspace is subsequently derived from the data set presented in a delayed coordinate. With the proposed order selection criteria, the number of structural modes is determined, resulting in the modal properties. This system identification framework is applied to a real-world bridge for exploring the feasibility in real-time applications. The results show that this improved system identification method significantly decreases computational time, while qualitative modal parameters are still attained.

  2. 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.

  3. 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.

  4. Identification and Validation of ISG15 Target Proteins.

    PubMed

    Durfee, Larissa A; Huibregtse, Jon M

    2010-01-01

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

  5. Identification of Secreted Candida Proteins Using Mass Spectrometry.

    PubMed

    Gómez-Molero, Emilia; Dekker, Henk L; de Boer, Albert D; de Groot, Piet W J

    2016-01-01

    Analysis of fungal secretomes using mass spectrometry is a useful technique in cell biology. Knowledge of the secretome of a human fungal pathogen may yield important information of host-pathogen interactions and may be useful for identifying vaccines candidates or diagnostic markers for antifungal strategies. In this chapter, with a main focus on sample preparation aspects, we describe the methodology that we apply for gel-independent batch identification and quantification of proteins that are secreted during growth in liquid cultures. Using these techniques with Candida and other yeast species, the majority of the identified proteins are classical secretory proteins and cell wall proteins containing N-terminal signal peptides for secretion, although dependent on sample preparation quality and the mass spectrometric analysis also usually, a number of nonsecretory proteins are identified. PMID:26519067

  6. Identification of PDC-109-like protein(s) in buffalo seminal plasma.

    PubMed

    Harshan, Hiron M; Sankar, Surya; Singh, L P; Singh, Manish Kumar; Sudharani, S; Ansari, M R; Singh, S K; Majumdar, A C; Joshi, P

    2009-10-01

    The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma. PMID:19117702

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

    PubMed

    Kubben, Nard; Voncken, Jan Willem; Demmers, Jeroen; Calis, Chantal; van Almen, Geert; Pinto, Yigal; Misteli, Tom

    2010-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  9. 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

  10. Identification of short peptide sequences in complex milk protein hydrolysates.

    PubMed

    O'Keeffe, Martina B; FitzGerald, Richard J

    2015-10-01

    Numerous low molecular mass bioactive peptides (BAPs) can be generated during the hydrolysis of bovine milk proteins. Low molecular mass BAP sequences are less likely to be broken down by digestive enzymes and are thus more likely to be active in vivo. However, the identification of short peptides remains a challenge during mass spectrometry (MS) analysis due to issues with the transfer and over-fragmentation of low molecular mass ions. A method is described herein using time-of-flight ESI-MS/MS to effectively fragment and identify short peptides. This includes (a) short synthetic peptides, (b) short peptides within a defined hydrolysate sample, i.e. a prolyl endoproteinase hydrolysate of β-casein and (c) short peptides within a complex hydrolysate, i.e. a Corolase PP digest of sodium caseinate. The methodology may find widespread utilisation in the efficient identification of low molecular mass peptide sequences in food protein hydrolysates. PMID:25872436

  11. Systematic identification of protein combinations mediating chromatin looping.

    PubMed

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

    2016-01-01

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

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

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

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

  13. 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.

  14. Identification of AOSC-binding proteins in neurons

    NASA Astrophysics Data System (ADS)

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

    2008-11-01

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

  15. 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.

  16. 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.

  17. Identification of Cryptic Anopheles Mosquito Species by Molecular Protein Profiling

    PubMed Central

    Müller, Pie; Pflüger, Valentin; Wittwer, Matthias; Ziegler, Dominik; Chandre, Fabrice; Simard, Frédéric; Lengeler, Christian

    2013-01-01

    Vector control is the mainstay of malaria control programmes. Successful vector control profoundly relies on accurate information on the target mosquito populations in order to choose the most appropriate intervention for a given mosquito species and to monitor its impact. An impediment to identify mosquito species is the existence of morphologically identical sibling species that play different roles in the transmission of pathogens and parasites. Currently PCR diagnostics are used to distinguish between sibling species. PCR based methods are, however, expensive, time-consuming and their development requires a priori DNA sequence information. Here, we evaluated an inexpensive molecular proteomics approach for Anopheles species: matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a well developed protein profiling tool for the identification of microorganisms but so far has received little attention as a diagnostic tool in entomology. We measured MS spectra from specimens of 32 laboratory colonies and 2 field populations representing 12 Anopheles species including the A. gambiae species complex. An important step in the study was the advancement and implementation of a bioinformatics approach improving the resolution over previously applied cluster analysis. Borrowing tools for linear discriminant analysis from genomics, MALDI-TOF MS accurately identified taxonomically closely related mosquito species, including the separation between the M and S molecular forms of A. gambiae sensu stricto. The approach also classifies specimens from different laboratory colonies; hence proving also very promising for its use in colony authentication as part of quality assurance in laboratory studies. While being exceptionally accurate and robust, MALDI-TOF MS has several advantages over other typing methods, including simple sample preparation and short processing time. As the method does not require DNA sequence information

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

    PubMed

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

    2012-01-01

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

  19. 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

  20. 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

  1. Identification of contractile vacuole proteins in Trypanosoma cruzi.

    PubMed

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

    2011-01-01

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

    PubMed

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

    2006-07-01

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

  3. Identification of Sequences Encoding Symbiodinium minutum Mitochondrial Proteins.

    PubMed

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

    2016-02-01

    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

  4. 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

  5. Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT

    PubMed Central

    Fonslow, Bryan R.; Carvalho, Paulo C.; Academia, Katrina; Freeby, Steve; Xu, Tao; Nakorchevsky, Aleksey; Paulus, Aran; Yates, John R.

    2011-01-01

    Ideally shotgun proteomics would facilitate the identification of an entire proteome with 100% protein sequence coverage. In reality, the large dynamic range and complexity of cellular proteomes results in oversampling of abundant proteins, while peptides from low abundance proteins are undersampled or remain undetected. We tested the proteome equalization technology, ProteoMiner, in conjunction with Multidimensional Protein Identification Technology (MudPIT) to determine how the equalization of protein dynamic range could improve shotgun proteomics methods for the analysis of cellular proteomes. Our results suggest low abundance protein identifications were improved by two mechanisms: (1) depletion of high abundance proteins freed ion trap sampling space usually occupied by high abundance peptides and (2) enrichment of low abundance proteins increased the probability of sampling their corresponding more abundant peptides. Both mechanisms also contributed to dramatic increases in the quantity of peptides identified and the quality of MS/MS spectra acquired due to increases in precursor intensity of peptides from low abundance proteins. From our large data set of identified proteins, we categorized the dominant physicochemical factors which facilitate proteome equalization with a hexapeptide library. These results illustrate that equalization of the dynamic range of the cellular proteome is a promising methodology to improve low abundance protein identification confidence, reproducibility, and sequence coverage in shotgun proteomics experiments, opening a new avenue of research for improving proteome coverage. PMID:21702434

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

    PubMed Central

    Xu, Ruifeng; Zhou, Jiyun; Liu, Bin; Yao, Lin; He, Yulan; Zou, Quan; Wang, Xiaolong

    2014-01-01

    DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97–9.52% in ACC and 0.08–0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83–16.63% in terms of ACC and 0.02–0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public. PMID:24977146

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

    PubMed

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

    2016-04-01

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

  8. Identification of major immunogenic proteins of Mycoplasma synoviae isolates.

    PubMed

    Bercic, Rebeka Lucijana; Slavec, Brigita; Lavric, Miha; Narat, Mojca; Bidovec, Andrej; Dovc, Peter; Bencina, Dusan

    2008-02-01

    Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight (M(w)) in the region above the lipoprotein MSPB (approximately 40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (approximately 47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M(w) of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively. PMID:17720337

  9. Identification, Analysis and Prediction of Protein Ubiquitination Sites

    PubMed Central

    Radivojac, Predrag; Vacic, Vladimir; Haynes, Chad; Cocklin, Ross R.; Mohan, Amrita; Heyen, Joshua W.; Goebl, Mark G.; Iakoucheva, Lilia M.

    2009-01-01

    Summary Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associated mutations. UbPred is available at http://www.ubpred.org PMID:19722269

  10. 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

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

    PubMed

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

    2014-06-01

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

  12. 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.

  13. 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.

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

    PubMed

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

    2016-01-01

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

  15. Precursor-ion mass re-estimation improves peptide identification on hybrid instruments.

    PubMed

    Luethy, Roland; Kessner, Darren E; Katz, Jonathan E; Maclean, Brendan; Grothe, Robert; Kani, Kian; Faça, Vitor; Pitteri, Sharon; Hanash, Samir; Agus, David B; Mallick, Parag

    2008-09-01

    Mass spectrometry-based proteomics experiments have become an important tool for studying biological systems. Identifying the proteins in complex mixtures by assigning peptide fragmentation spectra to peptide sequences is an important step in the proteomics process. The 1-2 ppm mass-accuracy of hybrid instruments, like the LTQ-FT, has been cited as a key factor in their ability to identify a larger number of peptides with greater confidence than competing instruments. However, in replicate experiments of an 18-protein mixture, we note parent masses deviate 171 ppm, on average, for ion-trap data directed identifications and 8 ppm, on average, for preview Fourier transform (FT) data directed identifications. These deviations are neither caused by poor calibration nor by excessive ion-loading and are most likely due to errors in parent mass estimation. To improve these deviations, we introduce msPrefix, a program to re-estimate a peptide's parent mass from an associated high-accuracy full-scan survey spectrum. In 18-protein mixture experiments, msPrefix parent mass estimates deviate only 1 ppm, on average, from the identified peptides. In a cell lysate experiment searched with a tolerance of 50 ppm, 2295 peptides were confidently identified using native data and 4560 using msPrefixed data. Likewise, in a plasma experiment searched with a tolerance of 50 ppm, 326 peptides were identified using native data and 1216 using msPrefixed data. msPrefix is also able to determine which MS/MS spectra were possibly derived from multiple precursor ions. In complex mixture experiments, we demonstrate that more than 50% of triggered MS/MS may have had multiple precursor ions and note that spectra with multiple candidate ions are less likely to result in an identification using TANDEM. These results demonstrate integration of msPrefix into traditional shotgun proteomics workflows significantly improves identification results. PMID:18707148

  16. 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

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

    PubMed Central

    Marasco, Daniela; Scognamiglio, Pasqualina Liana

    2015-01-01

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

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

    PubMed

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

    2007-01-01

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

  19. An overview of protein identification studies in cassava.

    PubMed

    Batista de Souza, Cláudia R; dos Reis, Sávio P; Castelo Branco Carvalho, Luiz J

    2015-01-01

    Cassava (Manihot esculenta Crantz) belongs to the Euphorbiaceae family and is originated from the Southern Amazon basin. The storage root is the most important product of cassava as food for more than 800 million people in Africa, Asia and Latin America. In this review, we present a retrospective of studies aiming the identification of cassava proteins, starting from the first investigations using SDS-PAGE and classical two-dimensional gel electrophoresis (2DE) to recent studies with advanced technologies such as high-resolution 2DE, mass spectrometry, and iTRAQ-based analysis that have contributed for characterization of cassava proteome. Several cassava proteins have been identified, including those involved in the storage root formation and post-harvest physiological deterioration processes. PMID:25707473

  20. Identification of protein coding regions in RNA transcripts

    PubMed Central

    Tang, Shiyuyun; Lomsadze, Alexandre; Borodovsky, Mark

    2015-01-01

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

  1. Shedding light on black boxes in protein identification.

    PubMed

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

    2014-05-01

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

  2. Ex vivo identification of protein-protein interactions involving the dopamine transporter.

    PubMed

    Hadlock, Gregory C; Nelson, Chad C; Baucum, Anthony J; Hanson, Glen R; Fleckenstein, Annette E

    2011-03-30

    The dopamine (DA) transporter (DAT) is a key regulator of dopaminergic signaling as it mediates the reuptake of extrasynaptic DA and thereby terminates dopaminergic signaling. Emerging evidence indicates that DAT function is influenced through interactions with other proteins. The current report describes a method to identify such interactions following DAT immunoprecipitation from a rat striatal synaptosomal preparation. This subcellular fraction was selected since DAT function is often determined ex vivo by measuring DA uptake in this preparation and few reports investigating DAT-protein interactions have utilized this preparation. Following SDS-PAGE and colloidal Coomassie staining, selected protein bands from a DAT-immunoprecipitate were excised, digested with trypsin, extracted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). From the analysis of the tryptic peptides, several proteins were identified including DAT, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) β, CaMKII δ, protein kinase C (PKC) β, and PKC γ. Co-immunoprecipitation of PKC, CaMKII, and protein interacting with C kinase-1 with DAT was confirmed by Western blotting. Thus, the present study highlights a method to immunoprecipitate DAT and to identify co-immunoprecipitating proteins using LC/MS/MS and Western blotting. This method can be utilized to evaluate DAT protein-protein interactions but also to assess interactions involving other synaptic proteins. Ex vivo identification of protein-protein interactions will provide new insight into the function and regulation of a variety of synaptic, membrane-associated proteins, including DAT. PMID:21291912

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

    PubMed

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

    2015-09-01

    As part of the Chromosome-Centric Human Proteome Project (C-HPP) mission, laboratories all over the world have tried to map the entire missing proteins (MPs) since 2012. On the basis of the first and second Chinese Chromosome Proteome Database (CCPD 1.0 and 2.0) studies, we developed systematic enrichment strategies to identify MPs that fell into four classes: (1) low molecular weight (LMW) proteins, (2) membrane proteins, (3) proteins that contained various post-translational modifications (PTMs), and (4) nucleic acid-associated proteins. Of 8845 proteins identified in 7 data sets, 79 proteins were classified as MPs. Among data sets derived from different enrichment strategies, data sets for LMW and PTM yielded the most novel MPs. In addition, we found that some MPs were identified in multiple-data sets, which implied that tandem enrichments methods might improve the ability to identify MPs. Moreover, low expression at the transcription level was the major cause of the "missing" of these MPs; however, MPs with higher expression level also evaded identification, most likely due to other characteristics such as LMW, high hydrophobicity and PTM. By combining a stringent manual check of the MS2 spectra with peptides synthesis verification, we confirmed 30 MPs (neXtProt PE2 ∼ PE4) and 6 potential MPs (neXtProt PE5) with authentic MS evidence. By integrating our large-scale data sets of CCPD 2.0, the number of identified proteins has increased considerably beyond simulation saturation. Here, we show that special enrichment strategies can break through the data saturation bottleneck, which could increase the efficiency of MP identification in future C-HPP studies. All 7 data sets have been uploaded to ProteomeXchange with the identifier PXD002255. PMID:26144840

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

    PubMed Central

    Huang, Bill X.; Kim, Hee-Yong

    2013-01-01

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

  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 protein secretion systems in bacterial genomes

    PubMed Central

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

    2016-01-01

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

  7. Retroactive Streaming Fails to Improve Concurrent Vowel Identification.

    PubMed

    Brandewie, Eugene J; Oxenham, Andrew J

    2015-01-01

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

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

    DOE PAGESBeta

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

    2013-03-07

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

  9. 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.

  10. 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.

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

    NASA Astrophysics Data System (ADS)

    Meyer, Jesse G.; Komives, Elizabeth A.

    2012-05-01

    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.

  12. De novo derivation of proteomes from transcriptomes for transcript and protein identification

    PubMed Central

    Evans, Vanessa C.; Barker, Gary; Heesom, Kate J.; Fan, Jun; Bessant, Conrad; Matthews, David A.

    2012-01-01

    Identification of proteins by tandem mass spectrometry requires a database of the proteins that could be in the sample. This is available for model species (e.g. humans) but not for non-model species. Ideally, for a non-model species the sequencing of expressed mRNA would generate a protein database for mass spectrometry based identification, allowing detection of genes and proteins using high throughput sequencing and protein identification technologies. Here we use human cells infected with human adenovirus as a complex and dynamic model to demonstrate this approach is robust. Our Proteomics Informed by Transcriptomics technique identifies >99% of over 3700 distinct proteins identified using traditional analysis reliant on comprehensive human and adenovirus protein lists. This facilitates high throughput acquisition of direct evidence for transcripts and proteins in non-model species. Critically, we show this approach can also be used to highlight genes and proteins undergoing dynamic changes in post transcriptional protein stability. PMID:23142869

  13. 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.

  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). PMID:26235816

  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. PMID:25881200

  16. 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)...

  17. Identification and validation of protein-protein interactions by combining co-immunoprecipitation, antigen competition, and stable isotope labeling.

    PubMed

    Sommer, Frederik; Mühlhaus, Timo; Hemme, Dorothea; Veyel, Daniel; Schroda, Michael

    2014-01-01

    Co-immunoprecipitation (coIP) in combination with mass spectrometry (MS) is a powerful tool to identify potential protein-protein interactions. However, unspecifically precipitated proteins usually result in large numbers of false-positive identifications. Here we describe a detailed protocol particularly useful in plant sciences that is based on (15)N stable isotope labeling of cells, (14)N antigen titration, and coIP/MS to distinguish true from false protein-protein interactions. PMID:25059616

  18. 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

  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. Detection and identification of sub-nanogram levels of protein in a nanoLC-trypsin-MS system.

    PubMed

    Slysz, Gordon W; Lewis, Darren F; Schriemer, David C

    2006-08-01

    Proteomic workflows involving liquid-based protein separations are an alternative to gel-based protein analysis, however the trypsin digestion procedure is usually difficult to implement, particularly when processing low abundance proteins from capillary column effluent. To convert the protein to peptides for the purpose of identification, current protocols require several sample handling steps, and sample losses become an issue. In this study, we present an improved system that conducts reversed-phase protein chromatography and rapid on-line tryptic digestion requiring sub-nanogram quantities of protein. This system employs a novel mirror-gradient concept that allows for dynamic titration of the column effluent to create optimal conditions for real-time tryptic digestion. The purpose behind this development was to improve the limits of detection of the online concept, to support flow-based alternatives to gel-based proteomics and to simplify the characterization of low abundance proteins. Using test mixtures of proteins, we show that peptide mass fingerprinting with high sequence representation can be easily achieved at the 20 fmol level, with detection limits down to 5 fmol (85 pg myoglobin). Limits of identification using standard data-dependent MS/MS experiments are as low as 10 fmol. These results suggest that the nanoLC-trypsin-MS/MS system could represent an alternative to the conventional "1D-gel to MS" proteomic strategy. PMID:16889418

  1. Identification of novel arthropod vector G protein-coupled receptors

    PubMed Central

    2013-01-01

    Background The control of vector-borne diseases, such as malaria, dengue fever, and typhus fever is often achieved with the use of insecticides. Unfortunately, insecticide resistance is becoming common among different vector species. There are currently no chemical alternatives to these insecticides because new human-safe classes of molecules have yet to be brought to the vector-control market. The identification of novel targets offer opportunities for rational design of new chemistries to control vector populations. One target family, G protein-coupled receptors (GPCRs), has remained relatively under explored in terms of insecticide development. Methods A novel classifier, Ensemble*, for vector GPCRs was developed. Ensemble* was validated and compared to existing classifiers using a set of all known GPCRs from Aedes aegypti, Anopheles gambiae, Apis Mellifera, Drosophila melanogaster, Homo sapiens, and Pediculus humanus. Predictions for unidentified sequences from Ae. aegypti, An. gambiae, and Pe. humanus were validated. Quantitative RT-PCR expression analysis was performed on previously-known and newly discovered Ae. aegypti GPCR genes. Results We present a new analysis of GPCRs in the genomes of Ae, aegypti, a vector of dengue fever, An. gambiae, a primary vector of Plasmodium falciparum that causes malaria, and Pe. humanus, a vector of epidemic typhus fever, using a novel GPCR classifier, Ensemble*, designed for insect vector species. We identified 30 additional putative GPCRs, 19 of which we validated. Expression of the newly discovered Ae. aegypti GPCR genes was confirmed via quantitative RT-PCR. Conclusion A novel GPCR classifier for insect vectors, Ensemble*, was developed and GPCR predictions were validated. Ensemble* and the validation pipeline were applied to the genomes of three insect vectors (Ae. aegypti, An. gambiae, and Pe. humanus), resulting in the identification of 52 GPCRs not previously identified, of which 11 are predicted GPCRs, and 19 are

  2. Harvest: an open-source tool for the validation and improvement of peptide identification metrics and fragmentation exploration

    PubMed Central

    2010-01-01

    Background Protein identification using mass spectrometry is an important tool in many areas of the life sciences, and in proteomics research in particular. Increasing the number of proteins correctly identified is dependent on the ability to include new knowledge about the mass spectrometry fragmentation process, into computational algorithms designed to separate true matches of peptides to unidentified mass spectra from spurious matches. This discrimination is achieved by computing a function of the various features of the potential match between the observed and theoretical spectra to give a numerical approximation of their similarity. It is these underlying "metrics" that determine the ability of a protein identification package to maximise correct identifications while limiting false discovery rates. There is currently no software available specifically for the simple implementation and analysis of arbitrary novel metrics for peptide matching and for the exploration of fragmentation patterns for a given dataset. Results We present Harvest: an open source software tool for analysing fragmentation patterns and assessing the power of a new piece of information about the MS/MS fragmentation process to more clearly differentiate between correct and random peptide assignments. We demonstrate this functionality using data metrics derived from the properties of individual datasets in a peptide identification context. Using Harvest, we demonstrate how the development of such metrics may improve correct peptide assignment confidence in the context of a high-throughput proteomics experiment and characterise properties of peptide fragmentation. Conclusions Harvest provides a simple framework in C++ for analysing and prototyping metrics for peptide matching, the core of the protein identification problem. It is not a protein identification package and answers a different research question to packages such as Sequest, Mascot, X!Tandem, and other protein identification

  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. PMID:12954162

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

    SciTech Connect

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

    1987-02-10

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

  5. 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...

  6. Learning score function parameters for improved spectrum identification in tandem mass spectrometry experiments

    PubMed Central

    Spivak, Marina; Bereman, Michael S.; MacCoss, Michael J.; Noble, William Stafford

    2012-01-01

    The identification of proteins from spectra derived from a tandem mass spectrometry experiment involves several challenges: matching each observed spectrum to a peptide sequence, ranking the resulting collection of peptide-spectrum matches, assigning statistical confidence estimates to the matches, and identifying the proteins. The present work addresses algorithms to rank peptide-spectrum matches. Many of these algorithms, such as PeptideProphet, IDPicker, or Q-ranker, follow similar methodology that includes representing peptide-spectrum matches as feature vectors and using optimization techniques to rank them. We propose a richer and more flexible feature set representation that is based on the parametrization of the SEQUEST XCorr score and that can be used by all of these algorithms. This extended feature set allows a more effective ranking of the peptide-spectrum matches based on the target-decoy strategy, in comparison to a baseline feature set devoid of these XCorr-based features. Ranking using the extended feature set gives 10–40% improvement in the number of distinct peptide identifications relative to a range of q-value thresholds. While this work is inspired by the model of the theoretical spectrum and the similarity measure between spectra used specifically by SEQUEST, the method itself can be applied to the output of any database search. Further, our approach can be trivially extended beyond XCorr to any linear operator that can serve as similarity score between experimental spectra and peptide sequences. PMID:22866926

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

    PubMed Central

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

    2016-01-01

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

  8. 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.

  9. 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. PMID:25561589

  10. Increased Protein Identification Capabilities Through Novel Tandem MS Calibration Strategies

    SciTech Connect

    Wu, Si; Kaiser, Nathan K.; Meng, Da; Anderson, Gordon A.; Zhang, Kai; Bruce, James E.

    2005-08-01

    High mass measurement accuracy is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is a unique technique which can provide unparalleled mass accuracy and resolving power. However, the mass measurement accuracy of FTICR-MS can be affected by space charge effects. Here we present a novel internal calibrant-free calibration method that corrects for space charge-induced frequency shifts in FTICR fragment spectra called Calibration Optimization on Fragment Ions (COFI). This new strategy utilizes the information from fixed mass differences between two neighboring peptide fragment ions (such as y1 and y2) to correct the frequency shift after data ollection. COFI has been successfully applied to LC-FTICR fragmentation data. Mascot MS/MS ion search data demonstrate that most of the fragments from BSA tryptic digested peptides can be identified using a much lower mass tolerance window after applying COFI to LC-FTICR-MS/MS of BSA tryptic digest. Furthermore, COFI has been used for multiplexed LC-CID-FTICR-MS which is an attractive technique because of its increased duty cycle and dynamic range. After the application of COFI to a multiplexed LC-CID-FTICR-MS of BSA tryptic digest, we achieved an average measured mass accuracy of 2.49 ppm for all the identified BSA fragments.

  11. Identification of HIV-1 Inhibitors Targeting The Nucleocapsid Protein

    PubMed Central

    Breuer, Sebastian; Chang, Max W.; Yuan, Jinyun; Torbett, Bruce E.

    2012-01-01

    The HIV-1 nucleocapsid (NC) is a RNA/DNA binding protein encoded within the Gag polyprotein, which is critical for the selection and chaperoning of viral genomic RNA during virion assembly. RNA/DNA binding occurs through a highly conserved zinc-knuckle motif present in NC. Given the necessity of NC-viral RNA/DNA interaction for viral replication, identification of compounds that disrupt the NC-RNA/DNA interaction may have value as an anti-viral strategy. To identify small molecules that disrupt NC-viral RNA/DNA binding a high-throughput fluorescence polarization assay was developed and a library of 14,400 diverse, drug-like compounds was screened. Compounds that disrupted NC binding to a fluorescence-labeled DNA tracer were next evaluated by differential scanning fluorimetry to identify compounds that must bind to NC or Gag to impart their effects. Two compounds were identified that inhibited NC-DNA interaction, specifically bound NC with nM affinity, and showed modest anti-HIV-1 activity in ex vivo cell assays. PMID:22587465

  12. Improved protein sequence coverage by on resin deglycosylation and cysteine modification for biomarker discovery.

    PubMed

    Kamada, Haruhiko; Fugmann, Tim; Neri, Dario; Roesli, Christoph

    2009-02-01

    Membrane proteins and secreted factors (soluble proteins or extracellular matrix components) are the targets of most monoclonal antibodies, which are currently in clinical development. These proteins are frequently post-translationally modified, e.g. by the formation of disulfide bonds or by glycosylation, which complicates their identification using proteomics technologies. Here, we describe a novel methodology for the on resin deglycosylation and cysteine modification of proteins after in vitro, in vivo or ex vivo biotinylation. Biotinylated proteins are captured on streptavidin resin and all subsequent modifications, as well as the proteolytic digestion, which yields peptides for MS analysis, are performed on resin. Using biotinylated bovine fetuin-A as a test protein, an improvement in sequence coverage from 7.9 to 58.7% could be shown, including the identification of all three glycosylation sites. Furthermore, a complex mixture derived from the ex vivo biotinylation of vascular structures in human kidney with cancer obtained by perfusion after surgical resection revealed almost a doubling of sequence coverage for all checked proteins when analyzed by LC-MALDI TOF/TOF. PMID:19137555

  13. Improving Peptide Identification Sensitivity in Shotgun Proteomics by Stratification of Search Space

    PubMed Central

    Alves, Gelio; Yu, Yi-Kuo

    2013-01-01

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

  14. Identification of local conformational similarity in structurally variable regions of homologous proteins using protein blocks.

    PubMed

    Agarwal, Garima; Mahajan, Swapnil; Srinivasan, Narayanaswamy; de Brevern, Alexandre G

    2011-01-01

    Structure comparison tools can be used to align related protein structures to identify structurally conserved and variable regions and to infer functional and evolutionary relationships. While the conserved regions often superimpose well, the variable regions appear non superimposable. Differences in homologous protein structures are thought to be due to evolutionary plasticity to accommodate diverged sequences during evolution. One of the kinds of differences between 3-D structures of homologous proteins is rigid body displacement. A glaring example is not well superimposed equivalent regions of homologous proteins corresponding to α-helical conformation with different spatial orientations. In a rigid body superimposition, these regions would appear variable although they may contain local similarity. Also, due to high spatial deviation in the variable region, one-to-one correspondence at the residue level cannot be determined accurately. Another kind of difference is conformational variability and the most common example is topologically equivalent loops of two homologues but with different conformations. In the current study, we present a refined view of the "structurally variable" regions which may contain local similarity obscured in global alignment of homologous protein structures. As structural alphabet is able to describe local structures of proteins precisely through Protein Blocks approach, conformational similarity has been identified in a substantial number of 'variable' regions in a large data set of protein structural alignments; optimal residue-residue equivalences could be achieved on the basis of Protein Blocks which led to improved local alignments. Also, through an example, we have demonstrated how the additional information on local backbone structures through protein blocks can aid in comparative modeling of a loop region. In addition, understanding on sequence-structure relationships can be enhanced through our approach. This has been

  15. 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

  16. A Method to Improve Mineral Identification Accuracy Based on Hyperspectral Data

    NASA Astrophysics Data System (ADS)

    Wang, Y. J.; Lin, Q. Z.; Wang, Q. J.; Chen, Y.

    2014-03-01

    To improve the mineral identification accuracy of the rapid quantificational identification model, the noise was filtered in fragment based on the wavelength of altered mineral absorption peak and the regional spectral library that fitted for the study area was established. The filtered spectra were analyzed by the method with regional spectral library. Compared with the originally mineral identification result, the average efficiency rate was improved by 5.1%; the average accuracy rate was improved by 17.7%. The results were optimized by the method based on the position of the altered mineral absorption peak. The average efficiency rate would be improved in the future to identify more accurate minerals.

  17. 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.

  18. EVEREST: automatic identification and classification of protein domains in all protein sequences

    PubMed Central

    Portugaly, Elon; Harel, Amir; Linial, Nathan; Linial, Michal

    2006-01-01

    Background Proteins are comprised of one or several building blocks, known as domains. Such domains can be classified into families according to their evolutionary origin. Whereas sequencing technologies have advanced immensely in recent years, there are no matching computational methodologies for large-scale determination of protein domains and their boundaries. We provide and rigorously evaluate a novel set of domain families that is automatically generated from sequence data. Our domain family identification process, called EVEREST (EVolutionary Ensembles of REcurrent SegmenTs), begins by constructing a library of protein segments that emerge in an all vs. all pairwise sequence comparison. It then proceeds to cluster these segments into putative domain families. The selection of the best putative families is done using machine learning techniques. A statistical model is then created for each of the chosen families. This procedure is then iterated: the aforementioned statistical models are used to scan all protein sequences, to recreate a library of segments and to cluster them again. Results Processing the Swiss-Prot section of the UniProt Knoledgebase, release 7.2, EVEREST defines 20,230 domains, covering 85% of the amino acids of the Swiss-Prot database. EVEREST annotates 11,852 proteins (6% of the database) that are not annotated by Pfam A. In addition, in 43,086 proteins (20% of the database), EVEREST annotates a part of the protein that is not annotated by Pfam A. Performance tests show that EVEREST recovers 56% of Pfam A families and 63% of SCOP families with high accuracy, and suggests previously unknown domain families with at least 51% fidelity. EVEREST domains are often a combination of domains as defined by Pfam or SCOP and are frequently sub-domains of such domains. Conclusion The EVEREST process and its output domain families provide an exhaustive and validated view of the protein domain world that is automatically generated from sequence data. The

  19. 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.

  20. 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. PMID:24259493

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

    PubMed

    Ursu, Andrei; Waldmann, Herbert

    2015-08-15

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

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

    SciTech Connect

    Dailey, D.C.

    1988-01-01

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

  3. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-01-01

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

  6. Improved Protocol for Rapid Identification of Certain Spa Types Using High Resolution Melting Curve Analysis

    PubMed Central

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

    2015-01-01

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

  7. Evaluation of parameters in peptide mass fingerprinting for protein identification by MALDI-TOF mass spectrometry.

    PubMed

    Lee, Kyunghee; Bae, Dongwon; Lim, Dongbin

    2002-04-30

    Protein identification by peptide mass fingerprinting, using the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), plays a major role in large proteome projects. In order to develop a simple and reliable method for protein identification by MALDI-TOF MS, we compared and evaluated the major steps in peptide mass fingerprinting. We found that the removal of excess enzyme from the in-gel digestion usually gave a few more peptide peaks, which were important for the identification of some proteins. Internal calibration always gave better results. However, for a large number of samples, two step calibrations (i.e. database search with peptide mass from external calibration, then the use of peptide masses from the search result as internal calibrants) were useful and convenient. From the evaluation and combination of steps that were already developed by others, we established a single overall procedure for peptide identification from a polyacrylamide gel. PMID:12018838

  8. 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…

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  10. Identification and isoprenylation of plant GTP-binding proteins.

    PubMed

    Biermann, B; Randall, S K; Crowell, D N

    1996-08-01

    To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies). PMID:8843944

  11. 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. PMID:26990122

  12. Improved Side Chain Dynamics in MARTINI Simulations of Protein-Lipid Interfaces.

    PubMed

    Herzog, Florian A; Braun, Lukas; Schoen, Ingmar; Vogel, Viola

    2016-05-10

    Specific interactions of protein side chains and lipid membranes regulate the localization, orientation, and activity of many peripheral proteins. Here, we introduce a modification of the coarse-grained MARTINI protein model, called 'side chain fix' (scFix), that was necessary and sufficient to correctly sample the side chain dynamics of β-strands in several globular proteins. When compared to μs long atomistic simulations or previous experimental findings, scFix MARTINI simulations reproduced all key interactions between the well-studied PLC-δ1 pleckstrin homology domain and a phosphatidylinositol-4,5-bisphosphate (PIP2) containing lipid membrane. Moreover, the extended runtime and higher sampling speed enabled the systematic mapping of the protein's rolling motion at the membrane, the identification of short-lived and stable binding orientations, as well as the verification and prediction of already known and of novel transient PIP2 binding sites. scFix also showed promise to maintain proper side chain orientation in other secondary structural motifs of the α-spectrin SH3 domain, the B1 domain of protein G, and the villin headpiece. This suggests that scFix improves on the predictive power of MARTINI simulations regarding protein-lipid and protein-ligand interactions. PMID:27042944

  13. IDENTIFICATION OF IMMUNOGENS OF 'MYCOPLASMA PNEUMONIAE' BY PROTEIN BLOTTING

    EPA Science Inventory

    Proteins of Mycoplasma pneumoniae were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet by blotting. Sera obtained from infected hamsters and immunized rabbits were then incubated with the nitrocellulose strips. Proteins which are capa...

  14. Discovery of active proteins directly from combinatorial randomized protein libraries without display, purification or sequencing: identification of novel zinc finger proteins

    PubMed Central

    Hughes, Marcus D.; Zhang, Zhan-Ren; Sutherland, Andrew J.; Santos, Albert F.; Hine, Anna V.

    2005-01-01

    We have successfully linked protein library screening directly with the identification of active proteins, without the need for individual purification, display technologies or physical linkage between the protein and its encoding sequence. By using ‘MAX’ randomization we have rapidly constructed 60 overlapping gene libraries that encode zinc finger proteins, randomized variously at the three principal DNA-contacting residues. Expression and screening of the libraries against five possible target DNA sequences generated data points covering a potential 40 000 individual interactions. Comparative analysis of the resulting data enabled direct identification of active proteins. Accuracy of this library analysis methodology was confirmed by both in vitro and in vivo analyses of identified proteins to yield novel zinc finger proteins that bind to their target sequences with high affinity, as indicated by low nanomolar apparent dissociation constants. PMID:15722478

  15. Improvement of protein structure comparison using a structural alphabet.

    PubMed

    Joseph, Agnel Praveen; Srinivasan, N; de Brevern, Alexandre G

    2011-09-01

    The three dimensional structure of a protein provides major insights into its function. Protein structure comparison has implications in functional and evolutionary studies. A structural alphabet (SA) is a library of local protein structure prototypes that can abstract every part of protein main chain conformation. Protein Blocks (PBs) is a widely used SA, composed of 16 prototypes, each representing a pentapeptide backbone conformation defined in terms of dihedral angles. Through this description, the 3D structural information can be translated into a 1D sequence of PBs. In a previous study, we have used this approach to compare protein structures encoded in terms of PBs. A classical sequence alignment procedure based on dynamic programming was used, with a dedicated PB Substitution Matrix (SM). PB-based pairwise structural alignment method gave an excellent performance, when compared to other established methods for mining. In this study, we have (i) refined the SMs and (ii) improved the Protein Block Alignment methodology (named as iPBA). The SM was normalized in regards to sequence and structural similarity. Alignment of protein structures often involves similar structural regions separated by dissimilar stretches. A dynamic programming algorithm that weighs these local similar stretches has been designed. Amino acid substitutions scores were also coupled linearly with the PB substitutions. iPBA improves (i) the mining efficiency rate by 6.8% and (ii) more than 82% of the alignments have a better quality. A higher efficiency in aligning multi-domain proteins could be also demonstrated. The quality of alignment is better than DALI and MUSTANG in 81.3% of the cases. Thus our study has resulted in an impressive improvement in the quality of protein structural alignment. PMID:21569819

  16. Towards the identification of cassava root protein genes.

    PubMed

    De Souza, C R B; Carvalho, L J C B; De Almeida, E R P; Gander, E S

    2002-01-01

    The protein population of cassava root layers was characterized by SDS-PAGE and bidimensional polyacrylamide gel electrophoresis. SDS-Page revealed the presence of a protein population in the molecular weight range between 94 and 20 kDa. The expression pattern of these proteins was well-defined within the different layers. Partial protein sequence analyses and preliminary results on the layer-specific expression pattern obtained with Northern analyses are presented. PMID:12602941

  17. Functional Module Search in Protein Networks based on Semantic Similarity Improves the Analysis of Proteomics Data*

    PubMed Central

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

    2014-01-01

    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. PMID:24807868

  18. Identification and Location of Brain Protein 4.1

    NASA Astrophysics Data System (ADS)

    Goodman, Steven R.; Casoria, Linda A.; Coleman, Diane B.; Zagon, Ian S.

    1984-06-01

    Protein 4.1 is a membrane skeletal protein that converts the low-affinity interaction between spectrin and actin into a high-affinity ternary complex of spectrin, protein 4.1, and actin that is essential to the structural stability of the erythrocyte. Pig brain was shown to contain an 87-kilodalton immunoreactive analog of protein 4.1 that has partial sequence homology with pig erythrocyte protein 4.1 and the same location as spectrin in the cortical cytoplasm of neuronal and glial cell types of the cerebellum.

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

    NASA Astrophysics Data System (ADS)

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

    1982-12-01

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

  20. Identification of Renibacterium salmoninarum surface proteins by radioiodination.

    PubMed

    Fredriksen, A; Bakken, V

    1994-09-01

    Surface exposed proteins of Renibacterium salmoninarum were identified by radiolabelling whole bacterial cells with 125I, followed by SDS-PAGE and autoradiography. The most prominent bands had molecular masses of approximately 57 kDa and 22 kDa; in addition, some less intensively labelled bands were detected. Polyclonal sera raised against the 22 kDa protein did not react with the 57 kDa protein. N-terminal amino acid sequence analysis of the purified 22 kDa protein showed no similarity with the sequence of the 57 kDa protein. PMID:7926685

  1. Identification of sumoylated proteins in the silkworm Bombyx mori.

    PubMed

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

    2014-01-01

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

  2. 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.

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

    PubMed

    Bidlingmaier, Scott; Liu, Bin

    2015-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology. PMID:26060076

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

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2016-01-01

    The identification of proteins that interact specifically with posttranslational modifications such as phosphorylation is often necessary to understand cellular signaling pathways. Numerous methods for identifying proteins that interact with posttranslational modifications have been utilized, including affinity-based purification and analysis, protein microarrays, phage display, and tethered catalysis. Although these techniques have been used successfully, each has limitations. Recently, yeast surface-displayed human proteome libraries have been utilized to identify protein fragments with affinity for various target molecules, including phosphorylated peptides. When coupled with fluorescently activated cell sorting and high throughput methods for the analysis of selection outputs, yeast surface-displayed human proteome libraries can rapidly and efficiently identify protein fragments with affinity for any soluble ligand that can be fluorescently detected, including posttranslational modifications. In this review we compare the use of yeast surface display libraries to other methods for the identification of interactions between proteins and posttranslational modifications and discuss future applications of the technology. PMID:26060076

  5. Development of a strategy for the identification of surface proteins in the pathogenic microsporidian Nosema bombycis.

    PubMed

    Zhao, Weixi; Hao, Youjin; Wang, Linglin; Zhou, Zeyang; Li, Zhi

    2015-06-01

    Parasite-host interactions mediated by cell surface proteins have been implicated as a critical step in infections caused by the microsporidian Nosema bombycis. Such cell surface proteins are considered as promising diagnostic markers and targets for drug development. However, little research has specifically addressed surface proteome identification in microsporidia due to technical barriers. Here, a combined strategy was developed to separate and identify the surface proteins of N. bombycis. Briefly, following (1) biotinylation of the spore surface, (2) extraction of total proteins with an optimized method and (3) streptavidin affinity purification of biotinylated proteins, 22 proteins were identified based on LC-MS/MS analysis. Among them, 5 proteins were confirmed to be localized on the surface of N. bombycis. A total of 8 proteins were identified as hypothetical extracellular proteins, whereas 7 other hypothetical proteins had no available function annotation. Furthermore, a protein with a molecular weight of 18·5 kDa was localized on the spore surface by western blotting and immunofluorescence analysis, even though it was predicted to be a nuclear protein by bioinformatics. Collectively, our work provides an effective strategy for isolating microsporidian surface protein components for both drug target identification and further diagnostic research on microsporidian disease control. PMID:25811320

  6. Identification of protein-protein interaction and topologies in living cells by chemical cross-linking and mass spectrometry

    SciTech Connect

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

    2008-10-20

    Current chemical cross-linking methods are commonly employed for mapping sites of interaction and three-dimensional structure in purified, known protein complexes. When applied in vivo in combination of co-immunoprecipitation methods, information on the sites of interaction between proteins are unattainable due to overwhelming sample complexity. We present results from a novel cross-linking strategy that allow simultaneous protein-protein interaction and surface topology measurement in vivo without any prior knowledge of the system. The strategy consists of: (i) cross-linking reaction: intact cell labeling with protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by 2D-LC/MS/MS; and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. This strategy was applied to Shewanella oneidensis MR-1 bacterial cells and successfully identified a protein-protein interaction between SecA and a small outer membrane lipoprotein as well as their sites of interaction in vivo.

  7. Identification of urinary protein biomarkers for tobacco smoking.

    PubMed

    Haniff, Aj Nabill; Gam, Lay-Harn

    2016-03-01

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

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

    PubMed

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

    2016-07-01

    As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii. PMID:27053129

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

    PubMed

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

    2014-07-01

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

  10. 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

  11. Identification of mRNA bound to RNA binding proteins by differential display.

    PubMed

    Carr-Schmid, Anne; Jiao, Xinfu; Kiledjian, Megerditch

    2006-01-01

    A large number of RNA binding proteins have recently been identified that influence various human genetic disorders. However, the specific function of many of these proteins and what role they may play in a particular disease remains unclear. Identification of the substrate mRNA bound by an RNA binding protein will provide insights into the function of that protein and how its aberrant expression could lead to a disease phenotype. We have developed a technique termed SNAAP, for isolation of specific nucleic acids associated with proteins, to identify natural mRNA substrates for an RNA binding protein. The technique couples affinity purification of specific mRNAs bound by an RNA binding protein, with the identification of that mRNA using differential display (DD). Methods are described herein for the isolation and identification of endogenous mRNAs bound by any RNA binding protein, as well as methodology to validate the specificity of the binding. The availability of technologies to isolate the cognate substrate mRNAs potentially bound and regulated by an RNA binding protein involved in genetic disorders will greatly expedite our etiological understanding of the disorder and provide modalities for intervention. PMID:16264238

  12. Proteomic identification of germline proteins in Caenorhabditis elegans

    PubMed Central

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

    2015-01-01

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

  13. Identification of ultramodified proteins using top-down spectra

    SciTech Connect

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

    2013-04-10

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

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

    SciTech Connect

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

    2013-11-05

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

  15. 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.

  16. Proteomic Profiling and Protein Identification by MALDI-TOF Mass Spectrometry in Unsequenced Parasitic Nematodes

    PubMed Central

    Millares, Paul; LaCourse, E. James; Perally, Samirah; Ward, Deborah A.; Prescott, Mark C.; Hodgkinson, Jane E.; Brophy, Peter M.; Rees, Huw H.

    2012-01-01

    Lack of genomic sequence data and the relatively high cost of tandem mass spectrometry have hampered proteomic investigations into helminths, such as resolving the mechanism underpinning globally reported anthelmintic resistance. Whilst detailed mechanisms of resistance remain unknown for the majority of drug-parasite interactions, gene mutations and changes in gene and protein expression are proposed key aspects of resistance. Comparative proteomic analysis of drug-resistant and -susceptible nematodes may reveal protein profiles reflecting drug-related phenotypes. Using the gastro-intestinal nematode, Haemonchus contortus as case study, we report the application of freely available expressed sequence tag (EST) datasets to support proteomic studies in unsequenced nematodes. EST datasets were translated to theoretical protein sequences to generate a searchable database. In conjunction with matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), Peptide Mass Fingerprint (PMF) searching of databases enabled a cost-effective protein identification strategy. The effectiveness of this approach was verified in comparison with MS/MS de novo sequencing with searching of the same EST protein database and subsequent searches of the NCBInr protein database using the Basic Local Alignment Search Tool (BLAST) to provide protein annotation. Of 100 proteins from 2-DE gel spots, 62 were identified by MALDI-TOF-MS and PMF searching of the EST database. Twenty randomly selected spots were analysed by electrospray MS/MS and MASCOT Ion Searches of the same database. The resulting sequences were subjected to BLAST searches of the NCBI protein database to provide annotation of the proteins and confirm concordance in protein identity from both approaches. Further confirmation of protein identifications from the MS/MS data were obtained by de novo sequencing of peptides, followed by FASTS algorithm searches of the EST putative protein database. This

  17. Identification of new palmitoylated proteins in Toxoplasma gondii.

    PubMed

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

    2016-04-01

    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 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

  18. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-07-01

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

  20. Identification of new modulators and protein alterations in non-apoptotic programmed cell death.

    PubMed

    Sperandio, Sabina; Poksay, Karen S; Schilling, Birgit; Crippen, Danielle; Gibson, Bradford W; Bredesen, Dale E

    2010-12-15

    This study describes the first proteomic analysis of paraptosis--a non-apoptotic form of programmed cell death. As with apoptosis, the first description of paraptosis was based on morphological criteria. Since there are no known markers for paraptosis, the purpose of this study was to dissect changes in the proteome profile occurring during paraptosis. Using one- and two-dimensional SDS-PAGE, Western analysis, and mass spectrometry, we show that during paraptosis, alterations occur mainly in cytoskeletal proteins, signal transduction proteins, mitochondrial proteins, and some metabolic proteins. We also report the identification of: (1) a paraptosis inhibitor, phosphatidylethanolamine binding protein (PEBP-1), and (2) a candidate mediator of paraptosis, prohibitin. Identification of specific paraptotic changes will ultimately lead to tools to detect this type of programmed cell death in in vivo systems and allow for its further characterization. PMID:20830744

  1. 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. PMID:25820699

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

    PubMed

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

    1997-04-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2014-12-01

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

  7. Identification of prognostic biomarkers for glioblastomas using protein expression profiling

    PubMed Central

    JUNG, YONG; JOO, KYEUNG MIN; SEONG, DONG HO; CHOI, YOON-LA; KONG, DOO-SIK; KIM, YONGHYUN; KIM, MI HYUN; JIN, JUYOUN; SUH, YEON-LIM; SEOL, HO JUN; SHIN, CHUL SOO; LEE, JUNG-IL; KIM, JONG-HYUN; SONG, SANG YONG; NAM, DO-HYUN

    2012-01-01

    A set of proteins reflecting the prognosis of patients have clinical significance since they could be utilized as predictive biomarkers and/or potential therapeutic targets. With the aim of finding novel diagnostic and prognostic markers for glioblastoma (GBM), a tissue microarray (TMA) library consisting of 62 GBMs and 28 GBM-associated normal spots was constructed. Immunohistochemistry against 78 GBM-associated proteins was performed. Expression levels of each protein for each patient were analyzed using an image analysis program and converted to H-score [summation of the intensity grade of staining (0–3) multiplied by the percentage of positive cells corresponding to each grade]. Based on H-score and hierarchical clustering methods, we divided the GBMs into two groups (n=19 and 37) that had significantly different survival lengths (p<0.05). In the two groups, expression of nine proteins (survivin, cyclin E, DCC, TGF-β, CDC25B, histone H1, p-EGFR, p-VEGFR2/3, p16) was significantly changed (q<0.05). Prognosis-predicting potential of these proteins were validated with another independent library of 82 GBM TMAs and a public GBM DNA microarray dataset. In addition, we determined 32 aberrant or mislocalized subcellular protein expression patterns in GBMs compared with relatively normal brain tissues, which could be useful for diagnostic biomarkers of GBM. We therefore suggest that these proteins can be used as predictive biomarkers and/or potential therapeutic targets for GBM. PMID:22179774

  8. Identification of Novel Amelogenin-Binding Proteins by Proteomics Analysis

    PubMed Central

    Fukuda, Takao; Sanui, Terukazu; Toyoda, Kyosuke; Tanaka, Urara; Taketomi, Takaharu; Uchiumi, Takeshi; Nishimura, Fusanori

    2013-01-01

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

  9. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Osterlund, Tobias; Liu, Zihe; Petranovic, Dina; Nielsen, Jens

    2013-04-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular stress response, which facilitates the cell recovery from many forms of stress, e.g., heat stress. In S. cerevisiae, HSR is regulated mainly by the transcription factor heat shock factor (Hsf1p) and many of its targets are genes coding for molecular chaperones that promote protein folding and prevent the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high level over-expression of HSF1-R206S increased heterologous α-amylase yield 25 and 70 % when glucose was fully consumed, and 37 and 62 % at the end of the ethanol phase, respectively. Moderate and high level over-expression also improved endogenous invertase yield 118 and 94 %, respectively. However, human insulin precursor was only improved slightly and this only by high level over-expression of HSF1-R206S, supporting our previous findings that the production of this protein in S. cerevisiae is not limited by secretion. Our results provide an effective strategy to improve protein secretion and demonstrated an approach that can induce ER and cytosolic chaperones simultaneously. PMID:23208612

  10. Identification of potential protein interactors of Lrrk2.

    PubMed

    Dächsel, Justus C; Taylor, Julie P; Mok, Su San; Ross, Owen A; Hinkle, Kelly M; Bailey, Rachel M; Hines, Jacob H; Szutu, Jennifer; Madden, Benjamin; Petrucelli, Leonard; Farrer, Matthew J

    2007-10-01

    Pathogenic substitutions in the Lrrk2 protein have been shown to be an important cause of both familial and sporadic parkinsonism. The molecular pathway involved in Lrrk2 dopaminergic neuron degeneration remains elusive. Employing a combination of Lrrk2-mediated protein precipitation and tandem mass spectrometry, we identified 14 potential Lrrk2 binding partners. The majority of these interactions may be subgrouped into three functional cellular pathways: (i) chaperone-mediated response, (ii) proteins associated with the cytoskeleton and trafficking and (iii) phosphorylation and kinase activity. Future investigation of these candidates is now warranted and may help resolve the pathomechanism behind Lrrk2 neurodegeneration. PMID:17400507

  11. Bar code technology improves positive patient identification and transfusion safety.

    PubMed

    Sandler, S G; Langeberg, A; Dohnalek, L

    2005-01-01

    As a result of human error, an estimated 1 in 12,000 blood transfusions is given to the wrong patient. The cause of nearly all of these errors is failure of hospital personnel to identify positively intended transfusion recipients, their blood samples for cross-matching, or their correct blood components. We describe our experience using a point-of-care bar code transfusion safety system that links patients' bar-coded wristbands, with bar-coded labels on blood sample tubes, blood component bags, and nurses' identification badges. The result was 100 % accuracy of matching patients, their blood samples, and components for transfusions. For verifying information before starting blood transfusions, nurses preferred bar code "double checks" to conventional visual "double checks" by a second nurse. Methods are needed to reinforce nurses' proficiency with technological approaches to transfusion safety, such as software-driven bar code scanning, in situations where transfusions are administered infrequently. PMID:16050151

  12. Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression.

    PubMed

    Kurotani, Atsushi; Takagi, Tetsuo; Toyama, Mitsutoshi; Shirouzu, Mikako; Yokoyama, Shigeyuki; Fukami, Yasuo; Tokmakov, Alexander A

    2010-04-01

    High-throughput cell-free protein synthesis is being used increasingly in structural/functional genomics projects. However, the factors determining expression success are poorly understood. Here, we evaluated the expression of 3066 human proteins and their domains in a bacterial cell-free system and analyzed the correlation of protein expression with 39 physicochemical and structural properties of proteins. As a result of the bioinformatics analysis performed, we determined the 18 most influential features that affect protein amenability to cell-free expression. They include protein length; hydrophobicity; pI; content of charged, nonpolar, and aromatic residues;, cysteine content; solvent accessibility; presence of coiled coil; content of intrinsically disordered and structured (alpha-helix and beta-sheet) sequence; number of disulfide bonds and functional domains; presence of transmembrane regions; PEST motifs; and signaling sequences. This study represents the first comprehensive bioinformatics analysis of heterologous protein synthesis in a cell-free system. The rules and correlations revealed here provide a plethora of important insights into rationalization of cell-free protein production and can be of practical use for protein engineering with the aim of increasing expression success.-Kurotani, A., Takagi, T., Toyama, M., Shirouzu, M., Yokoyama, S., Fukami, Y., Tokmakov, A. A. Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression. PMID:19940260

  13. Large-scale identification of potential drug targets based on the topological features of human protein-protein interaction network.

    PubMed

    Li, Zhan-Chao; Zhong, Wen-Qian; Liu, Zhi-Qing; Huang, Meng-Hua; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2015-04-29

    Identifying potential drug target proteins is a crucial step in the process of drug discovery and plays a key role in the study of the molecular mechanisms of disease. Based on the fact that the majority of proteins exert their functions through interacting with each other, we propose a method to recognize target proteins by using the human protein-protein interaction network and graph theory. In the network, vertexes and edges are weighted by using the confidence scores of interactions and descriptors of protein primary structure, respectively. The novel network topological features are defined and employed to characterize protein using existing databases. A widely used minimum redundancy maximum relevance and random forests algorithm are utilized to select the optimal feature subset and construct model for the identification of potential drug target proteins at the proteome scale. The accuracies of training set and test set are 89.55% and 85.23%. Using the constructed model, 2127 potential drug target proteins have been recognized and 156 drug target proteins have been validated in the database of drug target. In addition, some new drug target proteins can be considered as targets for treating diseases of mucopolysaccharidosis, non-arteritic anterior ischemic optic neuropathy, Bernard-Soulier syndrome and pseudo-von Willebrand, etc. It is anticipated that the proposed method may became a powerful high-throughput virtual screening tool of drug target. PMID:25847157

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

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Responsibilities resulting from identification for school improvement. 200.39 Section 200.39 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  15. 34 CFR 200.37 - Notice of identification for improvement, corrective action, or restructuring.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 34 Education 1 2012-07-01 2012-07-01 false Notice of identification for improvement, corrective action, or restructuring. 200.37 Section 200.37 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  16. 34 CFR 200.37 - Notice of identification for improvement, corrective action, or restructuring.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Notice of identification for improvement, corrective action, or restructuring. 200.37 Section 200.37 Education Regulations of the Offices of the Department of Education OFFICE OF ELEMENTARY AND SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING...

  17. 34 CFR 200.37 - Notice of identification for improvement, corrective action, or restructuring.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

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

  18. 34 CFR 200.37 - Notice of identification for improvement, corrective action, or restructuring.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

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

  19. Identification of novel sweet protein for nutritional applications

    PubMed Central

    Gnanavel, Mutharasu; Serva Peddha, Muthukumar

    2011-01-01

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

  20. Identification of novel sweet protein for nutritional applications.

    PubMed

    Gnanavel, Mutharasu; Serva Peddha, Muthukumar

    2011-01-01

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

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

    PubMed

    Wernisch, L; Hunting, M; Wodak, S J

    1999-05-15

    A novel automatic procedure for identifying domains from protein atomic coordinates is presented. The procedure, termed STRUDL (STRUctural Domain Limits), does not take into account information on secondary structures and handles any number of domains made up of contiguous or non-contiguous chain segments. The core algorithm uses the Kernighan-Lin graph heuristic to partition the protein into residue sets which display minimum interactions between them. These interactions are deduced from the weighted Voronoi diagram. The generated partitions are accepted or rejected on the basis of optimized criteria, representing basic expected physical properties of structural domains. The graph heuristic approach is shown to be very effective, it approximates closely the exact solution provided by a branch and bound algorithm for a number of test proteins. In addition, the overall performance of STRUDL is assessed on a set of 787 representative proteins from the Protein Data Bank by comparison to domain definitions in the CATH protein classification. The domains assigned by STRUDL agree with the CATH assignments in at least 81% of the tested proteins. This result is comparable to that obtained previously using PUU (Holm and Sander, Proteins 1994;9:256-268), the only other available algorithm designed to identify domains with any number of non-contiguous chain segments. A detailed discussion of the structures for which our assignments differ from those in CATH brings to light some clear inconsistencies between the concept of structural domains based on minimizing inter-domain interactions and that of delimiting structural motifs that represent acceptable folding topologies or architectures. Considering both concepts as complementary and combining them in a layered approach might be the way forward. PMID:10328269

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

    PubMed

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

    2015-03-13

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

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

    PubMed Central

    Moore, Rowan E; Knottenbelt, Derek; Matthews, Jacqueline B; Beynon, Robert J; Whitfield, Phillip D

    2008-01-01

    Background Ingestion of the poisonous weed ragwort (Senecio jacobea) by horses leads to irreversible liver damage. The principal toxins of ragwort are the pyrrolizidine alkaloids that are rapidly metabolised to highly reactive and cytotoxic pyrroles, which can escape into the circulation and bind to proteins. In this study a non-invasive in vitro model system has been developed to investigate whether pyrrole toxins induce specific modifications of equine blood proteins that are detectable by proteomic methods. Results One dimensional gel electrophoresis revealed a significant alteration in the equine plasma protein profile following pyrrole exposure and the formation of a high molecular weight protein aggregate. Using mass spectrometry and confirmation by western blotting the major components of this aggregate were identified as fibrinogen, serum albumin and transferrin. Conclusion These findings demonstrate that pyrrolic metabolites can modify equine plasma proteins. The high molecular weight aggregate may result from extensive inter- and intra-molecular cross-linking of fibrinogen with the pyrrole. This model has the potential to form the basis of a novel proteomic strategy aimed at identifying surrogate protein biomarkers of ragwort exposure in horses and other livestock. PMID:18691403

  4. 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.

  5. Process Correlation Analysis Model for Process Improvement Identification

    PubMed Central

    Park, Sooyong

    2014-01-01

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

  6. Affinity chromatographic selection of carbonylated proteins followed by identification of oxidation sites using tandem mass spectrometry.

    PubMed

    Mirzaei, Hamid; Regnier, Fred

    2005-04-15

    It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of a wide variety of routes by which proteins are oxidized is the generation of carbonyl groups. This paper reports a method for the isolation of oxidized proteins, which involves (1) biotinylation of oxidized proteins with biotin hydrazide and (2) affinity enrichment using monomeric avidin affinity chromatography columns. The selectivity of the method was validated by adding in vitro oxidized biotinylated BSA to a yeast lysate and showing that the predominant protein recovered was BSA. This method was applied to the question of whether large doses of 2-nitropropane produce oxidized proteins. A study of rat liver homogenates showed that animals dosed with 2-nitropropane produced 17 times more oxidized protein than controls in 6 h. Tryptic digestion of these oxidized proteins followed by reversed-phase chromatography and tandem mass spectrometry led to the identification of 14 peptides and their parent proteins. Nine of the 14 identified peptides were found to carry 1 or 2 oxidation sites and 5 of the 9 peptides were biotinylated. The significance of this affinity method is that it allows the isolation of oxidized proteins from the rest of the proteome and facilitates their identification. In some cases, it is even possible to identify the site of oxidation. PMID:15828771

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

    PubMed Central

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

    2013-01-01

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

  8. 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

  9. Non-targeted Identification of Prions and Amyloid-forming Proteins from Yeast and Mammalian Cells*

    PubMed Central

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

    2013-01-01

    The formation of amyloid aggregates is implicated both as a primary cause of cellular degeneration in multiple human diseases and as a functional mechanism for providing extraordinary strength to large protein assemblies. The recent identification and characterization of several amyloid proteins from diverse organisms argues that the amyloid phenomenon is widespread in nature. Yet identifying new amyloid-forming proteins usually requires a priori knowledge of specific candidates. Amyloid fibers can resist heat, pressure, proteolysis, and denaturation by reagents such as urea or sodium dodecyl sulfate. Here we show that these properties can be exploited to identify naturally occurring amyloid-forming proteins directly from cell lysates. This proteomic-based approach utilizes a novel purification of amyloid aggregates followed by identification by mass spectrometry without the requirement for special genetic tools. We have validated this technique by blind identification of three amyloid-based yeast prions from laboratory and wild strains and disease-related polyglutamine proteins expressed in both yeast and mammalian cells. Furthermore, we found that polyglutamine aggregates specifically recruit some stress granule components, revealing a possible mechanism of toxicity. Therefore, core amyloid-forming proteins as well as strongly associated proteins can be identified directly from cells of diverse origin. PMID:23926098

  10. High sensitivity identification of membrane proteins by MALDI TOF-MASS spectrometry using polystyrene beads.

    PubMed

    Bensalem, Noura; Masscheleyn, Sandrine; Mozo, Julien; Vallée, Benoit; Brouillard, Franck; Trudel, Stéphanie; Ricquier, Daniel; Edelman, Aleksander; Guerrera, Ida Chiara; Miroux, Bruno

    2007-04-01

    Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents. PMID:17355127

  11. Identification of cancer protein biomarkers using proteomic techniques

    SciTech Connect

    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.

  12. 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.

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

    SciTech Connect

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

    2010-08-01

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

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

    PubMed

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

    2010-08-01

    Disulfide bonds are a form of post-translational modification that often determines protein structure(s) and function(s). In this work, we report a mass spectrometry method for identification of disulfides in degradation products of proteins, specifically endogenous peptides in the human blood plasma peptidome. LC-Fourier transform tandem mass spectrometry (FT MS/MS) was used for acquiring mass spectra that were de novo sequenced and then searched against the IPI human protein database. Through the use of unique sequence tags (UStags), we unambiguously correlated the spectra to specific database proteins. Examination of the UStags' prefix and/or suffix sequences that contain cysteine(s) in conjunction with sequences of the UStags-specified database proteins is shown to enable the unambigious determination of disulfide bonds. Using this method, we identified the intermolecular and intramolecular disulfides in human blood plasma peptidome peptides that have molecular weights of up to approximately 10 kDa. PMID:20590115

  15. 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.

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

    PubMed Central

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

    2011-01-01

    Background Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now. Results By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141) of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system. Conclusions The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work. PMID:22140462

  17. Identification of diagnostic serum protein profiles of glioblastoma patients.

    PubMed

    Elstner, Anja; Stockhammer, Florian; Nguyen-Dobinsky, Trong-Nghia; Nguyen, Quang Long; Pilgermann, Ingo; Gill, Amanjit; Guhr, Anke; Zhang, Tingguo; von Eckardstein, Kajetan; Picht, Thomas; Veelken, Julian; Martuza, Robert L; von Deimling, Andreas; Kurtz, Andreas

    2011-03-01

    Diagnosis of a glioblastoma (GBM) is triggered by the onset of symptoms and is based on cerebral imaging and histological examination. Serum-based biomarkers may support detection of GBM. Here, we explored serum protein concentrations of GBM patients and used data mining to explore profiles of biomarkers and determine whether these are associated with the clinical status of the patients. Gene and protein expression data for astrocytoma and GBM were used to identify secreted proteins differently expressed in tumors and in normal brain tissues. Tumor expression and serum concentrations of 14 candidate proteins were analyzed for 23 GBM patients and nine healthy subjects. Data-mining methods involving all 14 proteins were used as an initial evaluation step to find clinically informative profiles. Data mining identified a serum protein profile formed by BMP2, HSP70, and CXCL10 that enabled correct assignment to the GBM group with specificity and sensitivity of 89 and 96%, respectively (p < 0.0001, Fischer's exact test). Survival for more than 15 months after tumor resection was associated with a profile formed by TSP1, HSP70, and IGFBP3, enabling correct assignment in all cases (p < 0.0001, Fischer's exact test). No correlation was found with tumor size or age of the patient. This study shows that robust serum profiles for GBM may be identified by data mining on the basis of a relatively small study cohort. Profiles of more than one biomarker enable more specific assignment to the GBM and survival group than those based on single proteins, confirming earlier attempts to correlate single markers with cancer. These conceptual findings will be a basis for validation in a larger sample size. PMID:20617365

  18. Proteomic identification of secreted proteins of Propionibacterium acnes

    PubMed Central

    2010-01-01

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

  19. 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

  20. Identification of the interaction between vimentin and nucleocapsid protein of transmissible gastroenteritis virus.

    PubMed

    Zhang, Xin; Shi, HongYan; Chen, JianFei; Shi, Da; Dong, Hui; Feng, Li

    2015-03-16

    Nucleocapsid (N) protein of transmissible gastroenteritis virus (TGEV) packages viral RNA genome to form a ribonucleoprotein complex. In addition to its function as a structural protein, N protein is involved in cell apoptosis or cell-cycle regulation. N protein possibly interacts with host factors to modulate cellular functions. To identify cellular proteins that interacted with N protein of TGEV, methods of GST pull-down and Co-IP were utilized to precipitate cellular proteins of swine testicular (ST). Bound cellular proteins were resolved by SDS-PAGE. Analysis of interacting proteins by mass spectrometry allowed identification of 15 cellular protein bands representative of 12 cellular proteins including vimentin that bound to N protein. Furthermore, the function of vimentin cytoskeleton in ST cells during TGEV infection was examined. Vimentin cytoskeleton was required for virus replication. The present study thus provides protein-related information about interaction of TGEV N protein with host cell that should be useful for understanding host cell response to coronavirus pathogenesis infection and the underlying mechanism of coronavirus replication. PMID:25533531

  1. 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

  2. Identification of three novel Toxoplasma gondii rhoptry proteins

    PubMed Central

    Camejo, Ana; Gold, Daniel A.; Lu, Diana; McFetridge, Kiva; Julien, Lindsay; Yang, Ninghan; Jensen, Kirk D. C.; Saeij, Jeroen P.J

    2013-01-01

    The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterize the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry candidate genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes across its cell cycle. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice. PMID:24070999

  3. 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

  4. Preliminary identification of differentially expressed tear proteins in keratoconus

    PubMed Central

    Wasinger, Valerie C.; Pye, David C.; Willcox, Mark D. P.

    2013-01-01

    Purpose To examine the proteins differentially expressed in the tear film of people with keratoconus and normal subjects. Methods Unstimulated tears from people with keratoconus (KC) and controls (C) were collected using a capillary tube. Tear proteins from people with KC and controls were partitioned using a novel in-solution electrophoresis, Microflow 10 (ProteomeSep), and analyzed using linear ion trap quadrupole fourier transform mass spectrometry. Spectral counting was used to quantify the individual tear proteins. Results Elevated levels of cathepsin B (threefold) were evident in the tears of people with KC. Polymeric immunoglobulin receptor (ninefold), fibrinogen alpha chain (eightfold), cystatin S (twofold), and cystatin SN (twofold) were reduced in tears from people with KC. Keratin type-1 cytoskeletal-14 and keratin type-2 cytoskeletal-5 were present only in the tears of people with KC. Conclusions The protein changes in tears, that is, the decrease in protease inhibitors and increase in proteases, found in the present and other previously published studies reflect the pathological events involved in KC corneas. Further investigations into tear proteins may help elucidate the underlying molecular mechanisms of KC, which could result in better treatment options. PMID:24194634

  5. An improved SPE method for fractionation and identification of phospholipids.

    PubMed

    Fauland, Alexander; Trötzmüller, Martin; Eberl, Anita; Afiuni-Zadeh, Somaieh; Köfeler, Harald; Guo, Xinghua; Lankmayr, Ernst

    2013-02-01

    This work reports an efficient and universal SPE method developed for separation and identification of phospholipids derived from complex biological samples. For the separation step, sequential combination of silica gel-aminopropyl-silica gel SPE cartridges is applied. This setup enables separation of phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, phosphatidylserine, cardiolipin, and sphingomyelin into four fractions according to the polarity of their headgroups. Sample acquisition of the SPE fractions is performed by a high-resolution LC-MS system consisting of a hybrid linear IT Fourier transform ion cyclotron resonance mass spectrometer coupled to RP-HPLC. The unequivocal advantage of our SPE sample preparation setup is avoidance of analyte peak overlapping in the determination step done by RP-HPLC. Overlapping phospholipid signals would otherwise exert adverse ion suppression effects. An additional benefit of this method is the elimination of polar and nonpolar (e.g. neutral lipids) contaminants from the phospholipid fractions, which highly reduces contamination of the LC-MS system. The method was validated with fermentation samples of organic waste, where 78 distinct phospholipid and sphingomyelin species belonging to six lipid classes were successfully identified. PMID:23349108

  6. Identification of an iron-binding protein of the Dps family expressed by Streptococcus thermophilus.

    PubMed

    Nicodème, Muriel; Perrin, Clarisse; Hols, Pascal; Bracquart, Patrice; Gaillard, Jean-Luc

    2004-01-01

    Streptococcus thermophilus PB18 can grow between 20 degrees and 52 degrees C and is resistant to various stresses such as heat, acidic or cold shock. During cold shock, a protein of 21.5 kDa was previously shown to be induced in S. thermophilus. In addition to its cold-shock induction, 2D-PAGE revealed that the 21.5-kDa protein was also expressed during the stationary phase of growth. The recent access to the genome sequence of S. thermophilus LMG18311 allowed the identification of a 173-amino acid protein displaying a strong homology between the 21.5-kDa protein and members of the Dps family of proteins. Specific staining of non-denaturing polyacrylamide gel electrophoresis (ND-PAGE) followed by two-dimensional PAGE (2D-PAGE) showed that the 21.5-kDa protein was an iron-binding protein. PMID:15018103

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

    PubMed Central

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

    2010-01-01

    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. PMID:20542033

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

    PubMed Central

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

    2014-01-01

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

  9. Mass Spectrometry-based Proteomics: Qualitative Identification to Activity-based Protein Profiling

    PubMed Central

    Cardoza, Job D.; Parikh, Jignesh R.; Ficarro, Scott B.; Marto, Jarrod A.

    2011-01-01

    Mass spectrometry has become the method of choice for proteome characterization, including multi-component protein complexes (typically tens to hundreds of proteins) and total protein expression (up to tens of thousands of proteins), in biological samples. Qualitative sequence assignment based on MS/MS spectra is relatively well-defined, while statistical metrics for relative quantification have not completely stabilized. Nonetheless, proteomics studies have progressed to the point whereby various gene-, pathway-, or network-oriented computational frameworks may be used to place mass spectrometry data into biological context. Despite this progress, the dynamic range of protein expression remains a significant hurdle, and impedes comprehensive proteome analysis. Methods designed to enrich specific protein classes have emerged as an effective means to characterize enzymes or other catalytically active proteins that are otherwise difficult to detect in typical discovery mode proteomics experiments. Collectively, these approaches will facilitate identification of biomarkers and pathways relevant to diagnosis and treatment of human disease. PMID:22231900

  10. Improved mutation tagging with gene identifiers applied to membrane protein stability prediction

    PubMed Central

    Winnenburg, Rainer; Plake, Conrad; Schroeder, Michael

    2009-01-01

    Background The automated retrieval and integration of information about protein point mutations in combination with structure, domain and interaction data from literature and databases promises to be a valuable approach to study structure-function relationships in biomedical data sets. Results We developed a rule- and regular expression-based protein point mutation retrieval pipeline for PubMed abstracts, which shows an F-measure of 87% for the mutation retrieval task on a benchmark dataset. In order to link mutations to their proteins, we utilize a named entity recognition algorithm for the identification of gene names co-occurring in the abstract, and establish links based on sequence checks. Vice versa, we could show that gene recognition improved from 77% to 91% F-measure when considering mutation information given in the text. To demonstrate practical relevance, we utilize mutation information from text to evaluate a novel solvation energy based model for the prediction of stabilizing regions in membrane proteins. For five G protein-coupled receptors we identified 35 relevant single mutations and associated phenotypes, of which none had been annotated in the UniProt or PDB database. In 71% reported phenotypes were in compliance with the model predictions, supporting a relation between mutations and stability issues in membrane proteins. Conclusion We present a reliable approach for the retrieval of protein mutations from PubMed abstracts for any set of genes or proteins of interest. We further demonstrate how amino acid substitution information from text can be utilized for protein structure stability studies on the basis of a novel energy model. PMID:19758467

  11. Identification of three transforming proteins encoded by cottontail rabbit papillomavirus.

    PubMed Central

    Meyers, C; Harry, J; Lin, Y L; Wettstein, F O

    1992-01-01

    Cottontail rabbit papillomavirus (CRPV) provides an animal model for human papillomaviruses associated with a high risk of cancer development. So far, nothing is known about the transforming functions of CRPV genes because of the lack of an assay system. We have recently developed two systems to assay for CRPV transforming functions. One is based on the finding that transformation of NIH 3T3 cells by CRPV is considerably increased by deleting sequences in open reading frame L2. The second one is based on the use of a cottontail rabbit skin epithelial cell line, sf1Ep (C. Meyers and F. O. Wettstein, Virology 181:637-646, 1991). Mutations were introduced which abolished expression of the full-length E6 protein (LE6), the short E6 protein (SE6) initiated at the second ATG of E6, the E7 protein, or the E5 protein. Mutations affecting LE6 or E7, but not SE6, reduced transformation of NIH 3T3 and sf1Ep cells. Transformed NIH 3T3 cell lines with mutations in LE6 and E7 did not grow in soft agar, while those with mutations in SE6 and E5 grew with a reduced efficiency. The cell lines with mutations in LE6, SE6, or E7 still did induce tumors in nude mice. These mutations, however, abolished the ability to induce papillomas in rabbits. When expressed individually with a retroviral vector, LE6, SE6, or E7, but not E5, conferred anchorage-independent growth. The level of viral protein expression in these cell lines was generally low, and a comparison of the abundance of virus-specific mRNA showed that cell lines contained 20 to 50 times less mRNA than a cottontail rabbit papilloma. These data demonstrate that CRPV encodes at least three transforming proteins. Images PMID:1310771

  12. Identification of outer membrane proteins of Mycobacterium tuberculosis.

    PubMed

    Song, Houhui; Sandie, Reatha; Wang, Ying; Andrade-Navarro, Miguel A; Niederweis, Michael

    2008-11-01

    The cell wall of mycobacteria includes an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the beta-barrel structure, the absence of hydrophobic alpha-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the beta-strand content and the amphiphilicity of the beta-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with beta-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis. PMID:18439872

  13. Immunochromatographic analysis of proteins. Identification, characterization and purity determination.

    PubMed

    Riggin, A; Sportsman, J R; Regnier, F E

    1993-02-19

    Antibodies specific to a protein and its structural variants were immobilized on a high-performance Protein G column. This column recognized and selectively subtracted specific molecules from a sample. When a size-exclusion column was coupled with this high-performance affinity column, a comparison between the elution profile before and after the antibody immobilization was used to study antigen components present in the sample. Various human growth hormone structural variants and aggregates were studied using this approach. The technique is simple, fast and does not involve the usage of radioactive material. PMID:8454718

  14. Genome engineering for improved recombinant protein expression in Escherichia coli.

    PubMed

    Mahalik, Shubhashree; Sharma, Ashish K; Mukherjee, Krishna J

    2014-01-01

    A metabolic engineering perspective which views recombinant protein expression as a multistep pathway allows us to move beyond vector design and identify the downstream rate limiting steps in expression. In E.coli these are typically at the translational level and the supply of precursors in the form of energy, amino acids and nucleotides. Further recombinant protein production triggers a global cellular stress response which feedback inhibits both growth and product formation. Countering this requires a system level analysis followed by a rational host cell engineering to sustain expression for longer time periods. Another strategy to increase protein yields could be to divert the metabolic flux away from biomass formation and towards recombinant protein production. This would require a growth stoppage mechanism which does not affect the metabolic activity of the cell or the transcriptional or translational efficiencies. Finally cells have to be designed for efficient export to prevent buildup of proteins inside the cytoplasm and also simplify downstream processing. The rational and the high throughput strategies that can be used for the construction of such improved host cell platforms for recombinant protein expression is the focus of this review. PMID:25523647

  15. 34 CFR 200.32 - Identification for school improvement.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... SECONDARY EDUCATION, DEPARTMENT OF EDUCATION TITLE I-IMPROVING THE ACADEMIC ACHIEVEMENT OF THE DISADVANTAGED... the same subject or meet the same other academic indicator for two consecutive years; but (B) May not... measurable objectives for the same subject or meet the same other academic indicator for the same...

  16. Efficiency of database search for identification of mutated and modified proteins via mass spectrometry.

    PubMed

    Pevzner, P A; Mulyukov, Z; Dancik, V; Tang, C L

    2001-02-01

    Although protein identification by matching tandem mass spectra (MS/MS) against protein databases is a widespread tool in mass spectrometry, the question about reliability of such searches remains open. Absence of rigorous significance scores in MS/MS database search makes it difficult to discard random database hits and may lead to erroneous protein identification, particularly in the case of mutated or post-translationally modified peptides. This problem is especially important for high-throughput MS/MS projects when the possibility of expert analysis is limited. Thus, algorithms that sort out reliable database hits from unreliable ones and identify mutated and modified peptides are sought. Most MS/MS database search algorithms rely on variations of the Shared Peaks Count approach that scores pairs of spectra by the peaks (masses) they have in common. Although this approach proved to be useful, it has a high error rate in identification of mutated and modified peptides. We describe new MS/MS database search tools, MS-CONVOLUTION and MS-ALIGNMENT, which implement the spectral convolution and spectral alignment approaches to peptide identification. We further analyze these approaches to identification of modified peptides and demonstrate their advantages over the Shared Peaks Count. We also use the spectral alignment approach as a filter in a new database search algorithm that reliably identifies peptides differing by up to two mutations/modifications from a peptide in a database. PMID:11157792

  17. 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

  18. 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. PMID:24217948

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

    PubMed Central

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

    2015-01-01

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

  20. 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.

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

    PubMed Central

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

    1993-01-01

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

  2. The identification of matrix Gla protein in cartilage.

    PubMed

    Hale, J E; Fraser, J D; Price, P A

    1988-04-25

    The vitamin K-dependent bone protein matrix gamma-carboxyglutamic acid (Gla) protein (MGP) has been identified by radioimmunoassay in the guanidine extract of rat cartilage. MGP was present in all cartilages tested at levels comparable to the MGP level in bone. Western blot analysis indicated that the molecular weight of cartilage MGP is the same as bone MGP, and Northern blot analysis revealed that MGP mRNA from cartilage is the same size as the MGP mRNA from bone. The structurally related vitamin K-dependent protein bone Gla protein could not be detected in cartilage by radioimmunoassay or by Northern blot analysis. The discovery that MGP is synthesized by growth plate cartilage could provide an explanation for the excessive growth plate mineralization disorder seen in rats treated with the vitamin K antagonist warfarin and the punctate mineralization of the growth plate seen in infants whose mothers received warfarin in the first trimester of pregnancy (the fetal warfarin syndrome). Both disorders appear to be caused by the inactivation of a vitamin K-dependent mineralization inhibitor in cartilage, an inhibitor which we suggest is MGP. PMID:3258600

  3. Systematic identification of signal integration by protein kinase A

    PubMed Central

    Filteau, Marie; Diss, Guillaume; Dubé, Alexandre K.; Schraffl, Andrea; Bachmann, Verena A.; Gagnon-Arsenault, Isabelle; Chrétien, Andrée-Ève; Steunou, Anne-Lise; Dionne, Ugo; Bisson, Nicolas; Stefan, Eduard; Landry, Christian R.

    2015-01-01

    Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein–protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA. PMID:25831502

  4. Identification of a Plasmodium falciparum Phospholipid Transfer Protein*

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2003-08-15

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

  6. Identification of components to optimize improvement in system reliability

    SciTech Connect

    Painton, L.; Campbell, J.

    1993-10-01

    The fields of reliability analysis and risk assessment have grown dramatically since the 1970s. There are now bodies of literature and standard practices which cover quantitative aspects of system analysis such as failure rate and repair models, fault and event tree generation, minimal cut sets, classical and Bayesian analysis of reliability, component and system testing techniques, decomposition methods, etc. In spite of the growth in the sophistication of reliability models, however, little has been done to integrate optimization models within a reliability analysis framework. That is, often reliability models focus on characterization of system structure in terms of topology and failure/availability characteristics of components. A number of approaches have been proposed to help identify the components of a system that have the largest influence on overall system reliability. While this may help rank order the components, it does not necessarily help a system design team identify which components they should improve to optimize overall reliability (it may be cheaper and more effective to focus on improving two or three components of smaller importance than one component of larger importance). In this paper, we present an optimization model that identifies the components to be improved to maximize the increase in system MTBF, subject to a fixed budget constraint. A dual formulation of the model is to minimize cost, subject to achieving a certain level of system reliability.

  7. 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...

  8. 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...

  9. A structural alphabet for local protein structures: improved prediction methods.

    PubMed

    Etchebest, Catherine; Benros, Cristina; Hazout, Serge; de Brevern, Alexandre G

    2005-06-01

    Three-dimensional protein structures can be described with a library of 3D fragments that define a structural alphabet. We have previously proposed such an alphabet, composed of 16 patterns of five consecutive amino acids, called Protein Blocks (PBs). These PBs have been used to describe protein backbones and to predict local structures from protein sequences. The Q16 prediction rate reaches 40.7% with an optimization procedure. This article examines two aspects of PBs. First, we determine the effect of the enlargement of databanks on their definition. The results show that the geometrical features of the different PBs are preserved (local RMSD value equal to 0.41 A on average) and sequence-structure specificities reinforced when databanks are enlarged. Second, we improve the methods for optimizing PB predictions from sequences, revisiting the optimization procedure and exploring different local prediction strategies. Use of a statistical optimization procedure for the sequence-local structure relation improves prediction accuracy by 8% (Q16 = 48.7%). Better recognition of repetitive structures occurs without losing the prediction efficiency of the other local folds. Adding secondary structure prediction improved the accuracy of Q16 by only 1%. An entropy index (Neq), strongly related to the RMSD value of the difference between predicted PBs and true local structures, is proposed to estimate prediction quality. The Neq is linearly correlated with the Q16 prediction rate distributions, computed for a large set of proteins. An "expected" prediction rate QE16 is deduced with a mean error of 5%. PMID:15822101

  10. Application of particle swarm optimization for improving the identification of image objects

    NASA Astrophysics Data System (ADS)

    Chiu, Nan-Hsing; Pu, Chang-En; Lin, Pei-Da; Wang, Shu-Shian

    2012-04-01

    Flight safety is very important issue for aviation industries. Analyzing the flight accidents on the basis of 2-dimensional image is hardly to illustrate the complex injuries of passengers in the flight cabin. However, how to illustrate the flight accident is a challenge from 2-dimensional space to 3-dimensional space. This study proposes a particle swarm optimization approach for improving the identification of objects from 2-dimensional image. The recognition results provide the information for building 3-dimensional systems for flight accident investigators. The experiments also show that it is a feasible approach for improving the identification of image objects.

  11. Computational Identification of MoRFs in Protein Sequences Using Hierarchical Application of Bayes Rule

    PubMed Central

    Malhis, Nawar; Wong, Eric T. C.; Nassar, Roy; Gsponer, Jörg

    2015-01-01

    Motivation Intrinsically disordered regions of proteins play an essential role in the regulation of various biological processes. Key to their regulatory function is often the binding to globular protein domains via sequence elements known as molecular recognition features (MoRFs). Development of computational tools for the identification of candidate MoRF locations in amino acid sequences is an important task and an area of growing interest. Given the relative sparseness of MoRFs in protein sequences, the accuracy of the available MoRF predictors is often inadequate for practical usage, which leaves a significant need and room for improvement. In this work, we introduce MoRFCHiBi_Web, which predicts MoRF locations in protein sequences with higher accuracy compared to current MoRF predictors. Methods Three distinct and largely independent property scores are computed with component predictors and then combined to generate the final MoRF propensity scores. The first score reflects the likelihood of sequence windows to harbour MoRFs and is based on amino acid composition and sequence similarity information. It is generated by MoRFCHiBi using small windows of up to 40 residues in size. The second score identifies long stretches of protein disorder and is generated by ESpritz with the DisProt option. Lastly, the third score reflects residue conservation and is assembled from PSSM files generated by PSI-BLAST. These propensity scores are processed and then hierarchically combined using Bayes rule to generate the final MoRFCHiBi_Web predictions. Results MoRFCHiBi_Web was tested on three datasets. Results show that MoRFCHiBi_Web outperforms previously developed predictors by generating less than half the false positive rate for the same true positive rate at practical threshold values. This level of accuracy paired with its relatively high processing speed makes MoRFCHiBi_Web a practical tool for MoRF prediction. Availability http://morf.chibi.ubc.ca:8080/morf/. PMID

  12. 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

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

    PubMed

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

    2013-01-01

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

  14. Retinal Identification Based on an Improved Circular Gabor Filter and Scale Invariant Feature Transform

    PubMed Central

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

    2013-01-01

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

  15. Computational identification of MoRFs in protein sequences

    PubMed Central

    Malhis, Nawar; Gsponer, Jörg

    2015-01-01

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

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

    PubMed

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

    2004-04-01

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

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

    PubMed

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

    2015-07-17

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

  18. 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%.

  19. Identification of Proteins Interacting with GTP Cyclohydrolase I

    PubMed Central

    Du, Jianhai; Xu, Hao; Wei, Na; Wakim, Bassam; Halligan, Brian; Pritchard, Kirkwood A.; Shi, Yang

    2009-01-01

    GTP cyclohydrolase I (GCH-1) is the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, an essential cofactor for nitric oxide synthase and aromatic amino acid hydroxylase. To explore the interactome of GCH-1, we established a HEK293 cell line stably expressing tetracycline-inducible FLAG-GCH-1. FLAG-GCH-1 and associated proteins were immunoprecipitated and analyzed by liquid chromatography/tandem mass spectrometry. Twenty-nine proteins, derived from different subcellular components such as cytosol, membranes, nucleus and mitochondria were identified to interact with GCH-1. Cell fractionation studies also showed that GCH-1 was present in the cytosol, membranes and nucleus. Gene ontology analysis revealed that GCH-1 interactome was involved in a variety of biological processes such as signal transduction, apoptosis, metabolism, transport and cell organization. To our knowledge, this study is the first to provide a comprehensive analysis of the GCH-1 interactome. Findings expand the number and diversity of proteins that are known to associate with GCH-1. PMID:19442649

  20. 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.

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

    PubMed

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

    2014-01-01

    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. PMID:25323771

  2. Identification of Cardiac Myofilament Protein Isoforms Using Multiple Mass Spectrometry Based Approaches

    PubMed Central

    Kirk, Jonathan A.; Ubaida-Mohien, Ceereena; Graham, David R.; Faber, Matthijs J.; Van Eyk, Jennifer E.

    2014-01-01

    Purpose The identification of protein isoforms in complex biological samples is challenging. We, therefore, used a mass spectrometry (MS) approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. Experimental design Three different workflows were used to isolate and fractionate rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (Mascot, X!Tandem, X!Tandem Kscore and OMSSA) with results combined via PepArML Meta-Search Engine, and a post-search analysis was performed by MASPECTRAS. Results The combination of multiple workflows and search engines resulted in a larger number of non-redundant proteins identified than with individual methods. A total of 102 myofilament annotated proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: 10 cardiac myofilament and 17 cardiac myofilament-associated proteins were identified with 39 isoforms and sub-isoforms. Conclusion and clinical relevance We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes in distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets. PMID:24974818

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

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

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

  4. Identification and Characterization of Citrulline-modified Brain Proteins by Combining HCD and CID Fragmentation

    PubMed Central

    Jin, Zhicheng; Fu, Zongming; Yang, Jun; Troncosco, Juan; Everett, Allen D.; Van Eyk, Jennifer E.

    2016-01-01

    Citrullination is a protein post-translational modification of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the central nervous system and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues has been limited. Using reversed-phase liquid chromatography and high resolution mass spectrometry, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP) and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has fourteen in vivo citrullination sites. Human neurogranin (NRGN/RC3) was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys-C, and Glu-C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer’s and control samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6% to 9.5%. Compared to collision-induced dissociation (CID), higher-energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests. PMID:23828821

  5. 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.

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

    PubMed

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

    2015-01-01

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

  7. Identification of Epstein-Barr Virus Replication Proteins in Burkitt’s Lymphoma Cells

    PubMed Central

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

    2015-01-01

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

  8. 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.

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

    PubMed

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

    2010-04-15

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

  10. Identification of an Ideal-like Fingerprint for a Protein Fold using Overlapped Conserved Residues based Approach

    PubMed Central

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

    2014-01-01

    Design of an efficient fingerprint that detects homologous proteins at distant sequence identity has been a great challenge. This paper proposes a strategy to extract an ideal-like fingerprint with high specificity and sensitivity from a group of sequences related to a fold. The approach is devised based on the assumptions that the critical residues for a protein fold may be conserved in three aspects, i.e. sequence, structure, and intramolecular interaction, and embedded in secondary structures. We hypothesized that the residues satisfying such conditions simultaneously may work as an efficient fingerprint. This idea was tested on protein folds of various classes, such as beta-strand rich, alpha + beta proteins and alpha/beta proteins with discrete sequence similarities. The fingerprint for each fold was generated by selecting the overlapped conserved residues (OCR) from the conserved residues obtained using independent three alignment methods, i.e. multiple sequence alignment, structure-based alignment, and alignment based on the interstrand hydrogen-bonds. The OCR fingerprints showed more than 90% detection efficiency for all the folds tested and were identified to be almost the minimal fingerprints composed of only critical residues. This study is expected to provide an important conceptual improvement in the identification or design of ideal fingerprints for a protein fold. PMID:25008052

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

    PubMed

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

    2012-01-01

    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. PMID:22370884

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

    PubMed

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

    2016-01-01

    Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae. PMID:26859666

  13. Identification and molecular characterization of BP75, a novel bromodomain-containing protein.

    PubMed

    Cuppen, E; van Ham, M; Pepers, B; Wieringa, B; Hendriks, W

    1999-10-15

    We here describe the identification and characterization of a novel bromodomain-containing protein, the bromodomain protein of 75 kDa (BP75). Initially, we identified BP75 in a two-hybrid screening for proteins that interact with the first PDZ (acronym for post-synaptic density protein PSD-95, Drosophila discs large tumor suppressor DlgA and the tight junction protein ZO-1) domain in protein tyrosine phosphatase-BAS-like (PTP-BL). We found that BP75 is expressed ubiquitously and show that both BP75 and a PTP-BL deletion mutant consisting of the first PDZ domain are located mainly in the nucleus, although cytoplasmic localization is also evident. Full-length PTP-BL, on the contrary, is predominantly localized in the cytoplasm, although some basal nuclear staining is observed. The described molecular interaction may reflect a mechanism of coupling submembraneous signalling events and nuclear events. PMID:10526152

  14. 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

  15. 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

  16. 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

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

    PubMed

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

    2016-05-01

    Covering: up to February 2016Identification of the target proteins of natural products is pivotal to understanding the mechanisms of action to develop natural products for use as molecular probes and potential therapeutic drugs. Affinity chromatography of immobilized natural products has been conventionally used to identify target proteins, and has yielded good results. However, this method has limitations, in that labeling or tagging for immobilization and affinity purification often result in reduced or altered activity of the natural product. New strategies have recently been developed and applied to identify the target proteins of natural products and synthetic small molecules without chemical modification of the natural product. These direct and indirect methods for target identification of label-free natural products include drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), and bioinformatics-based analysis of connectivity. This review focuses on and reports case studies of the latest advances in target protein identification methods for label-free natural products. The integration of newly developed technologies will provide new insights and highlight the value of natural products for use as biological probes and new drug candidates. PMID:26964663

  18. 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

  19. 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

  20. Targeting SR Proteins Improves SMN Expression in Spinal Muscular Atrophy Cells

    PubMed Central

    Hastings, Michelle L.

    2014-01-01

    Spinal muscular atrophy (SMA) is one of the most common inherited causes of pediatric mortality. SMA is caused by deletions or mutations in the survival of motor neuron 1 (SMN1) gene, which results in SMN protein deficiency. Humans have a centromeric copy of the survival of motor neuron gene, SMN2, which is nearly identical to SMN1. However, SMN2 cannot compensate for the loss of SMN1 because SMN2 has a single-nucleotide difference in exon 7, which negatively affects splicing of the exon. As a result, most mRNA produced from SMN2 lacks exon 7. SMN2 mRNA lacking exon 7 encodes a truncated protein with reduced functionality. Improving SMN2 exon 7 inclusion is a goal of many SMA therapeutic strategies. The identification of regulators of exon 7 inclusion may provide additional therapeutic targets or improve the design of existing strategies. Although a number of regulators of exon 7 inclusion have been identified, the function of most splicing proteins in exon 7 inclusion is unknown. Here, we test the role of SR proteins and hnRNP proteins in SMN2 exon 7 inclusion. Knockdown and overexpression studies reveal that SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF11, hnRNPA1/B1 and hnRNP U can inhibit exon 7 inclusion. Depletion of two of the most potent inhibitors of exon 7 inclusion, SRSF2 or SRSF3, in cell lines derived from SMA patients, increased SMN2 exon 7 inclusion and SMN protein. Our results identify novel regulators of SMN2 exon 7 inclusion, revealing potential targets for SMA therapeutics. PMID:25506695

  1. Genetic variability and improvement of seed proteins in wheat.

    PubMed

    Dhaliwal, H S

    1977-03-01

    Albumins, globulins, gliadins and glutenins presumably comprising 100 percent of the wheat seed proteins were sequentially extracted and electrophoresed on SDS-polyacrylamide gels. The SDS-electrophoretic patterns within each of the four fractions from T. boeotiaum, T. urartu, T. turgidum, T. timopheevii, T. aestivum, Ae. speltoides and Ae. squawosa were similar. They differed from one species to another only in a few minor components or density of certain components. Similarity in MW's of components, as indicated by the SDS-electrophoretic patterns, suggests that the wheats and Aegilops exhibit no variability for structural genes coding seed proteins. A minimum of 60 to 70 and a maximum of 360 to 420 structural genes with major or minor effects control the total seed protein in T. aestivum. Presumably, only one or the other homoeoallele was expressed in the polyploids. Different components of albumins and globulins presumably had distinct MW's and amino acid composition, while the components of gliadins and glutenins could be classified into a few groups each containing one or more components with the same MW and nearly identical amino acid composition. The genes for components with similar MW's and amino acid composition arose through multiplication of a single original gene and perhaps share the same regulatory mechanism. Seed protein content and quality in wheat might be improved through the incorporation of structural genes, coding for polypeptides with distinct MW's, from distantly related species, rather than by manipulation of the structural genes within the Triticum-Aegilops group. Regulatory mutants similar to opaque-2 of corn could be used to alter the proportion of gliadins in relation to albumins and globulins, to improve amino acid composition of wheat proteins. PMID:24317598

  2. 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

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

    PubMed

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

    2016-01-01

    Toxoplasma gondii maintains its intracellular life cycle using an extraordinary arsenal of parasite-specific organelles including the inner membrane complex (IMC), rhoptries, micronemes, and dense granules. While these unique compartments play critical roles in pathogenesis, many of their protein constituents have yet to be identified. We exploited the Vicia villosa lectin (VVL) to identify new glycosylated proteins that are present in these organelles. Purification of VVL-binding proteins by lectin affinity chromatography yielded a number of novel proteins that were subjected to further study, resulting in the identification of proteins from the dense granules, micronemes, rhoptries and IMC. We then chose to focus on three proteins identified by this approach, the SAG1 repeat containing protein SRS44, the rhoptry neck protein RON11 as well as a novel IMC protein we named IMC25. To assess function, we disrupted their genes by homologous recombination or CRISPR/Cas9. The knockouts were all successful, demonstrating that these proteins are not essential for invasion or intracellular survival. We also show that IMC25 undergoes substantial proteolytic processing that separates the C-terminal domain from the predicted glycosylation site. Together, we have demonstrated that lectin affinity chromatography is an efficient method of identifying new glycosylated parasite-specific proteins. PMID:26950937

  4. Identification of two integral membrane proteins of Plasmodium falciparum

    SciTech Connect

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

    1988-07-01

    The authors 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 M{sub r} 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a M{sub r} 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with ({sup 3}H)glucosamine and ({sup 3}H)myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the M{sub r} 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.

  5. Improving identification of dijet resonances at hadron colliders.

    PubMed

    Izaguirre, Eder; Shuve, Brian; Yavin, Itay

    2015-01-30

    The experimental detection of resonances has played a vital role in the development of subatomic physics. The overwhelming multijet backgrounds at the Large Hadron Collider (LHC) necessitate the invention of new techniques to identify resonances decaying into a pair of partons. In this Letter we introduce an observable that achieves a significant improvement in several key measurements at the LHC: the Higgs boson decay to a pair of b quarks; W±/Z0 vector-boson hadronic decay; and extensions of the standard model (SM) with a new hadronic resonance. Measuring the Higgs decay to b quarks is a central test of the fermion mass generation mechanism in the SM, whereas the W±/Z0 production rates are important observables of the electroweak sector. Our technique is effective in large parts of phase space where the resonance is mildly boosted and is particularly well suited for experimental searches dominated by systematic uncertainties, which is true of many analyses in the high-luminosity running of the LHC. PMID:25679886

  6. Improving the efficiency of attractor cycle identification in Boolean networks

    NASA Astrophysics Data System (ADS)

    Irons, David James

    2006-05-01

    Boolean network models provide a computationally efficient way of studying dynamical processes on networks and are most frequently used to study the dynamical properties of genetic regulatory networks. Presented here is a new and more efficient method for finding every attractor cycle (stable state) in a Boolean network. The critical part of this new method can be executed in polynomial time (O(v3)), as opposed to the exponential time taken for the standard exhaustive search (O(v2v)). The efficiency of this new method is dependent on the topology of the underlying network. In particular, efficiency significantly improves when the out-degree distribution is skewed, such as with a power law distribution. The findings also provide added insight into the dynamics on power law networks and make the method more applicable to biological networks, which are believed to have this property. This method can also be extended to some non-Boolean discrete models (e.g. cellular automata).

  7. Improving Identification of Dijet Resonances at Hadron Colliders

    NASA Astrophysics Data System (ADS)

    Izaguirre, Eder; Shuve, Brian; Yavin, Itay

    2015-01-01

    The experimental detection of resonances has played a vital role in the development of subatomic physics. The overwhelming multijet backgrounds at the Large Hadron Collider (LHC) necessitate the invention of new techniques to identify resonances decaying into a pair of partons. In this Letter we introduce an observable that achieves a significant improvement in several key measurements at the LHC: the Higgs boson decay to a pair of b quarks; W±/Z0 vector-boson hadronic decay; and extensions of the standard model (SM) with a new hadronic resonance. Measuring the Higgs decay to b quarks is a central test of the fermion mass generation mechanism in the SM, whereas the W±/Z0 production rates are important observables of the electroweak sector. Our technique is effective in large parts of phase space where the resonance is mildly boosted and is particularly well suited for experimental searches dominated by systematic uncertainties, which is true of many analyses in the high-luminosity running of the LHC.

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

    SciTech Connect

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

    1988-02-01

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

  9. 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

  10. 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

  11. Identification of Protein Markers Specific for Papillary Renal Cell Carcinoma Using Imaging Mass Spectrometry

    PubMed Central

    Na, Chan Hyun; Hong, Ji Hye; Kim, Wan Sup; Shanta, Selina Rahman; Bang, Joo Yong; Park, Dongmin; Kim, Hark Kyun; Kim, Kwang Pyo

    2015-01-01

    Since the emergence of proteomics methods, many proteins specific for renal cell carcinoma (RCC) have been identified. Despite their usefulness for the specific diagnosis of RCC, such proteins do not provide spatial information on the diseased tissue. Therefore, the identification of cancer-specific proteins that include information on their specific location is needed. Recently, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) based imaging mass spectrometry (IMS) has emerged as a new tool for the analysis of spatial distribution as well as identification of either proteins or small molecules in tissues. In this report, surgical tissue sections of papillary RCC were analyzed using MALDI-IMS. Statistical analysis revealed several discriminative cancer-specific m/z-species between normal and diseased tissues. Among these m/z-species, two particular proteins, S100A11 and ferritin light chain, which are specific for papillary RCC cancer regions, were successfully identified using LC-MS/MS following protein extraction from independent RCC samples. The expressions of S100A11 and ferritin light chain were further validated by immunohistochemistry of human tissues and tissue microarrays (TMAs) of RCC. In conclusion, MALDI-IMS followed by LC-MS/MS analysis in human tissue identified that S100A11 and ferritin light chain are differentially expressed proteins in papillary RCC cancer regions. PMID:26062552

  12. Ion soft-landing into liquids: Protein identification, separation, and purification with retention of biological activity.

    PubMed

    Gologan, Bogdan; Takáts, Zoltán; Alvarez, Jormarie; Wiseman, Justin M; Talaty, Nari; Ouyang, Zheng; Cooks, R Graham

    2004-12-01

    Protein ions, after mass spectrometric separation, can be soft-landed into liquid surfaces with preservation of their native structures. Retention of biological activity is strongly favored in glycerol-based surfaces but not in self-assembled monolayer solid surfaces. Soft-landing efficiency for multiply-charged hexokinase ions was found to be some four times higher for a glycerol/fructose liquid surface than for a fluorinated self-assembled monolayer surface. Soft-landing into liquid surfaces is also shown to allow (1) protein purification, (2) on-surface identification of the soft-landed material using MALDI, and (3) protein identification by in-surface tryptic digestion. Pure lysozyme was successfully isolated from different mixtures including an oxidized, partially decomposed batch of the protein and a partial tryptic digest. Liquid glycerol/carbohydrate mixtures could be used directly to record MALDI spectra on the soft-landed compounds provided they were fortified in advance with traditional MALDI matrices such as p-nitroaniline and alpha-cyano-4-hydroxycinnamic acid. Various proteins were soft-landed and detected on-target using these types of liquid surface. Soft-landing of multiply-charged lysozyme ions onto fluorinated self-assembled monolayer surfaces was found to occur with a limited amount of neutralization, and trapped multiply-charged ions could be desorbed from the surface by laser desorption. Initial data is shown for a new approach to protein identification that combines top-down and bottom-up approaches by utilizing protein ion soft-landing from a protein mixture, followed by tryptic digestion of the landed material and detection of characteristic tryptic fragments by MALDI. PMID:15589764

  13. Identification and characterization of antioxidant peptides from chickpea protein hydrolysates.

    PubMed

    Torres-Fuentes, Cristina; Contreras, María del Mar; Recio, Isidra; Alaiz, Manuel; Vioque, Javier

    2015-08-01

    Oxidative stress due to the excess of radical oxygen species (ROS) contribute to the development of different diseases. The use of antioxidants may prevent the development of these diseases by counteracting ROS levels. There is an increasing interest in natural antioxidants as they are safer for consumers than synthetic antioxidants. In this work, reducing power, free radical scavenging and cellular antioxidant activities of chickpea peptides fractions have been investigated. Peptide sequences included in fractions with antioxidant activity were identified. Main sequences, ALEPDHR, TETWNPNHPEL, FVPH and SAEHGSLH, corresponded to legumin, the main seed protein. Most peptides contained histidine, which has shown antioxidant activity. Two peptides also included tryptophan and phenylalanine, in which the phenolic group could also serve as hydrogen donor. These results show that legumin is a source of antioxidant peptides of high interest for food and pharmaceutical industries to develop new nutraceuticals and functional foods. PMID:25766818

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

    PubMed

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

    2012-09-01

    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

  15. Towards a Semen Proteome of the Dengue Vector Mosquito: Protein Identification and Potential Functions

    PubMed Central

    Sirot, Laura K.; Ribeiro, José M. C.; Kimura, Mari; Deewatthanawong, Prasit; Wolfner, Mariana F.; Harrington, Laura C.

    2011-01-01

    -mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti. PMID:21423647

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

    PubMed Central

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

    2012-01-01

    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. PMID:22442409

  17. 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.

  18. 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. PMID:27087524

  19. Identification of dengue RNA binding proteins using RNA chromatography and quantitative mass spectrometry.

    PubMed

    Ward, Alex M; Gunaratne, J; Garcia-Blanco, Mariano A

    2014-01-01

    A major challenge in dengue virus (DENV) research has been to understand the interaction of the viral RNA with host cell proteins during infection. Until recently, there were no comprehensive studies identifying host RNA binding proteins that interact with DENV RNA (Ward et al. RNA Biol 8 (6):1173-1186, 2011). Here, we describe a method for identifying proteins that associate with DENV RNA using RNA chromatography and quantitative mass spectrometry. The method utilizes a tobramycin RNA aptamer incorporated into an RNA containing the dengue 5' and 3' untranslated regions (UTRs) in order to reversibly bind RNA to a tobramycin matrix. The RNA-tobramycin matrix is incubated with SILAC-labeled cell lysates, and bound proteins are eluted using an excess of tobramycin. The eluate is analyzed using quantitative mass spectrometry, which allows direct and quantitative comparison of proteins bound to DENV UTRs and a control RNA-tobramycin matrix. This technique has the advantage of allowing one to distinguish between specific and nonspecific binding proteins based on the ratio of protein preferentially bound to the DENV UTRs versus the control RNA. This methodology can also be used for validation of quantitative mass spectrometry results using conventional Western blotting for specific proteins. Furthermore, though it was specifically developed to identify DENV RNA binding proteins, the RNA chromatography method described here can be applied to a broad range of viral and cellular RNAs for identification of interacting proteins. PMID:24696342

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

    SciTech Connect

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

    2012-01-01

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

  1. Identification of GABAC Receptor Protein Homeostasis Network Components from Three Tandem Mass Spectrometry Proteomics Approaches

    PubMed Central

    Wang, Ya-Juan; Han, Dong-Yun; Tabib, Tracy; Yates, John R.; Mu, Ting-Wei

    2013-01-01

    Gamma-amino butyric acid type C (GABAC) receptors inhibit neuronal firing primarily in retina. Maintenance of GABAC receptor protein homeostasis in cells is essential for its function. However, a systematic study of GABAC receptor protein homeostasis (proteostasis) network components is absent. Here, co-immunoprecipitation of human GABAC-ρ1 receptor complexes was performed in HEK293 cells overexpressing ρ1 receptors. To enhance the coverage and reliability of identified proteins, immunoisolated ρ1 receptor complexes were subjected to three tandem mass spectrometry (MS)-based proteomic analyses: namely, gel-based tandem MS (GeLC-MS/MS), solution-based tandem MS (SoLC-MS/MS), and multidimensional protein identification technology (MudPIT). From the 107 identified proteins, we assembled GABAC-ρ1 receptor proteostasis network components, including proteins with protein folding, degradation, and trafficking functions. We studied representative individual ρ1 receptor interacting proteins, including calnexin, a lectin chaperone that facilitates glycoprotein folding, and LMAN1, a glycoprotein trafficking receptor, and global effectors that regulate protein folding in cells based on bioinformatics analysis, including HSF1, a master regulator of the heat shock response, and XBP1, a key transcription factor of the unfolded protein response. Manipulating selected GABAC receptor proteostasis network components is a promising strategy to regulate GABAC receptor folding, trafficking, degradation and thus function to ameliorate related retinal diseases. PMID:24079818

  2. Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein*

    PubMed Central

    Bruck, Serawit; Huber, Tobias B.; Ingham, Robert J.; Kim, Kyoungtae; Niederstrasser, Hanspeter; Allen, Paul M.; Pawson, Tony; Cooper, John A.; Shaw, Andrey S.

    2008-01-01

    CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X)6P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton. PMID:16707503

  3. 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.

  4. 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.

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

    SciTech Connect

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

    1995-11-20

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

  6. 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

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

    PubMed

    Tong, Mingsi; Song, John; Chu, Wei

    2015-01-01

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

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

    DOE PAGESBeta

    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

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

    PubMed Central

    2014-01-01

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

  10. 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. PMID:26323838

  11. Identification of a GPER/GPR30 antagonist with improved estrogen receptor counterselectivity.

    PubMed

    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, Larry A; Hathaway, Helen J; Arterburn, Jeffrey B; Prossnitz, Eric R

    2011-11-01

    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

  12. 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. PMID:25815775

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

    SciTech Connect

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

    1986-05-01

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

  14. Identification of unique SUN-interacting nuclear envelope proteins with diverse functions in plants.

    PubMed

    Zhou, Xiao; Graumann, Katja; Wirthmueller, Lennart; Jones, Jonathan D G; Meier, Iris

    2014-06-01

    Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain-interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions. PMID:24891605

  15. Subproteomics: identification of plasma membrane proteins from the yeast Saccharomyces cerevisiae.

    PubMed

    Navarre, Catherine; Degand, Hervé; Bennett, Keiryn L; Crawford, Janne S; Mørtz, Ejvind; Boutry, Marc

    2002-12-01

    As a consequence of their poor solubility during isoelectric focusing, integral membrane proteins are generally absent from two-dimensional gel proteome maps. In order to analyze the yeast plasma membrane proteome, a plasma membrane purification protocol was optimized in order to reduce contaminating membranes and cytosolic proteins. Specifically, the new fractionation scheme largely depleted the plasma membrane fraction of cytosolic proteins by deoxycholate stripping and ribosomal proteins by sucrose gradient flotation. The plasma membrane complement was resolved by two-dimensional electrophoresis using the cationic detergent cetyl trimethyl ammonium bromide in the first, and sodium dodecyl sulfate in the second dimension, and fifty spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectometry. In spite of the presence of still contaminating ribosomal proteins, major proteins corresponded to known plasma membrane residents, the ABC transporters Pdr5p and Snq2p, the P-type H(+)-ATPase Pma1p, the glucose transporter Hxt7p, the seven transmembrane-span Mrh1p, the low affinity Fe(++) transporter Fet4p, the twelve-span Ptr2p, and the plasma membrane anchored casein kinase Yck2p. The four transmembrane-span proteins Sur7p and Nce102p were also present in the isolated plasma membranes, as well as the unknown protein Ygr266wp that probably contains a single transmembrane span. Thus, combining subcellular fractionation with adapted two-dimensional electrophoresis resulted in the identification of intrinsic plasma membrane proteins. PMID:12469340

  16. 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

  17. 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

  18. High-Throughput Identification of IMCD Proteins Using LC-MS/MS

    PubMed Central

    Pisitkun, Trairak; Bieniek, Jared; Tchapyjnikov, Dmitry; Wang, Guanghui; Wu, Wells W.; Shen, Rong-Fong; Knepper, Mark A.

    2006-01-01

    The inner medullary collecting duct (IMCD) is an important site of vasopressin-regulated water and urea transport. Here we have used protein mass spectrometry to investigate the proteome of the IMCD cell, and how it is altered in response to long-term vasopressin administration in rats. IMCDs were isolated from inner medullas of rats, and IMCD proteins were identified by liquid chromatography/tandem mass spectrometry (LC-MS/MS). We present a WWW-based “IMCD Proteome Database”, containing all IMCD proteins identified in this study (n = 704) and prior MS-based identification studies (n = 301). We used the isotope-coded affinity tag (ICAT) technique to identify IMCD proteins that change in abundance in response to vasopressin. dDAVP or vehicle was infused subcutaneously in Brattleboro rats for 3 days and IMCDs were isolated for proteomic analysis. dDAVP and control samples were labeled with different cleavable ICAT reagents (mass difference 9 amu) and mixed. This was followed by 1-D SDS-PAGE separation, in-gel trypsin digestion, biotin-avidin affinity purification, and LC-MS/MS identification and quantification. Responses to vasopressin for a total of 165 proteins were quantified. Quantification based on semiquantitative immunoblotting of 16 proteins for which antibodies were available showed a high degree of correlation with ICAT results. In addition to aquaporin-2 and γ -ENaC, five of the immunoblotted proteins were substantially altered in abundance in response to dDAVP, viz. syntaxin-7, Rap1, GAPDH, HSP70, and cathepsin D. A 28-protein vasopressin signaling network was constructed using literature-based network analysis software focusing on the newly identified proteins, providing several new hypotheses for future studies. PMID:16449382

  19. 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. PMID:17057345

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

    PubMed Central

    Ramelot, Theresa A.; Raman, Srivatsan; Kuzin, Alexandre P.; Xiao, Rong; Ma, Li-Chung; Acton, Thomas B.; Hunt, John F.; Montelione, Gaetano T.; Baker, David; Kennedy, Michael A.

    2010-01-01

    The structure of human protein HSPC034 has been determined by both solution NMR spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta.1 Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints, have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the RPF scores. Upon further examination, the additional MR-performance shortfall for NMR refined structures as compared to the X-ray structure MR performance were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures, demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of non-bivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance PMID:18816799

  1. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

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

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

    PubMed Central

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

    2014-01-01

    Royal jelly (RJ) intake lowers serum cholesterol levels in animals and humans, but the active component in RJ that lowers serum cholesterol level and its molecular mechanism are unclear. In this study, we set out to identify the bile acid-binding protein contained in RJ, because dietary bile acid-binding proteins including soybean protein and its peptide are effective in ameliorating hypercholesterolemia. Using a cholic acid-conjugated column, we separated some bile acid-binding proteins from RJ and identified the major RJ protein 1 (MRJP1), MRJP2, and MRJP3 as novel bile acid-binding proteins from RJ, based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified MRJP1, which is the most abundant protein of the bile acid-binding proteins in RJ, exhibited taurocholate-binding activity in vitro. The micellar solubility of cholesterol was significantly decreased in the presence of MRJP1 compared with casein in vitro. Liver bile acids levels were significantly increased, and cholesterol 7α-hydroxylase (CYP7A1) mRNA and protein tended to increase by MRJP1 feeding compared with the control. CYP7A1 mRNA and protein levels were significantly increased by MRJP1 tryptic hydrolysate treatment compared with that of casein tryptic hydrolysate in hepatocytes. MRJP1 hypocholesterolemic effect has been investigated in rats. The cholesterol-lowering action induced by MRJP1 occurs because MRJP1 interacts with bile acids induces a significant increase in fecal bile acids excretion and a tendency to increase in fecal cholesterol excretion and also enhances the hepatic cholesterol catabolism. We have identified, for the first time, a novel hypocholesterolemic protein, MRJP1, in RJ. Interestingly, MRJP1 exhibits greater hypocholesterolemic activity than the medicine β-sitosterol in rats. PMID:25144734

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

    PubMed Central

    Krehenbrink, Martin; Downie, J Allan

    2008-01-01

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

  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. 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

  7. Residues Coevolution Guides the Systematic Identification of Alternative Functional Conformations in Proteins.

    PubMed

    Sfriso, Pedro; Duran-Frigola, Miquel; Mosca, Roberto; Emperador, Agustí; Aloy, Patrick; Orozco, Modesto

    2016-01-01

    We present here a new approach for the systematic identification of functionally relevant conformations in proteins. Our fully automated pipeline, based on discrete molecular dynamics enriched with coevolutionary information, is able to capture alternative conformational states in 76% of the proteins studied, providing key atomic details for understanding their function and mechanism of action. We also demonstrate that, given its sampling speed, our method is well suited to explore structural transitions in a high-throughput manner, and can be used to determine functional conformational transitions at the entire proteome level. PMID:26688214

  8. Bayesian identification of protein differential expression in multi-group isobaric labelled mass spectrometry data.

    PubMed

    Jow, Howsun; Boys, Richard J; Wilkinson, Darren J

    2014-10-01

    In this paper we develop a Bayesian statistical inference approach to the unified analysis of isobaric labelled MS/MS proteomic data across multiple experiments. An explicit probabilistic model of the log-intensity of the isobaric labels' reporter ions across multiple pre-defined groups and experiments is developed. This is then used to develop a full Bayesian statistical methodology for the identification of differentially expressed proteins, with respect to a control group, across multiple groups and experiments. This methodology is implemented and then evaluated on simulated data and on two model experimental datasets (for which the differentially expressed proteins are known) that use a TMT labelling protocol. PMID:25153608

  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. 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. The cassava (Manihot esculenta Crantz) root proteome: protein identification and differential expression.

    PubMed

    Sheffield, Jeanne; Taylor, Nigel; Fauquet, Claude; Chen, Sixue

    2006-03-01

    Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots. PMID:16421938

  12. Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

    PubMed

    Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

    2001-08-01

    A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG. PMID:11565788

  13. Mass spectrometry-based protein identification with accurate statistical significance assignment

    PubMed Central

    Alves, Gelio; Yu, Yi-Kuo

    2015-01-01

    Motivation: Assigning statistical significance accurately has become increasingly important as metadata of many types, often assembled in hierarchies, are constructed and combined for further biological analyses. Statistical inaccuracy of metadata at any level may propagate to downstream analyses, undermining the validity of scientific conclusions thus drawn. From the perspective of mass spectrometry-based proteomics, even though accurate statistics for peptide identification can now be achieved, accurate protein level statistics remain challenging. Results: We have constructed a protein ID method that combines peptide evidences of a candidate protein based on a rigorous formula derived earlier; in this formula the database P-value of every peptide is weighted, prior to the final combination, according to the number of proteins it maps to. We have also shown that this protein ID method provides accurate protein level E-value, eliminating the need of using empirical post-processing methods for type-I error control. Using a known protein mixture, we find that this protein ID method, when combined with the Sorić formula, yields accurate values for the proportion of false discoveries. In terms of retrieval efficacy, the results from our method are comparable with other methods tested. Availability and implementation: The source code, implemented in C++ on a linux system, is available for download at ftp://ftp.ncbi.nlm.nih.gov/pub/qmbp/qmbp_ms/RAId/RAId_Linux_64Bit. Contact: yyu@ncbi.nlm.nih.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25362092

  14. Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis.

    PubMed

    Agallou, Maria; Athanasiou, Evita; Samiotaki, Martina; Panayotou, George; Karagouni, Evdokia

    2016-01-01

    Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests. PMID:26906226

  15. Identification of Immunoreactive Leishmania infantum Protein Antigens to Asymptomatic Dog Sera through Combined Immunoproteomics and Bioinformatics Analysis

    PubMed Central

    Samiotaki, Martina; Panayotou, George; Karagouni, Evdokia

    2016-01-01

    Leishmania infantum is the etiologic agent of zoonotic visceral leishmaniasis (VL) in countries in the Mediterranean basin, where dogs are the domestic reservoirs and represent important elements in the transmission of the disease. Since the major focal areas of human VL exhibit a high prevalence of seropositive dogs, the control of canine VL could reduce the infection rate in humans. Efforts toward this have focused on the improvement of diagnostic tools, as well as on vaccine development. The identification of parasite antigens including suitable major histocompatibility complex (MHC) class I- and/or II-restricted epitopes is very important since disease protection is characterized by strong and long-lasting CD8+ T and CD4+ Th1 cell-dominated immunity. In the present study, total protein extract from late-log phase L. infantum promastigotes was analyzed by two-dimensional western blots and probed with sera from asymptomatic and symptomatic dogs. A total of 42 protein spots were found to differentially react with IgG from asymptomatic dogs, while 17 of these identified by Coommasie stain were extracted and analyzed. Of these, 21 proteins were identified by mass spectrometry; they were mainly involved in metabolism and stress responses. An in silico analysis predicted that the chaperonin HSP60, dihydrolipoamide dehydrogenase, enolase, cyclophilin 2, cyclophilin 40, and one hypothetical protein contain promiscuous MHCI and/or MHCII epitopes. Our results suggest that the combination of immunoproteomics and bioinformatics analyses is a promising method for the identification of novel candidate antigens for vaccine development or with potential use in the development of sensitive diagnostic tests. PMID:26906226

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

    PubMed Central

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

    2015-01-01

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

  17. Identification of a DNA binding protein cooperating with estrogen receptor as RIZ (retinoblastoma interacting zinc finger protein).

    PubMed

    Medici, N; Abbondanza, C; Nigro, V; Rossi, V; Piluso, G; Belsito, A; Gallo, L; Roscigno, A; Bontempo, P; Puca, A A; Molinari, A M; Moncharmont, B; Puca, G A

    1999-11-01

    Double-stranded DNA fragments were selected from a random pool by repeated cycles of estrogen receptor-specific immunoprecipitation in the presence of a nuclear extract and PCR amplification (cyclic amplification and selection of target, CAST, for multiple elements). Fragments were cloned and sequence analysis indicated the 5-nucleotide word TTGGC was the most recurrent sequence unrelated to the known estrogen responsive element. Screening a HeLa cell expression library with a probe designed with multiple repeats of this sequence resulted in the identification of a 1700-aa protein showing a complete homology with the product of the human retinoblastoma-interacting zinc-finger gene RIZ. In transfection experiments, RIZ protein was able to bestow estrogen inducibility to a promoter containing an incomplete estrogen responsive element and a TTGGC motif. RIZ protein present in MCF-7 cell nuclear extract retarded the TTGGC-containing probe in an EMSA. Estrogen receptor was co-immunoprecipitated from MCF-7 cell extract by antibodies to RIZ protein and vice versa, thus indicating an existing interaction between these two proteins. PMID:10544042

  18. Phosphotyrosine in proteins: chemical and immunological identification and isolation of phosphotyrosyl proteins

    SciTech Connect

    Martensen, T.M.

    1986-05-01

    Proteins which contain phosphotyrosine (Tyr-P) residues can be identified and purified by utilizing the chemical and immunological properties of the Tyr-P moiety. The stability of Tyr-P to basic conditions which cleave most ser/thr phosphoryl bonds enabled the analysis of cellular phosphoproteins after Na dodecyl SO/sub 4/ gel electrophoresis. Analyses were simplified using a nylon electroblot of the gel which is stable to 1 hr incubation in 1 N NaOH at 65/sup 0/C. Autoradiograms of nylon blots before and after base treatment were performed on /sup 32/P-labeled phosphoproteins of normal and retrovirus transformed fibroblasts. Conditions for optimal labeling of cellular proteins were studied by protein tyrosine kinase activity measurements using poly glu/sub 4/tyr. Differences in base resistant phosphoprotein patterns could be seen for each transformed cell line. Confirmation that certain base resistant phosphoproteins contained Tyr-P was achieved by immunostaining with affinity-purified sheep anti-Tyr-P antibodies (Ab). These Ab served as useful reagents to study Tyr-P proteins since they inhibit their dephosphorylation by phosphatases, and precipitate them in the presence of rabbit anti-sheep IgG Ab. Tyr-P protein was immobilized when mixed with anti-Tyr-P Ab and incubated with immobilized protein A sepharose rabbit anti-sheep IgG Ab complex. Selective elution of the Tyr-P protein was achieved with free Tyr-P.

  19. 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

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

    PubMed Central

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

    2015-01-01

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

  1. 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.

  2. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.

    PubMed Central

    Trifillis, P; Day, N; Kiledjian, M

    1999-01-01

    Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions. PMID:10445881

  3. Proteomics-Based Identification of Differentially Abundant Proteins from Human Keratinocytes Exposed to Arsenic Trioxide

    PubMed Central

    Udensi, Udensi K; Tackett, Alan J; Byrum, Stephanie; Avaritt, Nathan L; Sengupta, Deepanwita; Moreland, Linley W; Tchounwou, Paul B; Isokpehi, Raphael D

    2014-01-01

    Introduction Arsenic is a widely distributed environmental toxicant that can cause multi-tissue pathologies. Proteomic assays allow for the identification of biological processes modulated by arsenic in diverse tissue types. Method The altered abundance of proteins from HaCaT human keratinocyte cell line exposed to arsenic was quantified using a label-free LC-MS/MS mass spectrometry workflow. Selected proteomics results were validated using western blot and RT-PCR. A functional annotation analytics strategy that included visual analytical integration of heterogeneous data sets was developed to elucidate functional categories. The annotations integrated were mainly tissue localization, biological process and gene family. Result The abundance of 173 proteins was altered in keratinocytes exposed to arsenic; in which 96 proteins had increased abundance while 77 proteins had decreased abundance. These proteins were also classified into 69 Gene Ontology biological process terms. The increased abundance of transferrin receptor protein (TFRC) was validated and also annotated to participate in response to hypoxia. A total of 33 proteins (11 increased abundance and 22 decreased abundance) were associated with 18 metabolic process terms. The Glutamate--cysteine ligase catalytic subunit (GCLC), the only protein annotated with the term sulfur amino acid metabolism process, had increased abundance while succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial precursor (SDHB), a tumor suppressor, had decreased abundance. Conclusion A list of 173 differentially abundant proteins in response to arsenic trioxide was grouped using three major functional annotations covering tissue localization, biological process and protein families. A possible explanation for hyperpigmentation pathologies observed in arsenic toxicity is that arsenic exposure leads to increased iron uptake in the normally hypoxic human skin. The proteins mapped to metabolic process terms and

  4. 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.

  5. 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...

  6. 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.

  7. Improved protein quality in transgenic soybean expressing a de novo synthetic protein, MB-16.

    PubMed

    Zhang, Yunfang; Schernthaner, Johann; Labbé, Natalie; Hefford, Mary A; Zhao, Jiping; Simmonds, Daina H

    2014-06-01

    To improve soybean [Glycine max (L.) Merrill] seed nutritional quality, a synthetic gene, MB-16 was introduced into the soybean genome to boost seed methionine content. MB-16, an 11 kDa de novo protein enriched in the essential amino acids (EAAs) methionine, threonine, lysine and leucine, was originally developed for expression in rumen bacteria. For efficient seed expression, constructs were designed using the soybean codon bias, with and without the KDEL ER retention sequence, and β-conglycinin or cruciferin seed specific protein storage promoters. Homozygous lines, with single locus integrations, were identified for several transgenic events. Transgene transmission and MB-16 protein expression were confirmed to the T5 and T7 generations, respectively. Quantitative RT-PCR analysis of developing seed showed that the transcript peaked in growing seed, 5-6 mm long, remained at this peak level to the full-sized green seed and then was significantly reduced in maturing yellow seed. Transformed events carrying constructs with the rumen bacteria codon preference showed the same transcription pattern as those with the soybean codon preference, but the transcript levels were lower at each developmental stage. MB-16 protein levels, as determined by immunoblots, were highest in full-sized green seed but the protein virtually disappeared in mature seed. However, amino acid analysis of mature seed, in the best transgenic line, showed a significant increase of 16.2 and 65.9 % in methionine and cysteine, respectively, as compared to the parent. This indicates that MB-16 elevated the sulfur amino acids, improved the EAA seed profile and confirms that a de novo synthetic gene can enhance the nutritional quality of soybean. PMID:24435987

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

    PubMed Central

    Saritas-Yildirim, Banu; Pliner, Hannah A.; Ochoa, Angelica; Silva, Elena M.

    2015-01-01

    Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development. PMID:26327321

  9. Phosfinder: a web server for the identification of phosphate-binding sites on protein structures.

    PubMed

    Parca, Luca; Mangone, Iolanda; Gherardini, Pier Federico; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2011-07-01

    Phosfinder is a web server for the identification of phosphate binding sites in protein structures. Phosfinder uses a structural comparison algorithm to scan a query structure against a set of known 3D phosphate binding motifs. Whenever a structural similarity between the query protein and a phosphate binding motif is detected, the phosphate bound by the known motif is added to the protein structure thus representing a putative phosphate binding site. Predicted binding sites are then evaluated according to (i) their position with respect to the query protein solvent-excluded surface and (ii) the conservation of the binding residues in the protein family. The server accepts as input either the PDB code of the protein to be analyzed or a user-submitted structure in PDB format. All the search parameters are user modifiable. Phosfinder outputs a list of predicted binding sites with detailed information about their structural similarity with known phosphate binding motifs, and the conservation of the residues involved. A graphical applet allows the user to visualize the predicted binding sites on the query protein structure. The results on a set of 52 apo/holo structure pairs show that the performance of our method is largely unaffected by ligand-induced conformational changes. Phosfinder is available at http://phosfinder.bio.uniroma2.it. PMID:21622655

  10. Identification and characterization of intracellular proteins that bind oligonucleotides with phosphorothioate linkages

    PubMed Central

    Liang, Xue-hai; Sun, Hong; Shen, Wen; Crooke, Stanley T.

    2015-01-01

    Although the RNase H-dependent mechanism of inhibition of gene expression by chemically modified antisense oligonucleotides (ASOs) has been well characterized, little is known about the interactions between ASOs and intracellular proteins that may alter cellular localization and/or potency of ASOs. Here, we report the identification of 56 intracellular ASO-binding proteins using multi-step affinity selection approaches. Many of the tested proteins had no significant effect on ASO activity; however, some proteins, including La/SSB, NPM1, ANXA2, VARS and PC4, appeared to enhance ASO activities, likely through mechanisms related to subcellular distribution. VARS and ANXA2 co-localized with ASOs in endocytic organelles, and reduction in the level of VARS altered lysosome/ASO localization patterns, implying that these proteins may facilitate ASO release from the endocytic pathway. Depletion of La and NPM1 reduced nuclear ASO levels, suggesting potential roles in ASO nuclear accumulation. On the other hand, Ku70 and Ku80 proteins inhibited ASO activity, most likely by competition with RNase H1 for ASO/RNA duplex binding. Our results demonstrate that phosphorothioate-modified ASOs bind a set of cellular proteins that affect ASO activity via different mechanisms. PMID:25712094

  11. Identification of repetitive units in protein structures with ReUPred.

    PubMed

    Hirsh, Layla; Piovesan, Damiano; Paladin, Lisanna; Tosatto, Silvio C E

    2016-06-01

    Over the last decade, numerous studies have demonstrated the fundamental importance of tandem repeat (TR) proteins in many biological processes. A plethora of new repeat structures have also been solved. The recently published RepeatsDB provides information on TR proteins. However, a detailed structural characterization of repetitive elements is largely missing, as repeat unit annotation is manually curated and currently covers only 3 % of the bona fide TR proteins. Repeat Protein Unit Predictor (ReUPred) is a novel method for the fast automatic prediction of repeat units and repeat classification using an extensive Structure Repeat Unit Library (SRUL) derived from RepeatsDB. ReUPred uses an iterative structural search against the SRUL to find repetitive units. On a test set of solenoid proteins, ReUPred is able to correctly detect 92 % of the proteins. Unlike previous methods, it is also able to correctly classify solenoid repeats in 89 % of cases. It also outperforms two recent state-of-the-art methods for the repeat unit identification problem. The accurate prediction of repeat units increases the number of annotated repeat units by an order of magnitude compared to the sequence-based Pfam classification. ReUPred is implemented in Python for Linux and freely available from the URL: http://protein.bio.unipd.it/reupred/ . PMID:26898549

  12. SCNProDB: A database for the identification of soybean cyst nematode proteins

    PubMed Central

    Natarajan, Savithiry; Tavakolan, Mona; Alkharouf, Nadim W; Matthews, Benjamin F

    2014-01-01

    Soybean cyst nematode (Heterodera glycines, SCN) is the most destructive pathogen of soybean around the world. Crop rotation and resistant cultivars are used to mitigate the damage of SCN, but these approaches are not completely successful because of the varied SCN populations. Thus, the limitations of these practices with soybean dictate investigation of other avenues of protection of soybean against SCN, perhaps through genetically engineering of broad resistance to SCN. For better understanding of the consequences of genetic manipulation, elucidation of SCN protein composition at the subunit level is necessary. We have conducted studies to determine the composition of SCN proteins using a proteomics approach in our laboratory using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) to separate SCN proteins and to characterize the proteins further using mass spectrometry. Our analysis resulted in the identification of several hundred proteins. In this investigation, we developed a web based database (SCNProDB) containing protein information obtained from our previous published studies. This database will be useful to scientists who wish to develop SCN resistant soybean varieties through genetic manipulation and breeding efforts. The database is freely accessible from: http://bioinformatics.towson.edu/Soybean_SCN_proteins_2D_Gel_DB/Gel1.aspx PMID:25097385

  13. 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

  14. Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator*

    PubMed Central

    Wright, James C.; Collins, Mark O.; Yu, Lu; Käll, Lukas; Brosch, Markus; Choudhary, Jyoti S.

    2012-01-01

    Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques. PMID:22493177

  15. Improvement in separation characteristics of protein precipitates by acoustic conditioning.

    PubMed

    Hoare, M; Titchener, N J; Foster, P R

    1987-01-01

    The effect of acoustic conditioning on the particle size distribution of isoelectric and calcium-ion-precipitated soya protein has been examined in low-residence-time chambers. In a previous study a beat frequency of 5 Hz obtained using a dual-source system of opposing vibrators was determined as giving optimal improvement in particle-settling characteristics for isoelectric soya protein precipitate. In this study the effect of amplitude of vibration, a measure of acoustic power input, and residence time of acoustic conditioning has been examined. Acoustic power input changed the flow pattern in the conditioning chamber from laminar streamline flow to a well-mixed, turbulent pattern. Such a mixing effect promoted the rapid aggregation of fine particles, a process that was modeled on the basis of orthokinetically controlled collisions. The rate of removal of fine particles due to acoustic conditioning was shown to be proportional to a mixing effect that was related to the acoustic power dissipated per unit volume. The consequences of fine-particle aggregation on the centrifugal recovery of the precipitate are discussed. PMID:18561125

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

    PubMed Central

    Walz, Alexander; Mujer, Cesar V; Connolly, Joseph P; Alefantis, Tim; Chafin, Ryan; Dake, Clarissa; Whittington, Jessica; Kumar, Srikanta P; Khan, Akbar S; DelVecchio, Vito G

    2007-01-01

    relevant in elucidation of the progression of pathogenicity, identification of therapeutics and diagnostic markers, and vaccine development. This study also adds to the continuously growing list of identified Bacillus anthracis secretome proteins. PMID:17662140

  17. Identification of DNA-binding and protein-binding proteins using enhanced graph wavelet features.

    PubMed

    Zhu, Yuan; Zhou, Weiqiang; Dai, Dao-Qing; Yan, Hong

    2013-01-01

    Interactions between biomolecules play an essential role in various biological processes. For predicting DNA-binding or protein-binding proteins, many machine-learning-based techniques have used various types of features to represent the interface of the complexes, but they only deal with the properties of a single atom in the interface and do not take into account the information of neighborhood atoms directly. This paper proposes a new feature representation method for biomolecular interfaces based on the theory of graph wavelet. The enhanced graph wavelet features (EGWF) provides an effective way to characterize interface feature through adding physicochemical features and exploiting a graph wavelet formulation. Particularly, graph wavelet condenses the information around the center atom, and thus enhances the discrimination of features of biomolecule binding proteins in the feature space. Experiment results show that EGWF performs effectively for predicting DNA-binding and protein-binding proteins in terms of Matthew's correlation coefficient (MCC) score and the area value under the receiver operating characteristic curve (AUC). PMID:24334394

  18. 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.

  19. Proteomic Identification of Novel Differentiation Plasma Protein Markers in Hypobaric Hypoxia-Induced Rat Model

    PubMed Central

    Ahmad, Mohammad Faiz; Sharma, Manish; Garg, Iti; Bhargava, Kalpana

    2014-01-01

    Background Hypobaric hypoxia causes complex changes in the expression of genes, including stress related genes and corresponding proteins that are necessary to maintain homeostasis. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of complex and dynamic changes that occur during the hypobaric hypoxia. Methods In this study we investigated the temporal plasma protein alterations of rat induced by hypobaric hypoxia at a simulated altitude of 7620 m (25,000 ft, 282 mm Hg) in a hypobaric chamber. Total plasma proteins collected at different time points (0, 6, 12 and 24 h), separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption ionization time of flight (MALDI-TOF/TOF). Biological processes that were enriched in the plasma proteins during hypobaric hypoxia were identified using Gene Ontology (GO) analysis. According to their properties and obvious alterations during hypobaric hypoxia, changes of plasma concentrations of Ttr, Prdx-2, Gpx -3, Apo A-I, Hp, Apo-E, Fetub and Nme were selected to be validated by Western blot analysis. Results Bioinformatics analysis of 25 differentially expressed proteins showed that 23 had corresponding candidates in the database. The expression patterns of the eight selected proteins observed by Western blot were in agreement with 2-DE results, thus confirming the reliability of the proteomic analysis. Most of the proteins identified are related to cellular defense mechanisms involving anti-inflammatory and antioxidant activity. Their presence reflects the consequence of serial cascades initiated by hypobaric hypoxia. Conclusion/Significance This study provides information about the plasma proteome changes induced in response to hypobaric hypoxia and thus identification of the candidate proteins which can act as novel biomarkers. PMID:24842778

  20. Identification of O-linked β-D-N-acetylglucosamine-modified proteins from Arabidopsis.

    PubMed

    Xu, Shou-Ling; Chalkley, Robert J; Wang, Zhi-Yong; Burlingame, Alma L

    2012-01-01

    The posttranslational modification of proteins with O-linked β-D: -N-acetylglucosamine (O-GlcNAc) on serine and threonine residues occurs in all animals and plants. This modification is dynamic and ubiquitous, and regulates many cellular processes, including transcription, signaling and cytokinesis and is associated with several diseases. Cycling of O-GlcNAc is tightly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Plants have two OGTs, SPINDLY (SPY) and SECRET AGENT (SEC); disruption of both causes embryo lethality. Despite O-GlcNAc modification of proteins being discovered more than 20-years ago, identification and mapping of protein GlcNAcylation is still a challenging task. Here we describe the use of lectin affinity chromatography combined with electron transfer dissociation mass spectrometry to enrich and to detect O-GlcNAc modified peptides from Arabidopsis. PMID:22576084

  1. Phytochemical-mediated Protein Expression Profiling and the Potential Applications in Therapeutic Drug Target Identifications.

    PubMed

    Wong, Fai-Chu; Tan, Siok-Thing; Chai, Tsun-Thai

    2016-07-29

    Many phytochemicals derived from edible medicinal plants have been investigated intensively for their various bioactivities. However, the detailed mechanism and their corresponding molecular targets frequently remain elusive. In this review, we present a summary of the research works done on phytochemical-mediated molecular targets, identified via proteomic approach. Concurrently, we also highlighted some pharmaceutical drugs which could be traced back to their origins in phytochemicals. For ease of presentation, these identified protein targets were categorized into two important healthcare-related fields, namely anti-bacterial and anti-cancer research. Through this review, we hope to highlight the usefulness of comparative proteomic as a powerful tool in phytochemical-mediated protein target identifications. Likewise, we wish to inspire further investigations on some of these protein targets identified over the last few years. With contributions from all researchers, the accumulative efforts could eventually lead to the discovery of some target-specific, low-toxicity therapeutic agents. PMID:26193174

  2. 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. PMID:25776061

  3. Improved single-letter identification after whole-word training in pure alexia.

    PubMed

    Ablinger, Irene; Domahs, Frank

    2009-06-01

    Pure alexia is characterised by a very time-consuming letter-by-letter reading strategy due to an impaired identification or integration of single letters. So far, therapy interventions have addressed impaired letter identification using specific single-letter training approaches. In the present study, we report patient KA with pure alexia and letter-by-letter reading. Contrary to common approaches, we investigated whether a whole-word reading approach can be successful despite severe impairments at the level of single-letter identification. As a first step, auditory-visual verification tasks were used to familiarise the patient with the training items, which in a second step were read with limited exposure duration. After a four week intervention, KA's word reading performance improved significantly for trained and control items in terms of speed and accuracy. Although not specifically addressed during our training programme, even single-letter identification and text reading performance increased significantly. However, the patient's reading was still based on a letter-by-letter strategy. PMID:19191066

  4. SHIFTX2: significantly improved protein chemical shift prediction.

    PubMed

    Han, Beomsoo; Liu, Yifeng; Ginzinger, Simon W; Wishart, David S

    2011-05-01

    A new computer program, called SHIFTX2, is described which is capable of rapidly and accurately calculating diamagnetic (1)H, (13)C and (15)N chemical shifts from protein coordinate data. Compared to its predecessor (SHIFTX) and to other existing protein chemical shift prediction programs, SHIFTX2 is substantially more accurate (up to 26% better by correlation coefficient with an RMS error that is up to 3.3× smaller) than the next best performing program. It also provides significantly more coverage (up to 10% more), is significantly faster (up to 8.5×) and capable of calculating a wider variety of backbone and side chain chemical shifts (up to 6×) than many other shift predictors. In particular, SHIFTX2 is able to attain correlation coefficients between experimentally observed and predicted backbone chemical shifts of 0.9800 ((15)N), 0.9959 ((13)Cα), 0.9992 ((13)Cβ), 0.9676 ((13)C'), 0.9714 ((1)HN), 0.9744 ((1)Hα) and RMS errors of 1.1169, 0.4412, 0.5163, 0.5330, 0.1711, and 0.1231 ppm, respectively. The correlation between SHIFTX2's predicted and observed side chain chemical shifts is 0.9787 ((13)C) and 0.9482 ((1)H) with RMS errors of 0.9754 and 0.1723 ppm, respectively. SHIFTX2 is able to achieve such a high level of accuracy by using a large, high quality database of training proteins (>190), by utilizing advanced machine learning techniques, by incorporating many more features (χ(2) and χ(3) angles, solvent accessibility, H-bond geometry, pH, temperature), and by combining sequence-based with structure-based chemical shift prediction techniques. With this substantial improvement in accuracy we believe that SHIFTX2 will open the door to many long-anticipated applications of chemical shift prediction to protein structure determination, refinement and validation. SHIFTX2 is available both as a standalone program and as a web server ( http://www.shiftx2.ca ). PMID:21448735

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

    PubMed

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

    2011-07-20

    Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain. PMID:21612301

  6. Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)*

    PubMed Central

    Champasa, Kanokwan; Longwell, Scott A.; Eldridge, Aimee M.; Stemmler, Elizabeth A.; Dube, Danielle H.

    2013-01-01

    Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the

  7. 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...

  8. 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...

  9. Improving Information Technology Adoption and Implementation Through the Identification of Appropriate Benefits: Creating IMPROVE-IT

    PubMed Central

    Sittig, Dean F

    2007-01-01

    This paper describes the objectives of a collaborative initiative that attempts to provide the evidence that increased information technology (IT) capabilities, availability, and use lead directly to improved clinical quality, safety, and effectiveness within the inpatient hospital setting. This collaborative network has defined specific measurement indicators in an attempt to examine the existence, timing, and level of improvements in health outcomes that can be derived from IT investment. These indicators are in three areas: (1) IT costs (which includes both initial and ongoing investment), (2) IT infusion (ie, system availability, adoption, and deployment), and (3) health performance (eg, clinical efficacy, efficiency, quality, and effectiveness). Herein, we outline the theoretical framework, the methodology employed to create the metrics, and the benefits that can be obtained. PMID:17513287

  10. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  11. A Pseudo MS3 Approach for Identification of Disulfide-Bonded Proteins: Uncommon Product Ions and Database Search

    NASA Astrophysics Data System (ADS)

    Chen, Jianzhong; Shiyanov, Pavel; Schlager, John J.; Green, Kari B.

    2012-02-01

    It has previously been reported that disulfide and backbone bonds of native intact proteins can be concurrently cleaved using electrospray ionization (ESI) and collision-induced dissociation (CID) tandem mass spectrometry (MS/MS). However, the cleavages of disulfide bonds result in different cysteine modifications in product ions, making it difficult to identify the disulfide-bonded proteins via database search. To solve this identification problem, we have developed a pseudo MS3 approach by combining nozzle-skimmer dissociation (NSD) and CID on a quadrupole time-of-flight (Q-TOF) mass spectrometer using chicken lysozyme as a model. Although many of the product ions were similar to those typically seen in MS/MS spectra of enzymatically derived peptides, additional uncommon product ions were detected including ci-1 ions (the ith residue being aspartic acid, arginine, lysine and dehydroalanine) as well as those from a scrambled sequence. The formation of these uncommon types of product ions, likely caused by the lack of mobile protons, were proposed to involve bond rearrangements via a six-membered ring transition state and/or salt bridge(s). A search of 20 pseudo MS3 spectra against the Gallus gallus (chicken) database using Batch-Tag, a program originally designed for bottom up MS/MS analysis, identified chicken lysozyme as the only hit with the expectation values less than 0.02 for 12 of the spectra. The pseudo MS3 approach may help to identify disulfide-bonded proteins and determine the associated post-translational modifications (PTMs); the confidence in the identification may be improved by incorporating the fragmentation characteristics into currently available search programs.

  12. Improving the representation of peptide-like inhibitor and antibiotic molecules in the Protein Data Bank.

    PubMed

    Dutta, Shuchismita; Dimitropoulos, Dimitris; Feng, Zukang; Persikova, Irina; Sen, Sanchayita; Shao, Chenghua; Westbrook, John; Young, Jasmine; Zhuravleva, Marina A; Kleywegt, Gerard J; Berman, Helen M

    2014-06-01

    With the accumulation of a large number and variety of molecules in the Protein Data Bank (PDB) comes the need on occasion to review and improve their representation. The Worldwide PDB (wwPDB) partners have periodically updated various aspects of structural data representation to improve the integrity and consistency of the archive. The remediation effort described here was focused on improving the representation of peptide-like inhibitor and antibiotic molecules so that they can be easily identified and analyzed. Peptide-like inhibitors or antibiotics were identified in over 1000 PDB entries, systematically reviewed and represented either as peptides with polymer sequence or as single components. For the majority of the single-component molecules, their peptide-like composition was captured in a new representation, called the subcomponent sequence. A novel concept called "group" was developed for representing complex peptide-like antibiotics and inhibitors that are composed of multiple polymer and nonpolymer components. In addition, a reference dictionary was developed with detailed information about these peptide-like molecules to aid in their annotation, identification and analysis. Based on the experience gained in this remediation, guidelines, procedures, and tools were developed to annotate new depositions containing peptide-like inhibitors and antibiotics accurately and consistently. PMID:24173824

  13. Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

    PubMed Central

    Ferrell, J E; Martin, G S

    1990-01-01

    We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C. Images PMID:1692963

  14. Identification of angiotensin-converting enzyme inhibitory proteins from mycelium of Pleurotus pulmonarius (oyster mushroom).

    PubMed

    Ibadallah, Badjie Xietaqieuallah; Abdullah, Noorlidah; Shuib, Adawiyah Suriza

    2015-01-01

    Pleurotus pulmonarius (grey oyster mushroom) has been acknowledged as a recuperative agent for many diseases in addition to its recognition as a nutritious provision. We performed a study on P. pulmonarius mycelium for an antihypertensive effect via the angiotensin-converting enzyme inhibitory activity. The preliminary assay on the mycelial water extract demonstrated that the angiotensin-converting enzyme inhibitory activity had an IC50 value of 720 µg/mL. Further protein purifications via ammonium sulphate precipitation and RP-HPLC resulted in 60× stronger angiotensin-converting enzyme inhibitory activity than that of the mycelial water extract (IC50 = 12 µg/mL). Protein identification and characterisation by MALDI-TOF/TOF, later corroborated by LC-MS/MS, indicated three proteins that are responsible for the blood pressure lowering effects via different mechanisms: serine proteinase inhibitor-like protein, nitrite reductase-like protein, and DEAD/DEAH box RNA helicase-like protein. PMID:25590365

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

    PubMed

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

    2015-02-01

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

  16. Identification of peptides that inhibit regulator of G protein signaling 4 function.

    PubMed

    Wang, Yuren; Lee, Yan; Zhang, Jie; Young, Kathleen H

    2008-01-01

    Regulators of G protein signaling (RGS) are a family of GTPase-activating proteins (GAP) that interact with heterotrimeric G proteins in the negative regulation of G-protein-coupled receptor (GPCR) signaling. RGS4, the first identified mammalian member of the RGS family, has been implicated in many GPCR signaling pathways involved in disease states. We report herein the identification of a 16-amino-acid peptide (P17) as an inhibitor of RGS4. The peptide was found by screening a random peptide library using RGS4 as 'bait' in a yeast two-hybrid system. This peptide inhibited RGS4 GAP activity on Galpha(i1)in a GTPase assay, and blocked the interaction between RGS4 and Galpha(i1)in a pull-down assay. The peptide displayed dose-dependent inhibition of RGS4 and Galpha-interacting protein (GAIP) GAP activities, yet showed no substantial effect on RGS7. Electrophysiological studies in Xenopus oocytes demonstrated that P17 attenuates RGS4 modulation of M(2) muscarinic receptor stimulation of GIRK (G-protein-mediated inwardly rectifying potassium) channels. Deletion of an arginine at the N terminus of P17 abolished its ability to inhibit RGS4 GAP activity, as did deletions of C-terminal residues. The P17 peptide showed no similarity to any known peptide sequence. Further investigation and optimization of the peptide may provide unique information for the development of RGS4 inhibitors for future therapeutic application. PMID:18547979

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

    PubMed Central

    Nahm, Francis Sahngun; Park, Zee-Yong; Kim, Yong Chul

    2014-01-01

    Background. Complex regional pain syndrome (CRPS) is a rare but debilitating pain disorder. Although the exact pathophysiology of CRPS is not fully understood, central and peripheral mechanisms might be involved in the development of this disorder. To reveal the central mechanism of CRPS, we conducted a proteomic analysis of rat cerebrum using the chronic postischemia pain (CPIP) model, a novel experimental model of CRPS. Materials and Methods. After generating the CPIP animal model, we performed a proteomic analysis of the rat cerebrum using a multidimensional protein identification technology, and screened the proteins differentially expressed between the CPIP and control groups. Results. A total of 155 proteins were differentially expressed between the CPIP and control groups: 125 increased and 30 decreased; expressions of proteins related to cell signaling, synaptic plasticity, regulation of cell proliferation, and cytoskeletal formation were increased in the CPIP group. However, proenkephalin A, cereblon, and neuroserpin were decreased in CPIP group. Conclusion. Altered expression of cerebral proteins in the CPIP model indicates cerebral involvement in the pathogenesis of CRPS. Further study is required to elucidate the roles of these proteins in the development and maintenance of CRPS. PMID:25313364

  18. 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. PMID:7482398

  19. 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

  20. Screening and identification of the differential proteins in kidney with complete unilateral ureteral obstruction

    PubMed Central

    Zhao, Qi; Yang, Yi; Wang, Chang-Lin; Hou, Ying; Chen, Hui

    2015-01-01

    Obstructive nephropathy is a major cause of renal failure, particularly in infants and children, and indications for therapeutic intervention remain highly controversial. There is a great need for the development of new methods to monitor patients, and the biomarker research field is a promising approach for this purpose to be used as prognostic tools for early disease detection and the choice of the optimal treatment and monitoring. Here, we presented our comparative proteomics study of rat kidney with complete unilateral ureteral obstruction (CUUO). Proteins from the groups of CUUO and corresponding sham rat kidney tissues were subjected to 2-D gel electrophoresis, and then protein identification by mass spectrometry. We identified 39 proteins with differential expression between kidney tissues from sham operated group and those with CUUO. These identified proteins were reported to be involved in cell apoptosis, energy metabolism and injuries of mitochondrion and oxidative stress, and so on. We confirmed 3 identified proteins by immunoblot analysis and immunofluorescence staining and assessed their mRNA levels in renal tissues. Our results demonstrate protein alterations that reflect the pathological situation of the obstructed kidneys, which may help understand the relationship between oxidative stress and obstructive nephropathy. PMID:26045767

  1. 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.

  2. Comprehensive identification of substrates for F-box proteins by differential proteomics analysis.

    PubMed

    Yumimoto, Kanae; Matsumoto, Masaki; Oyamada, Koji; Moroishi, Toshiro; Nakayama, Keiichi I

    2012-06-01

    Although elucidation of enzyme-substrate relations is fundamental to the advancement of biology, universal approaches to the identification of substrates for a given enzyme have not been established. It is especially difficult to identify substrates for ubiquitin ligases, given that most such substrates are immediately ubiquitylated and degraded as a result of their association with the enzyme. We here describe the development of a new approach, DiPIUS (differential proteomics-based identification of ubiquitylation substrates), to the discovery of substrates for ubiquitin ligases. We applied DiPIUS to Fbxw7α, Skp2, and Fbxl5, three of the most well-characterized F-box proteins, and identified candidate substrates including previously known targets. DiPIUS is thus a powerful tool for unbiased and comprehensive screening for substrates of ubiquitin ligases. PMID:22524983

  3. 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.

  4. Improved Sequence Tag Generation Method for Peptide Identification in Tandem Mass Spectrometry

    PubMed Central

    Cao, Xia; Nesvizhskii, Alexey I.

    2013-01-01

    The sequence tag-based peptide identification methods are a promising alternative to the traditional database search approach. However, a more comprehensive analysis, optimization, and comparison with established methods are necessary before these methods can gain widespread use in the proteomics community. Using the InsPecT open source code base (Tanner et al., Anal Chem. 2005, 77:4626–39), we present an improved sequence tag generation method that directly incorporates multi-charged fragment ion peaks present in many tandem mass spectra of higher charge states. We also investigate the performance of sequence tagging under different settings using control datasets generated on five different types of mass spectrometers, as well as using a complex phosphopeptide-enriched sample. We also demonstrate that additional modeling of InsPecT search scores using a semi-parametric approach incorporating the accuracy of the precursor ion mass measurement provides additional improvement in the ability to discriminate between correct and incorrect peptide identifications. The overall superior performance of the sequence tag-based peptide identification method is demonstrated by comparison with a commonly used SEQUEST/PeptideProphet approach. PMID:18785767

  5. Improvements to the Percolator algorithm for peptide identification from shotgun proteomics data sets

    PubMed Central

    Spivak, Marina; Weston, Jason; Bottou, Léon; Käll, Lukas; Noble, William Stafford

    2009-01-01

    Shotgun proteomics coupled with database search software allows the identification of a large number of peptides in a single experiment. However, some existing search algorithms, such as SEQUEST, use score functions that are designed primarily to identify the best peptide for a given spectrum. Consequently, when comparing identifications across spectra, the SEQUEST score function Xcorr fails to discriminate accurately between correct and incorrect peptide identifications. Several machine learning methods have been proposed to address the resulting classification task of distinguishing between correct and incorrect peptide-spectrum matches (PSMs). A recent example is Percolator, which uses semi-supervised learning and a decoy database search strategy to learn to distinguish between correct and incorrect PSMs identified by a database search algorithm. The current work describes three improvements to Percolator. (1) Percolator’s heuristic optimization is replaced with a clear objective function, with intuitive reasons behind its choice. (2) Tractable nonlinear models are used instead of linear models, leading to improved accuracy over the original Percolator. (3) A method, Q-ranker, for directly optimizing the number of identified spectra at a specified q value is proposed, which achieves further gains. PMID:19385687

  6. Identification of a Nosema bombycis (Microsporidia) spore wall protein corresponding to spore phagocytosis.

    PubMed

    Cai, Shunfeng; Lu, Xingmeng; Qiu, Haihong; Li, Mingqian; Feng, Zhenzhen

    2011-08-01

    Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion. PMID:21756420

  7. Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment.

    PubMed

    Dasari, Surendra; Chambers, Matthew C; Martinez, Misti A; Carpenter, Kristin L; Ham, Amy-Joan L; Vega-Montoto, Lorenzo J; Tabb, David L

    2012-03-01

    Spectral libraries have emerged as a viable alternative to protein sequence databases for peptide identification. These libraries contain previously detected peptide sequences and their corresponding tandem mass spectra (MS/MS). Search engines can then identify peptides by comparing experimental MS/MS scans to those in the library. Many of these algorithms employ the dot product score for measuring the quality of a spectrum-spectrum match (SSM). This scoring system does not offer a clear statistical interpretation and ignores fragment ion m/z discrepancies in the scoring. We developed a new spectral library search engine, Pepitome, which employs statistical systems for scoring SSMs. Pepitome outperformed the leading library search tool, SpectraST, when analyzing data sets acquired on three different mass spectrometry platforms. We characterized the reliability of spectral library searches by confirming shotgun proteomics identifications through RNA-Seq data. Applying spectral library and database searches on the same sample revealed their complementary nature. Pepitome identifications enabled the automation of quality analysis and quality control (QA/QC) for shotgun proteomics data acquisition pipelines. PMID:22217208

  8. l2 Multiple Kernel Fuzzy SVM-Based Data Fusion for Improving Peptide Identification.

    PubMed

    Jian, Ling; Xia, Zhonghang; Niu, Xinnan; Liang, Xijun; Samir, Parimal; Link, Andrew J

    2016-01-01

    SEQUEST is a database-searching engine, which calculates the correlation score between observed spectrum and theoretical spectrum deduced from protein sequences stored in a flat text file, even though it is not a relational and object-oriental repository. Nevertheless, the SEQUEST score functions fail to discriminate between true and false PSMs accurately. Some approaches, such as PeptideProphet and Percolator, have been proposed to address the task of distinguishing true and false PSMs. However, most of these methods employ time-consuming learning algorithms to validate peptide assignments [1] . In this paper, we propose a fast algorithm for validating peptide identification by incorporating heterogeneous information from SEQUEST scores and peptide digested knowledge. To automate the peptide identification process and incorporate additional information, we employ l2 multiple kernel learning (MKL) to implement the current peptide identification task. Results on experimental datasets indicate that compared with state-of-the-art methods, i.e., PeptideProphet and Percolator, our data fusing strategy has comparable performance but reduces the running time significantly. PMID:26394437

  9. Immunochemical Methods Applied to Art-Historical Materials: Identification and Localization of Proteins by ELISA and IFM.

    PubMed

    Cartechini, Laura; Palmieri, Melissa; Vagnini, Manuela; Pitzurra, Lucia

    2016-02-01

    Despite the large diffusion of natural organic substances in art-historical materials, their characterization presents many challenges due to the chemical complexity and instability with respect to degradation processes. Among natural products, proteins have been largely used in the past as binders but also as adhesives or additives in coating layers. Nevertheless, biological identification of proteins in art-historical objects is one of the most recent achievements obtained in heritage science thanks to the development of specifically tailored bio-analytical strategies. In the context of this active emerging discipline, immunological methods stand out for sensitivity, specificity and versatility for both protein recognition and localization in micro-samples. Furthermore, the growing use of immunological techniques for advanced diagnostics and clinical applications ensures continuous improvement in their analytical performance. Considering such, this review provides an overview of the most recent applications of enzyme linked immunosorbent assay and immunofluorescence microscopy techniques in the field of heritage materials. Specifically, the main strengths and potentials of the two techniques as well as their limits and drawbacks are presented and discussed herein. PMID:27572988

  10. A GTPase-activating protein for the G protein Galphaz. Identification, purification, and mechanism of action.

    PubMed

    Wang, J; Tu, Y; Woodson, J; Song, X; Ross, E M

    1997-02-28

    A GTPase-activating protein (GAP) specific for Galphaz was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. Gz GAP from bovine brain is a membrane protein that is refractory to solubilization with most detergents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of Mr approximately 22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble Galphaz, the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min -1 at 15 degrees C, or to >/=20 min-1 at 30 degrees C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric Gz and m2 muscarinic receptor, Gz GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the Km of the GAP for Galphaz-GTP is 2 nM. Its activity is inhibited by Galphaz-guanosine 5'-O-thiotriphosphate (Galphaz-GTPgammaS) or by Galphaz-GDP/AlF4 with Ki approximately 1.5 nM for both species; Galphaz-GDP does not inhibit. G protein betagamma subunits inhibit Gz GAP activity, apparently by forming a GTP-Galphazbetagamma complex that is a poor GAP substrate. Gz GAP displays little GAP activity toward Galphai1 or Galphao, but its activity with Galphaz is competitively inhibited by both Galphai1 and Galphao at nanomolar concentrations when they are bound to GTPgammaS but not to GDP. Neither phospholipase C-beta1 (a Gq GAP) nor several adenylyl cyclase isoforms display Gz GAP activity. PMID:9038185

  11. 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

  12. Identification of polymer surface adsorbed proteins implicated in pluripotent human embryonic stem cell expansion.

    PubMed

    Hammad, Moamen; Rao, Wei; Smith, James G W; Anderson, Daniel G; Langer, Robert; Young, Lorraine E; Barrett, David A; Davies, Martyn C; Denning, Chris; Alexander, Morgan R

    2016-08-16

    Improved biomaterials are required for application in regenerative medicine, biosensing, and as medical devices. The response of cells to the chemistry of polymers cultured in media is generally regarded as being dominated by proteins adsorbed to the surface. Here we use mass spectrometry to identify proteins adsorbed from a complex mouse embryonic fibroblast (MEF) conditioned medium found to support pluripotent human embryonic stem cell (hESC) expansion on a plasma etched tissue culture polystyrene surface. A total of 71 proteins were identified, of which 14 uniquely correlated with the surface on which pluripotent stem cell expansion was achieved. We have developed a microarray combinatorial protein spotting approach to test the potential of these 14 proteins to support expansion of a hESC cell line (HUES-7) and a human induced pluripotent stem cell line (ReBl-PAT) on a novel polymer (N-(4-Hydroxyphenyl) methacrylamide). These proteins were spotted to form a primary array yielding several protein mixture 'hits' that enhanced cell attachment to the polymer. A second array was generated to test the function of a refined set of protein mixtures. We found that a combination of heat shock protein 90 and heat shock protein-1 encourage elevated adherence of pluripotent stem cells at a level comparable to fibronectin pre-treatment. PMID:27466628

  13. Improved method for simultaneous isolation of proteins and nucleic acids.

    PubMed

    Chey, Soroth; Claus, Claudia; Liebert, Uwe Gerd

    2011-04-01

    Guanidinium thiocyanate-phenol-chloroform extraction (GTPC extraction) is widely used in molecular biology for isolating DNA, RNA, and proteins. Protein isolation by commercially available GTPC solutions is time consuming and the resulting pellets are only incompletely soluble. In this study ethanol-bromochloropropane-water was used for precipitation of proteins from the phenol-ethanol phase after GTPC extraction of RNA and DNA. The precipitated proteins can be readily dissolved in 4% SDS for subsequent analysis. This technique allows a fast (30min) and efficient (with a protein recovery of up to 95%) extraction of proteins for the study of transcriptional and posttranscriptional events from the same sample. PMID:21094121

  14. Separation and identification of the light harvesting proteins contained in grana and stroma thylakoid membrane fractions.

    PubMed

    Timperio, Anna Maria; Huber, Christian G; Zolla, Lello

    2004-06-18

    This paper presents the results of a study performed to develop a rapid and straightforward method to resolve and simultaneously identify the light-harvesting proteins of photosystem I (LHCI) and photosystem II (LHCII) present in the grana and stroma of the thylakoid membranes of higher plants. These hydrophobic proteins are embedded in the phospholipid membrane, and their extraction usually requires detergent and time consuming manipulations that may introduce artifacts. The method presented here makes use of digitonin, a detergent which causes rapid (within less than 3 min) cleavage of the thylakoid membrane into two subfractions: appressed (grana) and non-appressed (stroma) membranes, the former enriched in photosystem II and the latter containing mainly photosystem I. From these two fractions identification of the protein components was performed by separating them by reversed-phase high-performance liquid chromatography (RP-HPLC) and determining the intact molecular mass by electrospray ionization mass spectrometry (ESI-MS). By this strategy the ion suppression during ESI-MS that normally occurs in the presence of membrane phospholipids was avoided, since RP-HPLC removed most phospholipids from the analytes. Consequently, high quality mass spectra were extracted from the reconstructed ion chromatograms. The specific cleavage of thylakoid membranes by digitonin, as well as the rapid identification and quantification of the antenna composition of the two complexes facilitate future studies of the lateral migration of the chlorophyll-protein complexes along thylakoid membranes, which is well known to be induced by high intensity light or other environmental stresses. Such investigations could not be performed by sodium dodecylsulfate-polyacrylamide gel electrophoresis because of insufficient resolution of the proteins having molecular masses between 22,000 and 25,000. PMID:15248427

  15. Identification of pleckstrin-homology-domain-containing proteins with novel phosphoinositide-binding specificities.

    PubMed Central

    Dowler, S; Currie , R A; Campbell , D G; Deak, M; Kular, G; Downes, C P; Alessi, D R

    2000-01-01

    The second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is generated by the action of phosphoinositide 3-kinase (PI 3-kinase), and regulates a plethora of cellular processes. An approach for dissecting the mechanisms by which these processes are regulated is to identify proteins that interact specifically with PtdIns(3,4,5)P(3). The pleckstrin homology (PH) domain has become recognized as the specialized module used by many proteins to interact with PtdIns(3,4,5)P(3). Recent work has led to the identification of a putative phosphatidylinositol 3,4,5-trisphosphate-binding motif (PPBM) at the N-terminal regions of PH domains that interact with this lipid. We have searched expressed sequence tag databases for novel proteins containing PH domains possessing a PPBM. Surprisingly, many of the PH domains that we identified do not bind PtdIns(3,4,5)P(3), but instead possess unexpected and novel phosphoinositide-binding specificities in vitro. These include proteins possessing PH domains that interact specifically with PtdIns(3,4)P(2) [TAPP1 (tandem PH-domain-containing protein-1) and TAPP2], PtdIns4P [FAPP1 (phosphatidylinositol-four-phosphate adaptor protein-1)], PtdIns3P [PEPP1 (phosphatidylinositol-three-phosphate-binding PH-domain protein-1) and AtPH1] and PtdIns(3,5)P(2) (centaurin-beta2). We have also identified two related homologues of PEPP1, termed PEPP2 and PEPP3, that may also interact with PtdIns3P. This study lays the foundation for future work to establish the phospholipid-binding specificities of these proteins in vivo, and their physiological role(s). PMID:11001876

  16. Characterization of quinoa seed proteome combining different protein precipitation techniques: Improvement of knowledge of nonmodel plant proteomics.

    PubMed

    Capriotti, Anna Laura; Cavaliere, Chiara; Piovesana, Susy; Stampachiacchiere, Serena; Ventura, Salvatore; Zenezini Chiozzi, Riccardo; Laganà, Aldo

    2015-03-01

    A shotgun proteomics approach was used to characterize the quinoa seed proteome. To obtain comprehensive proteomic data from quinoa seeds three different precipitation procedures were employed: MeOH/CHCl3 /double-distilled H2 O, acetone either alone or with trichloroacetic acid; the isolated proteins were then in-solution digested and the resulting peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. However, since quinoa is a nonmodel plant species, only a few protein sequences are included in the most widely known protein sequence databases. To improve the data reliability a UniProt subdatabase, containing only proteins of Caryophillales order, was used. A total of 352 proteins were identified and evaluated both from a qualitative and quantitative point of view. This combined approach is certainly useful to increase the final number of identifications, but no particular class of proteins was extracted and identified in spite of the different chemistries and the different precipitation protocols. However, with respect to the other two procedures, from the relative quantitative analysis, based on the number of spectral counts, the trichloroacetic acid/acetone protocol was the best procedure for sample handling and quantitative protein extraction. This study could pave the way to further high-throughput studies on Chenopodium Quinoa. PMID:25580831

  17. 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.

  18. Identification of two bvg-repressed surface proteins of Bordetella pertussis.

    PubMed Central

    Stenson, T H; Peppler, M S

    1995-01-01

    Bordetella pertussis, the etiological agent of whooping cough, has the ability to modulate its phenotype in response to environmental conditions by using the BvgAS sensory transduction system which is encoded by the vir locus (now known as bvg). The BvgAS system is part of a large family of two-component sensory transduction systems which are common to a number of pathogenic bacteria. Although much is known about the proteins which exist in the B. pertussis virulent (X-mode or phase I) phenotype, relatively little is known about the proteins produced in the avirulent (C-mode or phase III) phenotype. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing techniques to demonstrate the existence of at least 22 vir-repressed molecules which are increased in the avirulent phenotype. In addition, a series of monoclonal antibodies which are specific for the surface of avirulent B. pertussis were developed. Using immunological and protein techniques, we characterized two of these antigens as surface-exposed proteins. One of these antigens is expressed only in B. pertussis but not in the related species B. parapertussis and B. bronchiseptica. The other antigen is also present in B. parapertussis and B. bronchiseptica but is expressed at lower levels which are not regulated by bvg. The identification and characterization of vir-repressed proteins (and the genes which encode and regulate them) may help elucidate a physiological role for modulation of this obligate human pathogen. PMID:7558280

  19. Identification and analysis of copine/BONZAI proteins among evolutionarily diverse plant species.

    PubMed

    Zou, Baohong; Hong, Xuexue; Ding, Yuan; Wang, Xiang; Liu, He; Hua, Jian

    2016-08-01

    Copines are evolutionarily conserved calcium-dependent membrane-binding proteins with potentially critical biological functions. In plants, the function of these proteins has not been analyzed except for in Arabidopsis thaliana where they play critical roles in development and disease resistance. To facilitate functional studies of copine proteins in crop plants, genome-wide identification, curation, and phylogeny analysis of copines in 16 selected plant species were conducted. All the identified 32 plant copines have conserved features of the two C2 domains (C2A and C2B) and the von Willebrand factor A (vWA) domain. Different from animal and protozoa copines, plant copines have glycine at the second residue potentially acquiring a unique protein myristoylation modification. Phylogenetic analysis suggests that copine was present as one copy when evolving from green algae to basal flowering plants, and duplicated before the divergence of monocots and dicots. In addition, gene expression and protein localization study of rice copines suggests both conserved and different properties of copines in dicots and monocots. This study will contribute to uncovering the role of copine genes in different plant species. PMID:27484220

  20. Bio-guided identification of proteins for the diagnosis of cysticercosis in swine.

    PubMed

    Nativel, Priscilla; Rahantamalala, Anjanirina; Ramiandrisoa, Sitraka; Rasoamampianinaa, Virginie; Duchateau, Magalie; Chamot-Rooke, Julia; Guebey, Remy; Rasamoelina-Andriamanivo, Harentsoaniaina; Jambou, Ronan

    2016-04-15

    Neurocysticercosis (NCC) is one of the most prevalent parasitic infection of the brain and the most common cause of seizures in adults in tropical countries. Cysticercosis is caused by larvae of Taenia solium, a human tapeworm. Pig or humans are infected by ingestion of eggs in food contaminated by human feces. Diagnosis and treatment of pigs is a pillar of the control of the disease in a country. However current diagnostic tests are based on ELISA and/or Western blot using native antigens needing laboratory facilities not available in rural areas. Development of a pen side diagnostic test for swines, makes sense. Immunochromatographic test should be adapted for this purpose. To design it we started a bio-guided identification of new proteins in cysticercus fluid. Proteins were analyzed using ion exchange chromatography and 2D separation and were selected by Western blot analysis using sera from infected/non infected pigs. Spots from the Coomassie-stained gel corresponding to these proteins were then analyzed by mass spectroscopy and proteins were identified using a bank of Expressed Sequence Tags (EST) of T. solium. Eighteen new proteins of interest were identified and nine were selected for further development. PMID:26995717

  1. Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites

    PubMed Central

    Bassi, Roberto; Croce, Roberta; Cugini, Daniela; Sandonà, Dorianna

    1999-01-01

    The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a–Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins. PMID:10468561

  2. Identification of major Toxoneuron nigriceps venom proteins using an integrated transcriptomic/proteomic approach.

    PubMed

    Laurino, Simona; Grossi, Gerarda; Pucci, Pietro; Flagiello, Angela; Bufo, Sabino Aurelio; Bianco, Giuliana; Salvia, Rosanna; Vinson, S Bradleigh; Vogel, Heiko; Falabella, Patrizia

    2016-09-01

    Endoparasitoids in the order Hymenoptera are natural enemies of several herbivorous insect pest species. During oviposition they inject a mixture of factors, which include venom, into the host, ensuring the successful parasitism and the development of their progeny. Although these parasitoid factors are known to be responsible for host manipulation, such as immune system suppression, little is known about both identity and function of the majority of their venom components. To identify the major proteins of Toxoneuron nigriceps (Hymenoptera: Braconidae) venom, we used an integrated transcriptomic and proteomic approach. The tandem-mass spectrometric (LC-MS/MS) data combined with T. nigriceps venom gland transcriptome used as a reference database resulted in the identification of a total of thirty one different proteins. While some of the identified proteins have been described in venom from several parasitoids, others were identified for the first time. Among the identified proteins, hydrolases constituted the most abundant family followed by transferases, oxidoreductases, ligases, lyases and isomerases. The hydrolases identified in the T. nigriceps venom glands included proteases, peptidases and glycosidases, reported as common components of venom from several parasitoid species. Taken together, the identified proteins included factors that could potentially inhibit the host immune system, manipulate host physiological processes and host development, as well as provide nutrients to the parasitoid progeny, degrading host tissues by specific hydrolytic enzymes. The venom decoding provides us with information about the identity of candidate venom factors which could contribute to the success of parasitism, together with other maternal and embryonic factors. PMID:27388778

  3. Identification of a new transmembrane adaptor protein that constitutively binds Grb2 in B cells

    PubMed Central

    Liu, Yan; Zhang, Weiguo

    2008-01-01

    Transmembrane adaptor proteins couple antigen receptor engagement to downstream signaling cascades in lymphocytes. One example of these proteins is the linker for activation of T cells (LAT), which plays an indispensable role in T cell activation and development. Here, we report identification of a new transmembrane adaptor molecule, namely growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT), which is expressed in B cells and myeloid cells. Similar to LAT, GAPT has an extracellular domain, a transmembrane domain, and a cytoplasmic tail with multiple Grb2-binding motifs. In contrast to other transmembrane adaptor proteins, GAPT is not phosphorylated upon BCR ligation but associates with Grb2 constitutively through its proline-rich region. Targeted disruption of the gapt gene in mice affects neither B cell development nor a nitrophenylacetyl-specific antibody response. However, in the absence of GAPT, B cell proliferation after BCR cross-linking is enhanced. In aged GAPT−/− mice, the number of marginal zone (MZ) B cells is increased, and other B cell subsets are normal. The serum concentrations of IgM, IgG2b, and IgG3 are also elevated in these mice. These data indicate that GAPT might play an important role in control of B cell activation and proper maintenance of MZ B cells. PMID:18559951

  4. Germins: A Diverse Protein Family Important For Crop Improvement

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The germin protein family is comprised of two main subgroups in plants, oxalate oxidases (OXOs) and germin-like proteins (GLPs). These proteins are implicated in a variety of plant processes including germination, development, pollen formation, and response to abiotic and biotic stress. Here, we exa...

  5. Fluorescently Labeled Peptide Increases Identification of Degenerated Facial Nerve Branches during Surgery and Improves Functional Outcome

    PubMed Central

    Hussain, Timon; Mastrodimos, Melina B.; Raju, Sharat C.; Glasgow, Heather L.; Whitney, Michael; Friedman, Beth; Moore, Jeffrey D.; Kleinfeld, David; Steinbach, Paul; Messer, Karen; Pu, Minya; Tsien, Roger Y.; Nguyen, Quyen T.

    2015-01-01

    Nerve degeneration after transection injury decreases intraoperative visibility under white light (WL), complicating surgical repair. We show here that the use of fluorescently labeled nerve binding probe (F-NP41) can improve intraoperative visualization of chronically (up to 9 months) denervated nerves. In a mouse model for the repair of chronically denervated facial nerves, the intraoperative use of fluorescent labeling decreased time to nerve identification by 40% compared to surgeries performed under WL alone. Cumulative functional post-operative recovery was also significantly improved in the fluorescence guided group as determined by quantitatively tracking of the recovery of whisker movement at time intervals for 6 weeks post-repair. To our knowledge, this is the first description of an injectable probe that increases visibility of chronically denervated nerves during surgical repair in live animals. Future translation of this probe may improve functional outcome for patients with chronic denervation undergoing surgical repair. PMID:25751149

  6. Imbalance in chemical space: How to facilitate the identification of protein-protein interaction inhibitors

    PubMed Central

    Kuenemann, Mélaine A.; Labbé, Céline M.; Cerdan, Adrien H.; Sperandio, Olivier

    2016-01-01

    Protein-protein interactions (PPIs) play vital roles in life and provide new opportunities for therapeutic interventions. In this large data analysis, 3,300 inhibitors of PPIs (iPPIs) were compared to 17 reference datasets of collectively ~566,000 compounds (including natural compounds, existing drugs, active compounds on conventional targets, etc.) using a chemoinformatics approach. Using this procedure, we showed that comparable classes of PPI targets can be formed using either the similarity of their ligands or the shared properties of their binding cavities, constituting a proof-of-concept that not only can binding pockets be used to group PPI targets, but that these pockets certainly condition the properties of their corresponding ligands. These results demonstrate that matching regions in both chemical space and target space can be found. Such identified classes of targets could lead to the design of PPI-class-specific chemical libraries and therefore facilitate the development of iPPIs to the stage of drug candidates. PMID:27034268

  7. 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.

  8. Identification of Proteins in Promastigote and Amastigote-like Leishmania Using an Immunoproteomic Approach

    PubMed Central

    Coelho, Vinicio T. S.; Oliveira, Jamil S.; Valadares, Diogo G.; Chávez-Fumagalli, Miguel A.; Duarte, Mariana C.; Lage, Paula S.; Soto, Manuel; Santoro, Marcelo M.; Tavares, Carlos A. P.; Fernandes, Ana Paula; Coelho, Eduardo A. F.

    2012-01-01

    Background The present study aims to identify antigens in protein extracts of promastigote and amastigote-like Leishmania (Leishmania) chagasi syn. L. (L.) infantum recognized by antibodies present in the sera of dogs with asymptomatic and symptomatic visceral leishmaniasis (VL). Methodology/Principal Findings Proteins recognized by sera samples were separated by two-dimensional electrophoresis (2DE) and identified by mass spectrometry. A total of 550 spots were observed in the 2DE gels, and approximately 104 proteins were identified. Several stage-specific proteins could be identified by either or both classes of sera, including, as expected, previously known proteins identified as diagnosis, virulence factors, drug targets, or vaccine candidates. Three, seven, and five hypothetical proteins could be identified in promastigote antigenic extracts; while two, eleven, and three hypothetical proteins could be identified in amastigote-like antigenic extracts by asymptomatic and symptomatic sera, as well as a combination of both, respectively. Conclusions/Significance The present study represents a significant contribution not only in identifying stage-specific L. infantum molecules, but also in revealing the expression of a large number of hypothetical proteins. Moreover, when combined, the identified proteins constitute a significant source of information for the improvement of diagnostic tools and/or vaccine development to VL. PMID:22272364

  9. A Novel Dual-Pressure Linear Ion Trap Mass Spectrometer Improves the Analysis of Complex Protein Mixtures

    PubMed Central

    Pekar, Tonya; Blethrow, Justin D.; Schwartz, Jae C.; Merrihew, Gennifer E.; MacCoss, Michael J.; Swaney, Danielle L.; Russell, Jason D.; Coon, Joshua J.; Zabrouskov, Vlad

    2009-01-01

    The considerable progress in high throughput proteomics analysis via liquid chromatography-electrospray ionization-tandem mass spectrometry over the last decade has been fueled to a large degree by continuous improvements in instrumentation. High throughput identification experiments are based on peptide sequencing and are largely accomplished through the use of tandem mass spectrometry, with ion trap and trap-based instruments having become broadly adopted analytical platforms. To satisfy increasingly demanding requirements for depth of characterization and throughput, we present a newly developed dual-pressure linear ion trap mass spectrometer (LTQ Velos) that features increased sensitivity, afforded by a new source design, and demonstrates practical cycle times two times shorter than that of an LTQ XL, while improving or maintaining spectral quality for MS/MS fragmentation spectra. These improvements resulted in a substantial increase in the detection and identification of both proteins and unique peptides from the complex proteome of Caenorhabditis elegans, as compared to existing platforms. The greatly increased ion flux into the mass spectrometer in combination with improved isolation of low-abundance precursor ions resulted in increased detection of low-abundance peptides. These improvements cumulatively resulted in a substantially greater penetration into the baker’s yeast (Saccharomyces cerevisiae) proteome compared to LTQ XL. Alternatively, faster cycle times on the new instrument allowed for higher throughput for a given depth of proteome analysis, with more peptides and proteins identified in 60 min using an LTQ Velos than in 180 min using an LTQ XL. When mass analysis was carried out with resolution in excess of 25,000 FWHM, it became possible to isotopically resolve a small intact protein and its fragments, opening possibilities for top down experiments. PMID:19689114

  10. 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.

  11. Identification of immunoreactive antigens in membrane proteins enriched fraction from Francisella tularensis LVS.

    PubMed

    Janovská, Sylva; Pávková, Ivona; Hubálek, Martin; Lenco, Juraj; Macela, Ales; Stulík, Jirí

    2007-02-15

    Francisella tularensis is a Gram-negative, facultative intracellular bacterium causing disease in many mammalian species. The low infectious dose of F. tularensis and the ease of air-borne transmission are the main features responsible for the classification of this bacterium as a potential biological weapon. The live attenuated strain of F. tularensis live vaccine strain (LVS) is currently only effective vaccine against tularemia, however, this type of vaccine has not been approved for human use. In the presented study, sub-immunoproteome analysis was performed to search for new immunogenic proteins of Francisella tularensis LVS grown under different conditions. By this approach 35 immunoreactive antigens were identified, 19 of them showed to be novel immunogens. In conclusion, sub-immunoproteome analysis resulted in successful identification of novel immunoreactive proteins. PMID:17241671

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

    PubMed Central

    Zaki, Nazar; Mora, Antonio

    2014-01-01

    High-throughput AP-MS methods have allowed the identification of many protein complexes. However, most post-processing methods of this type of data have been focused on detection of protein complexes and not its subcomplexes. Here, we review the results of some existing methods that may allow subcomplex detection and propose alternative methods in order to detect subcomplexes from AP-MS data. We assessed and drew comparisons between the use of overlapping clustering methods, methods based in the core-attachment model and our own prediction strategy (TRIBAL). The hypothesis behind TRIBAL is that subcomplex-building information may be concealed in the multiple edges generated by an interaction repeated in different contexts in raw data. The CACHET method offered the best results when the evaluation of the predicted subcomplexes was carried out using both the hypergeometric and geometric scores. TRIBAL offered the best performance when using a strict meet-min score. PMID:24584908

  13. 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

  14. 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.

  15. 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.

  16. Improving photoelectron counting and particle identification in scintillation detectors with Bayesian techniques

    NASA Astrophysics Data System (ADS)

    Akashi-Ronquest, M.; Amaudruz, P.-A.; Batygov, M.; Beltran, B.; Bodmer, M.; Boulay, M. G.; Broerman, B.; Buck, B.; Butcher, A.; Cai, B.; Caldwell, T.; Chen, M.; Chen, Y.; Cleveland, B.; Coakley, K.; Dering, K.; Duncan, F. A.; Formaggio, J. A.; Gagnon, R.; Gastler, D.; Giuliani, F.; Gold, M.; Golovko, V. V.; Gorel, P.; Graham, K.; Grace, E.; Guerrero, N.; Guiseppe, V.; Hallin, A. L.; Harvey, P.; Hearns, C.; Henning, R.; Hime, A.; Hofgartner, J.; Jaditz, S.; Jillings, C. J.; Kachulis, C.; Kearns, E.; Kelsey, J.; Klein, J. R.; Kuźniak, M.; LaTorre, A.; Lawson, I.; Li, O.; Lidgard, J. J.; Liimatainen, P.; Linden, S.; McFarlane, K.; McKinsey, D. N.; MacMullin, S.; Mastbaum, A.; Mathew, R.; McDonald, A. B.; Mei, D.-M.; Monroe, J.; Muir, A.; Nantais, C.; Nicolics, K.; Nikkel, J. A.; Noble, T.; O'Dwyer, E.; Olsen, K.; Orebi Gann, G. D.; Ouellet, C.; Palladino, K.; Pasuthip, P.; Perumpilly, G.; Pollmann, T.; Rau, P.; Retière, F.; Rielage, K.; Schnee, R.; Seibert, S.; Skensved, P.; Sonley, T.; Vázquez-Jáuregui, E.; Veloce, L.; Walding, J.; Wang, B.; Wang, J.; Ward, M.; Zhang, C.

    2015-05-01

    Many current and future dark matter and neutrino detectors are designed to measure scintillation light with a large array of photomultiplier tubes (PMTs). The energy resolution and particle identification capabilities of these detectors depend in part on the ability to accurately identify individual photoelectrons in PMT waveforms despite large variability in pulse amplitudes and pulse pileup. We describe a Bayesian technique that can identify the times of individual photoelectrons in a sampled PMT waveform without deconvolution, even when pileup is present. To demonstrate the technique, we apply it to the general problem of particle identification in single-phase liquid argon dark matter detectors. Using the output of the Bayesian photoelectron counting algorithm described in this paper, we construct several test statistics for rejection of backgrounds for dark matter searches in argon. Compared to simpler methods based on either observed charge or peak finding, the photoelectron counting technique improves both energy resolution and particle identification of low energy events in calibration data from the DEAP-1 detector and simulation of the larger MiniCLEAN dark matter detector.

  17. MALDI-in source decay applied to mass spectrometry imaging: a new tool for protein identification.

    PubMed

    Debois, Delphine; Bertrand, Virginie; Quinton, Loïc; De Pauw-Gillet, Marie-Claire; De Pauw, Edwin

    2010-05-15

    Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) imaging is a powerful technique giving access to the distribution of a large range of biomolecules directly from a tissue section, allowing, for example, the discovery of new pathological biomarkers. Nevertheless, one main difficulty lies in the identification of the detected species, especially proteins. MALDI-in source decay (ISD) is used to fragment ions directly in the mass spectrometer ion source. This technique does not require any special sample treatment but only the use of a specific MALDI matrix such as 2,5-dihydroxybenzoic acid or 1,5-diaminonaphthalene. MALDI-ISD is generally employed on classical, purified samples, but here we demonstrate that ISD can also be performed directly on mixtures and on a tissue slice leading to fragment ions, allowing the identification of major proteins without any further treatment. On a porcine eye lens slice, de novo sequencing was even performed. Crystallins not yet referenced in databases were identified by sequence homology with other mammalian species. On a mouse brain slice, we demonstrate that results obtained with ISD are comparable and even better than those obtained with a classical in situ digestion. PMID:20397712

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

    PubMed

    Delmonico, Lucas; Areias, Vivian Rabello; Pinto, Rodrigo César; Matos, Cintia Da Silva; Rosa, Marco Felipe Franco; De Azevedo, Carolina Maria; Alves, Gilda

    2015-07-01

    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

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

    PubMed

    Wöhlbrand, Lars; Trautwein, Kathleen; Rabus, Ralf

    2013-10-01

    The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell. Within the past decade, proteomic methods became increasingly accessible to microbiologists mainly due to the robustness of analytical methods (e.g. 2DE), and affordability of mass spectrometers and their relative ease of use. This review provides an overview on the complex portfolio of state-of-the-art proteomics and highlights the basic principles of key methods, ranging from sample preparation of laboratory or environmental samples, via protein/peptide separation (gel-based or gel-free) and different types of mass spectrometric protein/peptide analyses, to protein identification and abundance determination. PMID:23894077

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

    PubMed Central

    Liu, Pei; Zhang, Huoming; Yu, Boying; Xiong, Liming; Xia, Yiji

    2015-01-01

    Accumulation of reactive oxygen species (ROS) is one of the early defense responses against pathogen infection in plants. The mechanism about the initial and direct regulation of the defense signaling pathway by ROS remains elusive. Perturbation of cellular redox homeostasis by ROS is believed to alter functions of redox-sensitive proteins through their oxidative modifications. Here we report an OxiTRAQ-based proteomic study in identifying proteins whose cysteines underwent oxidative modifications in Arabidopsis cells during the early response to salicylate or flg22, two defense pathway elicitors that are known to disturb cellular redox homeostasis. Among the salicylate- and/or flg22-responsive redox-sensitive proteins are those involved in transcriptional regulation, chromatin remodeling, RNA processing, post-translational modifications, and nucleocytoplasmic shuttling. The identification of the salicylate-/flg22-responsive redox-sensitive proteins provides a foundation from which further study can be conducted toward understanding biological significance of their oxidative modifications during the plant defense response. PMID:25720653

  1. 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.

  2. A clickable glutathione approach for identification of protein glutathionylation in response to glucose metabolism.

    PubMed

    Samarasinghe, Kusal T G; Munkanatta Godage, Dhanushka N P; Zhou, Yani; Ndombera, Fidelis T; Weerapana, Eranthie; Ahn, Young-Hoon

    2016-07-19

    Glucose metabolism and mitochondrial function are closely interconnected with cellular redox-homeostasis. Although glucose starvation, which mimics ischemic conditions or insufficient vascularization, is known to perturb redox-homeostasis, global and individual protein glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. In this report, we use our clickable glutathione approach, which forms clickable glutathione (azido-glutathione) by using a mutant of glutathione synthetase (GS M4), for detection and identification of protein glutathionylation in response to glucose starvation. We found that protein glutathionylation is readily induced in HEK293 cells in response to low glucose concentrations when mitochondrial reactive oxygen species (ROS) are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Proteomic and biochemical analysis identified over 1300 proteins, including SMYD2, PP2Cα, and catalase. We further showed that PP2Cα is glutathionylated at C314 in a C-terminal domain, and PP2Cα C314 glutathionylation disrupts the interaction with mGluR3, an important glutamate receptor associated with synaptic plasticity. PMID:27216279

  3. 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

  4. 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.

  5. Characterization of the human submandibular/sublingual saliva glycoproteome using lectin affinity chromatography coupled to Multidimensional Protein Identification Technology

    PubMed Central

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

    2011-01-01

    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. PMID:21936497

  6. 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. PMID:24795876

  7. 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

  8. Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling

    PubMed Central

    Gookin, Timothy E; Kim, Junhyong; Assmann, Sarah M

    2008-01-01

    Background The classic paradigm of heterotrimeric G-protein signaling describes a heptahelical, membrane-spanning G-protein coupled receptor that physically interacts with an intracellular Gα subunit of the G-protein heterotrimer to transduce signals. G-protein coupled receptors comprise the largest protein superfamily in metazoa and are physiologically important as they sense highly diverse stimuli and play key roles in human disease. The heterotrimeric G-protein signaling mechanism is conserved across metazoa, and also readily identifiable in plants, but the low sequence conservation of G-protein coupled receptors hampers the identification of novel ones. Using diverse computational methods, we performed whole-proteome analyses of the three dominant model plant species, the herbaceous dicot Arabidopsis thaliana (mouse-eared cress), the monocot Oryza sativa (rice), and the woody dicot Populus trichocarpa (poplar), to identify plant protein sequences most likely to be GPCRs. Results Our stringent bioinformatic pipeline allowed the high confidence identification of candidate G-protein coupled receptors within the Arabidopsis, Oryza, and Populus proteomes. We extended these computational results through actual wet-bench experiments where we tested over half of our highest ranking Arabidopsis candidate G-protein coupled receptors for the ability to physically couple with GPA1, the sole Gα in Arabidopsis. We found that seven out of eight tested candidate G-protein coupled receptors do in fact interact with GPA1. We show through G-protein coupled receptor classification and molecular evolutionary analyses that both individual G-protein coupled receptor candidates and candidate G-protein coupled receptor families are conserved across plant species and that, in some cases, this conservation extends to metazoans. Conclusion Our computational and wet-bench results provide the first step toward understanding the diversity, conservation, and functional roles of plant

  9. Identification and Characterization of a Cleavage Site in the Proteolysis of Orf Virus 086 Protein

    PubMed Central

    Wang, Xiaoping; Xiao, Bin; Zhang, Jiafeng; Chen, Daxiang; Li, Wei; Li, Ming; Hao, Wenbo; Luo, Shuhong

    2016-01-01

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

  10. 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

  11. Easier, Better, Faster, Stronger: Improved Methods for RNA-Protein Interaction Studies.

    PubMed

    Haque, Nazmul; Hogg, J Robert

    2016-06-01

    The RNA field has been revolutionized by methods that allow genome-scale identification of RNA-protein interaction sites. Two reports now introduce more efficient approaches, opening the technology to wider adoption (Van Nostrand et al., 2016; Zarnegar et al., 2016). PMID:27259196

  12. Identification of differentially expressed proteins from primary versus metastatic pancreatic cancer cells using subcellular proteomics.

    PubMed

    McKinney, Kimberly Q; Lee, Jin-Gyun; Sindram, David; Russo, Mark W; Han, David K; Bonkovsky, Herbert L; Hwang, Sun-Il

    2012-01-01

    Pancreatic cancer is an aggressive disease with nearly equal yearly rates of diagnosis and death. Current therapies have failed to improve outcomes due to rapid disease progression and late stage at presentation. Recently, pathways involved in progression and metastasis have been elucidated; however, new knowledge has not generated more effective therapies. We report on the use of subcellular fractionation and liquid chromatography (LC)-mass spectrometry to identify 3,907 proteins in four pancreatic cancer cell lines, 540 of which are unique to primary cancer cells, and 487 unique to cells derived from metastatic sites. Statistical analysis identified 134 proteins significantly differentially expressed between the two populations. The subcellular localization of these proteins was determined and expression levels for four targets were validated using western blot techniques. These identified proteins can be further investigated to determine their roles in progression and metastasis and may serve as therapeutic targets in the development of more effective treatments for pancreatic cancer. PMID:22990105

  13. Utilization of HPASubC for the Identification of Sinusoid-Specific Proteins in the Liver.

    PubMed

    Anene, Divine-Favour; Rosenberg, Avi Z; Kleiner, David E; Cornish, Toby C; Halushka, Marc K

    2016-05-01

    Mass spectrometry-based proteomes of human organs and tissues are powerful tools but fail to capture protein localization and expression at the cellular level. For example, the proteome signal in liver represents the combined protein expression across diverse cellular constituents that include hepatocytes, Kupffer cells, endothelial cells, and others. We utilized HPASubC and the Human Protein Atlas (HPA) to identify the sinusoidal component of protein liver expression to further subset and organize this homogeneous signal. We evaluated 51 109 liver images covering 13 197 proteins from the HPA and discovered 1054 proteins that were exclusive to sinusoidal cells. Sinusoidal staining patterns were identified in a Kupffer cell (n = 247), endothelial cell (n = 358), or lymphocyte (n = 86) specific pattern. Two-hundred and thirty-nine of these proteins were not present in the NextProt or Human Proteome Map liver data sets, potentially expanding our knowledge of the liver proteome. We additionally demonstrate unique endothelial cell expression patterns that distinguish between portal vein, hepatic artery, capillary sinusoids, and central vein regions. These findings significantly improve our understanding of the liver proteome with insight into the endothelial complexity across the hepatic vascular network. PMID:27005832

  14. Improving Protein Fold Recognition by Deep Learning Networks

    PubMed Central

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-01-01

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl’s benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold. PMID:26634993

  15. Improving Protein Fold Recognition by Deep Learning Networks.

    PubMed

    Jo, Taeho; Hou, Jie; Eickholt, Jesse; Cheng, Jianlin

    2015-01-01

    For accurate recognition of protein folds, a deep learning network method (DN-Fold) was developed to predict if a given query-template protein pair belongs to the same structural fold. The input used stemmed from the protein sequence and structural features extracted from the protein pair. We evaluated the performance of DN-Fold along with 18 different methods on Lindahl's benchmark dataset and on a large benchmark set extracted from SCOP 1.75 consisting of about one million protein pairs, at three different levels of fold recognition (i.e., protein family, superfamily, and fold) depending on the evolutionary distance between protein sequences. The correct recognition rate of ensembled DN-Fold for Top 1 predictions is 84.5%, 61.5%, and 33.6% and for Top 5 is 91.2%, 76.5%, and 60.7% at family, superfamily, and fold levels, respectively. We also evaluated the performance of single DN-Fold (DN-FoldS), which showed the comparable results at the level of family and superfamily, compared to ensemble DN-Fold. Finally, we extended the binary classification problem of fold recognition to real-value regression task, which also show a promising performance. DN-Fold is freely available through a web server at http://iris.rnet.missouri.edu/dnfold. PMID:26634993

  16. 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.

  17. 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

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

    PubMed Central

    Walsh, Naomi; O'Donovan, Norma; Kennedy, Susan; Henry, Michael; Meleady, Paula; Clynes, Martin; Dowling, Paul

    2009-01-01

    Background Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer. Materials and methods Using 2D-DIGE followed by MALDI-TOF MS, two clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with high and low invasive capacities were incubated on matrigel 24 hours prior to analysis to stimulate cell-ECM contact and mimic in vivo interaction with the basement membrane. Results Sixty proteins were identified as being differentially expressed (> 1.2 fold change and p ≤ 0.05) between Clone #3 and Clone #8. Proteins found to have higher abundance levels in the highly invasive Clone #3 compared to the low invasive Clone #8 include members of the chaperone activity proteins and cytoskeleton constituents whereas metabolism-associated and catalytic proteins had lower abundance levels. Differential protein expression levels of ALDH1A1, VIM, STIP1 and KRT18 and GAPDH were confirmed by immunoblot. Using RNAi technology, STIP1 knockdown significantly reduced invasion and proliferation of the highly invasive Clone #3. Knockdown of another target, VIM by siRNA in Clone #3 cells also resulted in decreased invasion abilities of Clone #3. Elevated expression of STIP1 was observed in pancreatic tumour tissue compared to normal pancreas, whereas ALDH1A1 stained at lower levels in pancreatic tumours, as detected by immunohistochemistry. Conclusion Identification of targets which play a role in the highly invasive phenotype of pancreatic cancer may help to understand the biological behaviour, the rapid progression of this cancer and may be of importance in the development of new therapeutic strategies for pancreatic cancer. PMID:19216797

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

    PubMed Central

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

    2013-01-01

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

  20. 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

  1. Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin.

    PubMed

    Beyeler, Michael; Schild, Christof; Lutz, Roman; Chiquet, Matthias; Trueb, Beat

    2010-04-15

    Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell

  2. P185-M Protein Identification and Validation of Results in Workflows that Integrate over Various Instruments, Datasets, Search Engines

    PubMed Central

    Hufnagel, P.; Glandorf, J.; Körting, G.; Jabs, W.; Schweiger-Hufnagel, U.; Hahner, S.; Lubeck, M.; Suckau, D.

    2007-01-01

    Analysis of complex proteomes often results in long protein lists, but falls short in measuring the validity of identification and quantification results on a greater number of proteins. Biological and technical replicates are mandatory, as is the combination of the MS data from various workflows (gels, 1D-LC, 2D-LC), instruments (TOF/TOF, trap, qTOF or FTMS), and search engines. We describe a database-driven study that combines two workflows, two mass spectrometers, and four search engines with protein identification following a decoy database strategy. The sample was a tryptically digested lysate (10,000 cells) of a human colorectal cancer cell line. Data from two LC-MALDI-TOF/TOF runs and a 2D-LC-ESI-trap run using capillary and nano-LC columns were submitted to the proteomics software platform ProteinScape. The combined MALDI data and the ESI data were searched using Mascot (Matrix Science), Phenyx (GeneBio), ProteinSolver (Bruker and Protagen), and Sequest (Thermo) against a decoy database generated from IPI-human in order to obtain one protein list across all workflows and search engines at a defined maximum false-positive rate of 5%. ProteinScape combined the data to one LC-MALDI and one LC-ESI dataset. The initial separate searches from the two combined datasets generated eight independent peptide lists. These were compiled into an integrated protein list using the ProteinExtractor algorithm. An initial evaluation of the generated data led to the identification of approximately 1200 proteins. Result integration on a peptide level allowed discrimination of protein isoforms that would not have been possible with a mere combination of protein lists.

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

    PubMed Central

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

    2016-01-01

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

  4. Identification of a tripartite import signal in the Ewing Sarcoma protein (EWS)

    SciTech Connect

    Shaw, Debra J.; Morse, Robert; Todd, Adrian G.; Eggleton, Paul; Lorson, Christian L.; Young, Philip J.

    2009-12-25

    The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.

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

    PubMed

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

    2016-01-01

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

  6. Identification of LACTB2, a metallo-β-lactamase protein, as a human mitochondrial endoribonuclease

    PubMed Central

    Levy, Shiri; Allerston, Charles K.; Liveanu, Varda; Habib, Mouna R.; Gileadi, Opher; Schus